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Patent Searching and Data


Title:
HUMAN TOXICOLOGICALLY RELEVANT GENES AND ARRAYS
Document Type and Number:
WIPO Patent Application WO/2003/016500
Kind Code:
A2
Abstract:
The invention provides for a set of toxicologically relevant human genes which may be used to predict toxicological responses on a cellular, organ, or system level. Methods of identifying, selecting, isolating, and evaluating toxicologically relevant human genes are provided. Arrays comprising human genes that are useful for toxicological screening of drugs, pharmaceutical compounds, or chemicals are also provided. The human array provides a method by which toxicological responses can analyzed in a rapid and efficient manner. Methods of identifying and isolating human genes for a human array are also provided. Further, methods of making a human gene array and using a human gene array are disclosed. These methods are also useful for isolating human genes and for discovery of novel human genes. Also provided is a database of human toxicological responses which are useful for predicting toxicological response to agents on a cellular level, organ-by-organ, and system-by-system. Methods of obtaining such databases containing human toxicological data and methods of use are also provided herein. Further, algorithms which allows for evaluation and/or 1 analysis of toxicological data are also provided herein.

Inventors:
NEFT ROBIN E (US)
DUNN ROBERT T II (US)
ADKINS KARISSA (US)
PICKETT GAVIN G (US)
KIER LARRY D (US)
SCHMEISER KATJA (BE)
ALEN PHILLIPPE (BE)
Application Number:
PCT/US2002/026514
Publication Date:
February 27, 2003
Filing Date:
August 16, 2002
Export Citation:
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Assignee:
PHASE 1 MOLECULAR TOXICOLOGY I (US)
NEFT ROBIN E (US)
DUNN ROBERT T II (US)
ADKINS KARISSA (US)
PICKETT GAVIN G (US)
KIER LARRY D (US)
SCHMEISER KATJA (BE)
ALEN PHILLIPPE (BE)
International Classes:
C12Q1/68; C12Q1/6883; (IPC1-7): C12N/
Foreign References:
US6160105A2000-12-12
US20020192671A12002-12-19
Other References:
See also references of EP 1427850A2
Attorney, Agent or Firm:
Johnston, Madeline I. (755 Page Mill Road Palo Alto, CA, US)
Download PDF:
Claims:
CLAIMS
1. A method for determining a toxicological response to an agent, the method comprising: (a) exposing cells to an agent or isolating cells from a human subject who was exposed to an agent; (b) obtaining a test expression profile of one or more human toxic response genes in the cells selected from the group consisting of the genes corresponding to the gene sequences in Tables 1,2 or 5; and (c) comparing the test expression profile to a reference gene expression profile of human toxic response genes indicative of toxicity, thereby to determine the presence of a toxic response to the agent.
2. The method of claim 1, wherein the toxicity of the agent is evaluated by determining if there is a significant correlation between the test expression profile and the reference expression profile.
3. The method of claim 1, wherein the toxicity of the agent is evaluated by determining if there is a pattern of expression that is predictive of specific toxicity endpoints as determined by predictive models.
4. The method of claim 1, wherein the cells are derived from pancreas, thyroid, liver, lung, heart, kidney, spleen, testes, thymus, skin, bone, muscle, gastrointestinal tract, brain or nucleated cells present in the blood.
5. The method of claim 1, wherein the reference gene expression profile indicative of toxicity is stored in a database.
6. The method of claim 1, wherein the cells are from an organ, tissue, or cell culture, and wherein the test expression profile of human toxic response genes is compared to the reference gene expression profile to determine the presence of a toxicological response in the organ, tissue or cell culture.
7. The method of claim 1, wherein the agent is a pharmaceutical or industrial composition, and wherein the method comprises a method of screening the agent to determine a toxicological response of the pharmaceutical or industrial agent in the cells.
8. The method of claim 1, wherein the cells exposed in step a) are cells obtained from a human tissue, blood, sweat, saliva, urine or fecal sample.
9. The method of claim 1, wherein the agent is exposed to the cells at various concentrations or for various amounts of time or cells are derived from individuals exposed the agent at various concentrations by various routes of exposure for various amounts of time.
10. The method of claim 1, wherein, in step b) the test expression profile of at least two human toxic response genes in the cells is obtained.
11. The method of claim 1, wherein, in step b) the test expression profile of at least 10 human toxic response genes in the cells is obtained.
12. The method of claim 1, wherein, in step b) the test expression profile of at least 20 human toxic response genes in the cells is obtained.
13. The method of claim 1, wherein, in step b) the test expression profile of at least 50 human toxic response genes in the cells is obtained.
14. The method of claim 1, wherein, in step b) the test expression profile of at least 200 human toxic response genes in the cells is obtained.
15. The method of claim 1, wherein, in step b) the test expression profile of at least 500 human toxic response genes in the cells is obtained.
16. The method of claim 1, wherein the gene expression profile is obtained using realtime polymerase chain reaction, Rnase protection, Northern blot, electrochemical hybridization detection, branchedchain to quantitatively detect levels of messenger RNA.
17. The method of claim 1, wherein the gene expression profile is obtained by detecting protein expression.
18. An array comprising one or more polynucleotides selected from the group consisting of the genes corresponding to the partial sequences in Tables 1,2, or 5, or fragments of at least 20 nucleotides thereof.
19. The array of claim 18, wherein genes corresponding to the partial gene sequences are responsive in cells derived from kidney, liver, spleen, heart, lung, testis, thymus, skin, bone, muscle, gastrointestinal tract, pancreas, thymus or brain.
20. The array of claim 18, wherein the array includes at least 25 of the polynucleotides.
21. The array of claim 18, wherein the array includes at least 50 of the polynucleotides.
22. The array of claim 18, wherein the array includes at least 200 of the polynucleotides.
23. The array of claim 18, wherein the array includes at least 500 of the polynucleotides.
24. The array of claim 18, wherein the polynucleotide fragment comprises at least 300 nucleotides.
25. An array comprising one or more polynucleotides which are homologous to the polynucleotides of the array of claim 18.
26. The method of claim 1, wherein the gene expression profile is obtained using an array of claim 18.
27. A method of determining if a gene putatively identified to be a toxic response gene plays a definite role in toxic response pathways by determining the expression profile of that gene after exposure of cells or a human subject to a known toxic pharmaceutical or industrial agent. The method comprising: (a) exposing cells to an agent or isolating cells from a human subject who was exposed to an agent; (b) obtaining the test gene expression profile for a putatively identified toxic response gene after exposure to a known toxic pharmaceutical or industrial agent c) comparing the test profile to the expression profile for a gene with a similar function or comparing the test profile to the expression profile of that gene after exposure to other known toxic compounds. Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 1 Table 2 Size of Size of Human Human left Rat gene Target the Human Target the right primer Gene name Rat whole Rat right Rat left Gene name primer (5'end) rat accession region PCR accession Length region PCR, size primer primer human (3 end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation agcgcacacct acacacacagt progesterone ttctgtatgtccc tgcaccaagag gttcctgtcctg ggccccaaatg binding tccctcatttca caaaggatcaa 25DX U63315 1885 10231486 464 g protein XM010363 1878 10001484 485 ctt (HPR6. 6) like 25DX 8ttgatttgggatg tgcagcagcttc gggaaggtgct gctatcgtgccc oxo uanine g gg g gagg 8oxoguanine glycosylase glycosylase DNA glycosylase glycosylase Aflatoxin cttggtcaaagg accctctgcaa aflatoxin aatgccaggctt ctccaactatgc DNA g at,, t glycosylaseSlycosylase . . cttggtcaaagg accctctgcaa aflatoxin aatgccaggctt ctccaactatgc Bl aldehyde X74673 1269 5181007 490 gt ggat reductase, 12067 1247 S101045 536 gattaaaggcgtagcctgggaag réductase member A3 ggctacatctgc actcagtggatg Homo caatataatggc acatcaacott ttatctttggccc gtgtaccagtg sapiens tcccacgtgctg gactotgcgga Ciliary ca gcaa ciliary g g neurotrophi X17457 1101 229682 454 neurotrophic NM000614 603 140583 444 c factor factor (CNTF), mRNA, Alphatggagatgma tcagcagagct Alphactcccaaagsa agotgaccts fetoprotein X02361 1990 13591868 510 aacocccaaag gatagacctga fetoprotein XM 003498 2032 13691872 504 gcacgagtttttcggagctgatg cctc ca Complemen gctccacccac ggacttatccag Complement ggccagtgctc ccagcctggag t component X52477 5066 44494929 481 gtgtcct ccgggg component XM 009010 5067 44764955 480 cacccaagtgt cagtcaagg C3 C3 c ac Table 2 Size of Size of Human Human left Rat gene Target the Human Target the right primer Ratgene .., Target the ,... ., Human Target the j""\ Gene name Rat whole Rat right Rat left Gene name primer (5'end) accession region PCR accession Length region Per number (rat) product number (human) product reverse orientation () p (human) product (rat) (human) orientation orientation Aquaporinatgcactaagc ccatcatcggct gtgcaggctctc catcctgggctc 2 D13906 1393 7191221 503 acagtccccca ccctcctctaca Aquaporin2 M006751 1443 7061214 509 cagagtttccct cctcctctacaa gaagg act c c tctcatggtctcc ccaaggagctc Homo tctcatggtctcc ccaaggagctc Endothelingacctggtttgt cagaaacagca sapiens gacctggtttgt cagaaacagca M64711 check 242735 494 gtc endothelin 1 p01955 1251 381885 505 gtc (DNA), mRNA cttccagatggt ggggcctttttgt ggtgagcactc ggggcccttttg Bax (alpha) U49729 579 67567 501 gagcgagg tacaggg Bax (alpha) L22473 579 67546 480 ccgccacaaag cttcagggttt at Canalicular tagaaggaacc tagatgttgcct ttctcaatgcca gacctccgaga multispecifi tctgttggacag ccattggactgCagcc gar g gg g gg g gcttccttagcc gaagctgacc multispecific c organic D86086 4880 41114609 499 c U63970 4868 41514653 503 a tca organic anion transporter Carbonic aattccccacca aggtccatgctg aagaggcactg taggtcaatgci : anhydrase M22413 988 391895 505 gaggcac agaggtgg anhydrase IIr XM_005207 2356 9061401 496 ggggctcgttct gagagggggt cut act Dopamine Dopamine receptor D2 x53278 1596 10981580 483 agaaggccaa atgggcacgeceptor D2 X51362 2625 11321611 480 gaggccgatcc gaagaatgggti ac a' gcttcttgccaat atgccgattgct ttgggtctttgca actgttagcca, cR Caspase 6 AF025670 1146 5171008 492 tgcacct tcctgtgt Caspase 6 NM 001226 1545 391880 490 aaagtccactc goagatgooga : tt Table 2 Size of Size of Human Human left Rat gene Target the Human Target the right primer Gene name Rat whole Rat right Rat left Gene name primer (5'end) accession region PCR accession Length region PCR, rat size primer primer human (3 end) forward number (rat) product number (human) production reverse orientation orientation ccteagcgcgt ggtacattgagc ggctgctcttta tgaaccatcctg Cathepsin S L03201 1330 7981206 409 ccttgcttacag tccccttcggc Cathepsin S M002028 1257 8101229 420 actcctggcctc tactcagaatgt a gaa Cellular tagtctgcccct tgctaaaagctc Cellular gacccccagaa attgctcaaagt retinoic acid ctctggca tgggggtg retinoic acid gtgactggggt gctgggggtga bmdmg bmdmgaag at protein 2 rotwein 3 tacttcagtgcc caaagaagcca ccgccagitca cctctcaaagcc protein M83176 693 134627 494 cgccagtt ctggaagcc Creactive p01859 1631 228707 480 ggacattagga ttcactgtgtgc protein protein ct ca caagcctcgta cgtggatgtcac gcacagaggc cccagaggtca |P4502Ct I U33173 1856 9681477 510 aaaaggggg agctaaagtcc I'on XM011937 1473 9481438 491 aaatccattgac cagctaaagts arc can aac cagg gaggcacttgc gttgctggggg cgggaagaact ggaatgttccac nase 2 U04300 1815 12581751 494 gttgatggt aaggaatgt XM001734 4465 14051888 484 tgcattgatggt ccgcagtacagi go axa tccaccccgttt atccctgacatc cctttggagag catctttgcc CXCR4 U90610 1050 496999 504 ccctttggaaa atcttcgccga CXCR4 XM002327 1582 507986 480 gatcttgagget cgtcagtgaggf gg c 1433 zeta vector vector 1433 zeta gccatcatctca gggatcattg AA858662 620 1620 620 primer primer BC003623 1872 12241738 515 agttatttccctg tccagtcacag prutcin Droteiii,,. s tut go Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (e' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation ADP vector vector ADP actgagtgaag ctggagcactgt ribosylation primer primer ribosylation gatgaggccca cctctaacgcca factorlie AA817697 424 1424 424 factorlie XM_006528 900 400868 469 cag t protein protein ARL184 ARL184 vector vector agcctgaccta ataaaatggca Alpha Alpha AA925421 420 1420 420 primer primer M58569 4330 18312314 484 aatgtcaactga gatgaggccgg fibrinogen fibrinogen gaca aag vector vector tgttcacacata agtgacacgc Alpha Alpha AA875070 560 1560 560 primer primer XM_002247 956 436934 499 catcaaacagg gctctccaccac prothymosin prothymosin cca vector vector cccactttattag tcaagggacttg Arginisnosuc Argininosucc AA818673 514 1514 514 primer primer XM_012714 1502 10101496 487 ggggcaggtgt ccagcacctae cinate lyase inate lyase g aa Arginosucci vector vector Arginosuccin catttttataagct attgcagatcc nate AA924544 171 1171 171 primer primer ate XM_005578 1554 13471533 187 ccccaccgcag ccaagtacag synthetase 1 synthetase 1 c gc vector vector tgggtttttattctc ttctgtgacttgg Dynamin1 Dynamin1 AI071582 445 1445 445 primer primer XM_011757 1671 12001657 458 agccgaggga cagtggctccc (D100) (D100) c c vector vector cagcaggagtg ggaaatggaaa Arylsulfatas Arylsulfatase AI072268 465 1465 463 primer primer XM_003920 3528 26143099 486 ttgcagattttat ctcctcacggg e B B ca cta vector vector aggtcccttact aagcctttgtgg Cofilin AA859476 468 1468 468 primer primer Cofilin XM_006507 1060 5411045 505 ggtcctgcttcc gccccttctgg a Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (e' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Complemen vector vector tccaatatggca tgatacatgcat Complement t factor I AA900791 473 1473 473 primer primer Y00318 1965 14211936 516 taaactctgtgg cgtttctggctg factor I (CFI) (CFI) aga g D vector vector D aaagctggttat cgttcctggagc Dopachrome AA900788 449 1449 449 primer primer dopachrome NM_001355 666 131575 445 ctccaggccc tggacacgaatt tautomerase tautoerase ca t vector vector ccaaatgcag cttcctgttcgcc primer primer ccaaaacccttt tgcatctcctt Caveolin3 AI043968 424 1424 424 Caveolin3 XM_003178 1293 305813 509 ac vector vector Homo tggctacctagc tgactggagag primer primer sapiens tttgctttccaac aactggatgatg crystallin agc CDK108 AA965086 438 1438 438 (HUMAN NM_001888 1238 7231156 434 CDK108 DOES NOT EXIST UDP tcttcactcgac ttcatcacacac UDP ccatgctggttc glucuronosy ccttccccca tccggttccca glucuronosylt tgtgggctttctt ccatggtgtttat D83796 1593 10961567 472 2422 11941666 473 ltransferase ransferase NM_001072 aactcgccctt 1A6 1A6 aggatgggggt ttcactgtggaa cacacacagct agaacgggga Tyrosine Tyrosine M10244 1770 11281643 516 ccaagagg ttcgggc NM_000360 1816 11611676 516 gttgcgctgag ggtgaaggcc hydroxylase hydroxylase aag atg acagtgtgggt aggattggagc catggcactggt cagatcccttct Tryptophan Tryptophan X53501 3877 22442720 477gctgggaa acgtgtgg XM_006391 1335 7791263 485 tatgctcttggtg atacccaga hydroxylase hydroxylase ccag Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (e' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Transformin ggggtctaggg tgaagggcaga Transforming aaatgaacaca gggccagactg g growth U03491 2633 19582447 490 taggagggaa gtggtcagg growth XM_007417 2580 18602361 502 gggtcttggag gaagacacttc factorbeta3 factorbeta3 ggg aga vector vector caggctggtgg ccccaggccct Phospholipa Phospholips AA998338 186 1186 186 primer primer AF038440 3500 32173390 174 agggagttaat actacccattgtt se D e D gtg c tctgcagcgtcc tgtgaccccaa gctgcagcact ggtgccagatg Thrombomo Thrombomod AF022743 1813 13241804 481 XM_009595 4049 17502235 486 acctccttggaa ttttgcaaccag dulin ulin ac Sodium/glu acctgagacag ggaaggggtgct Sodium/gluc cacatggcttct aggaatcttcag cose acaacacaggg tcaggaaggca ose gattcttagtctg gagagcctatg D16101 2522 19662463 498 XM_009937 2450 18072289 483 cotransporte cctct tatgt cotransporter cc acc r 1 1 Sodium/bile cccattgtgggt tccaaaaggcc tcccagagctc tcctcaaatcca Sodium/bile acid acctttttcc acatatgtacc ctggaattgtttc aacggccacaa M77479 1633 6531144 492 acid XM_007466 1580 6061087 482 cotransporte t ta cotransporter r Senescence tatggagcaatt ccaagggtcctt Senescence ccgcatagga ccctctttcctga marker X69021 1594 477971 495 ccttgaccc gtactccttt marker NM_004683 1438 5591048 490 gtagggagcaa tcaccagtga protein30 pritein30 ttc a vector vector aaaggaggtca aactatgaccta. Selenoprotei Selenoprotei AA963445 454 1454 454 primer primer XM_011890 2001 14511911 461 ggtttatagggtt ggggtttctgt n P n P tgg gga vector vector aaaaattcaaac cggcccgggtc Ribosomal Ribosomal AA899769 435 1435 435 primer primer XM_07615 450 32411 380 aggtccccgag atcatagaaaa protein S17 protein S17 gc gta Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (e' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation vector vector gagcaaacaca aaagctgttgag Ribosomal Ribosomal AA924912 532 1532 532 primer primer BC004138 959 409869 461 gatcgcaggta ccacggcaaaa protein L6 protein L6 gcc a vector vector tcaaaacaaag gttgctgccgaa Ribosomal Ribosomal AA818880 420 1420 420 primer primer L05094 468 14450 437 catctaaaaccg atgggcaagtt protein L27 protein L27 ca Ribosomal ttctccacattctt cattgtggccaa gttcttctcggc tcgtggctaaac Ribosomal protein X68282 653 78575 498 ttctgcctg gcaggtact XM_011855 659 82569 488 ctgtttccgtag aggtactgctg protein L13 L13A c gg vector vector gcatgactgcc ggaccatcggc Ribosomal Ribosomal AA859756 420 1420 420 primer primer XM_012549 915 326725 400 gactgattccaa atttctgtggat protein L13 protein L13 gt Retinol vector vector Retinol ttcaggaaagg tgccaagttcaa binding primer primer binding caagcagattc gatgaagtactg AA858962 550 1550 550 XM_005907 925 391870 480 protein protein actg ggg (RBP) (RBP) Renal tgcttctgctgtt atccaggtgatt cttctggtgctct cctaatccggt Renal organic cctgctg ttcggtgc tgttgctgtcgv gatctttggtgct AF008221 2227 13611863 503 organic anion AF057039 2090 13481849 502 anion g transporter transporter Pyruvate vector vector Pyruvate aaaatggaagg ccattaggcca kinase, AA818951 491 1491 491 primer primer kinase, XM_+007659 2241 17152205 491 dtggagggtgga gcaacgcttgta muscle muscle gtg ga tgctagcctctg ctattccaatgtt ctagcctctgga tggcactgtgc PTEN/MM PTEN/MMA AF017185 1212 6051081 477 gatttgatgg cagtggcgg XM_005867 3153 16032110 508 tttgacggctcc tcacaagatg AC1 C1 t g Table 2 Size of Size of Human Human left Rat gene Target the Human Target the rlmer Srend Gene name Rat whole Rat right Rat left Gene name p ) accession region PCR accessi Length region Per rat size primer primer human (3'end) forward number (rat) product number (human) orientation (rat) (human) reverse orientation orientation Peroxisomal ataagcccgtct ggggtccctcct Peroxisomal ctccgtatgccc aggtcttatgca 3ketoacylgcctcg gacatc 3ketoacyl : tcttcccacgg gtggttggggtc CoA J2749 1580 9561231 276 CoA thiolase XM 002882 1695 10021299 298 thiolase 1 Protein gtttgtcagtgg cgctgactttgg Protein tatcagtgggg gccgattttggca kinase C M19007 2599 15392029 494 gagtcagttcc catgtgtaa kinase C NM002738 2574 15832072 490 gtcagttccaca tgtgtaaggaaa betal betal Protein tgggggaagaa ccaaggaagct Protein gaccctgaaca tgtccaaggagg kinase C X07286 3305 19042406 503 agttgatcact gtctccatctg kinase NM002737 2245 17092209 501 gttgatcacat ctgtttctatctgc alpha alpha Proteasome gatcatccccttt gggacgaaga Proteasome ctttgtttctccc agggggaggat activator 28 D45249 921 307776 470 gtctctccacggcgacaaaggtc activator28 XM007299 985 364827 464 ctgggcttcttg gaagacaaat alpha ctcc alpha cc Tissue vector vector Tissue aaaacagggtt ggaaacagga plasminoge AA924878 433 1433 433 primer primer plasminogen NM000931 2461 20412417 377 gtggcaacgaa ccacaaaa n activator activator aag aaagca vector vector acagggagtttc cagstggatg Osteopontm AA964431 420 1420 420 primer primer Osteopontin J04765 1447 7511197 447 catgaagccac ccagagtgctt aaa Ornithine attacggcagg ctgctgtgaga Omithine tgaaggacttga ccaaagattgi aminotransf M11842 1938 11551647 493 actctgcaggct gggaaagggtt aminotransfe NM000274 2013 12081672 465 tttagaggctgt atgottggaaggl erase gtat gctaa rase ctg Nucleosome vector vector Nucleosome tcaaatcgcttat cotggotcoc assembly AA899456 510 1510 510 primer primer assembly NM004537 1560 32431 400 caaatagaggc actagtcgcc protein protein tga Table 2 Size of Size of Human Human left Rat gene Target the Human Target the rig er Sri nd Gene name Rat whole Rat right Rat left Gene name p ) accession region PCR accession Length region PCR, rat size primer primer human (3 end) forward i number (rat) product number (human) product reverse orientation I (rat) (human) orientation Neutral caacaccactc tttcttcagcttct Neutral tcagcatcagtc agggtgattaaca endopeptida aagtgggtaaat tgggggaaatt endopeptidas aaagcaggtac gagagggcacc se 24. 11 M15944 3243 24202935 516 ggcg ca e 24. 11 XM003138 5725 25893128 540 aaca a (enkephalin (enkephalina ase) se) vector vector catgcattggta gtaccctccaa Neuropeptid AI045437 370 1370 370 primer primer Neuropeptide p04941 418 98399 302 ggatgggtgga gceggacaacc tt Neuronal vector vector cgggtatgtcct tcctgtcaacgcc cell primer primer Neurona ? cell ttaaatgtggag atgaattecttt adhesion ttaaat a at aattccttt adhesion AI144588 350 1350 350 rnolecule XM_004950 6219 39284298 371 ca molecule (NRCAM) (N°rleAw (NrCAM) s Nerve agagaccccca tgaagctgccc agtaaaaaggg gatgaagaaaag Nerve growth cccca aacc t c a growth gaaccaaacac ccagaagacta g 8 factor X05137 3259 25943091 498 atgct cagtg factor X1VI008437 3387 27173218 502 aaa g receptor. t Low density tcccatctggag gacatggctgg Low density ggaagaaacca aegttgctggiç lipoprotein X13722 3037 25303033 504 catggc cagaggg lipoprotein M009082 5174 24142867 454 aaatcccaacc gaggaaatgX receptor receptor caa a ^ NADPvector vector NAP_ gaaataaaaca actacctggag,. ; dependent primer primer ggccagcaga gtttgtcctctc. dépendent dedro AA925731 165 1165 165 isocitrate XM010837 1941 16961840 145 gt c dehydrogena ! ase, dehydrogena cytosolic se, cytosolic Table 2 ...,,., Human Humanteft Size of Size of Human Human left Rat gene Target the Human Target the right primer Gene name Rat whole Rat right Rat left Gene name primer (5'end) accession region PCR accession Length region PUR rat size primer primer human (3'end) forward number (rat) product number (human) product orientation orientation '''orientatton Na/K vector vector Na/K ccagtctatggg tttgtcatgggg ATPase AA956184 456 1456 456 primer primer ATPase betaXM_0015571 2149 15872019 433 gtactgcattgc gaactgcccttt becta1 Na/K aggggtaggtg cggtttcctgcc Na/K atgcagagtgt ttgtgattctggct ATPase M14511 3636 28653361 497 cagtgtgtggc ctttcacctgt ATPase BC003077 3680 28703379 510 gtggcctgatg gagaacggctt alpha1 a alpha1 ct Macrophage gggggagggc tggcgctctgg Macrophage gggacagggg ccatggctctctg inflammator tgaggaat aacgaag inflammatory aactctcagag caaccagttctc y protein1 proteinI caaa alpha alpha Monocyte ctcactcggtct tgatcagcatac Monocyte tgaagtcactcc gtggcccttatttt chemotactic gctgtctcc ttgtggccc chemotactic atccactgtctc ccacgaggatg protein U77349 1365 8151291 477 protein XM002924 1977 6491115 467 cc receptor receptor CCR2) (CCR2 Mitochondri vector vector Mitochondria catgcacacaat cagactctg al voltage primer primer 1 voltage tgcacagttttca ectggtgtg4gg dependent rependent anion AI030961 420 1420 420 on X1VI_005893 1372 8081217 410 channel channel channel channel (VDAC2) (VDAC2 MEC class I vector vector MHC class I ggagctcaggt cgaaccgtcetc . antigen 924062 490 1490 490 prer primer antigen pF016641 1089 19468 450 ctcgttcaggg etgctgctc RTl. A l ( RT1. A1 (fj ai hachain aI hachain Table 2 Size of Size of Human Human left Rat gene Target the Human Target the right primer Gene name Rat whole Rat right Rat left Gene name p () accession region PCR accession Length region Per rat size primer primer human (3'zend) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Methylenete ccttgggggac catctcctgccc Methylenetet trahydrofola U57049 1250 7391242 504 ttgcttttcaggt tatcctgcctgg rahydrofolate NM 005957 2196 7771255 479 ttgctcttcaggt agggctaccac te reductase reductase a Methylacylvector vector Methylacyltcaccatacaat ctgcacctctgc CoA primer primer CoA ttgtggcatttcc tgttaaacaccc AA818815 601 1601 601 M 003872 2048 10691644 576 racemase racemase al ha al ha catgctcggtag gcccgactgcc gcaaaggggtc ctcttcccttctc nein I J00750 389 15364 350 aaaacggg ttcttgtc Metallothion p12575 396 28347 320 aagattgtagca gcttgggaactc axa aa Membrane vector vector Membrane gcacactcgaa gttaaacaagc bound primeur primer bound aaatttgagcgc ctccggaac axa956323 407 1407 407 XM 008817 716 273622 350 ag aaa b5 b5 b5b5J Melanomavector vector Melanomatccgtttaattga cctggacagga° associated primer primer associated aaaacctggag tgcaggcagat antigen antigen ga tt ; ME491 ME491 tcacatttgatac ccaaaatccttg catttgataggg ccaaaatcctg Maspin U58857 3637 5771062 486 ggacactccctt tggttaatgctg Maspin U04313 2566 5451027 483 ccactcccttgg tggttaatgct gg cct t cc Malate vector vector agaatgcacac ggagaatttgte galate dehydrogen primer primer taacagcatga acgactgtgc _ AA900573 526 1526 526 dehydrogena XM 002358 1267 7161246 531 at c' ase,egat gc se, cytosolic Table 2 Human uman left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Macrophage ttcttcctaacaa tgggagtccag Macrophage acaccatttcta atttcttccttatg metalloelast X98517 1632 9651448 484 ccgaaccagct ccaccaacatta metalloelasta NM_002426 1778 9731434 462 acaaccaaacc gccaaccttgc ase cg ctt seage Macrophage tacagtgagctg gccagctcctc Macrophage ttccttctggtca cctgcagggaa inflammator gccaatgc aatgctgtact inflammatory gttggatttgcc ttcacctcaaga U45965 1064 50552 503 AF043340 234 42232 191 ac y protein2 protein2 alpha alpha Phosphatidy vector vector Phosphatidyl tcccagacagc gtggacgagct lethanolami primer primer ethanolamine tgctcgtacagt gggcaaagtgc AA955166 519 1519 519 :X85033 570 103562 460 nebinding binding tt t protein protein Peroxisome ccgtcttcttgat gcaagagatca Peroxisome ggagcgggtg cagagtatgcc proliferator cacatgcagta cagagtatgcc proliferator aagactcatgtc aaaagcattcct activated AB011365 1636 8821281 500 aa activated U79012 1608 10641579 516 tgt gg receptor receptor gamma gamma peroxisome tcgctgactgag ccccgtgcatc Peroxisome cagctcctgctc tttgatgaact assembly D63673 3169 24012905 505 ggctgc atcttcttt assembly D83703 3194 24742971 498 actgactgagg gactctttggcc factor 2 factor 2 gt c Peroxisome cctgctaaaag caactttgacag peroxisome gacagtggcta gcctcaaaacat assembly X57988 1401 6491168 520 ccacctacagat aacgcctccttg assembly : M862852 1630 9101408 499 ggagcacattc acgtgaagttg factor 1 gaca g factor 1 ctt gc Table 2 Human Human left size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Vesicle vector vector Vesicle tcatttgactgca gtctccactctc associated primer primer asscoiated aagtcccaggg ctctgggctcct membrane AI029578 337 1337 37 membrane XM_006955 856 522850 329 t c protein protein (VAMP1) (VAMP1) Very long vector vector Very long atgccttccctc acctagttatgc chein acyl primer primer chain acyl caacaagctca aaggccccggt AA925897 453 1453 453 AF096290 2344 18542311 458 CoA CoA gt tt synthetase synthetase Suppressor ggagtggagg gtgccccgcttt Suppressor of cttaaagcggg cagcgccactt of cytokine AF075383 683 357840 484 gcctgagg gactgt cytokine AF159854 682 226679 454 gcatcgtactgg cttcacgctcag signaling 3 signaling tc vector vector tttgcactcttgc tgaaagacaga Sulfotransfe Sulfotransfe AA926193 406 1406 406 primer primer AF026303 1041 6331020 388 catccaccttttt caccagattctc rase K2 rase K2 ttcc StearylCoA vector vector StearylCoA aatcccaccca ccgtgtgtccca desaturase, AI043833 140 1140 140 primer primer desaturase, Af097514 5221 49065070 65 atcacagaaaa gatgctgtcatt liver liver ggc a Sorbitol aggggttctggt tcactttgcttgt Sorbitol ggggatgagca tggtgactgatc dchydrogen X59037 1658 5831095 513 cattggggtca ggccaaagca dehydrogena NM_003104 1808 7431239 497 gagcccattaa tgtctgctaccc ase se cat g vector vector acaaggattttatt ctgaggttgg Ribosomal Ribosomal AA874997 490 1490 490 primer primer NM_001012 705 195658 464 tgcctttgcggg cgtggggaatt protein S8 protein S8 ct Table 2 Human Human left size of Size of right primer Rat gene Target the Human Target the Gane name Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (nrat) product number (human) product reverse orientation (rat) (human) orientation Retinol vector vector Retinol ggagtgggatt tttggggtgaag dehydrogen AA866390 479 1479 479 primer primer dehydrogena AF086735 2425 14411925 485 ggtgcctactga gtggctatgatt ase type III se type III cc g Retinal vector vector Retinal agctcctaccat agggtacctgg protein AA819052 423 1423 423 primer primer protein U40998 1381 9171321 405 gggctgcaatct tgtccccagtca (RRG4) (RRG4) c ag RAC vector vector catctcgtacat gcggaaggaa RAC protein protein AA818180 490 1490 490 primer primer M77198 1849 752132 481 gaccacaccca gtcatcattgcc kinase beta kinase beta gc aag vector vector gcagtcttgcta ggacgcacaca Zinc finger Zinc finger AA900368 560 1560 560 primer primer AB017493 1340 7821339 558 accacaaggaa ggagaaaagc protein protein aaa ctta Vascular cctctcccttcat agagatgagctt ctggtgagaga acatcttcaagc Vascular endothelial gtcaggct cctgcagca tctggttcccga catcctgtgtgc AF062644 469 16457 442 endothelial XM_004512 649 228648 421 growth aa c growth factor factor 3 gggcttctgtgt gacaggtccttg 3 ggacatagaag tcctagtccggc methyladeni ccattcccctg tccggcgactt methyladcnin cggaggggttt gacttcctaat X56420 963 330839 510 L10752 956 303824 522 ne DNA e DNA cc g glycosylase glycosylase vector vector gctgttttatttcc ggtccggccag Adenodoxi Adrenodoxin AA964367 449 1449 449 primer primer J03826 1830 13851823 439 agcatgttccca tctctttctcag n reductase reductase c tgcacttttcctc acagacaaaga tctccttaagcct gctggcacacc Adrenomed Adrenomedul U15419 1395 5441028 485 gctaggttttgg caaggacggc NM_001124 1449 513978 466 ggcaaacactc agatctaccag ullin lin a atggc a tca Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product) reverse orientation (rat) (human) orientation Alanine gcagccgcagt tctaagggctac Alanine ggtgctcagga agatgctgaag aminotransf D10354 1745 10501551 502 ttctccat atgggcga aminotransfe D10355 1653 10071487 481 gtactcgaggg ctgatgagtgtg erase rase tga cg Alcohol catgagtaatca agtcgccaagg Alcohol aagtaaaaggg tggctaagaagt dehydrogen M15327 1292 5951085 491 gcggctcca tgacccc dehydrogena X03350 2532 10821565 484 tcccctccactg tttcactggatg ase 1 se 1 gg cg Alpha1 vector vector Alpha1 tttatttggaccc aagaagagga microglobul primer primer microglobuli aggttgcttggc aggatcagggg in/bikunin AI043784 499 1499 499 n/bikunin XM_016890 1262 7231242 520 gtgg presursor precursor (Ambp) (Ambp) Alpha1,2 agttggtcttgg ttggccccaga Alpha1,2 ttcaggaactca ccgggaacag fucosyltrans AB015637 5435 48425344 503 aagggaataca gaaacttca fucosyltransf : AB004862 1098 5311010 480 gagtctggcag atccgcagaga ferse a erase gg gtt vector vector aggatttttattg atgaggctcttc Apolipoprote Apolipoprote AA955662 374 1374 374 primer primer NM_001645 417 55413 359 gtggcacctgg ctgtcgctccc ein C1 in C1 g Aryl agcagcagtct agctgtcctcag Aryl cggatgatgaa tgtccacagca hydrocarbon U09000 2574 20082499 492 gaaggtggc caggaacg hydrocarbon XM_004988 5493 26333142 510 gtggctgaaga agacccacac receptor receptor tgt aat Aryl gcgctcattctg cctgtccttgctc Aryl agcctgccatct aggtcaaggtc sulfotransfer X52883 1017 383878 496 ggctacag cctcaga sulfotransfern X78283 1250 5241018 495 tctccgcatagt gtctatgttgcc ase se c cg ccatcaccgttc agcgctaagaa aaaccagagac atgcagccaaa CalbindinD CalbindinD J02954 406 66250 185 ttatccagct atctcccgaag X65869 454 104384 281 tttgggggattc gaaggtgatcc (9K) (9K) ca aga Table 2 Human Human left Size of Size of right primer Rat gene Target Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product (human) product reverse orientation (rat) (human) orientation catctaccacca tgaggcgagtt aggatgttttatg gaattctgtgct Calcineurin Calcineurin L03554 530 20519 500 tctttttgtgga accctttgg M30773 2548 7921297 506 ttctgcttggca gttgtaggtggc B B ct aaaggcagaat gcctcttgaccg ccccacgaggc cagctgcagttg Caspase 2 U77933 3365 27403249 510 ttggcagca tgatgttga Caspase 2 XM_004692 2165 16572159 503 tcatttcagtaac aagagcctgac a aa CatecholO ctgaactccgat gaccctttctatg CatecholO acaatccagtgt aggacatcatc methyltransf M60753 1491 8311320 490 gcctctgc ctcctggg methyltransfe NM_007310 1067 489968 480 tgcagttcagag ccccagctgaa erase rase agg ga ccttctcaggcc cggtccacagg ccttggagggg cttgtgacacgg CCR5 U77350 1689 11381650 513 acagagcactt agaacaggaa CCR5 NM_000579 3665 14101938 529 aaatcacacatg actcaagtggg actg gtttct aa c Cholesterol agtctcagggta catagctgggg Cholesterol tatgatcacacc caccaggtcca 7alpha gggttctggga ccagagcttcat 7alpha cgaagaacccc atgttgttcacca J05509 3561 17142208 495 M93133 2901 16812195 515 hydroxylase ggtg cact hydroxylase ac g (P450 VII) (P450 (VII) cccactcggag tggaactgatga ccactggggag gcccttaagtgg Cyclin E D14015 1957 8751365 491 gaggagaaa tgatgaaggc Cyclin E M73812 1680 7181187 470 aggagaagcc cgtttaagtccc ctat c Diacylaglyce caccccaggcc aaggggaccat aagtgcagcag gagaaccggc rol kinase D78588 3560 29733481 509 atacagtcgga ggatcaacgag NM_003646 3490 28193329 509 gcgctgagtttc agcactaccag zeta a g atga tcagaacaaa acatcagcttct taaatggtgctt gatgctctttcaa Ecto Y11835 1430 7951290 496 gtcccactccg gagccgtggtt EctoATPase AF144748 1488 9801487 508 ggcagcttggc tggggtcttcca ATPase aatgg acca Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product) number (humer (human) product reverse orientation (rat) (human) orientation Epidermal tatgagcaggg acaaagggcct tgaccaaacca taagggctcctg Epidermal growth U04842 4760 40004491 492 gccacactt cagacctgc NM_001963 4877 39014437 537 gtgtgactgtct tccccaggtaat growth factor factor gc g vector vector tgaggctttacc agtcttttcagca Decorin AA859984 525 1525 525 primer primer Decorin L01131 785 218755 538 agtaatgatgg accggttccag gga t Diazepam vector vector Diazepam aggaaagtcag gggcactggga binding AA925794 510 1510 510 primer primer binding XM_002478 537 45491 447 taatcgttttagc cagaggctgag inhibitor inhibitor ccc ttt tgggaacccca gttccagtattat ggaatgcaaag catccctatggg Eselection L25527 3290 20282528 501 gatatacactct tgtcacaggga Eselection XM_001578 3833 25503072 523 cacatccatcttt attcagtgcttct c c aaggaatgtgct aaatgtgtagaa aaggaatgcga ttggacaggaa Estrogen Estrogen Y00102 2090 14701960 491 gaagtggagc ggcatggtgga NM_000125 6450 15882096 509 tgaagtagagc ccagggaaaat receptor receptor ccg gtg ttgagtcagtag acctttaagtca gctgtggatgat ccacctacgtac Fatty acid Fatty acid M76767 9135 85519040 490 actccaacgag ccctgaaggca NM_004104 7515 70047479 476 gctgatgatgg tggcctacaca synthase synthase c ac ca Fatty acyl ttaaggcttgtgt tcgttgaagatg tgttcagtgggg ggatatcaac Fatty acyl CoA J02752 3741 31213620 500 cgatgaccc gctgaagaaa : U07866 1983 14251927 503 agttcttagccc gccccgaaagc CoA oxidase oxidase a cta Fetuinlike vector vector Fetuinlike gatgcagctca ctaaccacgatt protein AA858896 373 1373 3737 primer primer protein AJ242928 1623 10891474 386 gggtacaaggc ccccagggag (IRL685) (IRL685) agt aaa Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Fumarylacet vecto vector Fumarylaceto acttcatccccg gtgccatgacg oacetate primer primer acetate tccagcagaaa agccctacacat AI044732 256 1256 256 XM_007704 1447 9171225 309 hydrolase hydrolase ct tt (FAH) (FAH) Gamma vector vector Gamma ggtggagacag tggaagcggttt actin, AA964496 494 1494 494 primer primer actin, X04224 2399 14121891 480 atgtatggagtc gcatttacacct cytoplasmic cytoplasmic aca g Gap vector vector gcgagagag agggagggtg Gap junction junction primer primer gccttggggac gccagcactag membrane membrane taga taaa AI029683 172 1172 172 channel XM_010141 1549 13491515 167 channel protein beta protein beta 1 protein beta (Gjb1) 1 (Gjb1) acctgggtggt tcacgattcttgc atgggagctga tctcgctggaat Gluckonase J04218 2297 17342219 486 ctcttggaggg ttcccaagcg Gluokinase XM_004994 2721 21062589 475 agatgtagggc caatttcccaga a tgg a Glucose cctcttcaccag ctgactggaatc Glucose ggagggcctgc gactggaattee regulated M14050 2383 15071996 490 ttggggg cctcctgc regulated AJ271729 2007 14651935 471 acttccatagag tcctgctcctcg@ protein 78 protein 78 tt Glutathione vector vector ggacttcctatg gaagtgcatcct@ Glutathione S primer primer attggtgggga ccttggctggat AA875380 369 1369 369 Stransferase : X56837 1222 8101168 359 transferase ctg t mu2 mu2 Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Glutathione ctgaggcgagc gccagagctgg cacatatgctga agagctggaag Glutathione S cacataggcag aaggaggaggt gagcagggg gaggaggtggt X02904 735 116593 478 Stransferase U30897 719 92555 646 transferase a g aac gac Pl Pl Glutathione acagtcgtgtaa gtgtggatgagt aatgctttgtgg cgtgtggatga Glutathione S tgcgagtggcc acctggcatgg actgctgagga gtacctggcatg X67654 914 281775 495 Stransferase NM_000853 1004 280771 492 transferase tg ca cg g theta1 theta1 vector vector aaggccacagt agggaattctct Heme Heme AA874884 516 1516 516 primer primer 1550 10101511 502 gccgttaaaca tggctggcttcc oxygenase oxygenase XM_009946 cct t Hepatocyte tcttgggatggc cgagagggagc cggagcgaca tgatgacaaga Hepatocyte growth aacagaga ccctccttat cattttacgttca ggagccccacc X96786 4149 38364121 286 growth factor XM_004968 4517 40684333 266 factor ca tta receptor receptor High vector vector tggggttctcc cgactgaagat affinity IgH primer priemr High affinity cttcccatatttt ccaagtgcgaa receptor IgE receptor agg gaama AA957422 400 1400 400 gamma chain NM_004106 591 158567 410 chain (FcERIgamm (FcERIgam a) ma) cgtattgtgtca cgtctgctttaca tatttgcctttgg aaagccaaga@ Histone 2A U95113 575 161547 387 ccagctggtc agggcaact Histone 2A XM_004453 393 40392 353 ccttgtggttgac ccgctcttctc@ Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation vector vector cgagcaagctg agactgctcgc Histoen H3 AI029542 412 1412 412 primer priemr Histone H3 XM_004419 411 17386 370 gatgtccttggg aagtccacggg t HMGCoA vector vector HMGCoA agaccaccatg agaggacatca synthase, primer primer synthase, gcataacgacc actccctgtgcc AI136048 297 1297 297 XM_001425 2045 347630 287 mitochondri mitochondria atc tg al 1 vector vector catgactccaat ctgactgacaa Hrev107 AA875291 322 1322 322 primer priemr Hrev107 XM_012008 654 150449 300 aaggctcatgg ggccatcgtga ctg aga vector vector tctgcccctttca atgctgataaca Ige binding Ige binding AA859797 468 1468 468 primer primer M57710 914 406786 381 gaattatatcatg attctgggcacg protein protein g g Insulinlike vector vector acacccaacact attgtgaccatc Insulinlike growth primer primer cttccaaccag gaggcttctacc growth factor factor AI059172 283 1283 283 XM_012161, 949 619915 297 cg g binding binding protein 6 protein 6 aggattttcacc gcttgtaagtgc cgtgtcccattt ggcatctgcga@ Integrin Integrin U12309 2894 18962390 495 XM_005799 3539 18562356 501 ggcattcattttc gtgtggtgtcg@ beta1 beta1 ta Interferon agagccgtcat gggatccctctc Interferon cagtggaagtc atccagaatcg@ inducible U22520 1129 152641 490 agctgcctg gcaagaac inducible X02530 1172 293799 507 catgaagtaaa aaggccatca@ protein 10 protein 10 gagca gaa Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Interferon vector vector ttctccaaacatet tccaacagaaa Interferon related primer priemr gctctcttatctc ccattaaatttgg related developmen g tcc AA955469 480 1480 480 development Y10313 1791 12721572 301 tal regulator at regualtor IFRD1 IFRD1 (PC4) (PC4) gtttccaagga agggctgcct ttctcaagggg cgagatgccttc Interleukin Interleukin L02926 682 133630 498 NM_000572 1601 163666 504 ctgggtcagcta agcagagtgaa 10 10 t ga agcatcatcttc tggctgcaatac ttatcatgtcctg tatttgtcgcag Interleukin Interleukin AJ222813 628 2504 503 XM_006289 1102 158617 460 ggacacttctct gaataaagatg 18 18 ga gctg tgagttggatgg ttcacaagtccg tgagttgtcatgt ccagtaccccc Interleukin M26744 1046 145641 497 tcttggtcc gagaggagac interleukin6 XM_004777 1125 147637 491 cctgcagccac aggagaagatt 6 t cca KAI1 ccaggggaca ccaccaatatg tggagtgcagt gaagaccctctt KAI1 metastasis ggggacag gggccct agcacgatctc gcccatcctga@ metastasis suppressor AF049882 1740 11341632 499 XM 012041 1624 10841593 510 agc ct suppressor gene gene (CD82) (CD82) cctgccagctttt ctctggtggcca gcttttgaatcg ccatccttctgtg Kcadherin D25290 3629 20272516 490 tgaaccg tccttctg kcadherin NM_004932 4315 19732249 477 aggtccccagt catcgtgatc@ c Keratinocyt cctttgacagga tgcttccacctc tcccctccgttgt tgccaactttgct Keratinocyte c growth X56551 693 160635 476 agccccttt gtctgtctt 3853 474930 457 gtgtccatttag ctacagatcat growth factor NM_002009 factor c Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation Lactate cctgagctgag ctgagcgggct Lactate tgcacttttcttg ccgcgtgattg dehydrogen U07181 1217 476979 504 cgacctcat acctaagca dehydrogena XM_006906 1289 5551047 493 agctgagcaac gaagtggatgt aseB seB c aat Lecithin:cho vector vector Lecithin:chol ggaaggtcttta gtatactgtcttt lesterol priemr priemr esterol ttcaggaggcg acggcgtgggc AA997322 353 1353 353 NM000229 1354 10141343 330 acyltransfer acyltransfera gg ct ase se Leptin vector vector Leptin tgaaattgtttca cccaatatttatg receptor AA998983 357 1357 357 primer primer receptor NM_002303 3800 26292950 322 ggctccaaaag gaggagtgg (fatty) (fatty) a ga CTP:phosph vector vector cTP:phospho ggtcttctttccc gaagtggggag ocholine primer priemr choline cagttgtctttcc gagaagtcccg AA925887 434 1434 434 m_005017 1286 8731283 411 cytidylyltra cytidylyltras agaa nsferase ferase Equilbrative vector vector Equilbrative agagagcctct gagggagcct@ nitrobenzylt primer priemr nitrobenzylth gaaggcacctg tggacggacag@ hioinosine ioinosine gtt t AI044510 468 1468 468 U81375 2162 17052134 430 sensitive sensitive nucleoside nucleoside transporter transporter Intracellular vector vector Calcium ccccacagcca ccaatactctgt calcium primer priemr binding agacagtttgac gaagctgggg inding protein A9 at ac AA900471 441 1441 441 XM_001683 575 105539 435 protein (calgranulin (MRP14) B) (S100A9), (RCT021) mRNA Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Length region PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation StearylCoA vector vector StearylCoA aatcccaccca ccactgacttgc desaturase, AI043833 140 1140 140 primer primer desaturase, AB032261 5329 48005183 384 atcacagaaaa tgtgtgaccctg liver liver ggc atcactgccag caaagccacgg peroxideduxi ggcttgatggta tcaaagccaca Heme gttccagc ctgttatgc n/Heme tcactgccaggt gotgttatgcca biding D30035 882 100601 502 XM_001393 937 104617 514 binding t ga protein 23 protein 23 Betaine vector vector ttgggattgcct tccttcgctgtca Betaine homocystel primer primer ttggagtcattg aagccagactt homocystein ne c AA901407 479 1479 479 e NM_017614 1708 10991612 514 methyltransf methyltransfe erase rase (BHMT) (BHMT) Insulinlike caatggctgcc ctgaaggcgct gggagaggct ctctgcgtcaac Insulinlike growth cgtacttgt gctgaatg gcccatacttat gctagtgccgt growth factor factor M33300 2058 349852 504 XM_004689 876 337818 482 cca binding binding protein 3 protein 3 Aldehyde vector vector Aldehyde tgaggttccata agtgggcaagg dehydrogen primer primer dehydrogen ccaggagcgtc ctgaagtaag@ AA956846 469 1469 469 u46689 3931 33963850 455 ase, se, at ggc microsomal microsomal tgaagcccctg ttgagcatccac Aquaporin Aquaporin3 AI045067 449 1449 449 primer primer XM_005543 1441 9821409 428 aaacatacaca tgactgtccaa@ 3 (AQP3) (AQP3) ccc g Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Lengthregion PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation vector vector aaacggggcttt gctgetgettttc Emerin AA963187 200 1200 200 primer primer Emerin XM_010157 1147 7421141 400 tattgtgtgtcca ctggtctttgtg Retinal vector vector Retinal agctcctaccat atgttgggagg protein AA819052 423 1423 423 primer primer protein U40998 1381 9051321 417 gggctgcaatct aagggtacctg (RRG4) (HRG4) c gtg vector vector aaggcaggcct atcggacttctc Squalene Squalene AA819300 390 1390 390 primer primer XM_005060 2275 68437 370 gagagaatatc gtcctgggaca epoxidase epoxidase cga ct PAR vector vector MYB agtggcagcgc gacgccaacca interacting AA899306 397 1397 397 primer primer bnding xm_008176 4521 37034127 425 cttagaaacag ccaagagtcca protein protein ctt g HMGCoA vector vector HMGCoA ggtactttcttgg tgtggagaagg cytosolic cytosolic a cta vector vector cctctacctagt cgtagccggga Betaalanine ureidopropio AI059491 407 1407 407 primer primer xm_009883 2005 10541433 380 ccccaaacattt tggactgctagt synthase nase cca tg vector vector CGI45 RCT020 AA899737 primer primer protein gaaacagcacg tctaccatcaga 583 1583 583 nm_015999 1842 79638 560 134 AA899452 (LOC51094) aaa g mRNA vector vector ethanolamine tcatctgcaaat atggccaattac RCT098 AT071448 529 1529 529 primer primer kinase KM_006871 2200 350892 543 cctgtgggaat atccacgtccc (EKI1) ga c Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Lengthregion PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation RCT vector vector gcagctttatca ctcggcttattcc AA858676/ 005/RCT primer primer gcagcttggca aggggtgtgttt AA859158/ 011/RCT albumin ac AA818004/ 575 1575 575 NM_000477 2216 81638 558 200269 (ALB) AA866234/ 105/preproa AA818342 Ibumin vector vector membrane gtttcatggcat atggtcctctttg primer primer protein of cagccaccttct acttcggcaac cholinergic c c RCT126 AI145194 604 1604 604 903 253838 586 synaptic XM_008209 vesicles (VATI) vector vector hemoglobin; ttattcaaagac cccacagactc RCT primer primer alpha 1 cacgggggtac agagagaaccc 004/hemogl AA858664 553 1553 553 NM_000558 576 13560 548 (Hba1), gg acc obin alpha 2 mRNA vector vector angiopoietin tgttggatggat gccatcagacc RCT128 AI058911 566 1566 566 primer primer like 3 NM_014495 1496 8051386 582 caacattttggtt cagcaactctc (ANGPTL3) g ag vector vector type I agacaccttgg caggacgtgca primer primer transmembra aaggttcccctg ctatggctcgg RCT050 AA957270 594 1594 594 ne protein XM_007981 986 27608 582 aa g Fn14(FN14), mRNA Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Lengthregion PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation vector vector tgttacaaaagc aaaaacccaaa RCT102 AI071578 539 1539 539 primer primer P311 protein XM_016675 1990 13591889 531 ctgcattttcagc acactggcatct ag ga vector vector carbamoyl gcacataggca atcagactggct RCT primer primer phosphate agcctagtcca caacgccaaca 104/carboa synthetase 1, cca at myl AI059983 580 1580 580 5207 42944879 586 mitochondria XM_010819 phosphate 1 (CPS1), synthase 1 mRNA vector vector agacagtgagg aaagtgccttca glypican 4 RCT114 AI071251 578 1578 578 primer primer XM_010339 3714 12851842 558 aggtaggcctg gtgctcgcttca (GPC4) tgc g vector vector O gtgacttgtccc aggaacctcag primer primer mannosyltran cgtaggtgagt cttcatggcgag RCT116 AI072418 577 1577 577 sferase 1 XM_005471 2973 16412205 565 gg at (POMT1), mRNA vector vector transmembra gtagtggccga acagtctcctgc primer primer nc, prostate tgacctcgctgt gaaaccaggca RCT129 AI058507 636 1636 636 XM_009671 1130 75709 635 androgen ag at induced RNA Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Lengthregion PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation vector vector electron tcctcctccacc attgagaggaat primer primer transferring ttcaggtaccac ggagccgtgga flavoprotein c ct dehydrogena se (ETFDH) RCT287 AI070723 547 1547 547 NM_004453 2124 15511953 403 nuclear gene encoding mitochondria I protein, mRNA vector vector hypothetical tcaacaagtgg atggtaatgag primer primer protein gggcaatatga gggccaccga RCT282 AA818555 583 1583 583 FLJ10578 XM_005795 11865771162 586 agtg gata (FLJ10578, mRNA vector vector KIAA1224 caaagacgatg agcttggaactc RCT276 AA819615 447 1447 477 primer primer protein, AB033050 5676 50285391 364 atggtttctgtgg catcacgtgga partial cds ga aa vector vector diubiquitin tgggaaatcatc cttgtctgcaga RCT267 AI030354 589 1589 589 primer primer (UBD), XM_004212 777 6605 600 agaagatgtgtt gatggctccca mRNA cgt at Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Lengthregion PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation vector vector cell death aaattcccagca acctctcagctc primer primer inducing acatatggccc tgaaccccagt DFFAlike ag ga RCT263 AI058382 581 1581 581 XM_007312 2256 11631744 582 effector b (CIDEB), mRNA vector vector interferon aggtagttgctg gtggccatgga primer primer stimulated tcccaaaaagc ctgcgagatgg RCT259 AI045075 594 1594 594 gene (20kD) NM_002201 679 37624 588 cg t (ISG20), mRNA vector vector fibrillarin gtcctccattac aggaaaaaga RCT258 AA955914 577 1577 577 primer primer (FBL), XM_009212 1079 258808 551 gcaggaaggtg ggaaaccagtc mRNA tg gggg vector vector glycine N ggaatgtaggtt gtgtggatgcc RCT249/ primer primer methyltransfe tggcctggcttg agtgacaagat glycine AI136103 591 1591 591 rase 888 251851 601 t gct methyltransf XM_011435 (GNMT), erase mRNA Table 2 Human ,,, . Human ! eft Re ion of Rat PCR ri ht H°man left Gene name EST Whole rat g Gene name Human Length Region of PCR g human ,. PCR prodnct. aceesston human PCR product prer (5'end), num er product size number ene (3'end), g product size forward reversé orientation orientation HIV1 Rev gaagatgccac attttggaggttt binding agaaggacca ccccacagcaa RCT215 Api060143 567 1567 567 protein Nom004504 2584 10041583 580 gcag g mRNA pM5 cagcagcaaag aagacaccgtg RCT213 Api030332 573 1573 573 protein, : m014287 4182 29783555 578 gaatgagcttgt acagacgaaga cl nuclear tacctgccttca ctgcgaagtatc localization gggggttggta tcgccagccct signal gt deleted in RCT212 AI043871 586 1586 586 velocardiofa Nom003776 760 28587 560 cial syndrome (NLVCF), mRNA Table 2 Human Human left Size of Size of right primer Rat gene Target the Human Target the Gene name Rat whole Rat right Rat left Gene name primer (5' end) accession region PCR accession Lengthregion PCR rat size primer primer human (3' end) forward number (rat) product number (human) product reverse orientation (rat) (human) orientation high ccacatcaatcc gtgtgaagaag RCT211/ mobility agctttggagac aggcgagaac high group a gacc mobility (nonhistone group17 AI058430 582 1582 582 chromosom NM_005517 1198 6591 586 mRNA, 3' al) protein untranslated 17 region, (HMG17), mRNA katanin p80 atgtctgtgatg agaagtgagcc subunit aggggcagaa cttccctgcacc RCT208 AA858769 588 1588 588 mRNA, AF052432 2324 12351827 593 acc complete cds RCT203/ gtgtcatcgcat ggtgtaccacc GSK3beta gaaaaacagac catccctgtggt axin interacting tcg ct (AXIN) protein AI059894 566 1566 566 AF009674 3411 28113320 510 mRNA, rAxin partial cds (Axin), mRNA Table 2 Human Human left Rat gene or right Region of Rat PCR Human Length Region of PCR human Gene name EST Whole rat Gene name primer PCR product human PCR product (5'end), rat accession or est size human (3' end), product size number gene product size forward number reverse orientation orientation ccagagggtgt gcagcggttga hypothetical gcaatgcatcat actcagaggag protein aa aaa DKFZp566I RCT199 AA956659 568 1568 568 NM_030938 2052 13211893 573 133 (DKFZP566 I133), mRNA ectodermal tcttcattagact tgtcagtcatga neural tggcctctccga caagctcccca cortex (with aa RCT194 AI058470 528 1528 528 BTBlike XM_003863 4785 17582266 509 domain) (ENC1), mRNA proteasome ggctctcgggt caggctgagaa (prosome, ggaatagatgtc gctggtgtccaa macropain) aa gt 26S subunit, RCT191 AA964001 596 1596 596 NM_002809 2177 9531572 620 non ATPase, 3 (PSMD3), mRNA Table 2 Human Human left Region of Rat PCR Human Length Region of PCR right human Gene name EST Whole rat Gene name primer rat accession or est size PCR product human accession human PCR product", (5'end), rat accessMn oreststze.,'. human.,.. (3 end),,, number product size number gene product size forward reverse orientation orientation onentatMn TARDNA ttttggttattacc ggcgctgtaca binding cgatgggcctg gaggacatgac RCT171 AA999071 469 1469 469 protein XM018397. 2739 6711148 478 tga (TARDBP), mRNA RCT163 /acetylcholi mRNA for ggcggcagcctgagctcgtgttt ne rece tor acetylcholin actttttcaaaat gaggggcaga epsilon A1059477 397 1397 397 e receptor X66403 2457 11851561 377 c 9 P (Chme), (epsilon subunit) collagen, tcatcagaagg atttcaagtgcc type XV, ccattacttccta aattatgagaag RCT155 AA923853 606 1606 606 alpha 1 m005592 5161 37124271 560 gcg cct (COL15A1) , MRNA vacuolar gggtttctcaaa atgggcgaagt proton gaaccagaaca gatgagagaag g g g ggg g RCT154 AA957163 599 1599 599 pp delta p07450 1610 333935 603 gga ctg polypeptide (VATD), MARNA Table 2 Human Human left Rat gene or right Region of Rat PCR Human Length Region of PCR human Gene name EST Whole rat Gene name primer PCR product human PCR product (5'end), rat accession or est size human (3' end), product size number gene product size forward number reverse orientation orientation ubiquinol ttggaattcaaa cgaggatgaag cytochrome aactccagccat atgtaaaagaa c reductase t gcca RCT153 AI028969 393 1363 363 binding XM_018456 515 146504 359 protein (UQCRB), mRNA eukaryotic aagcagtgatct ggcctccaggt translation gctcctccagc gctcaacgatta elongation at c RCT152 AI030272 603 1603 603 factor 1 beta XM_002668 958 266863 598 2 (EEF1B2), mRNA sirtuin ccaaagaacac tcaagtgttgttg (silent aatgtcgggctt gaagtggaggc mating type ca a information RCT150 AA859455 562 1563 562 regulation 2, NM_012239 1927 427986 560 S.cerevisiae, homolog) 3 (SIRT3), mRNA Table 2 Human Human left Rat gene or right Region of Rat PCR Human Length Region of PCR human Gene name EST Whole rat Gene name primer PCR product human PCR product (5'end), rat accession or est size human (3' end), product size number gene product size forward number reverse orientation orientation selenium cagatgtcaga tggtcagtgga binding gctacaatcgc gaaggtgatcc RCT148 AA819142 539 1539 539 protein 1 XM_001336 1416 8491408 560 ccc agg (SELENBP 1), mRNA KIAA0101 tgcctgttcaac gaatgcagttcg gene aaagctaattgg ttctctcctctcct RCT146 AA925567 562 1562 562 product XM_007736 836 20502 483 a c (KIAA0101 ), mRNA nucleolar atcatatgggct ccgatgaatcc protein p40; ttctcctgctgcc cttgtcacagac homolog of ag yeast RCT145 AA956268 579 1579 579 EBNA1 XM_002034 1322 180729 550 binding protein (P40), mRNA Table 2 Human Human left Rat gene or right Region of Rat PCR Human Length Region of PCR human Gene name EST Whole rat Gene name primer PCR product human PCR product (5'end), rat accession or est size human (3' end), product size number gene product size forward number reverse orientation orientation NADH gtacagcagct cttcaagatgcg dehydrogen cctcatggtct ctgcctgacca ase cc c (ubiquinone ) FeS protein 8 RCT143 AA925545 558 1558 558 XM_006097 779 86641 556 (23kD) (NADH coenzyme Q reductase) (NDUFS8), mRNA Table 2 human Size of Size of human left right rat gene target the human target the primer gene name rat whole rat right rat left gene name primer accession region PCR accession length region PCR (5' end) rat size primer primer human (3' end) number (rat) product number (human) product forward reverse (rat) (human) orientation orientation NADH dehydrogena se (ubiquinone) tttttattagggc gggggcacctc vector vector 1 beta RCT142 AA926306 371 1371 371 NM_005005 740 338691 354 aagtgcatgttc ctatgagagata primer primer subcomplex, tg cg 9 (22kD, B22) (NDUFB9), mRNA proteoglycan 4, (megakaryoc yte stimulating ggccttcttcca ccaaaccacca vector vector RCT141 AI113008 620 1620 620 factor, XM_001738 5011 32373875 639 gggcacttctgt ccagacctaac primer primer articular a aaa superficial zone protein) (PRG4), mRNA Table 2 human Size of Size of human left right rat gene target the human target the primer gene name rat whole rat right rat left gene name primer accession region PCR accession length region PCR (5' end) rat size primer primer human (3' end) number (rat) product number (human) product forward reverse (rat) (human) orientation orientation nectinlike protein 2 cacacgaatttc gggtgagagtc vector vector RCT140 AI029931 583 1583 583 (NECL2) AF132811 3512 8421422 581 tcgcaagttcca gatgatgaaatg primer primer mRNA, a cc complete cds TYPO protein tyrosine vector vector kinase gagagtattgg ggcccttacact RCT138 AA963886 560 1560 560 NM_003332 604 31550 520 ggagcggtctg gtggtgtccag primer primer binding gtc c protein (TYROBP), mRNA mitochondria 1 carrier ctggaagaagc vector vector gacctctgacct RCT136 AA926365 560 1560 560 protein CGI AF317711 1114 4841060 577 ttttgccgaactc primer primer ctacgcacccat 69 long form a mRNA, complete cds ggaaggggat gctcggactga RCT135/ AA926300vector vector stathmin x53305 1429 5568 564 ggggagaaagt gcaggactttcc stathmin (j04979) primer primer cagt tt Table 2 Size of Size of right human left rat gent target thé humain target the rimer primer gene name rat whole rat right rat left gene name accession len th re ion PCR p, (5'end) accession region PCR g g 3 end number (human roduct forward numbed (rat) product prer primer human) P reverse (rat) (human) orientation orientation aldoketo aldoketo RCT1581 reductase tcagtgttggca gacaccagcac aldosevector vector family 1, reduce" '' p meBlO M020299 1316 8341273 440 gagtttctgat catt aga c arc like protein (aldose reductase) CGI138 agggaggcaa gctggaaaccg vector vector protein RCT132 AA900081 563 1563 563 008155 1113 25581 557 gagaectttcgatagggagcatc primer primer (LOC51649), ctg tc mRNA for vector vector KIAAl184 ccgtgctaggc actcttgggtcg RCT278 AA819913 584 1584 584 AB033010 2951 23262897 572 gcagaaataca agatccctttgg primer primer protein, gag a partial cds serum amyloid A tgccatatctca gctacagcaca RCT149 AA997198 406 1406 406 (SAA) M26152 526 3492 490 gcttctctggac gatcagcacaa primer primer mRNA, a tga complete cds CpG binding vector vector protein RCT162 AA875643 513 1513 513 prer primer (CGBP), XM08699 2444 19362425 490 agggagagga gagctcac ca t mRNA annexin A2 cacaggagge tatgactcca AAA964578 450 1450 450 (ANXA2), NM 004039 1362 8721321 450 aaagtgtttcac aagggcaagg heavy chain primer primer atoca egg Table 2 human Size of Size of right human left human target the g primer rat gene target the rat right rat left gene name primer 5 end gene name rat whole accessi region PCR accession length region PCR (5'end) rat. size,,.,, primer primer human.... (3 end),. numbed (rat) product number (human) product'forward (rat) (human)..,. orientatio) (rat) orientation vector vector complement gggcaataggc acctggagaca RCT255 AI045144 582 1582 582 component 6 M011251 2472 11471731 585 agacacatttgg aggggatgtgg (C6), MRNA aa aat BCL2/adeno vector vector s E1B ttcaagaaatttg tggcactttgga RCT266 AI043589 584 1584 584 19KD NM_004330 2382 7961345 550 caaaccagtac gctattagtagc primer primer teracting agc aga mteractmg age aga protein 2 (BNIP2) nucleolar protein tttgtftttattgg aagaaaaagco RCT144 AA924405 452 1452 452 isjs/u 009574 1910 14481879 432 ggaacacggc aaagccccag primer primer repeat) a agg (NOP56), MARNA calpain 2, RCT157/ (m/II) large ccatggacaga tgcaaatgagt calpain 2 Ail 12943 605 1605 605 subunit XM 010682 3406 24543064 611 cactagtggtg gcttaaccciii ta a (Capn2) primer primer (CAtga a MARNA Human Human left Rat gene or right Region of Rat PCR Human Length Region of PCR human Gene name EST Whole rat Gene name primer PCR product human PCR product (5'end), rat accession or est size human (3' end), product size number gene product size forward number reverse orientation orientation cytochrome ttattgtctccat cgagattacatg P450, ggggtgggtga attcctgccaag subfamily a aca XIA (cholesterol Cytochro Cytochrome side chain NM_00 me P450 J05156 unknown unknown 1821 12571806 550 P450 11A1 cleavage) 0781 11A1 (CYP11A), nuclear gene encoding mitochondrial protein, Nhydroxy ttttgcactgga agacggagtcc 2 caagaagtcatt cgctctgtcgcc N ttt acetylaminof hydroxy luorene 2 sulfotransferas (ST1C1) acetylami L22339, e family, mRNA, vector vector NM_00 nofluorene AI030692 NM_03173 1363 1287 287 cytosolic, 1C, 2143 16222129 508 complete cds primers primers 6588 sulfotransf 2 member 2 or erase (SULT1C2), sulfotransfer ((ST1C1) ase family = C2) 1A member 2 Human Human left Region of Human Human right primer Original Rat gene Rat gene Rat PCR accessi Region of Whole gene PCR Rat right Rat left Gene name Lengt PCR primer (5' end), gene accession accession whole product (in on PCR name rat produ primer primer human h product (3' end forward ct size size reverse orientation gene) r orientation PC3 NGF gctcacagatc attcgcatcaac NGF inducible aagcacgcaac cacaagatgga inducible PC3 mRNA agt cc anti anti proliferative for NGF proliferative putative vector vector inducible PC3 ve AI146192 M60921 2519 1359 358 796 265784 520 secreted primer primer anti Y09943 putative protein proliferative secreted (PC3) protein protein mRNA, (PC3) complete cds iron agaaatgtgttc gccaatcctgta Iron responsive tcccaaaccgc ggcacaaaacc responsive element ct ag NM_01732 vector vector iron regulatory element AI045830 binding 3564 1576 576 Z11559 3498 29163467 552 1 primer primer factor binding protein protein (Ratireb), mRNA interalpha taatgagtggg gaagtgaccttc (globulin) actcttcctccc aagaacccccf Inter interalpha inhibitor H4 ca gg alpha inhibitor H4 (plasma inhibitor NM_01936 (plasma NM_01936 vector vector XM_00 AA901379 heavy chain 2958 1243 243 Kallikrein 2960 23802931 552 H4 heavy 9 primer primer (Ithih4), sensitive chain mRNA glycoprotein (Ithih4) (ITIH4), mRNA Human Humn left Region of Human Rat Human right primer Original Rate gene Rat gene Rat RCR accession Region of PCR Rat right Rat left Gene name Lengt PCR primer (5' end), gene accession accession whole product (in on PCR name rat produ primer primer human product (3' end) forward name rat number number size original number product ct size size reverse orientation gene) r orientation similar to ccagcaaatgc cgcaagccagc glycine agtgtatcgcaa agagttgtctta cleavage aa aa system protein CDK102 vector vector H XM_01 CDK102 AA999090 Y17321 1292 1416 416 1077 5741017 444 mRNA primer primer (aminomethyl 6378 carrier) (H. sapiens) (LOC82381), mRNA acacacacggc cctgcaggaca nm_01702 vector vector xm_009 MX1 MX1 NM_017028 278 mx1 2784 22012733 533 actcatgctcct aggacacctac 8 primer primer 773 aa agc aspartoacylase tgtgcttagatg gagaaagttgat aspartoacyla (aminoacylse cctaccgaataa tacccccggga Aspartoac NM_02439 vector vector XM_00 AI043719 se (Aspa), 1552 1499 499 2, Canavan 1418 8371240 404 ggc tg yease primer primer 8504 mRNA disease) (ASPA), encoding ggttgcagctat atacctactagg aldolase ctccttcccaac gtccaatgccc Aldolase (fructose aldolase B, c gc B, 1,6 vector vector fructose XM_00 functose AA957769 V01223 927 1413 413 1653 12111623 413 biphosphate primer primer biphosphate 5563 bisphosph aldolase, (ALDOB) ate subunit type B Human Human left Region of Human Rat Human right primer Original Rat gene Rat gene Rat PCR accessi Region of Whole gene PCR Rat right Rat left Gene name Lengt PCR Primer (5' end), gene accession accession whole product (in on PCR h product (3' end) forward name rat number number size original number product ct size size reverse orientation gene) r orientation 22kDa peroxisomal 22kDa tcaccattgaag cggccttcctca integral membrane peroxisomal aacctgcattat tgttgttcttcct peroxison protein 2,22 NM_03158 vector vector membrane cg AI137269 852 1256 256 NM_01 977 421957 537 al kDa primer primer proteinlike 8663 membrane (Pxmp2), (LOC55895), protein mRNA mRNA Major cgacccttgtac tcagctacgaat acute tcgcaggacctt tgcttccttctca phase a ca protein Tkiniogen tgtccctgga kininogen NM_01269 ctgtcttgcca XM_00 alpha1 M11661 (Kngt1), 1417 7021236 535 ggaggagg (KNG), 1481 7511275 525 cagccag 3195 (T mRNA a mRNA kininogen (Kngt1), mRNA acyl ttcacaaatgctt caagccactgtt Coenzyme A tccaaactgcaa tcagctccaga Acyl dehydrogenase aaa Coenzyme A AcylCoA , C4 to C12 dehydroge straight chain se, C40 to C NM_01698 vector vector NM_00 nase, AA925220 1866 1517 517 (ACADM0, 3387 26893122 434 12 straight 6 primer primer 0016 medium nuclear gene chain encoding (Acadm), mitochondrial mRNA protein, mRNA Human Human left Region of Human Rat Human right primer Original Rat gene PCR accessi Region of Whole gene PCR Rat right Rat left Gene name PCR primer (5' end), gene accession accession whole product (in on PCR name rat produ primer primer human product (3' end) forward number number size original number product et size size reverse orientation gene) r orientation hydroxyster hydroxydelta tggagctctagt accgcccttcaa 3beta oid 5steroid agtcaaaacga ccgccacatag hydroxyst dehydrogena dehydrogenase ccc eroid se6, NM_01726 vector vector , 3 beta and XM_00 AA923963 1634 1407 407 1605 10131512 500 dehydrogen delta<5>3 5 primer primer steroid elta 1544 name beta isomerase 1 (HSD3B1) (Hsd3b1) (HSD3B1), mRNA mRNA peroxisom peroxisomal Homo sapiens ttctaaggaaac ttgagtggcata al multifunctio hydroxysteroi aggacttgggc taaccaaaggc multifunct nal enzyme NM_02439 vector vector d (17beta) XM_00 aaa gg AA874974 2535 1542 542 2592 19952534 540 ional type II 2 primer primer dehydrogenase 3904 enzyme (Hsd17b4), 4 (HSD17B4), type II mRNA mRNA glycoprotein gcaggtcctaa gggcctcaact Osteoactiv osteoactivin vector vector (transmembra XM_0 agaaggggtg catggaagtga AA997841 AF184983 2320 1436 436 2658 7011116 416 in mRNA primer primer ne) nmb 4781 ggtt ctg (GPMB Receptor Protein aatgtgccaagt gtcggcagtte protein linked tyrosine tyrosine atttgaaggcg cagttcacaga protein phosphatase, gc ct NM_01914 vector vector phosphatase tyrosine AA926359 6469 1441 441 U407317 6000 54175989 573 rectpro 0 primer primer PTPsigma phosphata type, D (PTPsigma) sc (PTP (Ptprd), mRNA P1) mRNA Table 3 (+)cis3,5dimethyl2(3<BR> pyridyl)thiazolodin4<BR> (s)warfarin<BR> 1,2Dibromomethane<BR> 1chloro2nitrobenzene<BR> 2,4dinitrophenol<BR> 2,4dinitrotoluene<BR> 2acetylaminofluorene<BR> 2azido2deoxycytidine<BR> 2methylpentane<BR> 3methylpentane<BR> 4,4'methylene bis<BR> 4acetamidofluorene<BR> 5azacytidine<BR> 5chlorouracil<BR> 5fluorouracil<BR> 6mercaptopurine<BR> 7, 12dimethylbenz[a]anthracene<BR> acetaminophen<BR> acetaminophen/codeine<BR> acetohydroxamic acid<BR> acetone<BR> acetylalicylic acid<BR> acridine<BR> acrylamide<BR> acrylonitrile<BR> actinomycin<BR> actinomycin D<BR> acyclovir<BR> adenosine<BR> aflatoxin B1<BR> albuterol<BR> alendronate<BR> alendronate sodium<BR> alglucerase<BR> allopurinol<BR> allyl alcohol<BR> alosetron<BR> alprazolam<BR> alprostadil<BR> alteplase<BR> aluminum<BR> ambenonium<BR> amifostine<BR> amiloride<BR> aminobenzoate potassium<BR> aminoglutethimide<BR> benzene<BR> benzidine<BR> benzo(a)pyrene<BR> benzodiazepines benzoyl peroxide<BR> benztropine<BR> berylium<BR> beta carotene<BR> betamethasone<BR> betamethasone valerate<BR> bethanechol<BR> biphenyl<BR> bisacodyl<BR> bismuth subsalicylate plus<BR> bisoprolo/HCTZ<BR> bleomycin<BR> bradykinin antagonist<BR> bromfenac<BR> brominide tartrate<BR> bromobenzene<BR> bromocriptine<BR> bronchodilators<BR> btanapthylamine<BR> buclizine<BR> budesonide<BR> bumetanide<BR> bupropion HCL<BR> buspirone<BR> busulfan<BR> cadmium<BR> cadmium chloride<BR> caffeine<BR> calcipotriene<BR> calcitonin salmon<BR> calfactant<BR> camptothecin<BR> candesartan cilexetil<BR> capsaicin<BR> captopril<BR> carbamate(s)<BR> carbamazapine<BR> carbaryl<BR> carbenicillin<BR> carbidopa<BR> carbon disulfide<BR> carbon monoxide<BR> carbon tetrachloride<BR> carboplatin<BR> carisoprodol<BR> carmustine<BR> carvediol<BR> cefaclor<BR> cefepime<BR> cefprozil<BR> ceftibuten cefuroxime <BR> <BR> <BR> celecoxib<BR> <BR> <BR> <BR> <BR> cephalexin cephalosporins <BR> <BR> cerivastatin<BR> <BR> <BR> <BR> <BR> cetirizine chenodiol chlophedianol chloral hydrate chlorambucil chloramphenicol chloroform chloroquine chlorpromazine chlorpropamide chlorthalidone chlorzoxazone cholestyramine chromium VI cimetidine cinoxacin <BR> <BR> <BR> ciprofloxacin<BR> <BR> <BR> <BR> <BR> cisapride cisplatin <BR> <BR> citalopram<BR> <BR> <BR> <BR> <BR> clarithromycin clavulanate clavulanic acid <BR> <BR> <BR> clenbuterol<BR> <BR> <BR> <BR> <BR> clidinium clindamycin clofibrate clomiphene clonazepam clonidine <BR> <BR> <BR> clotrimoxazole<BR> <BR> <BR> <BR> <BR> cloxacillin<BR> <BR> <BR> <BR> <BR> <BR> clozapine<BR> <BR> <BR> <BR> CMC cobalt codeine colchicine colestipol collagenalginate conjugated estrogens copolymer1 copper corticosterone cortisone courmarin cromolyn cumene cyanamide cyanides cyclacillin<BR> <BR> cyclandelate<BR> <BR> cyclizine<BR> <BR> cyclobenzaprine cyclohexane cyclohexanone cycloheximide<BR> <BR> cyclopegic<BR> <BR> cyclopentolate<BR> <BR> cyclophosphamide<BR> <BR> cycloserine cyclosporin A cyclosporine cyclosporine A cytosin arabinoside cytoxin dacarbazine dalteparin injection danazol dantrolene dapsone daunomycin daunorubicin DDT DEEP dehydrocholic acid desmopressin desogestrel dexamethasome dextromethorphan dextrothyroxine diazepam diazoxon dichloralphenazone dichlorobenzene dichloromethane diclofenac <BR> <BR> diclofenacdihydrazine<BR> dicloxacillin<BR> <BR> <BR> dicyclomine didanosine dieldrin <BR> <BR> diethylamine<BR> <BR> diethylhexylphthalate<BR> <BR> diethylstilbestrol diethystilbesterol difenoxin<BR> <BR> diflunisal digitalis glycosides digitoxin digoxin dihydrazine dihydroergotamine mesylate<BR> <BR> <BR> <BR> dihydrolazine<BR> <BR> <BR> diltiazem dimethyl sulfoxide dimethylacetamide dimethylformamide <BR> <BR> dimethylhydrazine<BR> <BR> <BR> <BR> dimethylnitrosamine dinitroorthocresol dinoprostone dione dioxan <BR> <BR> diphenidol<BR> <BR> <BR> diphenoxylate<BR> <BR> <BR> <BR> dipyridamole<BR> <BR> <BR> disopyramide disulfiram divalproex divalproex sodium DNA docusate sodium <BR> <BR> dolasetron mesylate<BR> <BR> <BR> <BR> donepezil doxazosin doxercalciferol <BR> <BR> doxorubicin<BR> <BR> <BR> doxycycline<BR> <BR> <BR> <BR> enalapril endotoxin <BR> <BR> endrin<BR> <BR> <BR> enflurane<BR> <BR> <BR> <BR> enoxaparin entacapone ephedrine epirubicin eptifibatide ergoloid mesylates ergonovine erythromycin erythromycin estolate estradiol estramustine etanercept ethacrynic acid ethanol ethchlorvynol ethinamate ethinyl estradiol ethionamide ethyl methanesulfonate etliylbenzene ethylene oxide ethyleneglycol dinitrate ethyleneglycol (s) etidronate etoposide etretinate exemestane <BR> <BR> <BR> famciclovir<BR> <BR> <BR> <BR> <BR> famotidine<BR> <BR> <BR> <BR> <BR> felbamate<BR> <BR> <BR> <BR> <BR> felodipine felodipine SR fenofibrate <BR> <BR> <BR> fenoldopam mesylate fentanyl citrate fexofenadine <BR> <BR> <BR> fialuridine<BR> <BR> <BR> <BR> finasteride flavoxate flecainide acetate flosequinan fluconazole flunisolide fluoride fluoroquinolones fluorouracil <BR> <BR> <BR> fluoxetine<BR> <BR> <BR> <BR> <BR> flutamide fluticasone fluticasone propionate fluvastatin fluvoxamine maleate foscarnet sodium fosinopril fosphenytoin furazolidone furfural furfuryl alcohol <BR> <BR> furosemide<BR> <BR> <BR> <BR> <BR> <BR> gabapentin<BR> <BR> <BR> <BR> ganciclovir ganirelix acetate gemcitabine gemfibrozil gentamicin germanium <BR> <BR> glimepiride<BR> <BR> <BR> <BR> <BR> glipizide glucagon <BR> <BR> glyburide<BR> <BR> <BR> <BR> glycopyrrolate gold compounds gold sodium thiomalate granisetron grepafloxacin griseofulvin<BR> <BR> <BR> <BR> guaifenesin guanabenz <BR> <BR> guanadrel<BR> <BR> <BR> guanetliidine<BR> <BR> <BR> <BR> guanfacine guanine haloperidol halothane heparin hexachlorobenzene hexachlorobutadiene hismanol hydantoin <BR> <BR> hydralazine<BR> <BR> <BR> hydrochlorothiazide<BR> <BR> <BR> <BR> liydrocodone hydrocortisone hydroxychloroquine hydroxyurea <BR> <BR> hydroxyzine<BR> <BR> <BR> hyoscine<BR> <BR> <BR> <BR> hyoscyamine hyperozia ibuprofen ibutilide fumarate imiglucerase injection imiquimod 5% cream inactivated hepatitis A vaccine <BR> <BR> indapamide<BR> <BR> <BR> indinavir indomethacin insulin interferonbetala (recombinant) interferonbetalb (recombinant) iodinated glycerol <BR> <BR> iodoacetamide<BR> <BR> <BR> <BR> iodoquinol ipecac <BR> <BR> iphosphamide<BR> <BR> <BR> <BR> ipratropium irbesartan irinotecan isometheptene isoniazid isonicotinic acid isopropanol isopropylnitrate isoproterenol isosorbide mononitrate S. A. isotretinoin isoxsuprine isradipine itraconazole kanamycin ketoconazole <BR> <BR> ketorolac<BR> <BR> <BR> lactulose<BR> <BR> <BR> <BR> lamivudine, 3TC<BR> <BR> <BR> <BR> lamotrigine lansoprazole latamoxef latanoprost lead lead tetraethyl <BR> <BR> leflunomide<BR> <BR> <BR> <BR> letrozole leucovorin leuprolide levamisole levetiracetam levobupivacaine levocabastine <BR> <BR> levocamitine<BR> <BR> <BR> <BR> levodopa<BR> <BR> <BR> levofloxacin levonorgestrel levothyroxine lidocaine lincomycin <BR> <BR> lindane<BR> <BR> <BR> <BR> lipopolysaccharide liposomal amphotericin B lisinopril lispro insulin lithium 1norgestrel 1norgestrel/ethinyl estradiol <BR> <BR> lomustine<BR> <BR> <BR> <BR> loperamide loracarbef <BR> <BR> loratadine<BR> <BR> <BR> <BR> Loratidine/Pseudoephedrine lorazepam losartan <BR> <BR> lovastatin<BR> <BR> <BR> <BR> loxapine magnesium sulfate maleic anhydride manganese maprotiline masoprocol mazindol mecamylamine mechlorethamine meclizine medroxyprogesterone <BR> <BR> mefloquine<BR> <BR> <BR> <BR> melatonin<BR> <BR> <BR> <BR> <BR> melphalan menotropin meprobamate merbarone mercaptopurine mercury meropenem mesalamine metformin methanol methenamine methicillin methotrexate methyl methanesulfonate methylcellulose methylchloride methyldopa methylergonovine methylethylketone methylmercury <BR> <BR> methylphenidate<BR> <BR> <BR> <BR> <BR> methylprednisolone<BR> <BR> <BR> <BR> methyprylon<BR> <BR> <BR> <BR> <BR> methysergide<BR> <BR> <BR> <BR> <BR> metoclopramide<BR> <BR> <BR> <BR> <BR> metoprolol metronidazole metyrapone metyrosine mexiletine mibefradil miconazole cream 2% miglitol <BR> <BR> minocycline<BR> <BR> <BR> <BR> minoxidil<BR> <BR> <BR> <BR> <BR> misoprostol<BR> <BR> <BR> <BR> <BR> mitomycin C<BR> <BR> <BR> <BR> <BR> mitotane mitoxantrone mixed amphetamines <BR> <BR> moclobemide<BR> <BR> <BR> <BR> <BR> molindone mometasone <BR> <BR> monobromomethane<BR> <BR> <BR> <BR> <BR> monochlorobenzene moricizine <BR> <BR> <BR> moxifloxacin<BR> <BR> <BR> <BR> <BR> mupirocin<BR> <BR> <BR> <BR> nabilone nabumetone nafarelin<BR> <BR> <BR> <BR> <BR> nafcillin nalidixic acid naloxone naltrexone naproxen <BR> <BR> naratriptan<BR> <BR> <BR> <BR> natamycin navirapine nedocromil nefazodone neomycin <BR> <BR> neomycin/Polymx/HC<BR> <BR> <BR> <BR> <BR> neostigmine Nhexane nicardipine nickel nicorandil nicotine nifedipine nimodipine nitrobenzene nitrofurantoin nitroglycerin nitroglycerine nitrous oxide nizatidine NnitrosoNethylurea NnitrosoNmethylurea norethindrone norethindrone/ethinyl estradiol norgestimate norgestimate/ethinyl estradiol norgestrel norgestrel/ethinyl estradiol nylidrin nystatin <BR> <BR> ofloxacin<BR> <BR> <BR> <BR> <BR> oligomycin<BR> <BR> <BR> <BR> <BR> olsalazine omeprazole <BR> <BR> Organophosphorus<BR> <BR> <BR> <BR> <BR> orphenadrine<BR> <BR> <BR> <BR> otoluidine oxacillin oxaprozin <BR> <BR> oxtriphylline<BR> <BR> <BR> <BR> <BR> oxybutynin oxycodone oxymetazoline paclitaxel pancreatin pancrelipase papaverin paraldehyde paramethasone parathione paregoric paroxetine pediculisides <BR> <BR> pemoline<BR> <BR> <BR> penicillamine penicillin pentachlorophenol pentamidine pentoxifylline pepsin <BR> <BR> pergolide<BR> <BR> <BR> perhexiline perindopril perphenazine pexiganan acetate phenazopyridine phendimetrazine phenformin phenobarbital phenol phenolphthalein phenothiazines <BR> <BR> phentermine<BR> <BR> <BR> <BR> phenylephrine phenylhydrazine HCL phenylpropanolamine phenytoin phorbol 12myristate 13acetate diester phtalic anhydride pilocarpine pioglitazone piroxicam podophyllum poloxamer 188 polycarbophil calcium polychlorinated biphenyl polycyclic hydrocarbons polyethylene glycol polythiazide potassium chloride potassium iodide potassium phosphates pramipexole pravastatin prazosin prednisolone prednisone pregnenolone16alphacarbonitrile primaquine<BR> <BR> <BR> <BR> <BR> primethamine<BR> <BR> <BR> <BR> <BR> primidone probenecid probucol procainamide procarbazine proflavin progesterone progestins promethazine propafenone <BR> <BR> propantheline<BR> <BR> <BR> <BR> propoxyphene<BR> <BR> <BR> <BR> <BR> propranolol propulsid <BR> <BR> propyleneglycol<BR> <BR> <BR> <BR> <BR> pseudoephedrine psoralens psyllium puromycin pyridostigmine pyridoxine (vitamin b6) quinacrine <BR> <BR> quinapril<BR> <BR> <BR> <BR> quinidine quinine rabeprazole raloxifene <BR> <BR> <BR> ramipril<BR> <BR> <BR> <BR> ranitidine recombinant clotting factor vu recombinant interferon alpha2b recombinant OspA remoxipide reserpine rezulin <BR> <BR> <BR> ribavirin<BR> <BR> <BR> <BR> <BR> <BR> rifampicin<BR> <BR> <BR> <BR> rifampin<BR> <BR> <BR> <BR> <BR> rimantadine<BR> <BR> <BR> <BR> <BR> risedronate<BR> <BR> <BR> <BR> <BR> risperidone<BR> <BR> <BR> <BR> ritodrine<BR> <BR> <BR> <BR> <BR> rosiglitazone salicylates salmeterol saquinavir scopolamine seldane selegiline selenium sertraline sibutramine sildenafil citrate silver simethicone <BR> <BR> <BR> simvastatin<BR> <BR> <BR> <BR> <BR> <BR> <BR> smephenytoin sodium azide sodium ferric gluconate soman somatostatin sotalol spironolactone stanol esters streptozotocin styrene succinimide sucralfate sulfacytine sulfadoxine sulfamethoxazole sulfasalazine sulfinpyrazone sulfisoxazole sumatriptan synthetic pyrethroids tacrine <BR> <BR> <BR> tamoxifen<BR> <BR> <BR> <BR> <BR> tamsulosin Tbutylhydroperoxide TCDD tellerium telmisartan temazepam terazosin terbinafine HCl terbutaline sulfate terfenadine terpin hydrate tertbutylphenol testolactone tetrachloroethylene tetracycline tetracycline HUI thalium theophylline thiamine <BR> <BR> <BR> thiazide<BR> <BR> <BR> <BR> <BR> <BR> <BR> thioguanine<BR> <BR> <BR> <BR> <BR> <BR> thiopurine thiothixene <BR> <BR> <BR> tiagabine<BR> <BR> <BR> <BR> <BR> <BR> ticlopidine tienilic acid timolol<BR> <BR> <BR> <BR> <BR> tiopronin<BR> <BR> <BR> <BR> tirofiban<BR> <BR> <BR> <BR> <BR> tobramycin tobramycin/dexamethasone <BR> <BR> tocainide<BR> <BR> <BR> <BR> <BR> tolbumamide<BR> <BR> <BR> <BR> <BR> tolcapone<BR> <BR> <BR> <BR> tolterodine<BR> <BR> <BR> <BR> toluene toluene diisocyanate topotecan toremifene <BR> <BR> <BR> tramadol<BR> <BR> <BR> <BR> trandolapril<BR> <BR> <BR> <BR> <BR> transplatin trastuzumab trazodone tretinoin triamcinolone triamterene/HCTZ <BR> <BR> <BR> triamterine<BR> <BR> <BR> <BR> triazolam trichloroethane trichloroethylene triethylamine <BR> <BR> triethylbenzenes<BR> <BR> <BR> <BR> triethylenemelamine<BR> <BR> <BR> <BR> triethylenethiophosphoramide<BR> <BR> <BR> <BR> <BR> trihexyphenidyl<BR> <BR> <BR> <BR> trilostane<BR> <BR> <BR> <BR> trimeth/sulfameth<BR> <BR> <BR> <BR> trimethobenzamide<BR> <BR> <BR> <BR> <BR> trimethoprim<BR> <BR> <BR> <BR> <BR> troglitazone trovafloxacin uranium urokinase ursodiol valproic acid valsartan vanadium vancomycin <BR> <BR> venlafaxine<BR> <BR> <BR> <BR> verapamil vincristine vinyl chloride warfarin Wy 14, 643 xanthine xylene xylometazoline zafirlukast zalcitabine <BR> <BR> zidovudine zinc <BR> zolpidem Table 4 Human In Vitro Hepatocyte microarray data * Upregulated genes : Activating transcription factor 3 (amiodarone, chlorpromazine, paracetamol) activating transcription factor 4 (amiodarone, chlorpromazine, paracetamol) BRCA1 (tacrine, perhexiline) Phenol sulfotransferase (amiodarone, chlorpromazine) Hepatocyte nuclear factor 4 (amiodarone, paracetamol) Thioredoxin (chlorpromazine, paracetamol, perhexiline) Ferritin Hchain (chlorpromazine, paracetamol, perhexiline, amiodarone) Gadd 153 (amiodarone, chlorpromazine) Insulinlike growth factor binding protein 1 (chlorpromazine, paracetamol, perhexiline, amiodarone) Tcell cyclophilin (chlorpromazine, paracetamol) Cathepsin B (chlorpromazine, paracetamol, perhexiline, amiodarone, tacrine) Cathepsin L (amiodarone, paracetamol) Downregulated genes : Cytochrome c oxidase subunit II (amiodarone, chlorpromazine, perhexiline) Gammaglutaniyl transpeptidase (amiodarone, paracetamol) Glyceraldehyde 3phosphate dehydrogenase (paracetamol, amiodarone, chlorpromazine, perhexiline, tacrine) Apolipoprotein CIII (amiodarone, chlorpromazine, paracetamol) Fas antigen (amiodarone, paracetamol) Sterol carrier protein 2 (amiodarone, tacrine) Transferrin (amiodarone, chlorpromazine, paracetamol, tacrine) Tyrosine aminotransferase (paracetamole, amiodarone, tacrine) Table 5.
Description:
HUMAN TOXICQLQGICALLY RELEVANT GENES AND ARRAYS TECHNICAL FIELD [0001] This invention is in the field of toxicology. More specifically, the invention provides for methods to identify and isolate human genes which are indicative of toxicological responses, human genes which can be used to determine toxicological responses in vitro and in vivo to various agents, methods of making human microarrays, and methods of using human microarrays.

BACKGROUND OF THE INVENTION [0002] Every year, many new drugs and chemical compounds are discovered, produced, and introduced into the public domain. Guidelines set forth by the U. S. Food and Drug Administration (FDA) require toxicity studies to be conducted before a new drug or compound can be approved for human consumption or use. Toxicological studies are an important part of drug development but toxicity studies traditionally have required long time periods using a variety of animal models, and are quite often very expensive to conduct.

Additional information on safety and toxicity is obtained during clinical trials on humans which are also time consuming and can require long time periods to conduct. A two year toxicity study in rats can cost approximately $800,000. See, for example, Casarett and Doull's Toxicology, 4th Edition, M. O. Amdur et al., eds. Pergamon Press, New York, N. Y. p. 37 (1991). Further, traditional toxicology studies are not practical for assessing the toxicity of the large numbers of drug candidates derived from high throughput screening of combinatorial chemical libraries. Traditional toxicological methods have offered little insight into molecular mechanisms of toxicity, which makes extrapolation of toxicity results from animal models to humans difficult and often result in numerous failures in subsequent stages of development and post-launching of the drug.

[0003] Toxicogenomics allows for a better understanding of mechanisms of organ and system toxicity and facilitates prediction of deleterious outcomes prior to their detection by more laborious and time-consuming means. If toxicity manifested at the organism level is preceded by altered expression of related genes, then detection of altered gene expression may serve as an early warning for subsequent deleterious outcomes. Altered gene expression may precede organ or system outcomes by weeks, months or even years. To the extent that a causal or predictive relationship can be demonstrated between early alterations in gene expression and delayed manifestations of toxicity, measuring the alterations in gene expression may reduce reliance on observing delayed manifestations of toxicity. Better understanding of molecular mechanisms through toxicogenomics may also improve the predictive accuracy of animal models to humans, and in vitro systems to in vivo settings. A molecular approach to toxicology could save time, money, and animal resources. Some methods and kits for determining toxicity have been disclosed. See, for example, WO 00/47761, WO 01/32928, U. S. Patent Nos. 5,585, 232; 5, 589, 337; and 5,811, 231 ; and pending U. S. provisional patent applications 60/220, 057,60/254, 232,60/264, 933, and 60/308,161.

[0004] With the advent of molecular and recombinant technology, genomic and molecular analysis provides another method by which toxicity may be measured. Differential gene expression technology involves detecting the change in gene expression of cells exposed to various stimuli. The stimulus can be in the form of growth factors, receptor-ligand binding, transcription factors, or exogenous factors such as environmental agents, chemicals, or pharmaceutical compounds.

[0005] Several methods are available for detecting differential gene expression. One method is using an array of polynucleotides. A polynucleotide microarray may include genes for which full-length cDNAs have been accurately sequenced and genes which may be defined by high-throughput, single-pass sequencing of random cDNA clones to generate expressed sequence tags (ESTs).

Researchers focused on detecting changes in expression of individual mRNAs can use different methods to detect changes in gene expression e. g., microarray, gel electrophoresis, etc. Other methods have focused on using the polymerase chain reaction (PCR) and/or reverse transcriptase polymerase chain reaction (RT-PCR) to define tags and to attempt to detect differentially expressed genes. Many groups have used PCR methods to establish databases of mRNA sequence tags which could conceivably be used to compare gene expression among different tissues. See, for example, Williams, J. G. K., Nucl. Acids Res. 18: 6531,1990 ; Welsh, J. , et al. Nucl. Acids Res., 18: 7213,1990 ; Woodward, S. R. , Mamm.

Genome, 3: 73, 1992 ; and Nadeau, J. H. , Mamm. Genome 3: 55,1992. This method has also been adapted to compare mRNA populations in a process called mRNA differential display.

[0006] The process of isolating mRNA from cells or tissues exposed to a stimulus, e. g. , drugs or chemicals, and analyzing the expression with gel electrophoresis can be laborious and tedious. To that end, microarray technology provides a faster and more efficient method of detecting differential gene expression. Differential gene expression analysis by microarrays involves nucleotides immobilized on a substrate whereby nucleotides from cells which have been exposed to a stimulus can be contacted with the immobilized nucleotides to generate a hybridization pattern. This microarray technology has been used for detecting secretion and membrane-associated gene products, collecting pharmacological information about cancer, stage specific gene expression in Plasmodium falciparum malaria, translation products in eukaryotes, air-pollutant-induced lung injury, and a number of other scientific inquiries. See, for example, Diehn M, et al., Nat. Genet. 25 (1) : 58-62 (1993); Scherf, U. , et al., Nat Genet. 24 (3): 236-44 (1993); Hayward R. E. , et al., Mol. Microbiol. 35 (1) : 6- 14 (1993); Johannes G. , et al., Proc. Natl. Acad. Sci. 96 (23): 13118-23 (1993); and Nadadur, S. S. , et al., Inhal. Toxicol. 12 (12): 1239-1254 (2000). Microarray technology has also been used in exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis and in rats. See, for example, Wilson M. , et al. Proc Natl Acad Sci. 96 (22): 12833-8 (1999) ; Waring, J. F., Toxicol. Lett.

120 (1-3): 359-368 (2001) ; Cunningham, M. J., Ann. N. Y Acad. Sci. 919: 52-67 (2000); and Bulera, S. J., Hepatology 33 (5): 1239-1258 (2001).

[0007] There exists a need for methods that are fast, efficient, cost-effective, capable of generating large amounts of toxicology data, and could spare many animals from being the subjects of laboratory tests. There also exist a need for a method of effectively selecting genes which are toxicologically relevant to agents being tested and can predict toxicity on a cellular, organ, or system level. There also exists a need for a toxicological database of information whereby one can obtain information about one or more agents being tested and how that agent (s) affects a particular organ or system and algorithms which may be used to identify toxicologically relevant genes and correlate toxicity between agents and target genes.

[0008] Molecular toxicology analysis or toxicogenomics can provide a vast amount of information in the form of a database from a collection of toxicological response data that would be useful in toxicological analysis. The invention and its embodiments provided herein fulfill the aforementioned needs.

SUMMARY OF THE INVENTION [0009] Disclosed herein are methods of identifying and isolating human genes which are toxicologically relevant and methods of using these toxicologically relevant human genes to determine toxic responses to an agent. Further, arrays containing the human genes, methods of making these arrays, and methods of using these arrays are provided. Also disclosed herein are primer sequences for toxicologically relevant rat genes which are useful for obtaining the toxicologically relevant human homologues.

[0010] In one aspect, a method of identifying a toxicologically relevant human gene is disclosed whereby the gene expression profile of untreated human cells is obtained as well as a gene expression profile of human cells treated with an agent. The gene expression profile of untreated human cells is compared with the gene expression profile of the treated human cells to obtain a gene expression profile indicative of a toxicological response. In some aspects, human cells can be any type of cells including but not limited to biological samples from liver, lung, heart, kidney, spleen, testes, thymus, brain, cultured primary human cells, or cells lines obtained from commercial or other sources (e. g., ATCC). The agent can be any type of synthetic or non-synthetic compound including but not limited to agents listed in Table 3.

[0011] In another aspect, a method of isolating human genes indicative of a toxicological response to an agent is provided wherein sequences of mammalian, non-human genes associated with toxicological responses are provided, primers for human genes homologous to said mammalian, non-human genes associated with toxicological responses are provided; and the primers are used to amplify human gene sequences from human cDNA libraries.

[0012] In yet another aspect, a method for determining a toxicological response to an agent is provided wherein cells are exposed to an agent and a first gene expression profile is obtained and then compared to a gene expression profile of toxicologically relevant human genes to determine if the first gene expression profile is indicative of a toxicological response. In one aspect, the gene expression profiles of one or more toxicologically relevant human gene (s) are stored in a database. In another aspect, a database containing multiple gene expression profiles of toxicologically relevant human genes is used.

[0013] In yet another aspect, a method for determining a toxicological response to an agent in an organ is provided wherein cells from the organ are exposed to an agent and a gene expression profile is obtained and then compared to a gene expression profile of toxicologically relevant human genes to determine if the first gene expression profile is indicative of a toxicological response in an organ.

[0014] In another aspect, a method for screening an agent (e. g. , drug, medicament, or pharmaceutical composition) for potential toxicological responses is provided wherein cells are exposed to an agent; and a gene expression profile is obtained and then compared to a gene expression profile of toxicologically relevant human genes to determine if the first gene expression profile is indicative of a toxicological response in genes associated with toxicological responses. In one aspect, a database containing at least one gene expression profile of toxicologically relevant human genes is used for comparison.

[0015] In one aspect, the invention relates to methods of identifying human genes and gene sequences which are indicative of a toxicological response. These genes and their gene expression profiles are stored in a database. The database is useful for toxicological studies and analysis, particular when applied to the screening, development, and testing of potential new drugs. A panel of genes indicative of toxicity can vary between organs different in time of exposure to one or more agents, resulting effects of agent (s) and, different compounds.

[0016] In another aspect, a method for generating a human array comprising at least ten human genes which are indicative of a toxicological response is provided. Genes indicative of toxicological response are immobilized to a substrate.

[0017] In another aspect, an array is provided comprising at least ten human toxicological response genes or a portion thereof immobilized on a substrate. The human genes are assembled in an array such that at least 2 genes, more preferably at least 5 genes, more preferably at least 10 genes, more preferably at least 20 genes, more preferably at least 30 genes, even more preferably at least 40 genes, more preferably at least 50 genes, more preferably at least 100 genes, more preferably at least 250 genes, more preferably at least 350 genes, more preferably at least 400 genes, more preferably at least 500 genes, more preferably at least 600 genes, more preferably at least 750 genes, more preferably at least 850 genes, and more preferably at least 1000 genes are assembled on such array. In one aspect, the toxicologically relevant genes are attached to the array substrate by covalent linkage. In another aspect, the genes or portions thereof are capable of hybridization to expressed nucleic acids derived from a cell and are capable of indicating a toxicological response of the cell to said agent.

[0018] In yet another aspect, a method for obtaining a gene expression profile is provided whereby a population of cells is exposed to an agent, cDNA from the population of cells is obtained, labeled, and contacted with the array comprising toxicologically relevant genes.

[0019] In yet another aspect, a method for obtaining a human homologue of a toxicologically relevant non-human gene is provided whereby the sequence of a human homologue is obtained by using the sequence of said non-human gene in a sequence search; primers to the human homologue are provided; and primers to the human homologue are used to amplify a sequence of the human homologue from a human cDNA library.

[0020] In still another aspect of the invention, primer sequences that are used for identifying human genes are disclosed. These primer sequences can be used for probes, for PCR-related amplification, included on an array chip for identifying nucleotide sequences related to toxicological responses, or for identifying and isolating novel human genes. Sequences of such primers and methods of using thereof are disclosed herein and in Table 2.

[0021] In yet another aspect, toxicologically relevant human sequences are cloned and/or maintained in expression or cloning vectors.

[0022] In yet another aspect, expression or cloning vectors comprising human toxicologically relevant genes are maintained in suitable host cells.

[0023] A method for determining a toxicological response to an agent is provided, the method comprising: (a) exposing cells to an agent or obtaining cells derived from an individual exposed to an agent; (b) obtaining a test expression profile of one or more human toxic response genes in the cells, such as the genes identified in and corresponding to the full or partial gene sequences disclosed in the Tables herein, such as Table 1, 2 and 5; and (c) comparing the test expression profile to a reference gene expression profile of human toxic response genes indicative of toxicity, thereby to determine the presence of a toxic response to the agent. The cells may be derived, for example, from the liver, lung, heart, kidney, spleen, testes, thymus, skin, bone, muscle, gastrointestinal tract, skin, bone, blood, or brain, thyroid, muscle, nucleated cells of the blood, gastrointestinal tract or pancreas. Such cells may optionally be cultured cells. The cells may be from an organ or body fluid such as blood or cells in culture, and the test expression profile of human toxic response genes can be compared to the reference gene expression profile, to determine the presence of a toxicological response in the organ.

[0024] The cells in which a toxicological response is determined can be human. The cells may also may be primate, such as primates closely related to human.

[0025] The gene expression profile may be obtained by measuring RNA or protein levels. RNA levels may be measured by hydridization to an array, or other methods, such as real-time polymerase chain reaction, Rnase protection, Northern blot, electrochemical hybridization detection, or branched-chain, to quantitatively detect levels, for example, of messenger RNA.

[0026] The toxicity of the agent may be evaluated by determining if there is a significant correlation between the test expression profile and the reference expression profile. This correlation can be formally determined by a number of statistical correlation measures using computer assisted statistical analysis methods available in the art or other methods disclosed herein. An observed correlation can indicate that the agent has a similar expression profile to other agents in a database with the inference that the agent will have similar toxic properties. In addition to matching expression profiles for agents, the agent may correlate with expression profiles that are indicative of a specific toxic endpoint.

This would allow determination of specific toxic properties. The toxicity of the agent may also be evaluated by examining the profiles for expression specific marker genes using models that have been shown through analysis of gene expression databases to be predictive of specific toxicity endpoints. Methods which may be used in the practice of the invention, and examples of identification and use of predictive markers are described in U. S. Provisional Appl. Nos.

60/313,080, 60/361, 128 and 60/379,861. The reference gene expression profiles may be profiles obtained by previously exposing cells to a toxic agent or profiles obtained from cells of individuals previously exposed to a toxic agent. The reference gene expression profiles indicative of toxicity are, for example, stored in a database and will consist of expressions that can be categorized by agents as well as expressions that can be categorized by specific toxic endpoints. The reference gene expression profiles may be, for example, profiles obtained by previously exposing cells to a toxic agent at various doses and for various amounts of time or by other methods disclosed herein.

[0027] In one embodiment, the agent is a pharmaceutical composition such as a drug or diagnostic agent, and the method comprises a method of screening the agent to determine a toxicological response of the pharmaceutical agent in the cells. In one embodiment, rapid screening of multiple agents and multiple tissues can be implemented. This can be performed using automated or semi-automated equipment for high-throughput exposure of cells to multiple agents, processing of exposed cells and analysis of gene expression.

[0028] The cells exposed to the agent may be cells obtained from a human tissue or body fluid sample, or cultured cells, which are, for example, exposed in vitro to the agent, or the cells may be from a human subject who was exposed to a pharmaceutical or industrial agent. The agent may be exposed to the cells at various concentrations or for various amounts of time or by various routes of exposure. The test expression profile of at least two human toxic response genes in the cells is obtained, or, for example, at least 10, at least 20, at least 50, at least 200 or at least 500 human toxic response genes.

[0029] In another embodiment, an array comprising one or more polynucleotides that are the genes corresponding to the full or partial gene sequences disclosed herein, for example, in Tables 1,2, or 5, or fragments of at least 20 nucleotides thereof, or fragments that are, or are at least, 30,40, 50,100, 200,300, 400, 500 or 600 nucleotides long. The genes may be responsive, e. g., in kidney, liver, spleen, heart, brain, lung, testis thymus, blood, skin or brain cells.

The array may include, e. g. , at least 25,50, 200,500 or more of the polynucleotides.

[0030] In another embodiment, gene expression can be measured by any of a variety of methodologies for quantitative detection of specific RNA species coded for by the genes corresponding to the sequences herein, for example, in Tables 1, 2 or 5. These methods include real-time polymerase chain reaction, RNase protection, Northern blot, electrochemical hybridization detection, branched-chain or other methods to quantitatively detect levels of messenger RNA. The expression may be measured, for example, for at least 1, 2,10, 20,50, 100,200, 300,400, or 500 genes.

[0031] In another embodiment, gene expression can by measured by any of a variety of methodologies for quantitative detection of specific protein species coded for by genes corresponding to sequences herein including those identified in Tables 1,2 and 5. These methods would include use of specific antibodies in formats such as enzyme-linked immunoabsorbent assays, Western blots and mass- spectrometry methods.

[0032] The disclosure of all patents, patent applications, provisional applications and publications referred to herein are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE TABLES [0033] Table 1 is a list of toxicologically relevant human sequences and primers which may be used to obtain the toxicologically relevant human sequences.

[0034] Table 2 is a chart listing toxicologically relevant rat genes, primers which can be used for obtaining toxicologically relevant rat genes, and primers which were used to isolate human toxicologically relevant genes which are homologues of toxicologically relevant rat genes.

[0035] Table 3 is a list of agents which can be or are used in obtaining toxicologically relevant human genes.

[0036] Table 4 is a chart with human microarray data generated after exposure of human hepatocytes, in vitro, to 30mM amiodarone, 50mM chlorpromazine, 10mM paracetamol, 15mM perhexiline or 50mM tacrine. All genes were repressed or induced at least 2-fold. Some of the genes that are up-or down- regulated are known to be toxicologically relevant and others are not generally known to be toxicologically relevant.

[0037] Table 5 is a list of target sequences obtained from human gene sequences cloned using rat sequence-derived primers listed in Table 2 DETAILED DESCRIPTION OF THE INVENTION [0038] Throughout this disclosure, various publications, patents, patent applications, published patent specifications, and other references are indicated by an identifying citation. All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.

General Techniques [0039] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning : A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed. , 1984); Animal Cell Culture (R. I. Freshney, ed. , 1987); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds. ); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds. , 1987); Current Protocols in Molecular Biology (F. M.

Ausubel et al., eds. , 1987); PCR : The Polymerase Chain Reaction, (Mullis et al., eds. , 1994); Current Protocols in Immunology (J. E. Coligan et al., eds. , 1991); The Immunoassay Handbook (David Wild, ed. , Stockton Press NY, 1994); Antibodies : A Laboratory Manual (Harlow et al., eds. , 1987); Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993); Principals and Methods in Toxicology (A. Wallace Hayes, ed. , 2000) ; Analytical Methods in Toxicology (H. M. Stahr, 1991); and PCR Protocols in Molecular Toxicology (John P. Vanden Heuvel, ed. , 1997).

Definitions [0040]"Toxicity", as used herein, refers to the microscopic or macroscopic responses of cells, tissues, organs or systems to low, average, or high doses of an agent. Toxicity often results in toxic side effects that are different, in either degree or kind, from the response of the majority of patients at the recommended dose of a pharmaceutical compound. Manifestations of toxicity can include but are not limited to clinical symptoms (e. g., dizziness or nausea), abnormal serum chemistry, hematology or urinalysis values, changes detectable as histopathology results, or abnormal gross appearance of the tissues and organs at necropsy.

[0041] A"toxicological response"as used herein refers to a cellular, tissue, organ, or system level response to exposure to an agent and includes, but is not limited to, the differential expression of genes and/or proteins encompassing both the up-and down-regulation of expression of such genes; the up-or down- regulation of genes which encode proteins associated with the repair or regulation of cell damage; or the regulation of genes which respond to the presence of an agent.

[0042] The terms"toxicity gene (s)","toxicologically relevant gene (s) ", and "toxic response gene (s)" as used herein are interchangeable. These terms can be defined as a gene whose messenger RNA or protein level is altered by an agent (e. g., an adverse stimuli). The specific set of genes that are induced in cells is dependent upon, inter alia, the type of damage or toxic threat caused by the agent and which organs are most threatened. In addition to the up-regulation or down- regulation of genes which respond to specific toxic threat, genes which encode functions not appropriate under conditions of toxic injury may be down-regulated.

[0043] As used herein, the term"gene"refers to polynucleotide sequences which encode protein products and can encompass RNA, mRNA, cDNA, single stranded DNA, double stranded DNA, and fragments thereof. Genes can include introns and exons.

[0044] The term"gene sequence (s)" refers to gene (s), full-length genes or any portion thereof.

[0045] "Gene expression indicative of toxicological response"as used herein refers to the relative levels of expression of a toxicity gene or toxic response gene.

Profiles of gene expression profiles may be measured in a sample, such as samples comprising a variety of cell types, different tissues, different organs, or fluids (e. g., blood, urine, spinal fluid, sweat, saliva, or serum).

[0046] As used herein, the term"agent"means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, or antibody fragment. Physical agents, such as radiation, is also encompasses in this definition. Various compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term"agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Agents can be tested and/or used singly or in combination with one another. An"agent"to which an individual has a toxicological response can also be any substance to which an individual exhibits a toxicological response and includes, but is not limited to, drugs, pharmaceutical compounds, household chemicals, industrial chemicals, environmental chemicals, and other chemicals and compounds to which individuals may be exposed.

Exposure to an agent can constitute physical contact as well as secondary contact, such as inhalation and environmental exposure. As used herein,"agent"and "compound"may be used interchangeably.

[0047] As used herein, "array"and"microarray"are interchangeable and refer to an arrangement of a collection of nucleotide sequences in a centralized location. Arrays can be on a solid substrate, such as a glass slide, or on a semi- solid substrate, such as nitrocellulose membrane. The nucleotide sequences can be DNA, RNA, or any permutations thereof. The nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences.

[0048]"Hybridization"or"hybridize"refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding is sequence-specific, and typically occurs by Watson-Crick base pairing. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0049] Hybridization reactions can be performed under conditions of different "stringency". Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide, and the washing procedure. Higher stringency conditions are those conditions, such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form. Conditions that increase the stringency of a hybridization reaction are Widely known and published in the art: see, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989). When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, those polynucleotides are described as"complementary". A double-stranded polynucleotide can be"complementary"to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.

[0050] An"individual"is a vertebrate, preferably a mammal, for example a human. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice, and rats.

[0051] The term"sample"or"biological sample", as used herein, refers to substances supplied by an individual. The samples may comprise cells, tissue, parts of tissues, organs, parts of organs, or fluids (e. g., blood, urine, sweat, saliva, or serum). Samples include, but are not limited to, those of eukaryotic, mammalian or human origin.

[0052] The terms"protein","polypeptide", and"peptide"are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. It also may be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, myristylation, acetylation, alkylation, phosphorylation or dephosphorylation. Also included within the definition are polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids) as well as other modifications known in the art.

Methods of the Invention [0053] Human genes which are toxicologically relevant have been identified and are disclosed herein. Methods of identifying toxicologically relevant human genes or genes that are likely to be toxicologically relevant are described herein.

In addition, methods of isolating and using toxicologically relevant genes are disclosed. In one embodiment, toxicologically relevant genes are used to make arrays. The arrays can be used for drug screening purposes to determine toxicological response to an agent.

Identifying a set of toxicologically relevant genes [0054] Identification of a set of toxicologically relevant genes can be achieved by several methods. One method which can be used is to clone genes previously described to be relevant in toxicology or to clone genes putatively identified to be important for a toxicological response because of the known or suspected function of the gene or because of the functional relationship of that gene to other genes which play a role in toxicological responses. Using published sequences, for example in literature or from GenBank, primers can be made and then used to PCR amplify from a relevant library to obtain the toxicologically relevant gene of interest which can then be cloned into a plasmid or an expression vector, depending on the use desired. The gene can be placed amongst other toxicologically relevant genes in a microarray for high-throughput testing, as disclosed infra.

[0055] Alternatively, for replication to high copy numbers, a plasmid may be used to grow high copies of the toxicologically relevant gene of interest which can then be purified by any commercially available kit (e. g., from Qiagen or Promega). The purified toxicologically relevant gene may be used for"spotting" in a microarray or alternatively, the purified nucleic acid can then be inserted into an expression vector, transfected into mammalian cells, e. g., human cells, and then the cells can be exposed to a compound and observed for toxicological responses. Toxicity may be ascertained by any number of methods known to one of skill in the art such as observing changes in cell morphology or re-arrangement of cytoskeleton, which can be determined by examination under a microscope, or alternatively, cell apoptosis or necrosis, or biochemical changes such as leakage of enzymes or ions from the cell. In another alternative,"transcriptome profiling", described in greater detail below, may be used whereby nucleic acid can be isolated from both the exposed and unexposed cells and examined to determine which level of the compound causes the up-regulation or down- regulation of the toxicologically relevant gene of interest.

[0056] Another method which can be used to identify a set of toxicologically relevant genes is to test available human genes for the genes'response using tissues from human toxicity studies and select those with differential expression.

Differential expression may be assessed by any number of methods. One method which may be used is by microarray analysis. Provided herein are methods of using microarray analysis to determine differential gene expression. Another method of determining differential gene expression is by reverse transcriptase- polymerase chain reaction (RT-PCR), e. g., Taqmantk technology (Foster City, CA). Yet another method which could be used to detect differential gene expression is Invader technology, commercially available from Third Wave (Madison, WI). Yet another method which may be used to determine differential expression is Northern blot analysis.

[0057] Other methods which may be used include open systems such as cDNA-AFLP and SAGE (Klein, P. E. , et al. Genome Res. 10 (6): 789-807 (2000); Wang, X. and Feuerstein, G. Z., Cardiovasc Res. 35 (3): 414-21 (1997) ) Feuerstein, G. Z. and Wang X. Can J Physiol Pharmacol. 75 (6): 731-4 (1997) ; Hough, C. D. et al. , Cancer Res. 60 (22): 6281-7 (2000) ; Ye, S. Q. , et al., Anal Biochem.

287 (1) : 144-52 (2000) ). An"open system"allows the entire transcriptome to be analyzed instead of a defined set of genes.

[0058] Alternatively, comparisons between gene expression profiles from control human cells (or human cell lines) and human cells (or human cell lines) treated with an agent can be used to select responsive genes. This is referred to herein as"transcriptome profiling". This method empirically determines which genes are toxicologically relevant by analyzing differential gene expression. In this embodiment, experimental human cells are divided into two groups. One group is exposed to one agent at different concentrations for different lengths of time. Another group of human cells are not exposed to any agent and serve as the control group. Once the experimental group is exposed to at least one agent, then RNA of both groups is isolated and reverse transcribed in PCR reactions to generate cDNA which in turn is amplified to generate double stranded DNA. The PCR is performed in the presence of a radioactive DNA substrate that is incorporated into the double stranded DNA. On a polyacrylamide gel, the DNA derived from the treated cells is separated by length next to the DNA derived from untreated population. The intensity of the resulting band or bands is compared between the treated and untreated groups of cells. Bands that show different radioactive intensity are excised from the gel, amplified by PCR, cloned, and sequenced. The sequences are compared with known gene sequences in the public databases such as GenBank. In this manner, novel human genes, in addition to known human genes with varying degrees of similarity, which are toxicologically relevant can be discovered and identified.

[0059] If a partial sequence of a novel human gene is discovered, the technology, texts (see Sambrook et al. infta), and resources available to a skilled artisan would enable the sequencing of the of remainder of the gene and obtain a full-length gene without undue experimentation. One method of obtaining the remaining portion of a novel human gene is to make primers corresponding to the part of the novel human gene which are known combined with random primers and then use the primers in PCR reactions with a human cDNA library. The PCR reaction are run on a standard agarose gel and amplified bands are identified, excised from the gel, and sequenced.

[0060] Yet another method which may be utilized to identify a set of , toxicologically relevant genes is by obtaining human homologues to toxicologically genes of other species (e. g., rat). Methods for identifying and obtaining toxicologically relevant rat genes are disclosed in pending U. S. applications 60/264,933 and 60/308,161. Primers may be made from toxicologically relevant genes from non-human individuals and used in PCR reactions with human cDNA libraries to obtain a human homologue of a non- human toxicologically relevant gene.

[0061] In another alternative, sequences of human homologues of a toxicologically relevant non-human (e. g., rat) gene may be obtained by using the sequence of non-human gene in a sequence search (e. g., a BLAST search) to find known human sequences which have high homology to the non-human (e. g., rat) gene. Primers to the human homologue may be synthesized and then used to amplify a sequence of the human homologue from a human cDNA library.

Examples of primers which may be used are disclosed in Table 2 and the protocols which have been used are disclosed in Examples 1-5 and 9.

Successfully cloned human gene sequences using primers described in Table 2 are presented in Table 5. Methods of this embodiment are further detailed in the Examples section.

[0062] Each of these methods is disclosed in greater detail below. Other factors to consider in identifying toxicologically relevant genes include, but are not limited to, selection of one or more agent (s), the dosage amount to administer, and routes of administration.

Selection of agent (s) [0063] The agent to be tested can be selected on the basis of different criteria.

One method of selecting which compound to test is damage observed in specific organs. For example, cisplatin, amphotericin B and gentamicin have been observed to cause kidney tubular epithelial cell damage. Another example, liver peroxisome proliferation has been observed to be affected by clofibrate, gemfibrozil, and WY 14,643. Another basis for selection is molecular and biochemical action. For example, cisplatin causes apoptosis and reactive oxygen species, amphotericin B causes increased permeability of cell membranes to ions and renal vasoconstriction, and gentamicin causes phospholipid accumulation in lysosomes.

[0064] Other toxicants affect an organ in general, for example, some kidney toxicants include but are not limited to cisplatin, gentamicin, puromycin, and amphotericin B. Liver toxicant include but are not limited to chlorpromazine, clofibrate, diflunisal, tetracycline, erythromycin, and ethanol. Immunotoxicants include but are not limited to cyclosporin A, lipopolysaccharide (LPS), hydroxyurea, phenylhydrazine, dexamethasone, estradiol, and tamoxifen. Heart toxicant includes but is not limited to doxorubicin. Multiorgan toxicants include but are not limited to methotrexate and cadmium chloride.

[0065] Another criterion for selecting an agent is based on exposure to the agent, for example, those agents to which an individual might be exposed to on a regular basis, either in the environment (e. g., occupational exposure, accidental exposure, or voluntary exposure), by prescription, or over-the-counter drug can be selected for testing. Another criterion for selection of an agent is regulatory approval. For example, those agents which are required to be tested for toxicity for FDA-approval or alternatively for other toxicity requirements, for example in pre-clinical or clinical trials can be selected. Table 3 lists some agents which may be selected given the criteria above.

Determination of dosage [0066] Dosages to use in experiments with human cells or biological/clinical samples can be determined using several methods. One method is to use reported dosages (e. g. , obtained in pre-clinical or clinical studies or published in clinical reports) as a starting point and dose incrementally above and below the reported dosage. Increments can be at least 1%, 5%, 10%, 25%, 35%, 45%, 50%, 60%, 70%, 80%, 90%, or 95%. Alternatively, dosages which are known to affect non- human individuals (e. g., rats, primates, dogs, etc. ) which have similar gene expression profiles may also be used as a starting point and then dosages may be incrementally increased or decreased. If non-human individuals (e. g. , rats, primates, dogs, etc. ) are to be used for comparison purposes, the upregulation or downregulation of markers in the blood including but not limited to serum chemistry values and hematology values can be used to determine if toxicity has been reached. Alternatively, examining the histopathology of organs, in particular, organs which are the specific targets of the agent of interest, may be used to determine if a pathological change has occurred in response to administration of the agent. Another method which may be used is to determine the molecular changes by analyzing the gene expression in response to administration of different doses of a agent by the methods disclosed inf a.

[0067] Determination of the dosage experimentally using cell cultures is affected by many factors: the nature of the agent, its potency, mechanism of action, type of cell which is the target of the agent, and number of cells. To determine the dosage required experimentally, a low dosage level of the agent is added and then in a step-wise manner, the dosage is increased as well as length of time exposed to the agent. If the agent is lipophilic and easily crosses the lipid bilayer of cells, a lower initial concentration may be used and/or shorter length of time exposed to the agent. If the agent possesses a nature that would not cross the cell barrier easily and would need to be actively or passively transported across cell membranes, then a slighter higher initial concentration may be used and/or longer length of time exposed to the agent. Increasing dosage step-wise while monitoring toxicological response and morphology of the cells, rate of death of the cells, and growth patterns allows the skilled artisan to determine the dosage at which a toxicological response occurs. Toxicological responses may occur which are visible changes, including but not limited to, physical structure and integrity of the cells (e. g., morphology, growth pattern, etc. ). Monitoring for cellular toxic responses as well as molecular toxic responses, e. g., differential gene expression increases the likelihood of finding preferable dosages.

[0068] Changes in gene expression may be toxicologically significant. The point at which toxicologically relevant gene expression becomes even more relevant is at that dosage at which removal or diminishment of the treatment no longer results in a return to normalcy, e. g.,, the state of a cell, organ, or system that existed prior to the treatment with the agent. Treatments beyond a certain dosages or time period may commit the cell to a toxicologically-relevant fate.

This toxic dosage will be reflected by an identifiable gene expression pattern, which will be distinct from the pattern observed below the toxic dosage.

[0069] Dosage response is an important concept in toxicology. Depending on the dosage of a toxin or agent which may be toxic, the gene expression profile of a particular gene may vary. One way that this can be envisioned is by observing the changes in fold induction of a particular gene when analyzed using the arrays of this invention. The dosages determined in dose response curves may be useful in determining"threshold"levels of toxicity, for example for FDA approval.

Methods of analyzing gene expression and how to correlate gene expression data are provided herein.

Administration of an agent [0070] If non-human individuals (e. g., rats) are to be used for comparison purposes, administration of an agent to the non-human individual may be achieved by various routes. It will be readily appreciated by those skilled in the art that the route can vary, and can be intraperitoneal, intravenous, subcutaneous, transcutaneous, intramuscular, enteral, transdermal, transmucous, sustained release polymer compositions (e. g., a lactide polymer or co-polymer microparticle or implant), perfusion, pulmonary (e. g., inhalation), nasal, oral, etc. Injectables can be prepared in conventional forms, either as liquid solutions or suspension, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, aqueous dextrose, glycerol, ethanol or the like. Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990).

[0071] The carrier must be acceptable in the sense of being compatible with the agent to be tested and not deleterious (e. g.,, harmful) to the human to be treated. The composition or formulation to be administered can contain a quantity of the agent in an amount sufficient to effect one or more toxicological response in the human, either on a molecular level or on a physiological level.

Method of identifying toxicologically relevant genes using known human genes [0072] In one embodiment, sequences of human genes which are toxicologically relevant are known, either in the art or in a publicly available database, e. g., GenBank. The first method that is used to identify human genes involves searching a public database, for example GenBank, for human genes already known to have a toxicological response. Using the known toxicologically relevant human sequences, primers are designed and used in PCR reaction to amplify the human gene sequences from a cDNA library. The cDNA library can be made from different human cells. The generation of a cDNA involves reverse transcribing isolated RNA and is well-known in the art (see for example, Sambrook et al. supra). The human gene fragments, amplified by PCR, are cloned into any standard plasmid expression vector which can be obtained from numerous commercial sources (e. g., Promega, InVitrogen, New England BioLabs, etc. ) and sequenced. The resulting sequence information is then compared to the GenBank database to confirm that the cloned DNA is the specific human gene for which the primers were designed. Upon positive confirmation of the sequence, the amplified gene sequence is then added to the panel of genes to be included in the array. Methods of including toxicologically relevant genes are disclosed infra.

[0073] In another embodiment, human genes which are toxicologically relevant are known. This method to identify human genes utilizes known sequences of toxicologically relevant non-human genes (e. g., rat genes identified in pending U. S. applications 60/264,933 and 60/308,161 or canine genes identified in pending U. S. application 60/227,057 and the U. S. application claiming priority thereto). These toxicologically relevant non-human genes may be from a non-human species including, but not limited to rats, primates, and other mammals. Human homologues of the non-human genes can be identified through query of human sequence databases such as GenBank with comparative sequence analysis programs such as the Basic Local Alignment and Search Tool (Altschul, S. F. , et al., Nucleic Acids Res. 25: 3389-3402 (1997) ). Primers to these toxicologically relevant non-human genes or identified human homologues are designed, synthesized, and are subsequently used in PCR reaction with human cDNA libraries to amplify the homologous human gene. If non-human genes were used to design the primers, the homologous human gene may or may not be the exact sequence as the non-human gene with which the primers were designed.

Amplified human gene is then added to the panel of genes to be included in the array.

[0074] In yet another embodiment, target sequences for inclusion in a human array are obtained by de novo synthesis of polynucleotides. The polynucleotide may be synthesized directly on the slide or may be synthesized by other means and then attached to the slide. The target sequences are from genes which can indicate one or more toxicological responses.

Transcriptome profiling [0075] Transcriptome profiling is a generic term that can be applied to measurement of a large a variety of transcripts. Various methods can be used for the profiling such as microarray, PCR, and differential display, SAGE, Invader (D, etc. Several methods of differential display can be used to identify genes of interest. Differential gene expression can be observed by using techniques involving gel electrophoresis and polynucleotide microarrays or commercially available technologies, e. g., Invader or Taqman.

[0076] In one method, the results of PCR synthesis of mRNA (converted to cDNA before PCR) isolated from tissues of treated and control human cell lines are subjected to gel electrophoresis, and the bands produced by these mRNA populations are compared. Bands present on an image of one gel from one mRNA population, and not present or present with much less intensity on another, correspond to the presence of a particular mRNA in one population and absent or at much lower levels in the other, and thus indicate a gene that is likely to be differentially expressed. Messenger RNA derived from control and treated human or cell lines can be compared by using arbitrary oligonucleotide sequences (random 13-mers) as a 5'primer and a set of 3 oligonucleotides complimentary to the poly A tail as a 3'fluorescent labeled"anchor primer". These primers are then used to amplify partial sequences of mRNAs with the addition of deoxyribonucleotides. These amplified sequences are then resolved on a sequencing gel. The sequencing gels are then compared to each other to determine which amplified segments are expressed differentially. See, for example, Liang, P. et al. Science 257: 967,1992 ; Welsh, J. et al., Nucl. Acid Res.

20: 4965,1992 ; and Liang, P. , et al., Nucleic Acids Res. 21 (14): 3269-75 (1993)).

[0077] An open system may be used whereby human cells are exposed to drugs and/or chemicals at different concentration and then harvested at different time points. Human cells can be obtained from various sources including, but are not limited to, tissue samples, organs, blood, skin, biological fluids (e. g., urine, spinal fluid, semen, etc. ), and cell lines. Transcriptome profiling can also be done using tissues, biological fluids, etc. from humans who have been dosed in vivo during clinical trials or during one or more clinical treatment (s). Immortalized human cell lines may also be used and can be obtained from commercial sources, e. g., Gibco BRL Life Sciences or other sources, e. g., American Type Culture Collection (ATCC). Other methods of obtaining human cells include isolating cells obtained from tissue biopsies, blood, skin, or biological fluids, for example from humans dosed in vivo. In an alternative, non-human individuals (e. g., rats) may be used for comparison, as disclosed herein. Tissue samples, cells, or cell lines from a non-human individual may be utilized as well for transcriptome profiling.

[0078] As is well known to one of skill in the art, isolating cells from tissue samples can be achieved using any variety of techniques. In obtaining tissue samples, for example during necropsy, it is important to avoid conditions that would cause degradation of nucleic acids (e. g., RNA).

[0079] One method which may be used is to digest a tissue sample in an enzymatic solution to break up connective tissue and then agitate cells in the digested tissue to separate the cells from the connective tissue. Examples of other enzymes that can be used to digest tissue include neutral proteases, serine proteases including, but not limited to, trypsin, chymotrypsin, elastase, collagenase, and thermolysin. Another method is to homogenize the tissue sample or apply mechanical stress forces to the tissue sample to separate the cells from the basement membranes and allow the cells to become separated from within the tissue. In the alternative, DNA or RNA can be directly isolated from tissue samples, as exemplified in the examples disclosed herein. Isolating cells from blood can be achieved by layering blood over a gradient (e. g., Percoll or FicollTM), spinning the blood-gradient layer in a centrifuge, and extracting the layer of cells from serum.

[0080] Sources from which cells are obtained can be any number of organs, including but not limited to liver, lung, heart, kidney, spleen, thymus, and brain.

In one embodiment, liver cells may be used for toxicity studies where the agent to be administered is known or thought to induce liver malfunctions or liver toxicity.

In other embodiments, when the target of the action delivered by the agent is known, the use of cells deriving from the target organ may yield more beneficial information regarding toxicological responses than if a tissue were selected at random. In another embodiment where the agent to be tested has unknown effects, a panel of cells isolated from different sources may be used. In the alternative, liver cells may be used in the absence of knowledge of the agent's target of action because the liver is known to process many toxins. Although the toxicological responses may not be the most ideal compared to the results that one of skill in the art would obtain if the target tissue of the agent's action had been used, the benefits of using liver cells would be that toxicologically relevant genes may be identified and then subsequently tested on other organs to determine toxicity in the other organs or alternatively, to identify which organ (s) is the target for the agent.

[0081] Human cells obtained ex vivo or from a commercial or other source (e. g. , ATCC) can be used fresh or frozen for storage and then cultured in media at time of experimentation. A wide variety of basal cell-sustaining media that can be used to keep the pH of the liquid in a range that promotes survival of human cells.

Non-limiting examples include F12/DMEM, Ham's F10 (Sigma), CMRL-1066, Minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM, Sigma), and Iscove's Modified Eagle's Medium (IMEM). In addition, any of the basal nutrient media described in Ham and Wallace Meth. Enz., 58: 44 (1979), Barnes and Sato Anal. Biochem., 102: 255 (1980). Cells can be grown in plates or in flasks and expanded to an amount needed for DNA or RNA isolation. Cells can then be removed from the plate or flask to isolate DNA or RNA. If the cells are adherent, trypsin or another equivalent may be used to release the cells from the plate or flask. Methods of culturing cells and isolating nucleic acids from cells are well-known in the art.

[0082] Methods of exposing non-human individuals (e. g., rats) to toxic doses of agents include determination of the dosage and routes of administration of , agents as described above. Rats may be divided into treated rats that receive a specific concentration of the agent and the control rats that only receive the vehicle in which the agent is mixed (e. g., saline). At specified timepoints after administration of the agent, a set number of rats (control and treated) may be euthanized by any standard euthanization protocol known in the art and tissues may be collected. The method of collecting the tissues is important and ensures preserving the quality of the mRNA in the tissues. Each rat may be heavily sedated with a overdose of CO2 by inhalation and a maximum amount of blood drawn. Exsanguination of the rat by this drawing of blood kills the rat. The body of the rat may then be opened up and prosectors may rapidly remove the specified organs/tissues and immediately place them into liquid nitrogen. All of the organs/tissues should be completely frozen within 3 minutes of the death of the rat. The organs/tissues may then be packaged and stored at-80 degrees until needed for isolation of the mRNA from a portion of the organ/tissue sample.

[0083] Nucleotide sequences from tissue samples are isolated using any number of commercially available kits (e. g., from Qiagen, GenHunter@, Promega, etc. ). In general, a skilled artisan should take care to keep all reagents, tubes, and instruments sterile as to avoid contaminants which may affect how the results are interpreted. Once DNA or RNA has been isolated from cells which have been exposed to one or more agents, one or more toxicologically relevant genes are identified using the methods described above. The toxicologically relevant genes may be cloned into an expression vector, maintained in an expression vector or alternatively, the expression vector comprising the toxicologically relevant gene sequence may be transformed or transfected into a suitable host cell. Suitable host cells may be obtained from the ATCC or from commercial sources. Methods of isolating toxicologically relevant genes by cloning are further detailed in the Examples.

[0084] In some embodiments, the toxicologically relevant non-human gene may be used to find a homologue in another animal, for example, in humans. The homologue may be then be used as a target or to derive a target for drug development, toxicity screening, prognostic or diagnostic applications (e. g., antigen for antibody development or cellular regulation).

[0085] In other embodiments, human genes identified to be toxicologically relevant may be used to generate an array of toxicologically relevant human genes. In this case, the gene may be cloned to facilitate the process of generating an array.

Preparation of Microarray [0086] The isolated DNA or RNA is amplified to generate a product which can be attached to a substrate. In a preferred embodiment, the substrate is a solid substrate (e. g., a glass slide). The amplification process involves using primers which have a reactive group (e. g., amine group or derivative thereof) on one end of the primer, which is incorporated into the amplification product. One example of reactive primers that can be used is Amine Primers from Synthegen (Houston, TX ; catalog #5002). The gene fragments which are attached to the glass slide can vary in length. The more nucleotides of a gene that are in the array, the tighter the binding and the greater the specificity in binding can occur. However, it is important to consider that longer fragments are more difficult to amplify and may contain point mutations or other errors associated with amplification. Therefore, the desired length of a gene or a fragment thereof that is to be included in the array should take into consideration the balance between a high specificity of binding obtained with a long (e. g., >1 kb) gene sequence with the high mutational rate associated with a longer fragment.

[0087] The gene fragments attached to the glass slide are at least about 50 base pairs (bp) in length, more preferably at least about 100 bp in length, more preferably at least about 200 bp, even more preferably at least about 300 bp, even more preferably at least about 400 bp, even more preferably at least about 500 bp in length. In one embodiment, the gene fragments are about 500 bp in length.

The region of a gene that is used to attach to a solid substrate to generate an array can be any portion of the gene, coding, non-coding, 5'end, 3'end, etc. In one embodiment, about 500 base pairs of the 3'end of human gene related to toxicological responses are selected to be included in an array.

[0088] In some embodiments, the skilled artisan should be aware that the human homologues of toxicologically relevant rat genes to be attached to the array may have different lengths than the toxicologically relevant rat gene. For example, in the instance where the rat gene sequence is longer than the human homologue, the rat gene sequence will have 500 base pairs at the 3'end that the human sequence does not have. In another instance where the human homologue is longer than the rat gene sequence, the human sequence will have 500 base pairs at the 3'end that the rat gene sequence does not have. Either the 3'region can be used or regions of equivalent homology on the gene sequence should be used.

[0089] Several techniques are well-known to a skilled artisan for attaching a gene or a fragment thereof to a solid substrate such as a glass slide. One method is to attach an amine group, a derivative of an amine group, another group with a positive charge or another group which is reactive to one end of a primer that is used to amplify a gene or a gene fragment to be included in the array. Subsequent amplification of a PCR product will then incorporate this reactive group onto one end of the product. The amplified product is then contacted with a solid substrate, such as a glass slide, which is coated with an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified PCR product and become covalent attached to the glass slide. Other methods using amino propryl silicane surface chemistry and other methods of preparation of microarrays have been disclosed. See, for example, Nuwaysir, E. F. , et al.

Molecular Carcinogenesis, 24: 153-159 (1999); Kane, M. D. , et al. Nucleic Acids Res. 28 (22): 4552-7 (2000); MacBeath G. and Schreiber, S. L., Science 289 (5485): 1760-1763 (2000); Lockhart, D. J. and Winzeler, E. A., Nature 405 (6788) : 827-836 (2000); Cortese, J. D., The Scientist 14 (17): 25 (2000); and Cortese, J. D., The Scientist 14 (11): 26, (2000). Other methods using amino propryl silicane surface chemistry are disclosed by Corning Company (Corning, NY). Products made by Coming Company which include CMTTM Yeast-S288c gene arrays, CMTTM Human Cancer gene arrays, CMT-GAPSTM II coated slides, and CMTTM Hybridization chamber. The website of Coming Company discloses more information about how a skilled artisan may make microarrays. At present, the website may be accessed at <http://www. cmt. coming. com. >. Other methods for making microarrays are disclosed by Haab B. B. , et al Genome Biol. 2001 Jan 22; 2 (2): RESEARCH 0004.1-0004. 13; Sherlock G. , et al. Nucleic Acids Res.

2001 Jan 1; 29 (1) : 152-5; and the website of Dr. Pat Brown at Stanford University.

At present, the website may be accessed at <http ://cmgm. stanford. edu/pbrown>.

[0090] In one embodiment of the invention, fluorescence-labeled single strand (or"first strand") cDNA probe is made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microarray slides spotted with DNA specific for toxicologically relevant genes.

Methods for making the array and for labeling and making cDNA probes are disclosed in the Examples.

Algorithms for analysis and evaluation of toxicologically relevant genes [0091] A multi-step approach can be used in ranking candidate genes from human genes for possible inclusion on an array. First, three cutoff criteria can be specified for individual gene values from experiments results: 1) Fold Induction/Repression level, 2) Average Fluorescence level of the replicate spots (reflection of the expression level) and 3) Coefficient of Variation of the replicate spots. The initial screening to make the"cut"may be based on expression level and measurement quality.

[0092] Second, gene values that would made the cut can be aggregated into overall scores, and ranked for each gene, may be based on six ranking criteria: 1) Number of slides on which that gene met the cutoff criteria (NC), 2) Percent of consistency between slides (% of time the gene value made the cutoff criteria on the replicate slide for that initial slide) (CC), 3) Average magnitude (absolute value) of fold induction for all occurrences where that gene made the cutoff criteria (FI), 4) Coefficient of Variation of those fold induction scores (unlike all the other ranking criteria, lower is deemed better) (CV), 5) Average fluorescence value of all replicate spots of occurrences where that gene made the cutoff criteria (FL), and 6) Tissue consistency (what percent of cutoff-meeting occurrences of the gene were in the same tissue) (CT).

[0093] Each gene can be assigned a score between 0 and 100 for each ranking criterion. Each ranking criterion score can be computed as follows: The range of values for all genes is computed for the criterion by subtracting the lowest value present among all scores from the highest. The score for each gene is then calculated by subtracting the lowest value present from the value for that gene, then dividing by the range and multiplying by 100. In other words, the score for each gene is the percent above the minimum present toward the maximum. For example, if a gene's score was three-fourths of the way between the minimum present and the maximum for that criterion, its score would be 75%. Since for the CV factor (coefficient of variation of fold inductions) lower is deemed better, the score thus computed was subtracted from 100 to invert the percentage.

[0094] The final ranking score for each gene can be computed via a weighted combination of its score on the six ranking criteria. If a score could not be computed for a particular criterion, the entire value of that criterion would be removed from the equation, and ranking was based solely on the remaining factors.

Methods of using toxicological response data [0095] The set of toxicologically relevant human genes and methods of identifying toxicologically relevant genes in human may be used in several embodiments. In one embodiment, toxicity dosages and time of exposure which is required to reach a toxic dose are determined by using the methods disclosed above. In another embodiment, an individual (e. g., human) can be tested for toxic responses to a particular agent. The individual may be hypersensitive to the agent (e. g. , penicillin). Analyzing the individual's gene expression profile may determine if the agent has a toxic effect in the individual. Alternatively, the gene expression profile of the individual may be compared with other gene expression profile stored in a database. Methods of using gene expression profiles of toxicologically relevant human genes to generate a useful database is described in further detail below. In another embodiment, the gene expression profiles of toxic responses in non-human species (e. g., rats or mice or other animals) may be determined. This may assist in determining which species is best suited for animal models by assessing which species is most susceptible to toxic responses.

In yet another embodiment, the methods and set of toxicologically relevant genes disclosed herein can be used to predict and/or determine drug-drug interaction in an individual. As disclosed supra, gene expression profiles of toxicologically relevant human genes can be compared when dosed with one drug and then compared to a second gene expression profile when dosed with another drug. The toxicologically relevant data may be correlated using the algorithms disclosed herein. The effects of drug-drug interaction may induce a similar set of genes to be up-regulated or down-regulated. The effect may be additive or multiplicative.

Alternatively, the effects of the drug-drug interaction may induce different sets of genes which are not related in function. In another embodiment, the methods and set of toxicologically relevant genes disclosed herein allow target organs and toxic doses therein to be determined. This is useful in drug design where the drug may have an intended target of one organ but have toxic multi-organ effects. In another embodiment, the methods and set of toxicologically relevant genes may be used to predict toxic response to agents which may take repeated exposure over a period of time for symptoms of toxicity to appear. Examples of such agents are disclosed in Table 3 and can also include one-hit carcinogens (e. g., aflatoxin B1, dimethylnitrosamine, ENU, etc. ) or multi-dose carcinogens (e. g. , phenobarbital and WY14,643). The molecular toxic response to these carcinogens may be determined in advance of any macroscopic changes which may occur in response to exposure to these agents.

Method of using toxicological response data to generate a toxicological data, base and uses thereof [0096] By collecting many gene expression profiles from certain species, e. g., humans, in response to one or more agents, a database can be built with a collection of information about toxicological responses. With the database, it could be possible to predict toxicological response to specific agents or combinations thereof. The database can be stored on a computer and in a manner that allow for rapid searching when a comparison is desired. The database could store gene expression profiles for a particular toxin or alternatively, a group of toxins (e. g., kidney-specific toxins). The database could also store gene expression profiles for a group of genes known to be affected by a particular toxin. When a gene expression profile is obtained, it may be compared with the gene expression profiles stored in the database to determine what type of organ is likely to be affected, or alternatively, which genes could also be associated with the toxic response. One or more genes could be analyzed in this manner as well as one or more toxins. The database may be stored in a form that allows for rapid, access and analysis with compatible software programs.

[0097] The instant invention of human gene arrays provides an alternative to testing on live animals such as rats, mice, or dogs. The human gene array can provide answers concerning human response to a particular agent by examining the differential gene expression associated with that particular agent using an array or comparison with a human array database. Further, human gene arrays can provide answers about toxicological responses faster and more efficiently than testing in vivo.

[0098] Another use for a database of gene expression profile of toxicologically relevant genes is for comparisons across species. For example, a database comprising human gene expression profiles obtained from in vitro studies can be used to compare to rat gene expression profiles obtained from in vitro studies. If the human in vitro gene expression profile is similar to the rat in vitro gene expression profile in response to a particular agent, then it can be inferred that the rat model would be a good model to use for assessing in vivo responses. The rat in vivo response can be extrapolated to predict human in vivo responses.

[0100] The information generated from using human gene arrays can be used to predict cellular and pathological responses as well as histological changes induced by exposure to agents. This is accomplished by analyzing the differential gene expression observed when human gene arrays are used. Potential drugs or pharmaceutical agents can be tested and data gathered for FDA approval in an accelerated manner and can help pharmaceutical and biotechnology companies generate higher productivity with lower costs in research and development.

[0101] The human gene array can also generate information that can be used to predict downstream effects, such as which pathways are affected by certain agents. This is accomplished by looking at the differential gene expression and analyzing which pathways contain the toxicological response genes and also which pathways the genes can affect. This information in turn can be used to predict tissue responses and ultimately whole organ responses. Examples of whole organ responses include but are not limited to organ functions, inflammatory responses, and autoimmune responses. Those of skill in the art can determine when the normal functions of an organ are compromised by exposure to one or more agents which are toxic. For example, a kidney's ability to filter toxins is compromised after an individual has been exposed to an agent. The ability to predict whole organ responses has great potential in the development of drugs, pharmaceutical agents, and even in the use of chemicals.

[0102] The following Examples are provided to illustrate but not limit the present invention. It will be apparent to one of skill in the art that modifications can be made while keeping in the spirit and scope of the present invention.

[0103] EXAMPLE 1: PRIMER DESIGN [0104] Primers were designed by using the web page: http : //www- genome. wi. mit. edu (scroll down to find the link : WWW Primer Picking Primer3). Thus, Primer3 software was used to pick the primers based on inputted parameters such as melting temperature and length. The following factors were used for the design of the primers: [0105] 1. Global parameters, product size range, which depends on the size of the equivalent rat target on the CT array. For example: if it is known that the rat target is 500 bases long, then 480-520 should be filled in as the range for a possible size for the human target.

[0106] 2. Per sequence inputs, region included: The appropriate region for the human target sequence is known from the homology search between the human and the rat genes (full length). Fill in the base number where the computer should start looking for primers followed by the approximate length: e. g., 2697,550 [0107] 3. Global parameters, Tm minimum: 65°C [0108] 4. Global parameters, Tm maximum: 72°C [0109] 5. Global parameters, Tm optimum: 69°C [0110] 6. Global parameters, Minimum primer length: 23 [0111] 7. Global parameters, Maximum primer length : 27 [0112] 8. Global parameters, Optimum primer length: 25 Primer concentrations and storage [0113] Primers were ordered from Genset (Paris, France). Oligonucleotides were supplied as a lyophilized powder. The powder was dissolved in nanopure water to obtain a stock solution, and then the stock solution was diluted to a working concentration of lOpmol/u. 1 (lOuM).

[0114] The following procedures were followed: [0115] 1. Briefly spin tube containing the lyophilized powder to make sure all powder is at the bottom of the tube.

[0116] 2. Completely dissolve the oligonucleotide in the appropriate amount of nanopure water to obtain a stock solution of lnmol/l (lmM).

[0117] 3. Dilute 1 µl of this stock solution with 99, ul of nanopure water to obtain a working solution of lOpmol/u. 1 (lOuM).

[0118] 4. Label the tube with working solution with the oligo number and the concentration (10pM) and store at-20°C.

[0119] 5. Dry down the rest of the stock solution in the Speedvac. Store the dried powder at-20°C. For example: The original amount of oligo was dissolved in 10. 5µl of water to obtain the 1mM stock solution. This stock was then diluted 1 µl in 100µl to make the working solution and then dried down the rest of the stock. Next time the powder would be dissolved in 9.5 ul of water to prepare the 1mM stock solution.

EXAMPLE 2: POLYMERASE CHAIN REACTION (PCR) [0120] AdvanTaq PCR kit from Clontech was used. This kit contains the 10 X PCR buffer, 50 X dNTPs, PCR grade water and the 50 X Taq polymerase. The kit was stored at-20°C as recommended.

[0121] The following procedure was followed: [0122] 1. Place all the components on ice and allow them to thaw. Water and 10 X buffer can be thawed at room temperature.

[0123] 2. Combine the following reagents in a 0.5 ml PCR tube on ice: PCR grade water all 10 X PCR buffer all DNA template (Clontech QUICK-Clone cDNA) 11 Forward Primer (10pM) 2p1 Reverse Primer (10µM) 2µl 50 X dNTP Mix (lOmM each dNTP) 1p1 50 X Taq DNA polymerase 1 ! 11 TOTAL 50gel [0124] 3. Mix by pipeting up and down several times. If multiple PCR reaction will be performed simultaneously, a PCR master mix can be prepared.

[0125] 4. To separate labelled 0. 5ml PCR tubes, add the appropriate forward and reverse primers.

Forward Primer (10µM) 2µl Reverse Primer (10µM) 2µl [0126] 5. Per 10 reactions, combine the following reagents on ice: PCR grade water 399gl 10 X PCR buffer 52. 2ul DNA template 10µl 50 X dNTP Mix 10. 51l1 50 X Taq DNA polymerase 10. 5ut [0127] 6. Mix by pipeting up and down.

[0128] 7. To each tube, add 46 ul of the master mix. Mix by pipeting.

[0129] 8. Program the thermocycler to run the following program: 95°C for 1 minute # 35 cycles 95°C for 45 seconds o 63°C for 45 seconds # 68°C for 1 minute o 68°C for 10 minutes.

[0130] 9. Place the tubes in the thermocycler and start the program.

[0131] 10. When the PCR reaction is finished, an aliquot is run on an agarose gel to verify if the desired cDNA fragment was made. 0. 5M EDTA pH 8.0 (500ml) is made by dissolving 93g of Na2EDTA. 2H2O in 400ml of nanopure water, and adjusting pH to 8.0 with NaOH. EDTA will not dissolve completely until pH is adjusted. Add water to 50 ml. Autoclave. 10 X TBE (1 liter) is made by using 108g Tris base, 55g boric acid, 40ml of 0. 5M EDTA pH 8.0 (or 7.4g Na2EDTA. 2H20). 10 mg/ml ethidium bromide is made by dissolving one tablet of ethidium bromide (Merck) in 10 ml of nanopure water.

[0132] 11. While the PCR program is running, pour a 1.2% agarose gel. In an Erlenmeyer or glass bottle, add the following material: 1.8 g agarose, 15 ml 10 X TBE, and 135 ml water. Dissolve the agarose by heating the mixture in the microwave oven. Let cool down to approximately 65°C. Add 1.5-3 l of ethidium bromide (10 mg/ml). Pour the gel in the tray, insert combs, and let solidify at room temperature. When the PCR program is finished, take the tubes out of the machine and put on ice. From each tube, transfer all to a new-labelled tube and add 1 1ll 6 X Loading dye. Load the samples on the agarose gel. Include at least one lane with DNA molecular weight marker (Eurogentec Smart Ladder; use 5, u1 per lane). Immediately freeze the rest of the PCR reaction at-20°C. Run the gel in 1 X TBE at 100V until the blue dye has migrated a few centimeters in the gel. Analyze the gel on UV light and take a Polaroid picture. If you see multiple bands in a lane, then the PCR product needs to be gel-purified. If only one band is visible, the PCR can be used as such for cloning. If the PCR product needs to be purified, run another agarose gel and load 30 u. l of the PCR reaction (+ 6 p1 6 X loading buffer). Run the gel. Visualize band on UV light.

[0133] 12. Using a clean and sharp scalpel cut of the band corresponding to the desired length. Put the piece of agarose gel in an Eppendorf tube and either proceed immediately to the clean-up, or store the gel at-20°C.

DNA cleanup from agarose gel [0134] This procedure is only necessary when the PCR reaction contains multiple products. If only one band (of the correct length) is visible on agarose gel, this procedure can be omitted.

[0135] 1. Use QIAquick 8 PCR Purification kit and QIAGEN buffer QG with the QIAvac S6 manifold. Add ethanol to buffer PE before use. Label the bottle after ethanol is added.

[0136] 2. Excise the desired DNA fragment from the agarose gel using a clean scalpel.

[0137] 3. Weigh the gel slice.

[0138] 4. Add three volumes of buffer QG to one volume of gel (lOOmg = 100ml) [0139] 5. Incubate at 50°C for 10 minutes to dissolve the agarose. Vortex every 2-3 minutes to help dissolve the gel.

[0140] 6. In the meantime, assemble the QIAvac S6 manifold. Open the S6 lid and place the required amount of 8-well strips in the slots. Seal any unused slots with blanks and close the QIAvac S6 lid. Check the vacuum: it should be between-200 to-600mbar.

[0141] 7. After the gel is dissolved, make sure that the color of the mixture is yellow. If the color is orange or violet, add 10111 of 3M sodium acetate pH 5.0 to adjust the pH. The color of the mixture should now turn yellow.

[0142] 8. Add one gel volume of isopropanol to the sample and mix.

[0143] 9. Apply the samples to the wells of the QIAquick strips. Switch on the vacuum source. After all the liquid has been pulled through, switch off the vacuum.

[0144] 10. Add 0. 5ml of buffer QG. Apply vacuum. After all liquid has been pulled through, switch off vacuum.

[0145] 11. Add lml of buffer PE to each column. Pull through by applying vacuum.

[0146] 12. Repeat previous step. After buffer PE has been pulled through, apply maximum vacuum for an additional 5 minutes to dry the membrane. Switch off the vacuum source and ventilate the QIAvac S6 slowly. Remove the top plate of the QIAvac S6 (containing the 8-well strips) and vigorously rap the plate on a stack of absorbent papers, until no drops come out.

[0147] 13. Replace the waste tray with a rack of collection tubes in the QIAvac S6. Place the top plate (+ columns) back on the base.

[0148] 14. Add 60 ul of buffer EB or nanopure water to the center of the membrane in each column. Let stand for 1 minute. Switch on the vacuum for 5 minutes to elute the DNA.

[0149] 15. Ventilate the QIAvac slowly.

EXAMPLE 3: TOPO TA CLONING OF CDNA FRAGMENTS [0150] The PCR-amplified cDNA fragments is cloned in the Stratagene pCR@II-TOPO vector. If the PCR reaction contained only one cDNA fragment (of the desired length), it can be cloned without purification. If not, the desired band is purified prior to cloning using the QIAquick PCR purification kit. It is important to use a thermostable polymerase during PCR with a non-template- dependent terminal transferase activity that adds a single deoxyadenosine (A) to the PCR product. The vector supplied in the kit is linear and has a single T overhang. This allows the PCR product to ligate efficiently within the vector.

[0151] 1. The ligation reaction is accomplished at room temperature as follows: PCR product (total reaction or purified) 0.5 to 4. 0, u1 Salt solution lul Sterile water add to total Volume of 5p1 TOPO vectorlui Final volume 6p1 [0152] All the components are stored at-20°C, except the salt solution and the water, which can be stored at 4°C ; thaw the components on ice before use.

[0153] 2. Mix the reaction gently and incubate at room temperature for 5 minutes. Immediately proceed to the transformations. Alternatively, the reactions can be put on ice until all ligations are completed. The reactions can also be stored at-20°C overnight.

EXAMPLE 4: TRANSFORMATIONS [0154] Chemical transformation protocol was used. An alternative method of transformation which can be used is electroporation. Chemical transformation requires One Shoe Chemically competent E. coli strain TOP10 bacteria. The following solution were made and used for the transformation: Ampicillin stock solution (100mg/ml ! Dissolve 1 g of ampicillin (sodium salt) in 10ml of nanopure water Filter sterilize through a 0. 2µl filter Store at-20°C X-gal stock solution (4% w/v) . Dissolve 500mg of X-gal in 12. 5ml of DMF (dimethylformamide) Store at-20°C, protected from light (in a brown bottle) IPTG stock solution (looms Dissolve 238mg of IPTG in 10 ml of nanopure water Filter sterilize (0. 2um filter) and store in lml aliquots at-20°C LB/amp/X-gal plates (1L = +/- 30 plates) # Dissolve 40 grams of Luriah/agar medium in 950 ml of water. Add water to 1L.

Autoclave on liquid cycle for 20 minutes at 15 psi.

Allow the solution to cool down to 55°C.

Add ampicillin to a final concentration of 100pg/ml (1 ml of stock per liter).

Add 2 ml of X-gal (4%) to 1L of medium.

Pour into 10cm sterile petri dishes (approx. 3ml per plate).

# Allow cooling down.

Invert and store at 4°C.

[0155] 1. Be sure to have enough LB/amp/X-gal plates at 37°C.

[0156] 2. Equilibrate a water bath to 42°C.

[0157] 3. Warm the SOC medium to room temperature.

[0158] 4. Thaw on ice one vial of chemically competent One Shot cells per transformation.

[0159] 5. Add 2p1 of the TOPO cloning reaction to a vial of competent cells and mix gently. Do not mix by vortexing or pipeting up and down.

[0160] 6. Incubate on ice for 20 minutes.

[0161] 7. Heat shock the cells at 42°C for 30 seconds without shaking.

[0162] 8. Immediately put the tubes on ice and incubate for an additional 5 minutes.

[0163] 9. Add 25µl of room temperature SOC medium and shake the tube horizontally at 37°C (200rpm) for 1 hour.

[0164] 10. Spread 200p1 ofthe transformation reaction on selective plates.

Incubate overnight at 37°C.

EXAMPLE 5: ANALYSIS OF POSITIVE CLONES [0165] Using PCR, a minimum of three colonies from each transformation are analyzed for the correct cDNA insert. Either the M13 Forward and Reverse primer set supplied with the cloning kit is used, or the T7 and Sp6 primer set made in-house. These primers anneal to the vector and amplify any insert in the vector. The resulting PCR products are analyzed on an agarose gel and the length of the insert is compared to the length of the cloned fragment. Prior to PCR analysis, the colonies will be streaked on a separate selective LB/amp plate. The following steps are used: [0166] 1. Pick separate white or light blue colonies from the transformation plates (minimum 3 colonies per plate) with a sterile toothpick.

[0167] 2. Resuspend the clones individually in 20 l of sterile nanopure water.

[0168] 3. Streak the colonies on a fresh, correctly labelled LB/agar plate containing ampicillin.

[0169] 4. Incubate the plate overnight at 37°C.

[0170] 5. Incubate the tubes prepared in step 2 at 96°C for 10 minutes to lyse the cells and inactivate nucleases. Spin briefly.

[0171] 6. Prepare the PCR mix on ice. For each reaction, add the following components (multiply by the amount of reactions to be performed ; also be sure to prepare some in excess). The following materials are used: PCR water 36. 5111 400111 (for 10 reactions) 10 X PCR buffer 5ll1 55p1 (for 10 reactions) Forward primer (M13F) (10 µM) 1µl 11µl (for 10 reactions) Reverse Primer (M13R) (10 pM) 1p 1 (for 10 reactions) dNTP mix (10 mM each) 111 1 (for 10 reactions) Taq DNA polymerase 0. 5ul 5ul (for 10 reactions) [0172] 7. Mix by pipeting up and down.

[0173] 8. To a set of labelled PCR tubes, add 5 u. l of the resuspended clones.

[0174] 9. To each tube, add 45RI of the PCR mix.

[0175] 10. Place the tubes in a thermocycler, program the following parameters: 96°C for 3 minutes A 25 cycles of a). 96°C for 1 minute b).

55°C for 1 minute c). 72°C for 1 minute A 72°C for 10 minutes- hold at 4°C.

[0176] When the program is finished, add 4, u1 of 6 X loading buffer to 20, u1 of each sample and analyze the PCR product on a 1.2% agarose gel. The rest of the reaction is stored at-20°C.

EXAMPLE 6: IDENTIFYING AND ISOLATING GENES INVOLVED IN TOXICOLOGICAL RESPONSES [0177] Human liver cells exposed to a toxic dose of aflatoxin (1 mg/ml) or liver tissue from a human exposed to aflatoxin are used to determine the differentially expressed genes in a human exposed to a liver toxicant (aflatoxin).

Exposure to a toxicant can occur intentionally (e. g., clinical treatment) or unintentionally (e. g., occupational exposure or accidental exposure). RNA is isolated from both liver samples using an RNA isolation kit from Qiagen (RNeasy Midi kit) followed by use of a MessageClean kit from Genhunter@. The protocols from the MessageClean kit are modified to generate more optimal conditions for removing DNA contamination. Then, these ingredients are added: 50 u. l total RNA, 5.7 wl l Ox reaction buffer, 1. 0 gel DNase I (10 units/ l) for a total volume of 56. 7 ul. The ingredients are mixed well and incubated for 30 minutes at 37° Celsius. Then 40 1ll phenol/chloroform mixture (1: 1 volume) is added and the mixture is vortexed for 30 seconds and allowed to sit on ice for 10 minutes.

Then the tube containing the mixture is spun in an Eppendorf centrifuge at 4 degrees for 5 minutes at maximum speed. The upper phase is collected, transferred to a new tube and 5 ut off NaOAc and 200 il 95% ethanol is added to the upper phase. The mixture is allowed to sit for at least one hour at-80° C and then spun for about 10 minutes at 4° C. The supernatant is removed and the RNA dried for a few minutes. Subsequently, the RNA is suspended in 11 gel DEPC H20. 1 µl is used to measure A260/280 in 50 ul H20. The RNA is stored as 1- 2 llg aliquots at-80°C. Immediately prior to differential display, the appropriate amount of RNA is diluted to 0. 1 µg/µl with DEPC H20. It is important to avoid using the diluted RNA after freeze-thaw cycle.

[0178] RNAimage kits are used and protocols from the RNAimage# kits are altered to optimize more successful mRNA differential display. The following sections describe the methods by which this is accomplished: Reverse transcription [0179] In a tube, the following ingredients are added: 9.4 1 l dH2O, 4. 0 µl 5x RT buffer, 1.6 1 dNTP (250 µM), 2.0 al of 0.1 µg/µl freshly diluted total RNA that is DNase-free, 2.0 al H-TuM (2 µM) for a total volume of 19 µl. The ingredients are mixed well and incubated at 65°C for 5 minutes, 37°C for 60 minutes, 75°C for 5 minutes, and held at 4°C. After the tubes had been at 37°C for 10 minutes, and 1 zip of SuperScript II reverse transcriptase (Life Technologies Inc. ) is added to each reaction, and quickly mixed by finger tapping the tubes before the incubation continued. At the end of the reverse transcription, the tubes are spun briefly to collect condensation. The tubes are set on ice for PCR or stored at-20°C for later use.

PCR [0180] The following ingredients are used for a PCR reaction: 10 µl dH20, 2 al 10X PCR buffer, 1.6 ul dNTP (25 µM), 2 µl of 2 « uM H-AP primer, 2lof2 µM H-T11M, 2 µl RT-mix described above (must contain the same H-TllM used for PCR), 0.2 µl α-33P dATP (2000 Ci/mmole), 0. 2 µl Taq DNA polymerase from PE Biosystems for a total volume of 20 u. l. The tube containing all these ingredients are mixed well by pipeting up and down and placed in a thermocycler at 95°C for 5 minutes and then amplified for 40 cycles under the conditions of 94°C for 30 seconds, 40°C for 2 minutes, 72°C for 30 seconds and finally held at 4°C until the samples are removed from the thermocycler.

Gel electrophoresis [0181] A 6% denaturing polyacrylamide gel in TBE is prepared and allowed to polymerize for at least 2 hours before using. Then the gel is run for about 30 minutes before any samples are loaded. It is important for all the sample wells in the gel to be flushed and cleared of all urea prior to loading any samples in the wells. About 3. 5 u, l of each sample is mixed with 2 ul of loading dye and incubated at 80°C for 2 minutes immediately before loading onto the 6% gel. In this example, the loading dye is xylene and after the gel is loaded with the samples obtained from the rounds of PCR, the gel is run at 60 watts of constant power until the xylene dye is about 6 inches from the bottom of the gel. Once the power is turned off, the gel is blotted onto a large sheet of exposed autoradiograph film. The gel is covered with plastic wrap and under dark conditions, the gel is placed in a large autoradiograph cassette with a new sheet of unexposed film, marked for orientation, and the film is allowed to be exposed to the gel at-80°C.

The exposure period can be anywhere from overnight to 72 hours. Once the film has been developed, bands of interest are identified by alignment with the developed film and subsequently isolated by cutting the band of interest out of the polyacrylamide gel with a clean scalpel blade. The isolated band is placed in 100 111 of water and boiled at 95% for 5 minutes.

PCR to amplify gel band [0182] PCR is set up to amplify the gel band. The re-amplification should be done using the same primer set and PCR conditions except the dNTP concentrations should be at 20 RM. The following ingredients are combined for the PCR reaction: 20.4 pl H20, 4 Ill 10X PCR buffer, 3.2 pll of 250, uM dNTPs, 4 111 of 2 RM H-AP primers, 4ulof2 uM H-TuM, 4 Ill template (out of the 100 Ill containing gel band), and 0. 5 gel Taq polymerase for a total volume of 40 pi These ingredients are heated to 95°C for 5 minutes and then cycled for 40 cycles under the conditions of 94°C for 30 seconds, 40°C for 2 minutes, 72°C for 30 seconds followed by a final extension at 72°C for 5 minutes and finally held at 4°C until the samples are removed from the thermocycler. About 4 Ill of the PCR reaction is removed and run on a 1% agarose gel to ascertain the success of the PCR reaction.

Cloning amplified fragments [0183] To clone the amplified fragments, products from different sources (e. g., GenHunter@ or InVitrogen) may be used to achieve the desired cloned product. In this example, InVitrogen's TOPO TA Cloning Kit is used and the following material is combined in a reaction tube: 2 µl of freshly run PCR product, 2 lit of sterile H20, 1 Ill of PCRTOPO vector for a final volume of 5 gel.

The combined ingredients are mixed gently and incubated for 5 minutes at room temperature. Then 1 Ill of 6x TOPO Cloning Stop Solution is added and all combined ingredients are mixed for about 10 seconds at room temperature and then set on ice. One Shot cells are thawed on ice. 2 ul of the TOPO Cloning reaction is added to the One Shot cells, mixed, and incubated on ice for 30 minutes. The cells are heat shocked at 42°C for 30 seconds without shaking and incubated on ice for 2 minutes. Then 250 ul of room temperature SOC is added to the heat shocked cells and mixed. The cells are then placed at 37°C for 30 minutes. About 50-100 Ill of the cells are spread on 2 XYT plates containing 100 llg/ml ampicillin and X-gal. The plates are incubated overnight at 37°C and the next morning, 3 white colonies are selected for analysis.

Screening colonies for correct recombinant plasmids [0184] PCR is used to ascertain whether the white colonies selected contained the correct recombinant plasmid. The following ingredients are combined for the PCR reaction: 21, ul H20, 2.5 u. l 10X PCR buffer, 0. 12 ul of 1 OmM dNTPs, 1 pl of 25 ng/lll T7 primer, 1 u, l gene specific left or right primer at 25 ng/ptl, template (a toothpick is used to transfer colony from transformation plate to tube by swishing the toothpick around in the reaction mix), and 0.5 Ill Taq polymerase for a total volume of 25 Ill. The reaction mix is run at 95°C for 5 minutes and then cycled 35 times under the conditions of 95° C for 30 seconds, 45°C for 30 seconds, 72° C for 30 seconds, and followed by 72° C for 5 minutes and finally 4°C until samples are removed from the thermocycler. About 4 u, l of the PCR product is removed and run on a 1% agarose gel to ascertain the success of the PCR reaction.

Bacterial colonies corresponding to the colonies which yielded positive PCR results are grown overnight in LB media containing 100 llg/, ul ampicillin at 37° C with constant shaking. Plasmid DNA are isolated from the overnight cultures and sequenced using a T7 primer. Sequences are then compared to sequences in the GenBank database to confirm that the correct gene fragment is cloned. Gene fragments are then amplified by PCR from the plasmid DNA. The unincorporated primers and dNTPs are removed and the resulting gene fragments are arrayed on glass slides for the purposes of measuring differential gene expression using the Phase-1 Molecular Toxicology Microarray products.

EXAMPLE 7: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT GENES FROM HUMAN DATABASES [0185] One method that was used to identify and isolate toxicologically relevant genes for inclusion in a human array was to search a public database (e. g., GenBank) for toxicologically relevant human genes. Once these genes were identified, primers, for example, those listed in Table 2 were designed and used in an amplification process with a cDNA library made from human cells. As disclosed herein, a cDNA library can be made from a variety of sources including but not limited to liver, lymphocytes, spleen, lung, kidney, brain, thymus, heart, tissue culture cells, primary cells, lymph nodes, or obtained from a commercial source (e. g., Clontech QUICK-CloneTM Cat. No. 7109-1). The amplified product was cloned into an expression vector and sequenced to confirm that the sequence matched or was substantially similar to the gene sequence information obtained from GenBank. Confirmed amplified gene products were then incorporated into a human array using the methods disclosed herein to immobilize the gene product, or target sequence, to a glass slide.

EXAMPLE 8: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT GENES FROM RAT HOMOLOGUES [0186] One method that is used to identify and isolate toxicologically relevant genes for inclusion in a human array is to make primers to toxicologically relevant rat genes, for example, as disclosed in pending U. S. applications 60/264,933 and 60/308,161. Once toxicologically relevant rat (or other non-human species) genes are identified, human homologues are identified by searching human sequence database (e. g., GenBank) for human sequence homologous to the non-human gene sequences with sequence search tools such as the Basic Local Alignment and Search Tool (Alschul et. al. 1997). Primers are obtained and used in an amplification process with cDNA library made from human cells. As disclosed herein, cDNA library can be made from a variety of sources (e. g., liver, lymphocytes, etc. ). Confirmed amplified gene products are then incorporated into a human array using the methods disclosed herein to immobilize the gene product, or target sequence, to a glass slide. Primers which were used to obtain human toxicologically relevant genes which are homologues of toxicologically relevant rat genes are disclosed in Table 2.

EXAMPLE 9: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT HUMAN GENES USING RAT HOMOLOGUES [0187] Sequences of human homologues of a toxicologically relevant rat gene sequence were obtained by using the sequence of toxicologically relevant rat gene sequences in a sequence search (e. g., a BLAST search) to find human sequences which have high homology to the rat gene. Primers to the human homologue were synthesized and then used to amplify a sequence of the human homologue from a human cDNA library as detailed in previous examples. Table 2 lists the primers which were used to isolate the human homologues. Table 5 lists the sequences obtained from cloned human homologues of rat genes prepared by this approach.

EXAMPLE 10: IDENTIFYING AND ISOLATING TOXICOLOGICALLY RELEVANT GENES USING DE NOVO PRIMERS [0188] Toxicologically relevant genes are identified using a public database (e. g., GenBank) and sequences corresponding within these genes are synthesized de novo and used in amplification reactions. The amplified product was cloned into a cloning vector and sequenced to confirm that the sequence matched or was substantially similar to the gene sequence information obtained from GenBank.

Confirmed amplified gene products were then incorporated into a human array using the methods disclosed herein to immobilize the gene product, or target sequence, to a glass slide.

EXAMPLE 11: ATTACHING TOXICOLOGICALLY RELEVANT GENES TO GLASS SLIDES [0189] The genes to be attached to the glass slides can be amplified as provided herein. An important modification to the amplification process was the inclusion of amine primers, which can be obtained from any commercial source, e. g., Synthegen, such that a reactive amine group, a derivative thereof, or another reactive group was included in the amplified product. The amplified product was purified by any number of methods disclosed herein and immobilized or"spotted" onto a solid substrate, such as a glass slide, which can react with the amine group on the amplified product and form a covalent linkage.

MD Array Spotter Operation [0190] The terminology and equipment used in this example comprised the following: [0191] Spotter: MD Generation II Array Spotter main instrument [0192] Spotting Chamber: Area of spotter enclosed in glass which houses the pins, plates, trays and most spotter machinery.

[0193] Controller: Dedicated Dell Computer and Monitor to right of Spotter Unit [0194] Pins: (6) fine tubes in the Spotter Unit which pick-up and spot the Target [0195] Slides: Std. size glass microscope slides with a special coating on one side [0196] Plates: Plastic 96 well plates which hold the Target solution to be spotted [0197] Target: A solution of PCR product which the spotter deposits on the slides.

[0198] N2 Tank: 5 ft. high steel gas tank labeled"Nitrogen, Compressed" [0199] N2: The N2 gas from the N2 tank [0200] Air Conditioner: Kenmore air conditioner installed in window of spotting chamber [0201] Humidifier 1: Essick 2000 Evaporative Cooler against the window [0202] Humidifier 2: Bemis Airflow with white flexible duck into the Spotter Unit [0203] Humidifier 3: Bemis Airflow against the wall [0204] Humidifier 4: Kenmore QuietComfort 7 [, 0205] Vacuum Pump: Gast Laboratory Oilless Piston Vacuum Pump [0206] Dampbox: The plastic salable container containing an NaCI/water slurry [0207] Materials used for reagent solutions were: Nanopure water, 0.2 M KCI (1/10 dilution of Stock 2M KC1 in water), and 95% EtOH Reagent. The temperature control was adjusted to 60°C. The spotter chambers were adjusted to be greater than 39 % relative humidity and less than 65° C. The spotting pins were pre-washed for 20 cycles.

Slide Preparation/Loading: [0208] When the pre-wash was completed, the slides were first each blown with N2 gas for about 2 seconds per side. The slides were inserted into the Spotter following Array Spotter Run Values. The slides were aligned using a clean narrow rod orienting it on the center right edge of the slide and gently pushed to the left until the slide was aligned vertically against the metal pins.

After slides were loaded and straightened, a visual check was done to make sure no more debris had fallen. The humidity was confirmed to be greater than 39% relative humidity. The MD spotter recognizes 16 plates as a maximum for a run and will pause automatically after 8 plates. The MD spotter also advances sequentially to plates in an invariable order and is not programmable to accommodate unique plate sourcing scheme. Therefore, it was important to manually rotate (or shuffle) plates to accomplish the spotting for the human arrays.

Blocking (Slide Preparation post-spotting) [0209] This blocking procedure is important because it reduces the non- specific background signals. The amounts provided in this protocol are for 19 slides, however, a skilled artisan may make modifications accordingly. More staining dishes and slide racks will be required if more than 19 slides are to be blocked. A clean glass container was obtained and filled with Nanopure H20. The container was placed on a hot plate and heated to a high temperature. A blocking solution was made by adding 2.5 ml of 20% SDS to 500mL blocking solution bottle. The blocking solution was warmed in microwave for 2.5 minutes and checked to determine if the temperature had reached 50°C. If the temperature of the solution was not at yet 50°C, then the solution was warmed in the microwave at 10 second intervals until it reached the desired temperature. One staining dish was placed on an orbital shaker with 4x SSC solution and turned to an agitation speed of 75 rpm. Slides were placed in metal racks and placed in boiling water for several minutes (e. g., 2 minutes). The slides were taken out of boiling water and allowed to cool briefly. The slides were then transferred to staining container containing 4x SSC solution on orbital shaker for several minutes (e. g., 2 minutes), rinsed with nanopure water in a staining container, and then briefly placed in blocking solution for about 15 minutes. After 15 minutes, the slides were taken out of the blocking solution and rinsed three times by dipping into three separate containers with nanopure water each time. The tops of the slides were dabbed lightly with a tissue and the slides were placed in a centrifuge for about 5 minutes at a speed of 1000 rpm.

EXAMPLE 12: MICROARRAY RT REACTION [0210] Fluorescence-labeled first strand cDNA probe was made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra.

This probe is hybridized to microarray slides spotted with DNA specific for toxicologically relevant genes. The materials needed to practice this example are : total or messenger RNA, primer, Superscript II buffer, dithiothreitol (DTT), nucleotide mix, Cy3 or Cy5 dye, Superscript II (RT), ammonium acetate, 70% EtOH, PCR machine, and ice.

[0211] The volume of each sample that would contain 20g of total RNA (or 2gag of mRNA) was calculated. The amount of DEPC water needed to bring the total volume of each RNA sample to 14 nl was also calculated. If RNA is too dilute, the samples are concentrated to a volume of less than 14 gel in a Speedvac without heat. The Speedvac must be capable of generating a vacuum of 0 Milli- Torr so that samples can freeze dry under these conditions. Sufficient volume of DEPC water was added to bring the total volume of each RNA sample to 14, ul.

Each PCR tube was labeled with the name of the sample or control reaction. The appropriate volume of DEPC water and 8 ul of anchored oligo dT mix (stored at- 20°C) was added to each tube.

[0212] Then the appropriate volume of each RNA sample was added to the labeled PCR tube. The samples were mixed by pipeting. The tubes were kept on ice until all samples are ready for the next step. It is preferable for the tubes to kept on ice until the next step is ready to proceed. The samples were incubated in a PCR machine for 10 minutes at 70°C followed by 4°C incubation period until the sample tubes were ready to be retrieved. The sample tubes were left at 4°C for at least 2 minutes.

[0213] The Cy dyes are light sensitive, so any solutions or samples containing Cy-dyes should be kept out of light as much as possible (e. g., cover with foil) after this point in the process. Sufficient amounts of Cy3 and Cy5 reverse transcription mix were prepared for one to two more reactions than would actually be run by scaling up the following recipes: For labeling with Cy3 [0214] 8 ul 5x First Strand Buffer for Superscript II [0215] 4 ul 0. 1 M DTT [0216] 2 ul Nucleotide Mix [0217] 2 ul of 1: 8 dilution of Cy3 (i. e. , 0.125mM Cy3dCTP).

[0218] 2 ul Superscript II For labeling with Cy5 [0219] 8 ul 5x First Strand Buffer for Superscript II [0220] 4 ul 0. 1 M DTT [0221] 2 ul Nucleotide Mix [0222] 2 ul of 1: 10 dilution of Cy5 (i. e. , 0. lmM Cy5dCTP).

[0223] 2 ul Superscript II [0224] About 18 ul of the pink Cy3 mix was added to each treated sample and 18 ul of the blue Cy5 mix was added to each control sample. Each sample was mixed by pipeting. The samples were placed in a PCR machine for 2 hours at 45°C followed by 4°C until the sample tubes were ready to be retrieved. The samples were transferred to Eppendorf tubes containing 600 u. of ethanol precipitation mixture. Some of the EtOH precipitation mixture was used to rinse the PCR tubes. The tubes were inverted to mix. Samples were placed in-80°C freezer for at least 20-30 minutes. If desired, samples may be left at-20°C overnight or over the weekend.

[0225] The samples were centrifuged for 15 minutes at 20800 x g (14000 rpm in Eppendorf model 5417C) and carefully the supernatant was decanted. A visible pellet was seen (pink/red for Cy3, blue for Cy5). It is a preferable to centrifuge the tubes at a fixed position so the pellet will be at a known area in the tube. In some rare instances, the probe is seen spread on one side of the tube instead of a tight pellet. If the pellet is white or nonexistent, the reaction has not occurred to maximal efficiency.

[0226] Ice cold 70% EtOH (about 1 ml per tube) was used to wash the tubes and the tubes were subsequently inverted to clean tube and pellet. The tubes were centrifuged for 10 minutes at 20800 x g (14000 rpm in Eppendorf model 5417C), then the supernatant was carefully decanted. The tubes were flash spun and any remaining EtOH was removed with a pipet. The tubes were air dried for about 5 to 10 minutes. protected from light. The length of drying time will depend on the natural humidity of the environment. For example, an environment in Santa Fe would require about 2 to 5 minutes of drying time. It is preferable that the pellet are not overdried.

[0227] When the pellets were dried, they are resuspended in 80 ul nanopure water. The cDNA/mRNA hybrid was denatured by heating for 5 minutes at 95°C in a heat block and flash spun.

EXAMPLE 13: PURIFICATION OF CY-DYE LABELED CDNA [0228] To purify fluorescence-labeled first strand cDNA probes, the following materials were used: Millipore MAHV N45 96 well plate, v-bottom 96 well plate (Costar), Wizard DNA binding Resin, wide orifice pipette tips for 200 to 300 ul volumes, isopropanol, nanopure water. It is highly preferable to keep the plates aligned at all times during centrifugation. Misaligned plates lead to sample cross contamination and/or sample loss. It is also important that plate carriers are seated properly in the centrifuge rotor.

[0229] The lid of a"Millipore MAHV N45"96 well plate was labeled with the appropriate sample numbers. A blue gasket and waste plate (v-bottom 96 well) was attached. Wizard DNA Binding Resin (Promega catBA1151) was shaken immediately prior to use for thorough resuspension. About 160 ul of Wizard DNA Binding Resin was added to each well of the filter plate that was used. If this was done with a multi-channel pipette, wide orifice pipette tips would have been used to prevent clogging. It is highly preferable not to touch or puncture the membrane of the filter plate with a pipette tip. Probes were added to the appropriate wells (80 ul cDNA samples) containing the Binding Resin. The reaction is mixed by pipeting up and down-10 times. It is preferable to use regular, unfiltered pipette tips for this step. The plates were centrifuged at 2500 rpm for 5 minutes (Beckman GS-6 or equivalent) and then the filtrate was decanted. About 200 ul of 80% isopropanol was added, the plates were spun for 5 minutes at 2500 rpm, and the filtrate was discarded. Then the 80% isopropanol wash and spin step was repeated. The filter plate was placed on a clean collection plate (v-bottom 96 well) and 80 ul of Nanopure water, pH 8.0-8. 5 was added. The pH was adjusted with NaOH. The filter plate was secured to the collection plate with tape to ensure that the plate did not slide during the final spin. The plate sat for 5 minutes and was centrifuged for 7 minutes at 2500 rpm. If there are replicates of samples they should be pooled.

EXAMPLE 14: FLUORESCENCE READINGS OF CDNA PROBE AND HYBRIDIZATION ON THE MICROARRAY [0230] To semi-quantitatively assess the incorporation of fluorescence into cDNA probes and to concentrate probes prior to hybridization, the following material was used: 384 well, 100 nl assay plate (Falcon Microtest cat#35-3980) and Wallac Victor 1420 Multilabel counter (or equivalent).

[0231] It is preferable that a consistent amount of cDNA is pipeted into the 384-well plate wells because readings will vary with volume. Controls or identical samples should be pooled at this step, if required. The probes were transferred from the Millipore 96 well plate to every other well of a 384 well assay plate (Falcon Microtest). This was done using a multi-channel pipette. For replicate samples that have been pooled, 60 gel aliquots were transferred into wells of the assay plate.

[0232] The Cy-3 and Cy-5 fluorescence was analyzed using the Wallac 1420 workstation programmed for reading Cy3-Cy-5 in the 384-well format and the data was saved to disk. The typical range for Cy-3 (20, g) is 250-700,000 fluorescence units. The typical range for Cy-5 (20ug) is 100-250,000 fluorescence units. Settings for the Wallac 1420 fluorescence analyzer were as follows: Cy3 [0233] CW lamp energy = 30445 [0234] Lamp filter = P550 slot B3 [0235] Emission filter = D572 dysprosium slot A4 [0236] Emission aperture = normal [0237] Count time = 0. 1 s Cy5 [0238] CW lamp energy = 30445 [0239] Lamp filter = D642 samarium slot B7 [0240] Emission filter = D670 slot A8 [0241] Emission aperture = normal [0242] Count time = 0. 1 s Dry-down Process [0243] Concentration of the cDNA probes is highly preferable so that they can be resuspended in hybridization buffer at the appropriate volume. The volume of the control cDNA (Cy-5) was measured and divide by the number of samples to determine the appropriate amount to add to each test cDNA (Cy-3). Eppendorf tubes were labeled for each test sample and the appropriate amount of control cDNA was allocated into each tube. The test samples (Cy-3) were added to the appropriate tubes. These tubes were placed in a speed-vac to dry down, with foil covering any windows on the speed vac. At this point, heat (45°C) may be used to expedite the drying process. Time will vary depending on the machinery. The drying process takes about one hour for 150 ul samples dried in the Savant.

Samples may be saved in dried form at-20°C for up to 14 days.

Microarray Hybridization [0244] To hybridize labeled cDNA probes to single stranded, covalently bound DNA target genes on glass slide microarrays, the following material were used: formamide, SSC, SDS, 2 urn syringe filter, salmon sperm DNA, hybridization chambers, incubator, coverslips, parafilm, heat blocks. It is preferable that the array is completely covered to ensure proper hybridization.

[0245] About 30 Ill of hybridization buffer was prepared per sample. Slightly more than is what is needed should be made since about 100 ul can be lost during filtration.

Hybridization Buffer: for 100 50% Formamide 50 u. l fbrmamide 5X SSC 25 Ill 20X SSC 0.1% SDS 25 tl 0. 4% SDS [0246] The solution was filtered through 0.2 um syringe filter, then the volume was measured. About 1 u. l of salmon sperm DNA (lOmg/ml) was added per 100 ul of buffer. Materials used for hybridization were: 2 Eppendorf tube racks, hybridization chambers (2 arrays per chamber), slides, coverslips, and parafilm. About 30 µl of nanopure water was added to each hybridization chamber. Slides and coverslips were cleaned using N2 stream. About 30 u. l of hybridization buffer was added to dried probe and vortexed#gently for 5 seconds.

The probe remained in the dark for 10-15 minutes at room temperature and then was gently vortexed for several seconds and then was flash spun in the microfuge.

The probes were boiled for 5 minutes and centrifuged for 3 min at 20800 x g (14000 rpm, Eppendorf model 5417C). Probes were placed in 70 °C heat block.

Each probe remained in this heat block until it was ready for hybridization.

[0247] Pipette 25 ul onto a coverslip. It is highly preferable to avoid the material at the bottom of the tube and to avoid generating air bubbles. This may mean leaving about 1 u. l remaining in the pipette tip. The slide was gently lowered, face side down, onto the sample so that the coverslip covered that portion of the slide containing the array. Slides were placed in a hybridization chamber (2 per chamber). The lid of the chamber was wrapped with parafilm and the slides were placed in a 42°C humidity chamber in a 42°C incubator. It is preferable to not let probes or slides sit at room temperature for long periods. The slides were incubated for 18-24 hours.

Post-Hybridization Washing [0248] To obtain single stranded cDNA probes on the array, all non- specifically bound cDNA probe should be removed from the array. Removal of all non-specifically bound cDNA probe was accomplished by washing the array and using the following materials: slide holder, glass washing dish, SSC, SDS, and nanopure water. It is highly preferable that great caution be used with the standard wash conditions as deviations can greatly affect data.

[0249] Six glass buffer chambers and glass slide holders were set up with 2X SSC buffer heated to 30-34°C and used to fill up glass dish to 3/4th of volume or enough to submerge the microarrays. It is important to exercise caution in heating of the 2X SSC buffer since a temperature of greater than 35°C might strip off the probes. The slides were removed from chamber and placed in glass slide holders.

It is preferable that the slides are not allowed dry out. The slides were placed in 2X SSC buffer but it is recommended that no more than 4 slides be placed per dish. Coverslips should fall off within 2 to 4 minutes. In the event that the coverslips do not fall off within 2 to 4 minutes, very gentle agitation may be administered. The stainless steel slide carriers were placed in the second dish and filled with 2X SSC, 0.1% SDS. Then the slides were removed from glass slide holders and placed in the stainless steel holders submerged in 2X SSC, 0.1% SDS and soaked for 5 minutes. The slides were transferred in the stainless steel slide carrier into the next glass dish containing 0. 1X SSC and 0.1% SDS for 5 minutes.

Then the slides are transferred in the stainless steel carrier to the next glass dish containing only 0. 1X SSC for 5 minutes. The slides, still in the slide carrier, was transferred into nanopure water (18 megaohms) for 1 minute.

[0250] To dry the slides, the stainless steel slide carriers were placed on micro-carrier plates with a folded paper towel underneath. The top of the slides were gently dabbed with a tissue. Then the slides were spun in a centrifuge (Beckman GS-6 or equivalent) for 5 minutes at 1000 rpm. It is very important that the slides do not air dry, as this will lead to increased background.

EXAMPLE 15: USE OF ALGORITHMS TO IDENTIFY, SELECT, AND EVALUATE TOXICOLOGICALLY RELEVANT GENES [0251] A two-step approach has been used in ranking candidate genes from the human array.

[0252] First, three cutoff criteria were specified for individual gene values from experiments involving expression analysis of human genes: The three cutoff criteria were: fold induction/repression level, average fluorescence of the replicate spots used to calculate expression level, and coefficient of variation of the replicate spots.

[0253] To calculate fold induction/repression level, the stored induction/repression level of a gene in a particular experiment has been calculated as follows: [0254] 1). arrive at a treatment score for the gene. The treatment score was represented by the amount of Cy3 labeled cDNA from a treated source (e. g., human or non-human cells or laboratory animals dosed with an agent) that had bound to a complementary target DNA spot of the microarray slide. The amount of Cy3 labeled cDNA was detected by a microarray laser scanner at a wavelength of 532nm.

[0255] 2). arrive at a control score for the gene. The control score was represented by the amount of Cy5 labeled cDNA from an untreated source that had bound to a complementary target DNA spot of the microarray slide. The amount of Cy5 labeled cDNA was detected by a microarray laser scanner at a wavelength of 635nm. The unit of measure was the pixel intensity or the average of several pixel intensities reported by a microarray laser scanner at coordinate on a microarray slide. The pixel intensity at that location was proportional to the number of photons detected by a photomultiplier tube when a spot of target DNA labeled with fluorescent probe was illuminated by a laser with a wavelength to which the dye is sensitive. For an Axon Instruments Inc. GenePix 4000A MicroArray Scanner, which was used in these experiments, these values are between 0 and 65535.

[0256] 3). compute an un-normalized treated-to-control ratio by dividing the treatment score by the control score [0257] 4). compute a final, "normalized"induction score by dividing the un- normalized ratio by some normalization factor. For example, using the following 4-steps procedure above, calculation of Gene A is performed as follows: 1). Suppose that the average of its set of four raw treated values is 100,000 2). Suppose that the average of the set of its four raw control values is 25,000 3). The un-normalized ratio would thus be 4, and finally 4). If the normalization factor (designed to take into account environmental effects) turned out to be 2, one would end up with at a final, normalized induction score (often called fold induction) of 4 divided by 2, or 2. In this example, Gene A was induced two-fold.

In all experiments conducted with respect to this invention, there were four replicates of each gene, with each replicate having a treated and a control value.

Thus, the calculation of an expression level for each gene in an experiment involved aggregating eight data points.

[0258] The average fluorescence level of the replicate spots used to calculate the expression level is accomplished by a simple average of the four treated replicate values used in any experiment to calculate the expression level of a gene.

[0259] The coefficient of variation of the replicate spots was a conventional measure of variability, expressed as a percentage, that in this case was derived by dividing the standard deviation of the four replicate treated-to-control ratios by the average of the four replicated treated-to-control ratios. The latter criteria represent useful measures of data quality. Thus, initial screening is based on expression level and measurement quality.

[0260] Algorithms were written specifically to perform the entire process of ranking genes to be included on the human toxicity array. In the initial part of the program, the three criteria above were"ended"together, so that in order to"make the first cut"the gene would have to meet all three criteria. This was considered the first tier in the program.

[0261] The criteria are adjustable within the algorithms. For the actual ranking of the human toxicity array, the following criteria were used: induction/repression level: 2; fluorescence level: 400; and a coefficient of variation: 30%. To make the first cut and be selected as a potential toxicologically relevant gene, a gene only had to meet the 3 criteria within one experiment. Likewise, relevant values for the gene would have been included each time it met the criteria within a different experiment. The data for genes that made the cut (and hence were selected as potential toxicologically relevant genes) and each time the genes made the cut were stored in a separate, temporary data table for ranking (the second tier in the process). A gene's data could be included more than once: one time for each experiment in which that gene met the three criteria.

[0262] Each of the factors listed in the table below was given a relative weight, as indicated: RELATIVE WEIGHT OF EACH SCORING FACTOR (0-5) Number of Compounds Gene Avg. of Fold Induction Avg. Fluorescence 3 Made Cut 3 Magnitude 3 Consistency Across Duplicate COV of Fold Induction 3 Tissue Hit Consistency 3 Slides 3 [0263] Next, gene values that made the cut were aggregated into overall scores. It was necessary to"aggregate"the potentially more-than-one values for each gene into overall scores for the gene because data for a particular gene were included for each experiment in which that gene's values met the 3 cutoff criteria described above. Overall scores were aggregated for each six factors, as shown in the table above, and ranked for each gene, based on six ranking criteria: 1). Number of slides on which that gene met the cutoff criteria (NC = number of compounds). As noted, a gene could meet the 3 cutoff criteria described above in more than one of the experiments. NC is a simple count of the number of different compounds (not experiments, as duplicate experiments were performed for each compound) in which that gene met the 3 criteria, 2). Percent of consistency between slides (% time the gene value made the cutoff criteria on the replicated slide for that initial slide) (CC). As mentioned above, duplicate experiments were performed for each compound. CC is a count of the number of times the gene made the cutoff in both of the duplicate experiment pair. Thus, a gene could have an NC (number of compounds) of 3 but a CC (compound consistency) of 2, meaning that in two of the three compounds it made the cutoff in each of the duplicate experiments, and in one of the compounds, it did not.

3). Average magnitude (absolute value) of fold induction for all occurrences where that gene made the cutoff criteria (FI). This is a simple average of the magnitude of the expression/repression levels for each set of data values for a particular gene. 4). coefficient of variation of those fold induction scores (unlike all the other ranking criteria, a lower coefficient of variation is deemed better) (CV). The coefficient of variation applied to the set of expression values for a particular gene to assess the variability of its scores. 5). Average fluorescence value of all replicate spots of occurrences where that gene made the cutoff criteria (FL). This is a simple average of the fluorescence levels of the treated values for each occurrence of a gene that had made the cut. 6). Tissue consistency, i. e. , what percent of cutoff-meeting occurrences of the gene were in the same tissue (CT).

Expression levels were measured not only in several compounds but in several tissues as well (liver, kidney, etc. ) CT was calculated as the percentage of time the gene data values which made the cutoff were measured in the same tissue. For example, if a gene had made the cut in four experiments, twice in liver and twice in kidney, its tissue consistency would be 50%.

[0264] The following weightings were used in the actual process of ranking genes for inclusion on the human array: NC=3, CC=1, Fl=5, CV=0, FL=0, CT=0.

Thus, the final three criteria received no weight in this case.

[0265] Each gene was assigned a score between 0 and 100 for each ranking criterion. Each ranking criterion score was computed as follows: The range of values for all genes was computed for the criterion by subtracting the lowest value present among all scores from the highest. The temporary data table compiled from the experiments included scores for every gene that had met the initial 3 cutoff criteria. A gene would be present in the table with values for each experiment in which it had met or surpassed the 3 cutoff criteria. Scores were then aggregated from the gene occurrences of each gene into an overall score for that gene for each of the six criteria described above. For example, if Gene A made the cut in three experiments with respective induction scores of 4,6, and 8 then the aggregated induction score for Gene A would be 6 (the average of the three values). Further, for example, suppose the gene with the highest overall score on the induction factor had an overall score of 10, and the lowest an overall score of 2. Then, Gene A would receive a rating of 50%, because its score was halfway between the highest and the lowest. The score for each gene was then calculated by subtracting the lowest value present from the value for that gene, then dividing by the range and multiplying by 100. In other words, the score for each gene is the percent above the minimum present toward the maximum. For example, if a gene's score was three-fourths of the way between the minimum present and the maximum for that criterion, its score would be between the minimum present and the maximum for that criterion, its score would be 5%.

Since for the CV factor (coefficient of variation of fold inductions) lower was deemed better, the score thus computed was subtracted from 100 to invert the percentage.

[0266] The final ranking score for each gene was computed via a weighted combination of its score on the six ranking criteria. If a score could not be computed for a particular criterion, the entire value of that criterion was removed from the equation, and ranking was based solely on the remaining factors. Each of the ranking criteria could be weighted between 0 and 5, and weightings are relative, so that 2: 2: 2: 2: 2: 2 would be the same as 4: 4: 4: 4: 4: 4, etc. A zero weighting would drop the factor from the equation.

[0267] For example, suppose that Gene A had the following scores: NC: 75%, CC: 50%, FI : 80%, CV: 25%, FL: 50%, and CT: 30%. Using the weightings described above (3, 1, 5,0, 0,0) the final score for Gene A would be calculated as follows: [0268] (75*3 + 50*1 + 80*5 + 25*0 + 50*0 + 30*0)/9 (NOTE: 9 is the total number of"weightings"or 3+1+5) [0269] (225 + 50 + 400 + 0 +0 + 0)/9 [0270] 675/9 [0271] 75 would be the final score for Gene A.

[0272] A score was then computed for each discrete gene based on aggregating the individual values for that gene from one or more experiments where that gene had made the initial cut. The list of genes was then rank-ordered on the basis of final scores. An objective determination was made of how many genes to take from the top of the list to be added to the human toxicity array. The human toxicity gene array and sequences is shown in Table 1.