A NOVEL PROCESS AND REAGENT FOR RAPID GENETIC ALTERATIONS IN

EUKARYOTIC CELLS

[001]. Cross-reference to related application

[002]. This application claims benefit of, and priority from, U.S. provisional patent application No. 61/504,577, filed on 5 July 2011 , the contents of which are hereby incorporated herein by reference

[003]. Field

[004]. The present invention relates to the field of molecular and cell biology, more specifically genetic alterations in eukaryotic cells.

[005]. Background

[006]. The discovery of RNA interference (RNAi), sequence-specific gene silencing by double-stranded (ds) RNA molecules, has revolutionized the way we study gene function (1 , 2). Two altemitive molecules are routinely used to trigger RNAi in mammalian cells: (i) chemically or enzymatically synthesized/generated small interfering (si) RNAs and (ii) genetically encoded short hairpin (sh) RNAs that are converted into siRNAs by the cellular RNAi machinery (2-6). The relatively low cost and the possibility of inducing sustained and tunable silencing, combined with the minimized risk of off-target effects make the shRNA approach especially attractive for high through put loss-of-function screens and developing RNAi-based therapies (6-9).

[007]. Sequence-specific gene silencing by small hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider use of this approach in mammalian cell cultures has been hindered by the lack of straightforward techniques to achieve uniform expression and optimal performance of shRNAs.

[008]. However, unlike synthetic siRNAs that can be delivered into most cell populations like cultured mammalian cell lines in a virtually quantitative manner, equally straightforward methods for rapidly generating homogeneous cell populations expressing shRNAs are currently not available and have not been described/ Only few cell lines can be transiently transfected with plasmid DNA with efficiencies sufficient for penetrant RNAi. Similarly, a major limitation of stable expression approaches relying on plasmid- or viral vector mediated transgenesis is the random nature of the genomic integration, which often results in uneven expression or lowers the transgene expression levels due to epigenetic silencing (10-14) or position effect. As a consequence, obtaining populations of homogeneous shRNA-expressing cell normally entails time-consuming and labor intensive enrichment or cell cloning steps. For example, the recently published lenti viral toolkits for inducible shRNA expression generate populations containing substantial fractions of cells expressing shRNAs at low levels, which requires additional fluorescence-activated cell sorting (FACS) steps to improve the overall knockdown efficiency (10, 15). While feasible for experiments utilizing just a few shRNA molecules, FACS enrichment is not practical for medium- and high- throughput shRNA screens carried out in the arrayed format (7). In addition, shRNA-encoding virus vectors require considerable efforts to prepare high-titre stocks and importantly, are associated with biosafety concerns and a substantial bureaucratic burden.

[009]. Recombination-mediated cassette exchange (RMCE) utilizes the activity of site-specific recombinases to integrate a donor sequence flanked by self-compatible but mutually incompatible recombination sites at a predefined acceptor locus containing a similar recombination site pair (16). This method eliminates the genome position effect and allows uniform transgene expression in independent recombinant clones. RMCE can be employed to integrate

transgenes directionally into predefined genome sites. A common feature of the system is that each consists of a single polypeptide recombinase Cre, FLP or R and two identical or almost identical palendromic recognition sites lox, FRT or RS. For example using two identical but oppositely orientated RS sites. The donor vector containing the R recombinase gene. RMCE can use both the Cre//ox and FLPIFRT systems in animal cell cultures. [0010]. RMCE has recently been used for stable shRNA expression in HeLa cells (17-19). However, a relatively high incidence of unspecific integration events still called for additional cloning steps-to isolate shRNA-expressing cells from the original recombinant pools (17-19). DNA excision can subsequently happen between any pair of compatible sites and result in the restoration of the original two DNA molecules or the exchange of the intervening DNA segments between the two DNA molecules. Furthermore, the number of acceptor cell lines suitable for RMCE is rather limited.

[0011]. The present invention seeks to ameliorate at least one of the problems mentioned above.

[0012]. Summary

[0013]. Accordingly a first aspect of the invention includes a process for obtaining a nearly homogenous population of genetically altered eukaryotic cells, said process comprising:

a. transforming a first cell with a first isolated nucleic acid fragment

comprising a first selectable marker protein-coding sequence, wherein the isolated nucleic acid fragment is flanked by a first recombination site and a second non-identical recombination site;

b. introducing into the first cell a donor cassette, wherein said donor

cassette comprises a strong polyadenylation site operably linked to a second isolated nucleic acid fragment comprising a targeting nucleic acid site and a second selectable marker protein-coding sequence wherein the second isolated nucleic acid fragment is flanked by the first recombination site and the second non-identical recombination site; c. providing a recombinase that recognizes and implements

recombination at the non-identical recombination sites resulting in a transformed cell; and

d. Isolating the transformed cell in the presence of a selection agent. [0014]. Having a strong polyadenylation site upstream of the second isolated nucleic acid fragment comprising a targeting nucleic acid site and a second selectable marker protein-coding sequence wherein the second isolated nucleic ^" acid fragment is flanked by the first recombination site and the second non- identical recombination site may discourage unspecific integration events and generate high yields of cells with correct recombination with the first isolated nucleic acid fragment. Preferably, the strong polyadenylation site is a modified polyadenylation signal from thymidine kinase gene SEQ ID NO: 52.

[0015]. Preferably the targeting nucleic acid site comprises an shRNA directed to a 3' untranslated region of a target nucleic acid of interest. Some embodiments include an shRNA sequence selected from any one of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID numbers 54 to SEQ ID NO: 175 Listed in table 2.

[0016]. Preferably, the non-identical recombination sites are two similar or almost identical palendromic recognition sites such as lox, FRT or RS. Most preferably the non-identical recombination sites are Lox2272 (SEQ ID NO: 48), and LoxP (SEQ ID NO: 49).

[0017]. Preferably the recombinase comprises transiently expressing within the cell an expression plasmid comprising a polynucleotide encoding said

recombinase. Any recombinase enzyme known in the art to catalyse a site specific recombination event between two nucleic acid recognition sites would be suitable provided the recombinase is able to recognize the two nucleic acid recognition sites. Examples include a Cre recombinase with Lox recognition sites or a Flippase recombination enzyme (FLP) with Frt recognition sites. Preferably, the said recombinase is a Cre amino acid. In a preferred embodiment the Cre has been modified to include an N-terminal nuclear localization signal (SEQ ID NO: 50). This may enhance the efficiency of the recombination.

[0018]. The transgenic process of the invention ensures that transformed cells express a selectable marker. Cells that lack a functional selectable marker gene will be killed by the selection agent. Selectable marker genes include genes conferring resistance to antibiotics, herbicidal compounds. A specific selection agent may have one or more corresponding selectable marker genes. Likewise, a specific selectable marker gene may have one or more corresponding selection agents. Preferably, the first selectable marker protein-coding sequence encodes a blasticidin-resistant protein that has an amino acid sequence comprising SEQ ID NO: 51. Preferably, the second selectable marker protein-coding sequence encodes a puromycin-resistance protein that has an amino acid sequence comprising SEQ ID NO: 53 wherein the selection agent is puromycin.

[0019]. Preferably, the first isolated nucleic acid fragment further comprises at least one strong constitutive promoter operably linked to the targeting nucleic acid site. In one embodiment the at least one strong constitutive promoter operably linked to the targeting nucleic acid site is EF1 a (SEQ ID No. 47).

[0020]. Another aspect of the invention includes a nearly homogenous population of genetically altered eukaryotic cells, having stably incorporated in its genome a donor cassette comprises a strong polyadenylation site operably linked to a isolated nucleic acid fragment comprising a targeting nucleic acid site and a selectable marker protein-coding sequence wherein the isolated nucleic acid fragment is flanked by a first recombination site and a second non-identical recombination site.

[0021]. Preferably, the targeting nucleic acid site comprises an shRNA directed to a 3' untranslated region of a target nucleic acid of interest. The shRNA may be selected from any one of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID numbers 54 to 175 listed in table 2.

[0022]. Preferably the non-identical recombination sites are as described above.

[0023]. Preferably the strong polyadenylation site is as described above.

[0024]. Preferably the selectable marker protein-coding sequence is as described above.

[0025]. Another aspect of the invention includes a kit for obtaining a nearly homogenous population of genetically altered eukaryotic cells, comprising:

a. A vector comprising a first isolated nucleic acid fragment comprising a first selectable marker protein-coding sequence, wherein the isolated nucleic acid fragment is flanked by a first recombination site and a second non-identical recombination site;

b. a donor cassette, wherein said donor cassette comprises a strong

polyadenylation site operably linked to a second isolated nucleic acid fragment comprising a targeting nucleic acid site and a second selectable marker protein-coding sequence wherein the second isolated nucleic acid fragment is flanked by the first recombination site and the second non-identical recombination site;

c. a plasmid comprising a recombinase that recognizes the non-identical recombination sites; and

d. a selection agent.

[0026]. Preferably, the targeting nucleic acid site of the kit comprises an shRNA directed to a 3' untranslated region of a target nucleic acid of interest. The shRNA may be selected from any one of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID numbers 54 to 75 listed in table 2.

[0027]. Preferably, the non-identical recombination sites are as described above.

[0028]. Preferably, the strong polyadenylation site of the kit is as described above.

[0029]. Preferably, the selectable marker protein-coding sequence and the selection agent of the kit are as described above.

[0030]. Preferably, the recombinase of the kit is as described above.

[0031]. Preferably, the first isolated nucleic acid fragment of the kit further comprises at least one strong constitutive promoter operably linked to the targeting nucleic acid site. In one embodiment the at least one strong constitutive promoter operably linked to the targeting nucleic acid site is EF1a (SEQ ID No. 47)

[0032]. Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description, which proceeds with reference to the following illustrative drawings of preferred embodiments.

[0033]. Brief Description of the drawings

[0034]. Figure 1. Flowchart of a typical HILO-RMCE experiment.

[0035]. Figure 2. Diagram of the HILO-RMCE reaction using the pRD1 donor plasmid.

[0036]. Figure 3. (A) Southern blot analysis of the acceptor cell lines generated byJentiviral transduction of the HILO_RMCE acceptor locus (names containing a hyphen followed by "A" and a clone number) indicates that all acceptor lines contain only one lentivirus integration site. The parental cell controls have no lentiviral-specifiasequences, as expected (B) The acceptor cell lines were co-transfected in a 12-well (HEK293T-A2, HeLa-A12, HeLa-S3-A6, A549-A11 , HT1080-A4, U2OS-A13, L929-A6) or 6-well format (P19-A9, CAD- A13, N2a-5A) with a mixture containing 90% of pRD1 plasmid and 10% of the Cre-encoding plasmid pCAGGS-Cre. Following the puromycin selection, surviving colonies were stained with methylene blue and photographed.

[0037]. Figure 4. Diagram of the HILO-RMCE reaction using the pRD-RIPE donor plasmid.

[0038]. Figure 5. (A-B) Optimization of the HILO-RMCE protocol. (A)

HEK293T-A2 cells containing the RMCE acceptor locus were co-transfected in a 12-well format with mixtures containing pRD-RIPE plasmid and various amounts of Cre-encoding plasmids pCAGGS-Cre or pCAGGS-nlCre. Puromycin-resistant colonies were stained with methylene blue and photographed. (B) Same experiment as in (A) was repeated with the HeLa-A12 acceptor cells. Graph on the right shows the number of HeLa-A12 colonies per 1 //g of pRD-RIPE donor plasmid DNA as a function of Cre plasmid concentration. Note that in both HEK293T-A2 and HeLa-A 2 lines nICre performs better than the wild type Cre. (C) Genomic DNA was isolated from 3 parental cell lines (HEK293T, HeLa and A549, lanes labeled "parent") as well as the corresponding HILO-RMCE acceptor clones (Lanes labeled "A" followed by the clone number) and pooled clones obtained by the HILO-RMCE-mediated integration of the RIPE cassette (the "A+RIPE lanes) and analyzed by multiplex PCR reactions to assess

completeness of the RMCE reaction. PCR reactions using primers against GADPH gene were used as a loading control. Note that the spliced GAPDH pseudogene (ipGAPDH) product was detected in addition to a bona fide GAPDH band. (D) HILO-RMCE colonies produced by co-transfecting HEK293T-A2 and HeLa-A12 cells with pCAGGS-nlCre and either pRD1 or pRD-RIPE were pooled and incubated with 2 /vg/ml Dox for 48 hours or alternatively left untreated. The EGFP expression was then studied using fluorescence-activated cell sorting (FACS). Note that nearly all (>97%) cells become EGFP-positive in the pRD- RIPE samples treated with Dox but not in the corresponding Dox(-) controls. (E) HEK293T-A2 cells carrying RIPE cassettes with shRNA against either FLuc or LacZ were incubated with Dox for 36 hours or left untreated. The cells were then transfected with a mixture of plasmids encoding FLuc and RLuc and the activities of the two luciferases assayed 24 hours post transfection. The bars show FLuc activities normalized to the RLuc signal averaged from 6 transfection experiments ±SD.

[0039]. Figure 6. (A) HEK293T-A2 cells containing 4 different RIPE-shRNAs against the PTBP1 gene were incubated with Dox for 72 hours [Dox(+)] to induce the shRNA expression or left untreated [Dox(-)]. The efficiency of PTBP1 mRNA knock down was then assayed by RT-qPCR using HPRT mRNA levels as a normalization control. The graphs show residual PTBP1 expression in the Dox(+) samples as % of the corresponding Dox(-) controls. Data are averaged from 3RT- qPCR amplifications ±SD. (B) Mouse Ptbpl -specific RIPE-shRNAs integrated into N2a-A5 cells were induced with Dox and the Ago2 mRNA levels measured by RT-qPCR as in (A). shRNA against FLuc was used in this experiment as a negative control. (C) mouse Ago2-specific RIPE-shRNAs integrated into N2a-A5 cells were induced with Dox and the Ago2 mRNA levels measured by RT-qPCR as in (A). (D-E) HILO-RMCE was used to integrate a library of shRNAs against the TUTase family proteins into HEK293T-A2 cells, shRNA expression was induced by Dox and the knockdaown efficiency determined as in (A). Arrowheads indicate shRNAs reducing the expression of the corresponding TUTase mRNAs most efficiently.

[0040]. Figure 7. Using HILO-RMCE for rapid transgenesis in mammalian cell line. HEK293T-A2 cells were co-transfected with pCAGGS-nlCre and different pRD vector-based donor plasmids additionally encoding: (A) CAG promoter- driven intron-containing dTomato gene (i-dTom); (B) CAG-driven EGFP gene containing a nuclear localization sequence and preceded by an internal ribosomal entry site (IRES-nlEGFP); (C) CAG-driven bicistronic cassest containing both dTomato gene (dTom) and IRES-nlEGFP; and (D) constitutively expressed reverse tetracycline transactivator (rtTA3) gene and a tetracycline-induced tRFP gene. Recombinant cells were selected for 8 days with 5 μg/m\ puromycin, pooled and propagated for another 4 days. Cells in (A-C) were imaged

immediately using phase contrast and GFP or/and RFP epifluorescence, as appropriate. Cells in (D) were incubated with 2 /vg/ml Dox or left untreated for another 2 days prior to imaging. Maps of the corresponding donor constructs are shown on the top of each panel.

[0041]. Figure 8. Establishing the HILO-RMCE acceptor cell lines. (A)

Flowchart of a typical HILORMCE shRNA experiment. (B) Diagram of the HILO- RMCE reaction using the pRD1 donor plasmid. (C) The newly established 11 acceptor lines were co-transfected in a 12-well (HEK293T-A2, HeLa-A12, HeLa- S3-A6, A549-A11 , HT1080-A4, U2OS-A13, L929-A12, NIH 3T3-A7) or 6-well format (P19-A9, CAD-A13, N2a-A5) with a mixture containing 90% of pRD1 plasmid and 10% of a Cre-encoding plasmid (most cell lines, pCAGGS-Cre; NIH 3T3-A7, pCAGGS-nlCre) or the EGFP-encoding control plasmid pCIG. Following the puromycin selection, multiple colonies formed in the presence of Cre but not when Cre was substituted with EGFP.

[0042]. Figure 9. Developing the HILO-RMCE technology. (A) Diagram of the HILO-RMCE reaction using the pRD-RIPE donor plasmid. (B-C) Optimization of the HILO-RMCE protocol. (B) HEK293T-A2 cells containing the RMCE acceptor locus were co-transfected in a 12-well format with the pRD-RIPE plasmid and the indicated amounts of the pCAGGS-Cre or pCAGGS-nlCre plasmids. Puromycin-resistant colonies were stained with methylene blue and photographed. (C) The experiment described in (B) was repeated with the HeLa- A12 acceptor line. Graph on the right shows the relative number of the HeLa-A12 colonies as a function of the Cre plasmid concentration. Note that nICre performs better than the wild-type Cre in both cell lines. (D) Genomic DNA was isolated from 3 parental cell lines (HEK293T, HeLa and A549; lanes labeled "parent") as well as the corresponding HILO-RMCE acceptor clones ("A" followed by the clone number) and pooled clones obtained by the HILO-RMCE-mediated integration of the RIPE cassette ("A+RIPE") and analyzed by multiplex PCR using either the 5' junction primer mixture (EF, BR, and PR; Fig. 1 b) or the 3' junction mixture (GF, BF, and WR; Fig. 1 b). The mixtures were formulated so that the corresponding PCR product sizes were distinct for the original acceptor (5'-Bsd and Bsd-3') and the RIPE-targeted loci (5'-Pur and EGFP-3'). GAPDH-specific primers detecting both the bona fide gene (GAPDH) and a pseudogene (IJJGAPDH) were used as a control. (E) HILO-RMCE colonies produced by co-transfecting HEK293T-A2 and Hel_a-A12 cells with pCAGGS-nlCre and either pRD1 or pRD-RIPE were pooled and incubated with 2 yg/ml Dox for 48 hours or left untreated. The EGFP expression was then studied using FACS. Note that nearly all cells express EGFP in the Dox-treated pRD-RIPE samples. (F) HEK293TA2 cells carrying RIPE cassettes with shRNAs against either FLuc or LacZ were induced with Dox for 36 hours or left untreated. The cells were then transfected with a mixture of plasmids encoding the FLuc and RLuc luciferases and the FLuc activities normalized to the corresponding RLuc signals. Data are averaged from 6 transfection experiments ±SD.

[0043]. Figure 10. Silencing cell-encoded genes with HILO-RMCE. (A)

HEK293T-A2 cells containing four different RIPE-encoded shRNAs against human PTBP1 mRNA or the shFLuc shRNA were induced with Dox for 72 hours and the efficiency of the PTBP1 knockdown analyzed by reverse transcription- quantitative PCR (RT-qPCR) (top) and immunoblotting with PTBP1 -specific antibodies (bottom). The RT-qPCR graph shows relative PTBP1 expression levels normalized to the shFLuc control. In the immunoblot panel, anti-GAPDH antibody was used to control lane loading. (B-C) The experiment in (A) was repeated in N2a-A5 cells using shRNAs against mouse PTBP1 (B) or Ago2 (C) mRNAs. Note that co-expressing the two most potent Ago2-specific shRNAs from a single RIPE cassette further improves the protein knockdown (lane "sh1 +sh4" in C).

[0044]. Figure 11. Using HILO-RMCE to knock down the human TUT family members. (A) An shRNA library against the human TUT family was integrated into HEK293T-A2 cells and the knockdown efficiencies determined by RT-qPCR. The TUT expression levels in the Dox-treated samples are normalized to the corresponding Dox-negative controls. (B) The efficiencies of the TUT4- specific shRNAs were also studied by immunoblotting using anti-TUT4 antibody. (C) RT-qPCR analysis showing down-regulation of TUT2/GLD2 and

TUT4/ZCCHC11 by corresponding shRNAs in three additional human acceptor lines (Hel_a-A12, A549-1 1 , U20S- 13). Expression levels are normalized to the Dox-treated shFLuc controls as in (Fig. 3). In all RT-qPCR graphs, data are averaged from three amplifications experiments ±SD.

[0045]. Figure 12. HILO-RMCE can be readily adapted for rapid

engineering of transgenic cell pools. HEK293T-A2 cells were co-transfected with pCAGGS-nlCre and pRD1 -based donor plasmid (pEM705) containing a CAG promoter-driven bicistronic cassette encoding dTomato (dTom) (34) and a nuclear localized EGFP proteins. Recombinant cells were selected with

puromycin for 7 days, pooled and propagated for another 4 days. Cells were then imaged using phase contrast

[0046]. (PhC) and epiflourescence to detect the fluorescent protein expression. Diagram of pEM705 is shown on the top of the panel.

[0047]. Figure 13. Southern blot analysis of the human and mouse acceptor cell lines generated by lentiviral transduction of the HILO-RMCE acceptor locus (lanes marked with "A" and the clone number) were carried out using /Vcol-cut genomic DNAs and a lentivirus vector-specific 32Plabeled probe. The presence of a single band in the acceptor line samples corresponds to a single lentiviral integration site within the cellular genome. The parental cells contain no vectorspecific sequences, as expected.

[0048]. Figure 14. NLS-containing Cre is a more efficient HILO-RMCE recombinase than the wildtype Cre. L929-A12 cells containing the RMCE acceptor locus were co-transfected in a 12-well format with the pRD-RIPE plasmid and the indicated amounts of the pCAGGS-Cre or pCAGGSnlCre plasmids. Puromycin-resistant colonies were stained with methylene blue and photographed. Note that the well corresponding to the optimal pCAGGS-nlCre concentration (2.5%) contains noticeably larger number of colonies than the well with the optimal pCAGGSCre concentration (1 %). [0049]. Figure 15. The acceptor locus is uniformly rearranged in HILO- RMCE-generated cell pools. Genomic DNA was isolated from 3 parental cell lines (HEK293T, HeLa and A549; lanes labeled "parent"), the corresponding HILO-RMCE acceptor clones ("A" followed by the clone number) and pooled clones obtained by the HILO-RMCE-mediated integration of the RIPE cassette (the "A+RIPE" lanes). The DNA samples were analyzed by Southern blotting as in the Fig. 3A to confirm the uniform rearrangement of the acceptor locus as a result of the RMCE reaction. The data are consistent with the expected increase in the length of the acceptor locus-specific Nco\ fragment by 856 bp following the RIPE integration.

[0050]. Figure 16. Uniform induction of EGFP expression in HILO-RMCE cell pools. HEK293T-A2 and Hel_a-A12 cell pools containing the HILO-RMCE- integrated RIPE cassette were incubated with 2 /vg/ml doxycycline for 48 hours or alternatively left untreated. The EGFP expression was then studied using epifluorescence and phase contrast (PhC) microscopy. Note that all viable cells express EGFP in doxycycline-treated samples, whereas EGFP is not detected in the corresponding doxycycline-negative controls.

[0051]. Figure 17. HILO-RMCE generates virtually homogeneous cell populations expressing doxyciclin-inducible shRNAs. (A) Genomic DNAs from the parental HEK293T line, the acceptor HEK293T-A2 line, and pooled HILO-RMCE clones containing RIPE-encoded shFLuc or shLacZ shRNAs were analyzed by multiplex PCR to detect the changes at the 5' (primers EF, BR, and PR; see Fig. 2A and Table 1 ) and the 3' boundaries (GF, BF, and WR; Fig. 2A and Table 1 ) of the acceptor locus following the integration of the RIPE cassettes. Note that the DNA from the original acceptor line generates the 5'-Bsd and Bsd-3' PCR products whereas the HILO-RMCE-targeted cell pools give rise to the 5'-Pur and EGFP-3' products. No PCR products are formed in the reactions containing the parental HEK293T DNA, as expected. GAPDH, control amplifications with primers detecting the bona fide GAPDH gene and a pseudogene (lyGAPDH). (B- C) HILO-RMCE colonies produced by co-transfecting HEK293T-A2 with pCAGGS-nlCre and pRD1 or modified pRD-RIPE plasmids encoding either shFLuc or shLacZ shRNAs were pooled and incubated with 2 /vg/ml doxycycline for 48 hours or left untreated. The EGFP expression was then studied using either (B) FACS or (C) epifluorescence combined with the phase contrast (PhC) microscopy. Note that virtually all cells-become EGFP-positive in the pRD-RIPE samples treated with doxycycline but not in the corresponding doxycycline- negative controls.

[0052]. Figure 18. Knocking down Ptbpl by shRNAs changes alternative splicing patterns of the Ptbpl target genes. CAD-A13 cells containing RIPE- encoded shRNAs against mouse Ptbpl (sh2 or sh4) or the shFLuc shRNA were induced with 2 jc/g/ml doxycycline for 72 hours. After confirming the Ptbpl knockdown by RT-qPCR (not shown) we used the previously described RT-PCR procedure (36) to analyze the splicing patterns of three alternative cassette exons known to be repressed by the Ptbpl protein: exon 10 of the Ptbpl gene, exon N1 of the Src gene and exon 5 of the Cltb gene. The inclusion of these exons was indeed stimulated in the Ptbpl - knockdown samples as compared to the shFLuc control, i, exon-included splice form; s, exon skipped splice form. RT-PCR primers specific to the mouse Gapdh gene were used in control RT-PCR amplifications.

[0053]. Figure 19. Identifying an efficient human TUT6-specific shRNA. (a)

HEK293T-A2 cell pools containing four different RIPE-encoded shRNAs against human TUT6 mRNA or the shFLuc shRNA were induced with Dox for 72 hours or left untreated and the TUT6 knockdown efficiency was analyzed by RT-qPCR. The expression levels in the Dox-treated samples are normalized to the corresponding Dox-negative controls. Data are averaged from three

amplifications experiments ±SD. Note that of the four computationally designed candidates, only one shRNA (sh3) shows an adequate performance.

[0054]. Figure 20. Using HILO-RMCE for engineering of transgenic cell populations. HEK293T-A2 cells were co-transfected with pCAGGS-nlCre and either of the two pRD vector-based donor plasmids encoding (A) CAG-driven EGFP gene containing a nuclear localization sequence and preceded by an internal ribosomal entry site (IRES-nlEGFP) or (B) CAG promoter-driven introncontaining dTomato gene (i-dTom). Recombinant cells were selected with puromyein for 7 days, pooled and propagated for another 4 days. Images were taken using phase contrast and appropriate epifluorescence filters. Maps of the corresponding donor constructs (pEM652 and pEM689) are shown on the top of the panels

[0055]. Detailed Description

[0056]. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields genetically homogeneous cell populations containing doxicycline-inducible shRNA elements in a matter of days and with minimal effort. To ensure an immediate utility of this platform for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, Hel_a-S3 and U20S) and mouse (CAD, L929, N2a, NIH 3T3 and P19) cell lines to be compatible with the HILO-RMCE process. As a proof of principle, we used the newly established cell lines to optimize the shRNA mediated silencing of a range of cellular RNA- interacting proteins. Due to its technical simplicity and cost efficiency the new platform will be highly advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that this technology will facilitate a wide range of molecular and cell biology applications by allowing rapid engineering of cells expressing essentially any transgene of interest.

[0057]. We disclose a new process based on recombination mediated cassette exchange (RMCE) that yields virtually homogeneous cell populations containing desired genetic elements including but not limited to shRNA elements in a matter of days with minimal efforts. A further purpose includes modifying cell lines to obtain modified cell lines and novel genetic constructs that allow a person skilled in the art to practice the process. [0058]. Throughout the description the terms listed have the following meanings

[0059]. "A cell" includes one or more cells and equivalents thereof known to those skilled in the art.

[0060]. A " targeting nucleic acid site " comprises a nucleotide sequence flanked by two non-identical recombination sites. A targeting nucleic acid site targets or provides a "specific chromosomal site".

[0061]. A "transfer cassette" for use with a given target site comprises a nucleotide sequence flanked by the same two non-identical recombination sites present in the corresponding target site. The terms "transfer cassette", "donor cassette" and "targeting cassette" are used interchangeably herein.

[0062]. A target site and a transfer cassette may each comprise more than two non-identical recombination sites.

[0063]. A "donor construct" is a recombinant construct that contains a transfer cassette. The terms "donor construct" and "donor vector" are used

interchangeably herein.

[0064]. "Transgenic" refers to any cell, cell line, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0065]. "Genome" as it applies to cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial) of the cell.

[0066]. "Heterologous" with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.

[0067]. "Polynucleotide", "nucleic acid sequence", "nucleotide sequence", or "nucleic acid fragment" are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases. Nucleotides (usually found in their 5'- monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uhdylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.

[0068]. "Polypeptide", "peptide", "amino acid sequence" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" are also inclusive of

modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0069]. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell.

[0070]. "cDNA" refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.

[0071]. "Mature" protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and propeptides may be and are not limited to intracellular localization signals. [0072]. "Isolated" refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized

polynucleotides.

[0073]. "Recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the

manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0074]. "Recombinant" also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

[0075]. "Recombinant DNA construct" refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a

recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.

[0076]. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.

Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein. [0077]. "Promoter" refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.

[0078]. "Promoter functional in a cell" is a promoter capable of controlling transcription in cells whether or not its origin is from that cell.

[0079]. "Operably linked" refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.

[0080]. "Expression" refers to the production of a functional product. For example, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.

[0081]. "Introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic cell where the nucleic acid fragment may be

incorporated into the genome of the cell (e.g., chromosome, plasmid), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0082]. A "transformed cell" is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.

[0083]. "Transformation" as used herein refers to both stable transformation and transient transformation.

[0084]. "Stable transformation" refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.

[0085]. "Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance. [0086]. "Selection agent" refers to a compound which is toxic to non- transformed cells and which kills non-transformed cells when it is incorporated in the culture medium in an "effective amount", i.e., an amount equal to or greater than the minimal amount necessary to kill non-transformed cells. Cells can be transformed with an appropriate gene, such that expression of that transgene confers resistance to the corresponding selection agent, via de-toxification or another mechanism, so that these cells continue to grow and are subsequently able to regenerate cells. The gene conferring resistance to the selection agent is termed the "selectable marker gene", "selectable marker" or "resistance gene". Transgenic cells that lack a functional selectable marker gene will be killed by the selection agent. Selectable marker genes include genes conferring resistance to antibiotics, herbicidal compounds. A specific selection agent may have one or more corresponding selectable marker genes. Likewise, a specific selectable marker gene may have one or more corresponding selection agents. Examples of suitable selection agents, include but are not limited to, cytotoxic agents such as blasticidin, puromycin, hygromycin, sulfonylurea herbicides such as chlorsulfuron, nicosulfuron and hmsulfuron, and other herbicides which act by inhibition of the enzyme acetolactate synthase (ALS), glyphosate, bialaphos and phosphinothricin (PPT). It is also possible to use positive selection marker systems such as phospho-mannose isomerase and similar systems which confer positive growth advantage to transgenic cells.

[0087]. To overcome some of the limitations of the RMCE technology we developed a high efficiency and low background (HILO) RMCE technology for rapidly generating genetically homogenous cell populations without the labour- intensive cloning steps. Figure 1 depicts a flowchart of the HILO procedure. To establish RMCE acceptor lines, we assembled a lentiviral vector-encoded cassette consisting of a strong constitutive promoter from the EFIagene and a downstream blasticidin resistance gene (Bsd) flanked ("floxed") by mutually incompatible Cre recombinase-specific sites Lox2272 and LoxP (see Figure 2). Working at low multiplicity of infection we transduced a single copy of this cassette into 10 mammalian cell lines commonly used in molecular and cellular biology studies including 6 human cell lines (HEK293T, HeLa, Hel_a-S3, A549, HT1080 and U20S) and 4 mouse cell lines (L929, N2a, CAD and P19) (see figure 3A). We then constructed an RMCE donor plasmid (pRD1 ) containing a Lox2272 and Lox-floxed puromycin resistance gene (Pur) (see figure 2). We reasoned that substituting a promoter in front of the floxed Pur gene with a strong polyadenylation site should discourage unspecific integration events and generate puromycin-resistant colonies only in the case of correct recombination with the RMCE acceptor locus.

[0088]. The blasticidin-resistant acceptor cell lines were co-transfected in a 12- or 6- well format with a mixture of pRD1 and either a plasmid encoding wild type Cre recombinase under the strong constitutive promoter CAG (pCAGGS-Cre) or a control plasmid encoding a CAG-driven EGFP gene (pCIG). For all the 10 cell lines, multiple colonies appeared 5-10 days following puromycin selection in wells expressing Cre, whereas the EGFP wells had either none or just a single colony (see Figure 3B). Thus, the HILO-RMCE reaction proceeded efficiently and with a low background of Cre-independent integration events, as intended.

[0089]. To adapt this system for shRNA expression, we retrofitted pRD1 with a RIPE cassette containing a constitutively expressed reverse tetracycline transactivator (rtTA3) gene and a tetracycline-inducible segment that included an intron harboring a pre-miR-155 micro RNA precursor-based shRNA cloning site and an EGFP expression market (see Figure 4). Co-tansfecting the acceptor cell lines with the new pRD-RIPE plasmid and pRD1. To further enhance the RMCE efficiency, we modified pCAGGS-Cre to add an N-terminal nuclear localization signal to the Cre protein (pCAGGS-nlCre) and optimized the ratio between pRD- RIPE and the Cre expression plasmids (See Figure 5A&B). Genotyping the pooled recombinant colonies confirmed that the RIPE integration occurred in a precise and quantitative manner (see Figure 5C). Furthermore, the RIPE- encoded EGFP gene turned on in >97% of the recombinant cells in the presence but not in the absence of doxycycline (Dox) (see Figure 5D)> [0090]. To assess the RNAi performance of the RIPE cassette, we inserted a firefly luciferase (FLuc) specific shRNA (shFLuc) at the pre-miR-155 cloning site and generated a pool of HEK293T-A2 cells carrying the RIPE-shFLuc cassette using HILO-RMCE. The cells were either pre-incubated with Dox [Dox(+)] or left untreated [Dox(-)]. The two populations were then transfected with a mixture of plasmids encoding FLuc and Renilla luciferase (RLuc) and the relative FLuc expression measured as a ratio between the FLuc and the RLuc activities. The FLuc expression was reduced 3.45 fold (p=2.37x10 ^~7 ; t-test) in the Dox(+) samples expressing shFLuc and the EGFP marker as compared to the Dox(-) control thus indicating an efficient FLuc knockdown was substituted with a LacZ- specific shRNA (shLacZ) (see figure 5E).

[0091]. We then examined whether RIPE-encoded shRNAs can be used to knockdown endogenously encoded proteins. Using the above strategy we obtained HEK293T-A2 pools expressing shRNAs against human RNA-binding protein PTBP1. Satisfyingly, two out of four computationally designed shRNA substantially reduced PTBP1 expression in a Dox-dependent manner (see Figure 6A). Efficient knock-downs were also obtained in N2a cells using shRNA against mouse ptbpl (see Figure 6B) and mouse Argonaute family protein Ago2/Eif2c2, a critical RNAi component (see Figure 6C) and therefore a potentially problematic target using currently available technologies.

[0092]. To test the method performance in larger scale RNAi experiments, we turned to the family of human terminal uridyl transferases (TUTases) implicated in small RNA metabolism and designed an shRNA library containing 4-7 shRNAs against each of its 7 members (TUTase 1 to 7). Similar to the above results, we rapidly identified 1-2 potent shRNAs for each TUTase gene that reduced the mRNA abundance >2.5 fold (see Figure 6D&E). Notably, optimized shRNA for TUTase 2 (also known as GLD2 or PAPD4) and TUTase 4 (also known as ZCCHC1 1 or PAPD3) performed better than commercially available siRNA reagents. [0093]. HILO-RMCE allows one to induce stable shRNA-medicated RNAi in mammalian cells with ease and penetrance previously only possible using siRNA. Since different shRNA cassettes inserted at the same acceptor locus have comparable expression levels, this approach will substantially accelerate experimental validation and scoring of computationally designed sh RNAs. The cost efficiency and the technical simplicity of HILO-RMCE should make it a popular alternative to the retro and lentivirus vector-based high-throughput shRNA screens. Our data on the other types of transgenic cassettes (see figure 7) suggests that the method will facilitate a wide range of molecular and cell biology experiments by providing a possibility of rapid integration of transgenic sequence into a cell line of interest.

[0094]. The process allows yields of virtually homogeneous cell populations of genetically altered eukaryotic cells in a rapid and cost-effective manner. The new process comprised the steps of (a) delivering a nucleic acid sequence of interest operably linked with site-specific recombination sites into a eukaryotic cell; (b) integrating the sequence of interest at a predefined genetic locus through the activity of a site-specific recombinase and (c) obtaining nearly homogenous populations of cells with a correctly integrated nucleic acid sequence of interest using s selection procedure that does not require substantioal human

manipulations with the exception for minimal efforts such as changing culture medium.

[0095]. Preferably a eukaryotic cell is a mammalian cell. The term operably linked refers to a juxtaposition of components permitting them to function in an intended manner. For the purpose of this process the term nearly homogenous means preferably 90% to 100% homogenous, this may be 90% homogenous, 95% homogenous, 97% homogenous, 99% homogenous, or 100% homogenous.

[0096]. For use with the process new reagents are disclosed that comprise nucleic acid constructs with a site-specific recombination site and a site specific recombinase. The reagent may further comprise a nucleic acid sequence of interest.

[0097]. The resulting newly developed cell lines comprising nearly

homogenous populations of genetically identical clones that include a nucleic acid sequence of interest may also fall within the scope of the invention when they are obtained by the process disclosed.

[0098]. The process described is advantageous to transgenic technologies as it immediately yields nearly homogenous populations of genetically identical clones thus bypassing laborious cell cloning and enrichment steps commonly known in the field. The improvement is made possible through the combination of rationally designed genetic constructs and proprietary cell lines derived in our laboratory from parental cell lines by integrating a single copy of an acceptor cassette containing a eukaryotic promoter and a selection marker flanked by two site-specific recombination sites, which was followed by selecting cell clones with properties advantageous for the described art. The process offers considerable savings in terms of time, resources and human effort.

[0099]. The reagents can be used in the process for academic and commercial institutions performing molecular and cell biology work in eukaryotic cells. This includes but is not limited to RNAi experiments, gene overexpression studies, cell engineering and synthetic biology project. While it is primarily designed as a research and development tool it may also be used to create therapeutic constructs in vitro for use in gene therapy.

[00100]. Results and Discussion

[00101]. Here we developed a high-efficiency and low-background (HILO) RMCE platform that significantly accelerates engineering of mammalian cells expressing shRNAs and other genetic elements (Fig. 8A). To establish acceptor lines compatible with the HILO-RMCE procedure, we assembled a lentiviral vector-encoded cassette containing the human EF-1a promoter and a blasticidin resistance gene (Bsd) "floxed" by the mutually incompatible Cre recombinase specific sites Lox2272 and LoxP (Fig. 8B). A single copy of this cassette was transduced into 6 human (HEK293T, HeLa, HeLa-S3, A549, HT10 _. 80 and U20S) and 5 mouse (CAD, L929, N2a, NIH 3T3 and P19) commonly used cell lines (Fig. 13). We then constructed an RMCE donor plasmid (pRD1 ) containing a Lox2272- and LoxP-floxed puromycin (Pur) resistance gene (Fig. 1 B). We reasoned that substituting a promoter in front of the floxed Pur gene with a strong

polyadenylation signal (TK pA; derived from HS V thymidine kinase) should discourage unspecific integration events and generate puromycin-resistant colonies only in the case of correct recombination with the RMCE acceptor locus containing the strong promoter (Fig. 8B).

[00102]. The acceptor cell lines were co-transfected with a mixture of pRD1 and either the pCAGGS-Cre plasmid encoding a wild-type Cre recombinase or a control plasmid encoding EGFP (pCIG). In all cases, multiple colonies appeared 5-10 days following puromycin selection in the presence of Cre but not in the control wells expressing EGFP (Fig. 8C). Thus, the HILO-RMCE reaction proceeded efficiently and with a negligibly low background of Cre-independent integration, as intended.

[00103]. To adapt this system for inducible shRNA expression, we retrofitted pRD1 with a RIPE cassette containing a constitutively expressed reverse tetracycline transactivator gene {rtTA3) (20) and a tetracycline-inducible module that included an intron harboring a pre-miR-155 microRNA precursor-based shRNA cloning site and an EGFP expression marker (Fig. 9A). Co-transfecting the acceptor cell lines with the pRD-RIPE plasmid and pCAGGS-cre (but not the pCIG control) gave rise to multiple puromycin-resistant colonies, similar to pRD1 (not shown). To further optimize the RMCE efficiency, we added an N-terminal nuclear localization signal to the Cre protein (nICre; pCAGGS-nlCre plasmid). This modified recombinase performed better than the wild-type Cre in the tested human and mouse cell lines (Fig. 2B-C, Fig. S2 and not shown). Genotyping the pooled recombinants confirmed that the RIPE integration occurred in a precise and quantitative manner (Fig. 2D and Fig. S3). FACS and epifluorescent microscopy manalyses showed that the RIPE-encoded EGFP gene turned on in >97% of the recombinant cells in the presence but not in the absence of the rtTA3 inducer doxycycline (Dox) (Fig. 9E and Fig. 16), an indication of tightly controlled Dox-inducible expression.

[00104]. To assess the utility of the RIPE cassette for the RNAi, we inserted a firefly luciferase (FLuc) specific shRNA (shFLuc) at the pre-miR-155 cloning site and integrated the RIPEshFLuc cassette into the HEK293T-A2 cells using HILO- RMCE (Fig. 17). The cells were either pre-incubated with Dox or left untreated. The two populations were then transfected with a mixture of plasmids encoding FLuc and Renilla luciferase (RLuc) and the relative FLuc expression measured as a ratio between the Flue and the Rluc activities (Fig. 9F). The FLuc expression was knocked down 3.45 fold (p=2.37x 0-7, t-test) in the Dox-positive samples expressing shFLuc as compared to the Dox-negative control. As expected, LacZ- specific shRNA control (shLacZ) had no significant effect on the FLuc expression (Fig. 9F).

[00105]. We then examined if RIPE-encoded shRNAs can be used to knock down endogenous cellular proteins. Using the above strategy we obtained HEK293T-A2 pools expressing Dox inducible shRNAs against human RNA- binding protein PTBP1 (21 ). Satisfyingly, all four computationally designed shRNAs reduced PTBP1 expression at both the mRNA and the protein levels as compared to the shFLuc control (Fig. 10A-B). Efficient knockdowns were also obtained in N2a-A5 and CAD-A 3 cells using sets of shRNAs against mouse Ptbpl (Fig. 10C-D and not shown) and the Argonaute protein Ago2/Eif2c2 (22, 23), a critical RNAi component and therefore potentially problematic target (Fig. 10E-F and not shown). Knocking down mouse PTBP1 with shRNA was sufficient to enhance the inclusion of several neuron-specific alternative exons (Fig. 18), consistent with our previous results obtained by siRNA-mediated knockdown of PTBP1 in the CAD cells (24). Interestingly, the Ago2 protein down-regulation was further improved by co-expressing the two most potent Ago2-specific shRNAs from the same RIPE cassette (lane "sh1 +sh4" in Fig. 10F). [00106]. As a test of the method performance in a larger scale RNAi

experiment, we turned to the family of human terminal uridyl transferases (TUTs) implicated in various aspects of RNA metabolism including 3'-terminal

modifications of microRNA and their precursors (25, 26) and designed a RIPE- encoded shRNA library containing 4-7 shRNAs against each of its seven members (TUT1 to 7). Similar to the above results, the screen identified at least one potent shRNA for each TUT gene that substantially reduced the target expression in a Dox-dependent manner (Fig. 4A and Fig. 19). The efficiency of the TUT4-specific shRNAs was further confirmed using immunoblotting (Fig. 1 1 B). The optimized shRNAs against TUT2/GLD2 and TUT4/ZCCHC1 1 , the two TUTs involved in the microRNA pathway (25, 26), fared well in other human acceptor lines (Fig. 1 1 C). Notably, the sh3 TUT4/ZCCHC1 shRNA outperformed a previously published siRNA (25) and a commercially available siRNA mixture (ONTARGETplus, Dharmacon; not shown).

[00107]. We finally explored the possibility of using the HILO-RMCE platform for rapidly generating homogeneous cell pools expressing genetic elements other than shRNAs. For this purpose, we designed three pRD1 -based donor plasmids containing various transgenes under the control of a constitutive promoter (CAG) and co-transfected HEK293T-A2 cells with these constructs and the pCAGGS- nlCre plasmid (Fig. 12 and Fig. 20). In all three cases, the populations of puromycin-resistant recombinant cells expressed the integrated transgenes at readily detectable levels and in a homogeneous manner (Fig. 12 and Fig. 20).

[00108]. In conclusion, HILO-RMCE transforms the shRNA experiment into a user-friendly procedure that combines the possibility of long-term RNAi with the speed and convenience of siRNA-based approaches. The cost efficiency and the technical simplicity make HILO-RMCE an ideal platform for optimizing shRNA efficiency and other types of low-throughput projects in diverse laboratory settings, as well as a useful addition to the high-throughput RNAi screening toolbox. Moreover, our data on other types of transgenic cassettes suggest that the described platform should facilitate a wider range of molecular and cell biology experiments.

[00109]. Materials and Methods

[00110]. Plasmids

[00111]. To construct the RMCE acceptor lentiviral vector (pEM584), we modified the pHAGE backbone (27) by inserting a human EF-1a promoter from a derivative of the pEF-BOS vector (28) followed by a Lox2272- and LoxP-floxed blasticidin resistance gene (Bsd) from the pLenti6A/5-DEST plasmid (Invitrogen) between the cpPu {Trip) and WPRE elements. The RMCE donor vector (pRD1 ; alternative name ρΕΜΘΟ ) was generated by subcloning the polyadenylation signal-containing HSV thymidine kinase (TK) gene from a pEasyFlox derivative (29) into the pBluescript II KS(+) backbone. Immediately downstream of the TK polyadenylation signal, we introduced a Lox2272- and LoxP-floxed promoter-less puromycin resistance {Pur) gene from the pPUR plasmid (Clontech). pRD-RIPE plasmid (alternative name pEM791 ) was derived from pRD1 by removing the TK promoter and coding sequences and inserting a UBC promoter-driven reverse tetracycline transactivator gene {rtTA3) and the tet-inducible promoter TRE (both elements adapted from the pTRIPZ plasmid, Open Biosystems) between the Pur gene and the LoxP site. The TRE promoter was followed by an intron-EGFP module containing an intronic pre-miR-155-based shRNA cloning site with two BsmBl sequences (30). pCAGGS-nlCre (alternative name pEM784) was modified from pCAGGS-Cre (31 ) by substituting the original sequence 5'- ACTTTACTTAAAACCATTATCTGAGTGTGAAATG-3' in front of the Cre gene with the sequence 5'-CTAGACTCGACCATGCCCAAGAAGAAGAGGAAGGTG-3' encoding the N-terminal nuclear localization sequence (NLS; underlined) from the Large T antigen of the SV40 virus. Complete sequences and maps of all pEM plasmids are available upon request.

[00112]. Design and cloning of pre-miR-155 based shR As

[00113]. shRNAs were designed using the miR RNAi design option of the Block- iT RNAi Designer program (https://rnaidesigner.invitrogen.com/rnaiexpress/). Wherever possible, multiple shRNAs targeting the open reading frame (ORF) and 3' untranslated region (3'UTR) were chosen for a given target gene. For each shRNA, two 64 nt long complementary DNA oligonucleotides were ordered from Sigma Life Science, annealed and inserted into pRD-RIPE plasmid at the BsmBI sites (for detailed description of the pre-miR-155 based shRNA cloning strategy, see (30) and https://rnaidesigner.invitrogen.com/rnaiexpress/). For example, the firefly liciferase-specific shRNA (shFLuc) was engineered by annealing the following oligonucleotides: SEQ ID NO 1 : 5'-

TGCTGTATTCAGCCCATATCGTTTCAGTTTTGGCCACTGACTGACTGAAACG ATGGGCTGAATA-3' and SEQ ID NO 2: 5'-

CCTGTATTCAGCCCATCGTTTCAGTCAGTCAGTGGCCAAAACTGAAACGATA TGGGCTGAATAC-3'. Other oligonucleotides used to generate shRNA

sequences (SEQ ID numbers 54 through 175) are listed in Table 2. To improve ■ the mAgo2 knockdown efficiency, the mAgo2 sh4 shRNA element was amplified using the RIPE-specific primers shRNAdimer_F1 and shRNAdimer_R1 (Table 1 ), the PCR fragment was treated with Mfe\ (NEB; underlined sequence) and inserted into the mAgo2 sh1 -containing RIPE cassette at the EcoRI-EcoRV sites located downstream of the miR 0 155 element to create an intron-encoded sh1- sh4 tandem. Since the shRNAdimer_R1 primer restores both the EcoRI and the EcoRV sites (italicized sequences), this procedure can theoretically be repeated to generate tandem shRNA arrays of any desired length and complexity.

[00114]. Table 1. Oligonucleotides used in this study.

9 BF GCAACGGCTACAATCAACAG

10 WR GGGCCACAACTCCTCATAAA

11 hGAPDH_F1 CCTGACCTGCCGTCTAGAAA

12 hGAPDH_R1 CCCTGTTGCTGTAGCCAAAT

13 South584_F1 CCAAGCCTTTGTCTCAAGAA

14 South584_R1 GAGATCCGACTCGTCTGAGG

RT-PCR and RT-qPCR:

15 hHPRT1_F1 GTTTGTTGTAGGATATGCCCTTGA

16 hHPRT1_R1 ACTAAGCAGATGGCCACAGAACTA

17 hPTBP1_F1 TGACCAAGGACTACGGCAAC

18 hPTBP1_R1 CCATTG CTG G AAAACAG G AC

19 hTUT1_F1 TCGTGAGGTTCTCACACCAG

20 hTUT1_R1 GCACCGTACACTGAACACCA

21 hTUT2_F1 CGAAGATACTCAATGCCACCA

22 hTUT2_R1 CTCTCTGCAGCTGTGTTCGAC

23 hTUT3_F1 ACCGAGTAGGGTCGCAAGAT

24 hTUT3_R1 TGCTGGAAGAACGAAAGAGC

25 hTUT4_F1 GATGTGACATTGGGGATGCT

26 hTUT4_R1 AGCATTCCATCCATCAACCA

27 hTUT5_F1 TAGGGGTTGCTCCTGTTCCT

28 hTUT5_R1 GGACAGTTTCATGCCGTTGT

29 hTUT6_F1 CTTTCCCAGGGATGTGGATT

30 hTUT6_R1 TGTGCTGGGACTGTGACAAG

31 hTUT7_F1 GCTGGCCCCAAATGATAGAT

32 hTUT7_R1 AGGGCATCTTCCTGATCTCG

33 mAgo2_F1 TGCACACGCTCTGTGTCAAT

34 mAgo2_R1 CAGTGTGTCCTGGTGGACCT

35 mCltb_F1 GAAAGACCTGGAGGAGTGGA

36 mCltb_R1 CTAGCGGGACAGTGGTGTTT

37 mGapdh_F1 AAATGGGGTGAGGCCGGTGC

38 mGapdh_R1 ATCGGCAGAAGGGGCGGAGA

39 mHprt1_F1 CCAGACAAGTTTGTTGTTGGA 40 mHprt1_R1 TTTACTAGGCAGATGGCCACA

41 mPtbp1_F1 AGTGCGCATTACACTGTCCA

42 mPtbp1_R1 CTTGAGGTCGTCCTCTGACA

43 mPtbp2_F1 TGGCTATTCCAAATGCTGCT

44 mPtbp2_R1 TCCCATCAGCCATCTGTATC

45 mSrc_F1 ACCTTTGTGGCCCTCTATGA

46 mSrc_R1 CACATAGTTGCTGGGGATGT

[00115]. Table 2. Oligonucleotides used to generate shRNA sequences

SE

Q

ID: Name Primer sequence, 5' to 3'

hTUTasel _shRNA1_ TGCTGTAAAGTGCCACTGAGTATCAGGTTTTGGCCACTGACTGACCTGATACTGTGGC

54 FP ACTTTA

hTUTasel _shRNA1_ CCTGTAAAGTGCCACAGTATCAGGTCAGTCAGTGGCCAAAACCTGATACTCAGTGGCA

55 P CTTTAC

hTUTasel _shRNA2_ TGCTGTTTGATATCAGTACCAACCCAGTTTTGGCCACTGACTGACTGGGTTGGCTGATA

56 FP TCAAA

hTUTasel _shRNA2_ CCTGTTTGATATCAGCCAACCCAGTCAGTCAGTGGCCAAAACTGGGTTGGTACTGATAT

57 RP CAAAC

hTUTasel _shRNA3_ TGCTGCAAGGTGGAATTCTCCCATGCGTTTTGGCCACTGACTGACGCATGGGAATTCC

58 FP ACCTTG

hTUTasel _shRNA3_ CCTGCAAGGTGGAATTCCCATGCGTCAGTCAGTGGCCAAAACGCATGGGAGAATTCCA

59 RP CCTTGC

hTUTasel _shRNA4_ TGCTGAAGAAACGTGATCTGAATGGAGTTTTGGCCACTGACTGACTCCATTCATCACGT

60 FP TTCTT

hTUTasel _shRNA4_ CCTGAAGAAACGTGATGAATGGAGTCAGTCAGTGGCCAAAACTCCATTCAGATCACGT

61 RP TTCTTC

hTUTasel _shRNA5_ TGCTGCAAAGGGTCTGACTATGCAGTGTTTTGGCCACTGACTGACACTGCATACAGAC

62 FP CCTTTG

hTUTasel _shRNA5_ CCTGCAAAGGGTCTGTATGCAGTGTCAGTCAGTGGCCAAAACACTGCATAGTCAGACC

63 RP CTTTGC

hTUTasel _shRNA6_ TGCTGAACACAGACAGGATCTTCTGAGTTTTGGCCACTGACTGACTCAGAAGACTGTCT

64 FP GTGTT

hTUTasel _shRNA6_ CCTGAACACAGACAGTCTTCTGAGTCAGTCAGTGGCCAAAACTCAGAAGATCCTGTCT

65 RP GTGTTC

hTUTasel _shRNA7_ TGCTGCTGAGTATCAGACTGATCAAAGTTTTGGCCACTGACTGACTTTGATCACTGATA

66 FP CTCAG

hTUTasel _shRNA7_ CCTGCTGAGTATCAGTGATCAAAGTCAGTCAGTGGCCAAAACTTTGATCAGTCTGATAC

67 RP TCAGC

hTUTase2 _shRNA1_ TGCTGTGTTGTTGATGATTTGGAGTGGTTTTGGCCACTGACTGACCACTCCAACATCAA

68 FP CAACA

hTUTase2 _shRNA1_ CCTGTGTTGTTGATGTTGGAGTGGTCAGTCAGTGGCCAAAACCACTCCAAATCATCAAC

69 RP AACAC

hTUTase2 _shRNA2_ TGCTGCTATCTGGTTAACTACAGTTGGTTTTGGCCACTGACTGACCAACTGTATAACCA

70 FP GATAG

hTUTase2 _shRNA2_ CCTGCTATCTGGTTATACAGTTGGTCAGTCAGTGGCCAAAACCAACTGTAGTTAACCAG

71 RP ATAGC

hTUTase2 _shRNA3_ TGCTGAAATAACTCCAGTATCTGCTGGTTTTGGCCACTGACTGACCAGCAGATTGGAGT

72 FP TATTT

hTUTase2 _shRNA3_ CCTGAAATAACTCCAATCTGCTGGTCAGTCAGTGGCCAAAACCAGCAGATACTGGAGT

73 RP TATTTC

hTUTase2 _shRNA4_ TGCTGATCACCAGCACTAACGGACGAGTTTTGGCCACTGACTGACTCGTCCGTGTGCT

74 FP GGTGAT

hTUTase2 _shRNA4_ CCTGATCACCAGCACACGGACGAGTCAGTCAGTGGCCAAAACTCGTCCGTTAGTGCTG

75 RP GTGATC

hTUTase2 _shRNA5_ TGCTGATTACATGGAGCTTGATGTACGTTTTGGCCACTGACTGACGTACATCACTCCAT

76 FP GTAAT

hTUTase2 _shRNA5_ CCTGATTACATGGAGTGATGTACGTCAGTCAGTGGCCAAAACGTACATCAAGCTCCAT

77 RP GTAATC hTUTase2 shRNA6 TGCTGTACACATCTCTTCAGGATTAGGTTTTGGCCACTGACTGACCTAATCCTAGAGAT

78 FP GTGTA

hTUTase2 shRNA6 CCTGTACACATCTCTAGGATTAGGTCAGTCAGTGGCCAAAACCTAATCCTGAAGAGATG

79 RP TGTAC

hTUTase2 shRNA7 TGCTGAGTATTTCATGACTGCAGTGAGTTTTGGCCACTGACTGACTCACTGCACATGAA

80 FP ATACT

hTUTase2 shRNA7 CCTGAGTATTTCATGTGCAGTGAGTCAGTCAGTGGCCAAAACTCACTGCAGTCATGAAA

81 RP TACTC

hTUTase3 shRNAI TGCTGATCAAAGGCCTGCTTCACTTGGTTTTGGCCACTGACTGACCAAGTGAAAGGCC

82 FP TTTGAT

hTUTase3 shRNAI CCTGATCAAAGGCCTTTCACTTGGTCAGTCAGTGGCCAAAACCAAGTGAAGCAGGCCT

83 RP TTGATC

hTUTase3 shRNA2 TGCTGATATGTGGCAACTTCATCTGTGTTTTGGCCACTGACTGACACAGATGATTGCCA

84 FP CATAT

hTUTase3 shRNA2 CCTGATATGTGGCAATCATCTGTGTCAGTCAGTGGCCAAAACACAGATGAAGTTGCCA

85 RP CATATC

hTUTase3 shRNA3 TGCTGATTGCATGAAGGCTCAGGTCTGTTTTGGCCACTGACTGACAGACCTGACTTCAT

86 FP GCAAT

hTUTase3 shRNA3 CCTGATTGCATGAAGTCAGGTCTGTCAGTCAGTGGCCAAAACAGACCTGAGCCTTCAT

87 RP GCAATC

hTUTase3 shRNA4 TGCTGTTGAGTTGTACCTTGGAAGCCGTTTTGGCCACTGACTGACGGCTTCCAGTACA

88 FP ACTCAA

hTUTase3 shRNA4 CCTGTTGAGTTGTACTGGAAGCCGTCAGTCAGTGGCCAAAACGGCTTCCAAGGTACAA

89 RP CTCAAC

hTUTase3 shRNA5 TGCTGAACAGAGCCATACACATTGATGTTTTGGCCACTGACTGACATCAATGTATGGCT

90 FP CTGTT

hTUTase3 shRNA5 CCTGAACAGAGCCATACATTGATGTCAGTCAGTGGCCAAAACATCAATGTGTATGGCTC

91 RP TGTTC

hTUTase3 shRNA6 TGCTGTTCACAGTCACCTTAAAGCTGGTTTTGGCCACTGACTGACCAGCTTTAGTGACT

92 FP GTGAA

hTUTase3 shRNA6 CCTGTTCACAGTCACTAAAGCTGGTCAGTCAGTGGCCAAAACCAGCTTTAAGGTGACT

93 RP GTGAAC

hTUTase3 shRNA7 TGCTGTAATAACTACACCTCCTAGTCGTTTTGGCCACTGACTGACGACTAGGATGTAGT

94 FP TATTA

hTUTase3 shRNA7 CCTGTAATAACTACATCCTAGTCGTCAGTCAGTGGCCAAAACGACTAGGAGGTGTAGTT

95 RP ATTAC

hTUTase4 shRNAI TGCTGATACCCTTGAGACAGCAGGATGTTTTGGCCACTGACTGACATCCTGCTCTCAA

96 FP GGGTAT

hTUTase4 shRNAI CCTGATACCCTTGAGAGCAGGATGTCAGTCAGTGGCCAAAACATCCTGCTGTCTCAAG

97 RP GGTATC

hTUTase4 shRNA2 TGCTGTTCAGAGCAAATCTAGTCAGAGTTTTGGCCACTGACTGACTCTGACTATTTGCT

98 FP CTGAA

hTUTase4 shRNA2 CCTGTTCAGAGCAAATAGTCAGAGTCAGTCAGTGGCCAAAACTCTGACTAGATTTGCTC

99 RP TGAAC

hTUTase4 shRNA3 TGCTGTAAACCAGCTGGCTGTTTAAGGTTTTGGCCACTGACTGACCTTAAACACAGCTG

100 FP GTTTA

hTUTase4 shRNA3 CCTGTAAACCAGCTGTGTTTAAGGTCAGTCAGTGGCCAAAACCTTAAACAGCCAGCTG

101 RP GTTTAC

hTUTase4 shRNA4 TGCTGAAGAGATGAAGAGTCCTGTCCGTTTTGGCCACTGACTGACGGACAGGACTTCA

102 FP TCTCTT

hTUTase4 shRNA4 CCTGAAGAGATGAAGTCCTGTCCGTCAGTCAGTGGCCAAAACGGACAGGACTCTTCAT

103 RP CTCTTC

hTUTase4 shRNA5 TGCTGTGCAGATGCTGCATACTATCGGTTTTGGCCACTGACTGACCGATAGTACAGCA

104 FP TCTGCA

hTUTase4 shRNA5 CCTGTGCAGATGCTGTACTATCGGTCAGTCAGTGGCCAAAACCGATAGTATGCAGCAT

105 RP CTGCAC

hTUTase4 shRNA6 TGCTGATCAATAGCTGCATAAGTAGCGTTTTGGCCACTGACTGACGCTACTTACAGCTA

106 FP TTGAT

hTUTase4 shRNA6 CCTGATCAATAGCTGTAAGTAGCGTCAGTCAGTGGCCAAAACGCTACTTATGCAGCTAT

107 RP TGATC

hTUTase4 shRNA7 TGCTGAATAGCAGCTGACTGGGAAGAGTTTTGGCCACTGACTGACTCTTCCCACAGCT

108 FP GCTATT

hTUTase4 shRNA7 CCTGAATAGCAGCTGTGGGAAGAGTCAGTCAGTGGCCAAAACTCTTCCCAGTCAGCTG

109 RP CTATTC

hTUTase5 shRNAI TGCTGTAAAGGAGGACGCTCCCATTTGTTTTGGCCACTGACTGACAAATGGGAGTCCT

110 FP CCTTTA

hTUTase5 shRNAI CCTGTAAAGGAGGACTCCCATTTGTCAGTCAGTGGCCAAAACAAATGGGAGCGTCCTC

111 RP CTTTAC

hTUTase5 shRNA2 TGCTGTTGTCAAGGACTTTGATGGAAGTTTTGGCCACTGACTGACTTCCATCAGTCCTT

112 FP GACAA

113 hTUTase5 shRNA2 CCTGTTGTCAAGGACTGATGGAAGTCAGTCAGTGGCCAAAACTTCCATCAAAGTCCTT RP GACAAC

hTUTase5 shRNA3 TGCTGAATTCTTGATGAACTCCGCTGGTTTTGGCCACTGACTGACCAGCGGAGCATCA

114 FP AGAATT

hTUTase5 shRNA3 CCTGAATTCTTGATGCTCCGCTGGTCAGTCAGTGGCCAAAACCAGCGGAGTTCATCAA

115 RP GAATTC

hTUTase5 shRNA4 TGCTGAACTGTAGAAAGCTAATGGCCGTTTTGGCCACTGACTGACGGCCATTATTTCTA

116 FP CAGTT

hTUTase5 shRNA4 CCTGAACTGTAGAAATAATGGCCGTCAGTCAGTGGCCAAAACGGCCATTAGCTTTCTA

117 RP CAGTTC

hTUTase5 shRNA5 TGCTGAACAATGACCTCCCAAGCTTAGTTTTGGCCACTGACTGACTAAGCTTGAGGTCA

118 FP TTGTT

hTUTase5 shRNA5 CCTGAACAATGACCTCAAGCTTAGTCAGTCAGTGGCCAAAACTAAGCTTGGGAGGTCA

119 RP TTGTTC

hTUTase5 shRNA6 TGCTGTATACCTGTATGCTTCCGACGGTTTTGGCCACTGACTGACCGTCGGAAATACA

120 FP GGTATA

hTUTase5 shRNA6 CCTGTATACCTGTATTTCCGACGGTCAGTCAGTGGCCAAAACCGTCGGAAGCATACAG

121 RP GTATAC

hTUTase5 shRNA7 TGCTGATAACTTTCGGTCATCGAGAAGTTTTGGCCACTGACTGACTTCTCGATCCGAAA

122 FP GTTAT

hTUTase5 shRNA7 CCTGATAACTTTCGGATCGAGAAGTCAGTCAGTGGCCAAAACTTCTCGATGACCGAAA

123 RP GTTATC

hTUTase6 shRNA TGCTGCTCGGAAGCAACTTCCGCCGAGTTTTGGCCACTGACTGACTCGGCGGATTGCT

124 FP TCCGAG

hTUTase6 shRNAI CCTGCTCGGAAGCAATCCGCCGAGTCAGTCAGTGGCCAAAACTCGGCGGAAGTTGCT

125 RP TCCGAGC

hTUTase6 shRNA2 TGCTGACACAAGCACCTCTGCCACCAGTTTTGGCCACTGACTGACTGGTGGCAGGTGC

126 FP TTGTGT

hTUTase6 shRNA2 CCTGACACAAGCACCTGCCACCAGTCAGTCAGTGGCCAAAACTGGTGGCAGAGGTGC

127 RP TTGTGTC

hTUTase6 shRNA3 TGCTGATGCTAGGAAGTACTCAGAGAGTTTTGGCCACTGACTGACTCTCTGAGCTTCCT

128 FP AGCAT

hTUTase6 shRNA3 CCTGATGCTAGGAAGCTCAGAGAGTCAGTCAGTGGCCAAAACTCTCTGAGTACTTCCT

129 RP AGCATC

hTUTase6 shRNA4 TGCTGAGAAGAGGTCAAGATCACAGCGTTTTGGCCACTGACTGACGCTGTGATTGACC

130 FP TCTTCT

hTUTase6 shRNA4 CCTGAGAAGAGGTCAATCACAGCGTCAGTCAGTGGCCAAAACGCTGTGATCTTGACCT

131 RP CTTCTC

hTUTase6 shRNA5 TGCTGCCCACAAGCTTTATCATTTGTGTTTTGGCCACTGACTGACACAAATGAAAGCTT

132 FP GTGGG

hTUTase6 shRNA5 CCTGCCCACAAGCTTTCATTTGTGTCAGTCAGTGGCCAAAACACAAATGATAAAGCTTG

133 RP TGGGC

hTUTase6 shRNA6 TGCTGAGAACTTGACCACAGGGCGCCGTTTTGGCCACTGACTGACGGCGCCCTGGTC

134 FP AAGTTCT

hTUTase6 shRNA6 CCTGAGAACTTGACCAGGGCGCCGTCAGTCAGTGGCCAAAACGGCGCCCTGTGGTCA

135 RP AGTTCTC

hTUTase7 shRNAI TGCTGAGTTGAAGCCTGATCTACACTGTTTTGGCCACTGACTGACAGTGTAGAAGGCTT

136 FP CAACT

hTUTase7 shRNAI CCTGAGTTGAAGCCTTCTACACTGTCAGTCAGTGGCCAAAACAGTGTAGATCAGGCTT

137 RP CAACTC

hTUTase7 shRNA2 TGCTGATAACAGGAATTCTAGGTCCAGTTTTGGCCACTGACTGACTGGACCTAATTCCT

138 FP GTTAT

hTUTase7 shRNA2 CCTGATAACAGGAATTAGGTCCAGTCAGTCAGTGGCCAAAACTGGACCTAGAATTCCT

139 RP GTTATC

hTUTase7 shRNA3 TGCTGTTGAAACCCAATCTGCTACAGGTTTTGGCCACTGACTGACCTGTAGCATTGGGT

140 FP TTCAA

hTUTase7 shRNA3 CCTGTTGAAACCCAATGCTACAGGTCAGTCAGTGGCCAAAACCTGTAGCAGATTGGGT

141 RP TTCAAC

hTUTase7 shRNA4 TGCTGAACACAGGTTGACTATTTAGGGTTTTGGCCACTGACTGACCCTAAATACAACCT

142 FP GTGTT

hTUTase7 shRNA4 CCTGAACACAGGTTGTATTTAGGGTCAGTCAGTGGCCAAAACCCTAAATAGTCAACCTG

143 RP TGTTC

hTUTase7 shRNA5 TGCTGTAAAGTGGCAAGTCCCTCACAGTTTTGGCCACTGACTGACTGTGAGGGTTGCC

144 FP ACTTTA

hTUTase7 shRNA5 CCTGTAAAGTGGCAACCCTCACAGTCAGTCAGTGGCCAAAACTGTGAGGGACTTGCCA

145 RP CTTTAC

hTUTase7 shRNA6 TGCTGTACCGTAGGAGACTTGCCTTTGTTTTGGCCACTGACTGACAAAGGCAACTCCTA

146 FP CGGTA

hTUTase7 shRNA6 CCTGTACCGTAGGAGTTGCCTTTGTCAGTCAGTGGCCAAAACAAAGGCAAGTCTCCTA

147 RP CGGTAC

hTUTase7 shRNA7 TGCTGTTTAGTTCCTGGAAAGTCCTGGTTTTGGCCACTGACTGACCAGGACTTCAGGA

148 FP ACTAAA hTUTase7 shRNA7 CCTGTTTAGTTCCTGAAGTCCTGGTCAGTCAGTGGCCAAAACCAGGACTTTCCAGGAA

149 RP CTAAAC

hPTBPI shRNAI F TGCTGTTCTCTGGAATGATGGAAGTTGTTTTGGCCACTGACTGACAACTTCCAATTCCA

150 P GAGAA

hPTBPI shRNAI R CCTGTTCTCTGGAATTGGAAGTTGTCAGTCAGTGGCCAAAACAACTTCCATCATTCCAG

151 P AGAAC

hPTBPI shRNA2 F TGC ^' I G GTCATTTCCGTTTGCTGCAGGTT I TGGCCAC rGACTGACCTGCAGCACGGAA

152 P ATGACA

hPTBPI shRNA2 R CCTGTGTCATTTCCGTGCTGCAGGTCAGTCAGTGGCCAAAACCTGCAGCAAACGGAAA

153 P TGACAC

hPTBPI shRNA3 F TGCTGTATTGAACAGGATCTTCACGCGTTTTGGCCACTGACTGACGCGTGAAGCCTGT

154 P TCAATA

hPTBPI shRNA3 R CCTGTATTGAACAGGCTTCACGCGTCAGTCAGTGGCCAAAACGCGTGAAGATCCTGTT

155 P CAATAC

hPTBPI shRNA4 F TGCTGAATATTGCTAGGCACAGACGTGTTTTGGCCACTGACTGACACGTCTGTCTAGC

156 P AATATT

hPTBPI shRNA4 R CCTGAATATTGCTAGACAGACGTGTCAGTCAGTGGCCAAAACACGTCTGTGCCTAGCA

157 P ATATTC

mPTBPI shRNAI F TGCTGAGCATGAGAAGGTTGGTAACCGTTTTGGCCACTGACTGACGGTTACCACTTCT

158 P CATGCT

mPTBPI shRNAI R CCTGAGCATGAGAAGTGGTAACCGTCAGTCAGTGGCCAAAACGGTTACCAACCTTCTC

159 P ATGCTC

mPTBPI shRNA2 F TGCTGTCCACAATGATCCTGAGCACTGTTTTGGCCACTGACTGACAGTGCTCAATCATT

160 P GTGGA

mPTBPI shRNA2 R CCTGTCCACAATGATTGAGCACTGTCAGTCAGTGGCCAAAACAGTGCTCAGGATCATT

161 P GTGGAC

mPTBPI shRNA3 F TGCTGATGTATAGGCCACCTGGCTCAGTTTTGGCCACTGACTGACTGAGCCAGGGCCT

162 P ATACAT

mPTBPI shRNA3 R CCTGATGTATAGGCCCTGGCTCAGTCAGTCAGTGGCCAAAACTGAGCCAGGTGGCCTA

163 P TACATC

mPTBPI shRNA4 F TGCTGATACAAAGGTCACAATGAGGCGTTTTGGCCACTGACTGACGCCTCATTGACCT

164 P TTGTAT

mPTBPI shRNA4 R CCTGATACAAAGGTCAATGAGGCGTCAGTCAGTGGCCAAAACGCCTCATTGTGACCTT

165 P TGTATC

TGCTGAATCCAACTTGGTACACAATCGTTTTGGCCACTGACTGACGATTGTGTCAAGTT

166 mAgo2_shRNA1_FP GGATT

CCTGAATCCAACTTGACACAATCGTCAGTCAGTGGCCAAAACGATTGTGTACCAAGTTG

167 mAgo2 shRNAI RP) GATTC

TGCTGAGAAGTGCACTCTTACGTGAAGTTTTGGCCACTGACTGACTTCACGTAAGTGCA

168 mAgo2_shRNA2_FP CTTCT

CCTGAGAAGTGCACTTACGTGAAGTCAGTCAGTGGCCAAAACTTCACGTAAGAGTGCA

169 mAgo2 shRNA2 RP) CTTCTC

TGCTGATTCCTACCTGCCAGTCCCATGTTTTGGCCACTGACTGACATGGGACTCAGGT

170 mAgo2_shRNA3_FP) AGGAAT

CCTGATTCCTACCTGAGTCCCATGTCAGTCAGTGGCCAAAACATGGGACTGGCAGGTA

171 mAgo2 shRNA3 RP GGAATC

TGCTGCAAAGACGTCTCATGTTCGATGTTTTGGCCACTGACTGACATCGAACAAGACGT

172 mAgo2_shRNA4_FP CTTTG

CCTGCAAAGACGTCTTGTTCGATGTCAGTCAGTGGCCAAAACATCGAACATGAGACGT

173 mAgo2 shRNA4 RP CTTTGC

TGCTGAAATCGCTGATTTGTGTAGTCGTTTTGGCCACTGACTGACGACTACACATCAGC

174 lacZ shRNA FP GATTT

CCTGAAATCGCTGATGTGTAGTCGTCAGTCAGTGGCCAAAACGACTACACAAATCAGC

175 lacZ shRNA RP GATTTC

Cells

[00116]. Parental cell lines used in this study were from the ATCC except for the CAD line, which was kindly provided by the authors (32). A549, HEK293T, HeLa, HeLa-S3, HT1080, L929, N2a, NIH 3T3 and U2OS were routinely propagated in DMEM/high glucose medium (Hyclone) supplemented with 10% Fetal Bovine Serum (FBS, "characterized" grade; Hyclone), 1 mM sodium pyruvate (Invitrogen) and 1 * penicillin-streptomycin (100 U/ml penicillin, 100 //g/ml streptomycin; Invitrogen). For passaging, adherent cells were detached using 1 ^χ trypsin-EDTA (Invitrogen). In some experiments, regular FBS was substituted with certified tetracycline negative FBS (PAA). CAD cells were cultured in a similar medium except FBS was substituted

[00117]. with the FetalClone III serum (Hyclone). P19 cells were cultured in σΜΕΜ medium supplemented with 2.5% FBS, 7.5% Bovine Calf Serum (BCS, Hyclone) and 1 * penicillinstreptomycin. When required, media were

supplemented with 2.5-10 /vg/ml blasticidin S or 1 -16 yg/ml puromycin. To turn on the Tet-inducible expression, doxycycline was added to the final concentration of

[001 8]. RMCE acceptor cell lines

[00119]. To establish RMCE acceptor cell lines, -40% confluent cell cultures were incubated with serially dilutions of the pEM584 lentiviral stock (1 to 200 cfu per 10 cm plate) for 18 hours without polybrene. The medium was then changed and the cells were incubated for another 18 hours prior to the addition of blasticidin S to 5-10 /g/ml. The incubation was continued in the presence of blasticidin until non-infected cells died and visible blasticidin-resistant colonies formed. For each cell line, 2- 8 individual colonies were picked using 200 μ\ pipette tips and clonally expanded. Dishes containing >50 colonies were discarded to avoid multiple integration events and colony cross-contamination. Clones with optimal RMCE performance were maintained in the presence of 2.5- 5 g/ml blasticidin S. For long-term storage, cells were cryo-preserved in a mixture containing 90% of the appropriate culture medium and 10% DMSO.

[00120]. RMCE protocol

[00121]. RMCE acceptor cell lines were plated in 12-well plates at 1.0-1.5x105 cells per well in an antibiotic-free medium 12-18 hours prior to transfection except L929-A12 and P 9-A9 cells that were plated 3 hours before transfection. Cells were then co-transfected with an RMCE donor plasmid (pRD, pRD-RIPE or a derivative of pRD-RIPE containing a gene-specific shRNA sequence) blended with 0.5-10% (w/w) of a Cre-encoding plasmid (pCAGGS-Cre or pCAGGSnlCre). To transfect one well of a 12-well plate, 0.5 g of total DNA was mixed with 1.25 μ\ Lipofectamine 2000/32.5 μ\ Opti-MEM I (Invitrogen) following the manufacturer's protocol. Cells were incubated with the transfection mixtures overnight, after which the medium was changed and the incubation continued for another 24 hours before adding puromycin. For most cell lines, we used a three- step selection protocol beginning with 1/2 of the maximal puromycin

concentration for the first 24-48 hours of selection, followed by the maximal concentration (Hel_a-A12, 2 /yg/ml; A549-A1 1 , CAD-A13, HEK293T-A2, Hel_a-S3- A6, HT1080-A4, N2a-A5, NIH 3T3-A7 and U2OS-A13, 5 yg/ml) for several days until the puromycin-sensitive cells were eliminated and then returning to the 1/2 maximal concentration to accelerate the proliferation of the puromycin-resistant cells. L929-A12 and P19-A9 cells were immediately exposed to the maximal puromycin concentration (L929-A12, 16 vg/ml; P19-A9, 5 /yg/ml) followed by the ½ concentration step after the death of the puromycin-sensitive cells. The cultures were incubated until the appearance of visible puromycin-resistant colonies, which were either pooled and expanded in a medium containing 1/2 maximal puromycin concentration or alternatively stained with 0.1 % methylene blue in 50% methanol and photographed.

[00122]. Luciferase Assays

[00123]. HEK293T-A2 cells encoding RIPE-shRNAs against firefly luciferase or LacZ were pre-treated with 2 /vg/ml doxycycline for 36 hours and seeded into 96- well plates (Costar, #3610) at 4x104 cells/well in antibiotic-free medim. The suspended cells were immediately co-transfected with 60 ng of the Photinus pyralis firefly luciferase reporter plasmid pGL3-control (Promega) and 40 ng of the Renilla reniformis luciferase plasmid pTK-Renilla (a modified version of pGL4.74; Promega) per well using Lipofectamine 2000 as recommended

(Invitrogen). The medium was changed 6 hours post-transfection to include penicillin, streptomycin and doxycycline and the incubation continued for another 18 hours. The activities of the two luciferases were measured 24 hours post- transfection using Dual-Glo Luciferase Assay System (Promega) and a

Fluoroskan Ascent FL microplate fluorometer and luminometer (Thermo

Scientific). [00124]. RT-PCR and RT-qPCR

[00125]. Total RNA was purified using Trizol (Invitrogen) as recommended. RNA samples were treated with 50 units/ml of RQ1 DNase (Promega) at 37°C for 30 min to remove genomic DNA contamination. Reverse transcription (RT) was carried out using Superscript III (Invitrogen) and random decamer (N10) primers at 50°C for 1 hour. cDNA samples were analyzed by PCR using Taq DNA polymerase (KAPA Biosystems) or quantitative PCR using Fast SYBR Green Master Mix and a StepOnePlus Real-Time PCR System (Applied Biosystems) as recommended. The corresponding primer sequences are listed in the Table 1. The RT-PCR products were analyzed by electrophoresis in 2% NuSieve 3:1 (Lonza), 1 ^χ ΤΑΕ agarose gels. The qPCR reactions were carried out in triplicate and the data were normalized against the HPRT mRNA levels.

[00126]. Lentiviral stocks

[00127]. One well of a 6-well plate containing -1 *10 ⁶ HEK293T cells was co- transfected with the pEM584 plasmid (see above) and the Lenti-X HT packaging mix using Lentiphos HT transfection reagent as recommended (Clontech).

Medium was changed 12-16 hours post transfection and the plate was incubated for another 48 hours before collecting the supernatant containing the lentiviral particles. The supernatant was cleared from the cellular debris by centrifugation at 1000xg for 5 min and frozen at -80°C in small, single use aliquots. To titre the lentiviral stocks, serial dilutions were added to individual wells of 6-well plates containing -40% confluent HeLa cultures and incubated for 18 hours without polybrene. The medium was then changed and the incubation continued for another 18 hours prior to the addition of 5 jug/ml of blasticidin S. The medium was changed every 2-3 days until non-infected cells died and the infected cells containing the Bsd gene formed visible colonies. The colonies were stained with 0.1 % methylene blue in 50% methanol and counted. The titres were calculated as colony forming units (cfu) per milliliter by averaging the colony counts multiplied by the corresponding dilution factors. The titres were normally in the range of 0.5-1 *106 cfu/ml.

[00128]. Genomic DNA isolation [00129]. To isolate genomic DNA, 70% confluent cell cultures grown in 10 cm dishes were trypsinized and pelleted at 500xg for 5 min in 15 ml falcon tubes. The cell pellets were washed with 1.2 ml PBS and lysed in 0.6 ml of DNA extraction buffer containing 100 mM Tris-HCI, pH 7.4, 200 mM NaCI, 5 mM EDTA, 0.2 %SDS and 0.1 mg/ml proteinase K (Fermentas). The lysates were incubated overnight at 55°C followed by subsequent phenol, phenol-chloroform (1 :1 ) and chloroform extractions and genomic DNA precipitation with 0.7 volumes of isopropanol at room temperature. DNA pellets were washed with 70% ethanol and rehydrated in 10 mM Tris-HCI, pH 8.0.

[00130]. Multiplex PCR

[00131]. Genomic DNAs from the RMCE acceptor cell lines were analyzed by multiplex PCR using Taq DNA polymerase (KAPA Biosystems) and either the 5' (EF, BR, and PR; Table 1 ) or the 3' junction primer mixtures (GF, BF, and WR; Table 1 ). The mixtures were designed to generate distinct PCR products from the acceptor locus before and after inserting the RIPE cassette. The PCR program consisted of a 3 min 95°C step followed by 37 cycles of melting (94°C, 20 sec), annealing (56°C, 30 sec) and elongation (72°C, 90 sec). DMSO was added to the 5' junction reactions to the final concentration of 5% to facilitate the amplification of the GC-rich Pur gene. Control PCR reactions were carried out using primers hGAPDH_F1 and hGAPDH_R1 (Table 1 ) detecting both the bona fide GAPDH gene and a GAPDH pseudogene under the conditions employed.

[00132]. Southern blot analysis

[00133]. Fifteen μg of genomic DNA samples were digested with 70 units of the Nco\ (NEB) in 200 μ\ reactions at 37°C overnight. The samples were extracted once with phenol-chloroform (1 :1 ), precipitated with ethanol and rehydrated in 20 /vl of 10 mM Tris-HCI, pH 8.0. The samples were separated using 0.8% agarose gel electrophoresis in 1 *TAE buffer at 5 V/cm and the DNA was transferred to Hybond N+ membranes (GE Healthcare) as described (35). To prepare the probe, we amplified a ~1 kb fragment from pEM584 using KAPA HiFi DNA polymerase (KAPA Biosystems) and South584_F1 and South584_R1 primers

(Table 1 ). The PCR fragment was labeled using a Megaprime DNA labeling System (GE Healthcare) and [σ-32Ρ]- dCTP (Perkin Elmer) and purified from non-incorporated nucleotides using G-50 spin columns (Geneaid Biotech).

Hybridizations were carried out in ExpressHyb hybridization buffer (Cloiitech) as recommended and the radioactive signal was visualized using a Typhoon Trio imager (GE Healthcare).

[00134]. Microscopy and fluorescence-activated cell sorting (FACS)

[00135]. Cells cultured in 6-well plates were incubated with 2 /yg/ml doxycycline for 48 hours or left untreated. The expression of fluorescent proteins was then analyzed by either epifluorescence microscopy using an Eclipse Ti microscope (Nikon) equipped with a CoolSNAP HQ2 CCD camera (Photometries) or FACS using a LSR II Flow Cytometer (BD Biosciences). To prepare the FACS samples, the cells were trypsinized and resuspended in buffer containing 89% PBS, 10% FBS and 1mM sodium pyruvate.

[00136]. Immunoblotting

[00 37]. Proteins were extracted from PBS-washed cells with 20 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM EDTA, 10% glycerol, 1 % Nonidet P-40, 1 mM PMSF and 1 χ Complete EDTA-free protease inhibitor cocktail (Roche), separated by 4-20% gradient SDS-PAGE (Bio-Rad), and immunoblotted using the following primary antibodies: mouse monoclonal anti-PTBP1 (ZYMED; 1 :1000 dilution), rabbit monoclonal anti-Ago2 (Cell Signalling Technology; 1 :1000), goat polyclonal anti-TUT4/ZCCHC1 (Imgenex; 1 :500), or mouse monoclonal anti-GAPDH (Ambion; 1 :10,000). The protein bands were visualized using corresponding horseradish peroxidase-conjugated secondary antibodies (mouse and rabbit, GE Healthcare; goat, Santa Cruz Biotechnology) and ECL detection reagents from Millipore or Thermo Scientific.

[00138]. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. [00139]. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[00140]. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[00141], The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[00142]. The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

[00143]. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as

"comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[00144]. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[00145]. References'

[00146]. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, & Mello CC (1998) Nature 391, 806-81 1 .

[00147]. 2. Hannon GJ & Rossi JJ (2004) Nature 431 , 371-378.

[00148]. 3. Carthew RW & Sontheimer EJ (2009) Cell 136, 642-655.

[00149], 4. Ghildiyal M & Zamore PD (2009) Nat Rev Genet 10, 94-108.

[00150]. 5. Kim VN, Han J, & Siomi MC (2009) Nat Rev Mol Cell Biol 10, 126- 139.

[00151]. 6. Rao DD, Vorhies JS, Senzer N, & Nemunaitis J (2009) Adv Drug Deliv Rev 61 , 746-759.

[00152]. 7. Mohr S, Bakal C, & Perrimon N (2010) Annu Rev Biochem 79, 37- 64.

[00153]. 8. Grimm D (2009) Adv Drug Deliv Rev 61, 672-703.

[00154]. 9. Wiznerowicz M, Szulc J, & Trono D (2006) Nat Methods 3, 682-688.

[00155]. 10. Zuber J, McJunkin K, Fellmann C, Dow LE, Taylor MJ, Hannon GJ, & Lowe SW (201 1 ) Nat Biotechnol 29, 79-83.

[00156]. 1 1. Markstein M, Pitsouli C, Villalta C, Celniker SE, & Perrimon N (2008) Nat Genet 40, 476-483.

[00157]. 12. Ellis J, Hotta A, & Rastegar M (2007) Cell 131, 13-14.

[00158]. 13. Pikaart MJ, Recillas-Targa F, & Felsenfeld G (1998) Genes Dev M,

2852-2862.

[00159]. 14. Lund AH, Duch M, & Pedersen FS (1996) J Biomed Sci 3, 365-378.

[00160]. 15. Meerbrey KL, Hu G, Kessler JD, Roarty K, Li MZ, Fang JE,

Herschkowitz Jl, Burrows AE, Ciccia A, Sun T, et al. (201 1 ) Proc Natl Acad Sci U S A.

[00161]. 16. Wirth D, Gama-Norton L, Riemer P, Sandhu U, Schucht R, & Hauser H (2007) Curr Opin Biotechnol 18, 41 1-419.

[00162]. 7. Berger SM, Pesold B, Reber S, Schonig K, Berger AJ, Weidenfeld I, Miao J, Berger MR, Gruss OJ, & Bartsch D (2010) Nucleic Acids Res 38, e168. [00163]. 18. Saito F, Yokota H, Sudo Y, Yakabe Y, Takeyama H, & Matsunaga T (2008) Toxicol In Vitro 22, 077-1087.

[00164]. 19. Weidenfeld I, Gossen M, Low R, Kentner D, Berger S, Gorlich D, Bartsch D, Bujard H, & Schonig K (2009) Nucleic Acids Res 37, e50.

[00165]. 20. Das AT, Zhou X, Vink M, Klaver B, Verhoef K, Marzio G, &

Berkhout B (2004) J Biol Chem 279, 18776-18782.

[00166]. 21 . Sawicka K, Bushell M, Spriggs KA, & Willis AE (2008) Biochem Soc Trans 36, 641- 647.

[00167], 22. Hutvagner G & Simard MJ (2008) Nat Rev Mol Cell Biol 9, 22-32.

[00168], 23. Cheloufi S, Dos Santos CO, Chong MM, & Hannon GJ (2010) Nature 465, 584-589.

[00169], 24. Makeyev EV, Zhang J, Carrasco MA, & Maniatis T (2007) Mol Cell 27, 435-448.

[00170], 25. Heo I, Joo C, Kim YK, Ha M, Yoon MJ, Cho J, Yeom KH, Han J, & Kim VN (2009) Cell 138, 696-708.

[00171], 26. Katoh T, Sakaguchi Y, Miyauchi K, Suzuki T, Kashiwabara S, & Baba T (2009) Genes Dev 23, 433-438.

[00172]. 27. Mostoslavsky G, Fabian AJ, Rooney S, Alt FW, & Mulligan RC

(2006) Proc Natl Acad Sci U S A 103, 16406-16411.

[00173]. 28. Mizushima S & Nagata S (1990) Nucleic Acids Res 18, 5322.

[00174]. 29. Casola S (2004) Methods Mol Biol 271, 91-109.

[00175]. 30. Du G, Yonekubo J, Zeng Y, Osisami M, & Frohman MA (2006)

FEBS J 273, 5421-5427.

[00176]. 31. Araki K, Araki M, & Yamamura K (1997) Nucleic Acids Res 25, 868-872.

[00177]. 32. Qi Y, Wang JK, McMillian M, & Chikaraishi DM (1997) J Neurosci 17, 1217-1225.

[00178]. 33. Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME, Shcheglov AS, Fradkov AF, Gaintzeva A, Lukyanov KA, Lukyanov S, Gadella TW, et al. (2007) Nat Methods 4, 555- 557.

[00179]. 34. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, & Tsien RY (2004) Nat Biotechnol 22, 1567-1572. [00180]. 35. Sambrook J & Russell RW (2001 ) Molecular cloning: A laboratory manual, 3rd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[00181]. 36. Makeyev EV, Zhang J, Carrasco MA, & Maniatis T (2007) Mol Cell 27, 435-448.