Introduction

[0001] This application claims benefit of U.S. Provisional Patent Application Serial No. 61/181,832, filed May 28, 2009, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant numbers ROl HL083249 and ROl HL065418 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background of the Invention

[0003] Caveolin 1 (Cavl) is the gene product of the Cavl locus. Cavl is a member of the caveolin family of proteins along with Cav2 and Cav3 as well as some invertebrate caveolins. Cavl is a phosphoprotein with a structural role in the formation of caveolae, organelles with multiple but controversial assigned functions. Loss of Cavl results in severe dislipidemia both in mouse and humans. It also leads to a cardiovascular phenotype with dilative cardiomyopathy and pulmonary hypertension. In addition, loss of Cavl protein results in a type II diabetes syndrome with hyperglyceridemia, hyperinsulinemia and decreased tolerance to glucose. Caveolin 1 is involved in multiple signal transduction pathways. It is believed to be a tumor suppressor, as well as an angiogenesis inhibitor. Caveolin 1 is involved in mechanical flow sensing as well as in inflammation and permeability.

Summary of the Invention

[0004] The present invention is a transgenic mouse whose genome harbors a nucleic acid molecule encoding caveolin- 1 fused in-frame with a reporter, wherein said nucleic acid molecule further includes a selectable marker expression cassette flanked by recombinase target sites in such a manner that, upon exposure to recombinase, said selectable marker expression cassette is excised from said nucleic acid molecule. In one embodiment, the recombinase is Cre and the recombinase target sites are lox P sequences. In another embodiment, the recombinase is FIp and the recombinase target sites are Frt sequences . In certain embodiments, the reporter protein is a fluorescent protein. In particular embodiments the selectable marker expression cassette is neomycin expression cassette. Transgenic mice wherein the endogenous caveolin-1 gene has been knocked out are also embraced by this invention.

[0005] A method for generating a mouse expressing caveolin- 1 in a time- or tissue-specific manner is also provided by mating the transgenic Cavl-reporter mouse to a mouse whose genome harbors a nucleic acid molecule encoding a recombinase under the control of a time- or tissue-specific promoter, thereby generating a mouse expressing caveolin-1 in a time- or tissue-specific manner.

Detailed Description of the Invention

[0006] Using heterologous recombination in mice, a Caveolin 1 (Cavl) knock- in mouse has now been generated. The knock- in construct used in the generation of the transgenic mouse of the invention was composed of nucleic acids encoding Cavl fused in- frame with nucleic acids encoding the reporter protein GFP. The knock-in construct further included the marker expression cassette encoding the neomycin resistant gene flanked by loxP recombinase target sites, wherein the neomycin expression cassette disrupted the Cavl locus of the knock- in construct, thereby resulting in a Cavl null allele.

[0007] Using the transgenic mouse of the invention, Cavl- GFP expression can be controlled in a time- and/or tissue- specific manner by crossing the Cavl knock-in mouse with a transgenic mouse expressing a recombinase, such as Cre recombinase, the expression of which is under the control of a time- and/or tissue-specific promoter. Transgenic mice of the invention can express endogenous Cavl or alternatively lack endogenous Cavl expression so that rescue experiments can be performed, wherein the Cavl -GFP protein is expressed in time- and/or tissue-specific manner. [0008] The mice of the present invention find application in in vivo studies of vascular permeability and in analyzing cardiovascular functions and diseases where caveolae have been implicated. Such diseases include, but are not limited to atherosclerosis and tumor angiogenesis . In addition, caveolae have been implicated in the formation of fat (adipose) tissue, and the Cavl knock-in mice on could be used to evaluate the role of caveolin 1 in the regulation of fat deposit formation. Moreover, transgenic mice of the invention could be used in screening assays to identify agents that modulate the expression of caveolin 1. [0009] For the purposes of the present invention, caveolin 1 is the scaffolding protein that is the main component of the caveolae plasma membranes found in most cell types. The protein links integrin subunits to the tyrosine kinase FYN, an initiating step in coupling integrins to the Ras-ERK pathway and promoting cell cycle progression. The caveolin 1 gene is a tumor suppressor gene candidate and a negative regulator of the Ras-p42/44 MAP kinase cascade. The Cavl to be introduced into the transgenic mouse can be from any suitable source including, but not limited to, mouse, rat, human, monkey, and the like. Nucleic acids encoding these proteins are well-known in the art. For example, human Cavl is available under GENBANK Accession No. NP_001744, mouse Cavl is available under Accession No. NP_031642; and rat Cavl is available under Accession No. NP_113744. In particular embodiments, the mouse Cavl protein is employed. [00010] To facilitate detection of transgenic Cavl expression in the transgenic mouse of the invention, the nucleic acids encoding Cavl are fused in- frame with nucleic acids encoding a reporter protein thereby creating a Cavl- reporter fusion protein. In- frame fusions of nucleic acids to create fusion proteins are routinely practiced in the art and can be accomplished by conventional restriction enzyme and/or overlap PCR technologies .

[00011] A reporter protein of the invention includes fluorescent proteins and enzymes detectable by a histochemical process. Exemplary enzymes detectable by a histochemical process include, but are not limited to β- galactosidase, β-glucuronidase, alkaline phosphatase, luciferase, alcohol dehydrogenase, chloramphenicol -acetyl transferase, and peroxidase. The substrates to be used with these specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Substrate can be soluble or insoluble, added into the culture medium of a cell or tissue isolated from a transgenic mouse of the invention, or present in the host cell, depending upon the chosen method. For example, 5-bromo-4-chloro-3-indoyl phosphate/nitroblue tetrazolium is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1 , 2 -phenylenediamine-5-aminosalicylic acid, 3,3,5,5,- tetramethylbenzidine , tolidine or dianisidine are commonly used. Autofluorescent proteins such as green fluorescence protein (GFP) , the enhanced green fluorescence protein (eGFP) , the red fluorescence protein (RFP) , the blue fluorescence protein (BFP) , the yellow fluorescence protein (YFP) and fluorescent variants of these proteins offer particular advantages in the transgenic mouse of this invention in that substrates are not required. Accordingly, particular embodiments embrace fusion of Cavl to an autofluorescent reporter protein.

[00012] According to the invention, the Cavl open reading frame has inserted therein a selectable marker expression cassette to inactivate the Cavl -reporter transgene. For the purposes of the present invention, a selectable marker expression construct is a nucleic acid molecule coding for and capable of expressing a selectable marker protein. In this respect, the selectable marker expression construct includes all the necessary regulatory sequences (e.g., promoter, terminator, and the like) to regulate expression of the selectable marker protein. Selectable marker expression constructs which can be used in accordance with the present invention include, for example, drug-resistance genes, such as the neomycin-resistance gene (selected by G418 resistance) , the thymidine kinase gene (selected by ganciclovir), etc.; toxin genes, such as the diphteria toxin (DT) A gene, etc.; or combinations of these genes. [00013] So that Cavl-reporter expression can be reinstated at a selected time, the present invention features recombinase target sites flanking the 5' and 3' ends of the open reading frame of the selectable marker expression construct . The recited recombinase target sites refer to nucleotide sequences which undergo recombination (e.g., DNA cross-over and exchange) when catalyzed by a recombinase, such as Cre, FIp or another member of the Int family of recombinases (see, e.g., Argos, et al . (1986) EMBO J. 5:433) . Suitable target sequences include, for example, the well-known loxP sequences recognized by Cre recombinase, and the frt sequences recognized by FIp recombinase. [00014] In this respect, the term "recombinase" refers to any recombinase which catalyzes a site-specific recombination at a corresponding target site thereby excising the marker expression cassette from the Cavl open reading frame and restoring Cavl -reporter protein expression. Suitable recombinases include, for example, Cre recombinase (Sauer, et al . (1993) Methods in Enzymology 225:898), FIp recombinase (Buchholz, et al . (1996) Nucl . Acids Res. 24:4256-4262; Buchholz, et al . (1998) Nat. Biotechnol . 16:657-662), the R recombinase of Zygosaccharomyces rouxii pSRl, the A recombinase of Kluyveromyces drosophilarium pKDl , the A recombinase of Kluyveromyces waltii pKWl, the integrase λ Int, the recombinase of the GIN recombination system of the Mu phage or a variant thereof. For example, in many embodiments, the Cre/LoxP system is interchangeable with the FRT/FLP system, which has also been demonstrated to work in mice. Indeed, the Cre-loxP system has been routinely used to direct site- specific DNA recombination in transgenic animals (see, e.g., Orban, et al . (1992) Proc . Natl. Acad. Sci . USA 89(15) : 6861-5) . Other recombinases which may be adapted to the mouse herein include Rs, RAGl/2, etc. In addition, alternative recombinases with novel specificity may be generated using established mutation and selection ^• protocols, e.g. Buchholz, et al . (2001) Nat. Biotechnol. 19 (11) :1047-52.

[00015] Transgenic mice of the invention can be obtained by preparing vectors harboring the coding sequence of the Cavl-reporter disrupted by a selectable marker expression construct, wherein said vector is suitable for heterologous or homologous recombination. When homologous recombination is desired to create a Cavl knock out, desirably, the vector contains Cavl gene sequences sufficient to permit the homologous recombination of the vector into at least one allele of the Cavl gene resident in the chromosomes of the target or recipient cell (e.g., ES) cells. The insertion of nucleic acids into vectors can be carried out in vitro by using conventional DNA recombination techniques

(Sambrook, et al . (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) . Vectors for creating transgenic mice are well-known in the art and readily available from various commercial sources.

[00016] The introduction of the vector into cells, e.g., embryonic stem (ES) cells or somatic cells, can be performed by methods well-known to those skilled in the art, for example, the electroporation method, calcium phosphate co-precipitation method, protoplast or spheroplast fusion method, lipofection or DEAE-dextran- mediated transfection. Such introduction results in insertion of the Cavl-reporter nucleic acids at some random or otherwise designated location in the genome, or alternatively, recombination occurs between the endogenous Cavl gene and the corresponding region of the recombination vector in some population of the cells thereby creating a knock out or gene disruption. In this manner, the endogenous wild-type gene is substituted with the gene having the genotype constructed in the vector. Thus, it is possible to obtain cells having a Cavl-reporter gene in which a selectable marker gene and/or loxP sequences have been introduced into the endogenous Cavl locus .

[00017] In so far as the vector contains a selectable marker expression construct, cells can be selected based upon acquisition of the marker. For example, when a drug- resistance gene is used as the marker, cells in which the desired recombination has taken place can be selected by culturing the cells in the presence of the drug at a lethal level subsequent to the introduction of vector. In addition to the selectable marker expression construct that inactivates the Cavl transgene, the vector may also contain one or more additional selectable marker genes. When more than one selectable marker gene is employed, desirably one marker is a positive selectable marker (e.g., the neo gene) and the second is a negative selectable marker (e.g., the Herpes simplex virus tk (HSV-tk) gene) .

[00018] Desirably mice cells which have integrated said Cavl -reporter coding sequence are selected and implanted in mice embryos leading to adult mice harboring the transgene. According to one embodiment, ES cells are used and injected into blastocysts to prepare chimera embryos. The chimeric embryos are then transferred into the horn of uterus of pseudopregnant mammals to obtain newborns. The blastocysts to be used for the injection can be obtained by perfusing the uterus of a pregnant female. To determine whether or not the ES cell has been incorporated in the developing embryo after the creation of an individual mammal, it is preferable to select the type of blastocyst that gives different external characteristics (for example, fur color) to distinguish the origin of a cell, whether it is derived from the ES cell or blastocyst, in the created animal. Subsequently, newborns are obtained by mating the resulting chimeric animal with an animal of an appropriate strain of the same species .

[00019] When somatic cells other than ES cells are used in the present invention, it is possible to create a knock-in animal by using techniques for creating somatic cell cloned animals. For example, Cavl-reporter nucleic acids harboring the selectable marker expression construct are introduced into cells other than ES cells, e.g., fibroblast cells; an animal carrying the Cavl-reporter nucleic acids is created from this cell by using the method for creating somatic cell cloned animals (Wilmut, et al . (1997) Nature 385:810- 803; Wakayama, et al . (1998) Nature 394:369-374; and the resulting animal newborn carrying the Cavl-reporter corresponds to the Fl mouse of the method using the ES cell, and can be used thereafter according to a same manner as the ES cell. Transgenic mice obtained via ES cell or somatic cell methods have the phenotype Cavl-reporter ^+/~ /Marker ^+/~ or Cavl-reporter ^+/+ /Marker ^+/+ .

[00020] For conditional expression of the Cavl-reporter transgene, the present invention also embraces a transgenic mouse expressing Cavl in a time- or tissue-specific manner. Such a transgenic mouse can be obtained by mating a Cavl- reporter ^+/~ /Marker ^+/~ _O r Cavl-reporter ^+/+ /Marker ^+/+ transgenic mouse to a mouse whose genome contains a nucleic acid molecule encoding a recombinase under the control of a time-specific, phase-specific and/or tissue-specific promoter. The mating of a transgenic mouse having regulated recombinase expression with an Cavl-reporter ^"1" /Marker ^"1" or Cavl-reporter ^+/+ /Marker ^"l"/+ animal results in time-specific, phase-specific and/or tissue-specific removal of the marker expression construct from the Cavl transgene thereby allowing for expression of the Cavl-reporter fusion protein.

[00021] Transgenic mice having regulated recombinase expression can be obtained by the methods disclosed herein. It is contemplated that any time-specific, phase-specific and/or tissue-specific promoter can be employed to regulate expression of the recombinase and hence excision of the marker expression construct. The selection of the promoter used will be dependent upon the analysis to be conducted. In this respect, the wild-type caveolin-1 promoter can be employed to analyze wild-type expression of caveolin-1. In addition, it is contemplated that the promoter can be constitutive, to determine the effects of constitutive Cavl expression; or inducible {e.g., the metallothionein promoter, tet promoter, or a hormone inducible promoter) to control expression of Cavl in a time-specific manner by external stimuli. Additional exemplary promoters of use in this invention include, but are not limited to, the Tie2 promoter (Minami, et al . (2003) Arterioscl. Thromb Vascular Biol. 23:2041), which provides endothelial-specific expression and the Adipocyte Fatty Acid Binding Protein (aP2) promoter (Rival, et al . (2004) J. Pharmacol. Exp. Therap . 311:467-475), which provides adipocyte-specific expression .

[00022] In addition to the time- and tissue-specific analysis of Cavl expression, the knock-in mice of the invention find application in developing therapeutics for the treatment of a variety of diseases in which Cavl or caveolae have been implicated. For example, a test compound is administered to the knock- in mouse of the present invention, and the influence of the compound on Cavl expression and/or a particular phenotype is tested to select compounds exhibiting desired effects. Furthermore, cells prepared from knock-in mouse can be used for developing therapeutics. For example, cells are prepared from embryos and such from knock- in mice of the present invention, and then a test compound is added to the cells to determine the influence of the compound on Cavl expression or other phenotype thereby selecting compounds exhibiting the desired effect. The cells may be primary culture cells or established cell lines. The compounds screened are candidates for pharmaceutical agents.

[00023] In so far as up-regulation of Cavl has been shown to provide protection against lung fibrosis (U.S. Patent Application No. 20090075875) , the transgenic mouse of the invention can be used in the identification of agents for use in the treatment of lung fibrosis. Similarly, agents that elevate Cavl expression levels have been shown to reduce glucosylceramide accumulation (U.S. Patent Application No. 20080132508) . Thus, the transgenic mice of the invention can be used in the identification of agents useful in the treatment of diabetic retinopathy. Cavl expression has also been shown to increase in metastatic human prostate cancer cells as compared to primary prostate tumors (U.S. Patent Application No. 20050036986). Accordingly, certain cancers may be treated by suppressing expression of the Cavl gene in metastatic cells or cells predisposed to metastasis. Thus, agents inhibiting the expression of Cavl can also be identified using the transgenic mouse of the invention.

[00024] The animal testing herein can also be supplemented and confirmed by testing on human subjects. However, the present animal models allow the testing of a large number of compounds, both by the methods described above and other methods known in the art, in a system similar in many important respects to that in humans.

[00025] The invention is described in greater detail by the following non-limiting examples.

[00026] Example 1: Generation of Mouse' Line

[00027] The BAC clone containing the mouse Cavl exon 3 was obtained from the Sanger Center, UK. A targeting construct was generated by recombination in bacteria, wherein the GFP gene was inserted in- frame with Caveolin 1 followed by a Neomycin expression cassette (the neomycin gene driven by the PGK promoter) , wherein said cassette was flanked by loxP recombination sites. The Neomycin expression cassette disrupted the Cavl mRNA resulting in a Cavl null allele, allowed for selection of mutant ES cell clones, and destabilized the Cavl-GFPp mRNA thereby sharply decreasing its expression. A targeting plasmid was recovered and linearized. The construct was electroporated into V6.5 ES cells and candidate recombinants were selected antibiotic resistance. The resistant clones were screened for homologous recombination by Southern blot analysis. Two clones (#20 and #96) were aggregated with mouse blastocysts to obtain chimaera. Chimaera, detected by their agouti coat color, were crossed first with wild-type mice to check for germline transmission. Cavl-GFP ^+/~ ; Neo ^+/~ mice are viable, fertile and did not exhibit any overt phenotype . [00028] The offspring (genotype Cavl-GFP ^+/" ; Neo ^+/" ) were then crossed with CMV-Cre transgenic mice (The Jackson Laboratory) to excise the Neomycin expression cassette and obtain Cavl-GFP ^+/~ ;Neo ^~7~ mice (Cavl-GFP mice) . Equal amounts mouse lung total membrane protein from wild type and three Cavl-GFP mice were resolved by 12% SDS-PAGE, transferred to PVDF membrane and blotted with anti-caveolin 1 antibodies. The Cavl-GFP fusion protein was detected by the anti-Cavl antibody at the expected molecular weight of 40 kD. The resulting Cavl-GFP ^+/~ _/ Neo ^" ' ^" ; CMV-CRE ^+/" mice expressed Cavl- GFP at physiological levels in all the cells expressing Caveolin 1. However, endogenous Cavl levels were diminished in Cavl-GFP mice, whereas endothelial markers CD31 and PVl did not change .

[00029] Based on flow cytometry analysis of splenocytes, it was observed that Cavl-GFP was not expressed in mice that have no excision of the selectable marker cassette (i.e., PGK-Neo) .

[00030] Confocal fluorescence microscopy analysis of unfixed and unstained heart sections from wild type mice showed a lack of fluorescence. In contrast, GFP fluorescence in Cavl-GFP mice exhibited a pattern reminiscent of blood vessels in the heart where Cavl is specifically expressed. Moreover, Cavl-GFP was shown to colocalize with endogenous Cavl in the lung, heart and pancreas and with endogenous CD31 as a marker of endothelial cells in the aorta, lung and pancreas. It was noted, however, that Cavl-GFP was expressed in other cell types as well.

[00031] To demonstrate tissue-specific expression, the Neomycin expression cassette was excised in a tissue- dependent manner in endothelial cells and adipocytes by crossing Cavl-GFP ^+/+ ;Neo ^+/+ with Tie2-Cre or adipocyte- specific aP2 promoter/enhancer-Cre transgenic mice from The Jackson Laboratory.

[00032] In addition, Cavl-GFP ^+/+ ;Neo ^+/+ mice with the endogenous Cavl locus disrupted show the same phenotype as that published for Cavl knockout mice.