Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the Lim2 gene and cataractogenesis in the To3 mouse mutant

PURPOSE: Lim2 is the gene encoding the ocular lens-specific intrinsic membr ane protein MP19. We previously reported finding a single nonconservative G ->T transversion in exon two of the Lim2 gene. This mutation was linked to the cataract in the To3 (Total opacity number 3) mouse mutant, confirming L im2 as an ideal candidate gene for the To3 cataract. The aim of the present study was to substantiate a causative relationship between the mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. To this end a Lim2(To3) transgene cassette was engineered and introduced into fertilized normal mouse embryos to test its ability to induce cataractogenic lens deve lopment. METHODS: A Lim2 genomic clone was isolated and purified from a murine 129/S vJ genomic library. A restriction endonuclease map of the gene was generate d using classical Southern techniques. The murine Lim2 promoter was charact erized by transfecting primary chicken lens epithelial cells with Lim2 prom oter-CAT reporter constructs and assaying promoter activity and specificity . This genomic clone was then used in conjunction with PCR to generate a Li m2(To3) transgene cassette. After sequencing of the PCR engineered portion, the Lim2(To3) transgene was then used to generate Lim2(To3) transgenic mic e via pronuclear injection. Founder mice and their offspring from outcrosse s and intercrosses were characterized by ophthalmic examination, PCR and So uthern DNA analysis, RT-PCR mRNA analysis, and histology of lens sections. RESULTS: Two mice, from independent microinjections, were identified as pos itive for presence of the Lim2(To3) transgene cassette as well as presence of bilateral congenital cataracts and reduced eye size and mass. One of the se founders was incapable of germline transmission of the transgene to offs pring and was not characterized further. The other was capable of germline transmission and was characterized as described above. PCR DNA analysis rev ealed a perfect concordance between presence of the Lim2(To3) transgene cas sette and congenital cataract in offspring of this founder. Transgenic hemi zygotes exhibited cataract and a reduction in eye and lens size and mass, w hile transgenic "homozygotes" presented with a more severe cataract and mic rophthalmic reduction in eye and lens size and mass. Southern analysis reve aled approximately 2 copies of the transgene cassette integrated into a sin gle chromosomal site in the founder and all hemizygous offspring. RT-PCR an alysis revealed a very low ratio of Lim2(To3) transgenic mRNA compared to e ndogenous normal Lim2. Finally, histology revealed that lens development wa s abnormal in mutant transgenic animals by embryonic day E15. By E19, just prior to birth, gross disorganization of secondary fibers was observed in m utants. CONCLUSIONS: These transgenic experiments firmly establish a causative rela tionship between the previously identified mutation in the Lim2 gene and ca taractogenesis in the To3 mouse mutant. The low levels of mutant mRNA produ ced by the transgene cassette as compared to endogenous levels of normal Li m2 mRNA provides evidence that this dominant mutation results in a mutant M P19 protein with altered function rather than simply loss of function.