Lim2To3 transgenic mice establish a causativerelationship between the mutation identified in the Lim2 gene andcataractogenesis in the To3 mouse mutant

[摘要] Purpose: Lim2 is the gene encoding the ocular lens-specificintrinsic membrane protein MP19. We previously reported finding a singlenonconservative G->T transversion in exon two of the Lim2 gene.This mutation was linked to the cataract in the To3 (Total opacitynumber 3) mouse mutant, confirming Lim2 as an ideal candidate genefor the To3 cataract. The aim of the present study was tosubstantiate a causative relationship between the mutation in theLim2 gene and cataractogenesis in the To3 mouse mutant. To thisend a Lim2To3 transgene cassette was engineered and introducedinto fertilized normal mouse embryos to test its ability to inducecataractogenic lens development.Methods: A Lim2 genomic clone was isolated and purified from amurine 129/SvJ genomic library. A restriction endonuclease map of thegene was generated using classical Southern techniques. The murineLim2 promoter was characterized by transfecting primary chicken lensepithelial cells with Lim2 promoter-CAT reporter constructs andassaying promoter activity and specificity. This genomic clone was thenused in conjunction with PCR to generate a Lim2To3 transgenecassette. After sequencing of the PCR engineered portion, theLim2To3 transgene was then used to generate Lim2To3transgenic mice via pronuclear injection. Founder mice and theiroffspring from outcrosses and intercrosses were characterized byophthalmic examination, PCR and Southern DNA analysis, RT-PCR mRNAanalysis, and histology of lens sections.Results: Two mice, from independent microinjections, were identifiedas positive for presence of the Lim2To3 transgene cassette aswell as presence of bilateral congenital cataracts and reduced eye sizeand mass. One of these founders was incapable of germline transmissionof the transgene to offspring and was not characterized further. Theother was capable of germline transmission and was characterized asdescribed above. PCR DNA analysis revealed a perfect concordance betweenpresence of the Lim2To3 transgene cassette and congenitalcataract in offspring of this founder. Transgenic hemizygotes exhibitedcataract and a reduction in eye and lens size and mass, while transgenic"homozygotes" presented with a more severe cataract and microphthalmicreduction in eye and lens size and mass. Southern analysis revealedapproximately 2 copies of the transgene cassette integrated into asingle chromosomal site in the founder and all hemizygous offspring.RT-PCR analysis revealed a very low ratio of Lim2To3transgenic mRNA compared to endogenous normal Lim2. Finally,histology revealed that lens development was abnormal in mutanttransgenic animals by embryonic day E15. By E19, just prior to birth,gross disorganization of secondary fibers was observed in mutants.Conclusions: These transgenic experiments firmly establish acausative relationship between the previously identified mutation in theLim2 gene and cataractogenesis in the To3 mouse mutant. The lowlevels of mutant mRNA produced by the transgene cassette as compared toendogenous levels of normal Lim2 mRNA provides evidence that thisdominant mutation results in a mutant MP19 protein with altered functionrather than simply loss of function.

[学科分类] 生物化学/生物物理