Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant.
Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant.
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DOI：
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发表时间：
2000-06
期刊：
影响因子：
2.2
通讯作者：
E. Steele;J. Wang;W. Lo;D. Saperstein;X. Li;R. Church
中科院分区：
文献类型：
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作者：
E. Steele;J. Wang;W. Lo;D. Saperstein;X. Li;R. Church
目的Lim 2是编码晶状体特异性内膜蛋白MP 19的基因。我们以前曾报道在Lim 2基因的第二外显子中发现一个非保守的G->T颠换。该突变与To 3（总混浊数3）小鼠突变体中的白内障相关，证实Lim 2是To 3白内障的理想候选基因。本研究的目的是证实Lim 2基因突变与To 3突变小鼠白内障发生之间的因果关系。为此，将Lim 2 To 3转基因盒工程化并引入受精的正常小鼠胚胎中以测试其诱导白内障发生性透镜发育的能力。方法从小鼠129/SvJ基因组文库中分离并纯化Lim 2基因组克隆。使用经典的Southern技术产生该基因的限制性内切酶图谱。通过用Lim 2启动子-CAT报告基因构建体转染原代鸡透镜上皮细胞并测定启动子活性和特异性来表征鼠Lim 2启动子。然后将该基因组克隆与PCR结合使用以产生Lim 2 To 3转基因盒。在PCR工程化部分测序后，然后使用Lim 2 To 3转基因通过原核注射产生Lim 2 To 3转基因小鼠。通过眼科检查、PCR和Southern DNA分析、RT-PCR mRNA分析和透镜切片的组织学来表征来自异交和互交的创始小鼠及其后代。结果两个小鼠，从独立的显微注射，被确定为阳性的Lim 2 To 3转基因盒的存在，以及存在双侧先天性白内障和眼睛大小和质量减少。这些创始人之一是不能生殖系的转基因传递到后代，并没有进一步的特点。另一种能够进行生殖系传播，并如上所述进行表征。PCR DNA分析揭示了Lim 2 To 3转基因盒的存在与该创始人后代的先天性白内障之间的完美一致性。转基因半合子表现出白内障以及眼睛和透镜大小和质量的减少，而转基因“纯合子”表现出更严重的白内障以及眼睛和透镜大小和质量的小眼减少。Southern分析揭示了大约2个拷贝的转基因盒整合到创始人和所有半合子后代的单个染色体位点中。RT-PCR分析显示，与内源性正常Lim 2相比，Lim 2 To 3转基因mRNA的比例非常低。最后，组织学显示，透镜发育异常的突变转基因动物胚胎天E15。到E19，就在出生前，在突变体中观察到二级纤维的严重解体。结论：这些转基因实验牢固地建立了先前鉴定的Lim 2基因突变与To 3突变小鼠白内障发生之间的因果关系。与正常Lim 2 mRNA的内源水平相比，转基因盒产生的低水平的突变mRNA提供了这样的证据，即这种显性突变导致功能改变而不是简单功能丧失的突变MP 19蛋白。
PURPOSE Lim2 is the gene encoding the ocular lens-specific intrinsic membrane 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 Lim2 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 Lim2To3 transgene cassette was engineered and introduced into fertilized normal mouse embryos to test its ability to induce cataractogenic lens development. METHODS A Lim2 genomic clone was isolated and purified from a murine 129/SvJ genomic library. A restriction endonuclease map of the gene was generated using classical Southern techniques. The murine Lim2 promoter was characterized by transfecting primary chicken lens epithelial cells with Lim2 promoter-CAT reporter constructs and assaying promoter activity and specificity. This genomic clone was then used in conjunction with PCR to generate a Lim2To3 transgene cassette. After sequencing of the PCR engineered portion, the Lim2To3 transgene was then used to generate Lim2To3 transgenic mice via pronuclear injection. Founder mice and their offspring from outcrosses and intercrosses were characterized by ophthalmic examination, PCR and Southern DNA analysis, RT-PCR mRNA analysis, and histology of lens sections. RESULTS Two mice, from independent microinjections, were identified as positive for presence of the Lim2To3 transgene cassette as well as presence of bilateral congenital cataracts and reduced eye size and mass. One of these founders was incapable of germline transmission of the transgene to offspring and was not characterized further. The other was capable of germline transmission and was characterized as described above. PCR DNA analysis revealed a perfect concordance between presence of the Lim2To3 transgene cassette and congenital cataract in offspring of this founder. Transgenic hemizygotes exhibited cataract and a reduction in eye and lens size and mass, while transgenic "homozygotes" presented with a more severe cataract and microphthalmic reduction in eye and lens size and mass. Southern analysis revealed approximately 2 copies of the transgene cassette integrated into a single chromosomal site in the founder and all hemizygous offspring. RT-PCR analysis revealed a very low ratio of Lim2To3 transgenic mRNA compared to endogenous normal Lim2. Finally, histology revealed that lens development was abnormal in mutant transgenic 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 a causative relationship between the previously identified mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. The low levels of mutant mRNA produced by the transgene cassette as compared to endogenous levels of normal Lim2 mRNA provides evidence that this dominant mutation results in a mutant MP19 protein with altered function rather than simply loss of function.