乙酰基亚硝基脲诱变的一种角膜混浊小鼠及其突变基因的染色体定位

Production of a corneal opacity mouse by N-ethyl-N-nitrosourea mutagenesis and chromosome mapping ofthe mutant gene

背景乙酰基亚硝基脲（ENU）诱导小鼠基因突变是研究基因功能及建立人类疾病动物模型的一种有效手段。目的研究ENU诱变获得的1只角膜混浊小鼠角膜组织形态变化，并对其突变基因进行染色体定位。方法选用8-10周龄的C57BL／6J（B6）雄鼠40只，腹腔内注射ENU。将注射后的雄鼠与同品系母鼠交配，在其后代小鼠中筛选眼部突变个体，并将突变个体与同品系小鼠配种以确定是否遗传。对ENU诱变获得1只角膜混浊小鼠的角膜行组织病理学观察。繁殖[（B6xD2）F1xB6]N2角膜混浊小鼠，用平均分布于小鼠染色体上的微卫星标记对N2小鼠染色体进行扫描。采用优势对数计分法（LOD）判定突变基因与微卫星是否连锁。结果将角膜呈现混浊小鼠与B6背景小鼠配种，在其59只后代中出现19只类似表型的突变小鼠。角膜组织病理学检查发现，该例角膜混浊小鼠角膜基质增厚，出现新生血管和炎性细胞浸润，成纤维细胞明显增多。突变基因与微卫星间的连锁分析发现，26个N2样品中突变基因与位于小鼠2号染色体63．42eM处的D2Mi307位点发生3例交换，LOD值为3．79，该突变基因位于小鼠第2号染色体。结论成功获得角膜混浊小鼠并将其突变基因初步定位于2号染色体，有望为人类角膜混浊疾病研究提供一种新的小鼠模型。

Background N-ethyl-N-nitrosourea （ENU）-induced mouse mutagenesis is a powerful approach for the study of gene function and the generation of human disease models. Objective This study was to create the corneal morphologic change and map the mutant gene of a kind of corneal opacity in ENU mutagenesis in mouse. Methods ENU was intraperitoneally injected in forty C57BL/ 6J （B6） male mice aged 8-10 weeks old. The male mice were mated with the same strain female mice. Their progenies were screened for visible eye mutation, and the mutant mice were mated with the same strain mice to confirm the heredity of mutation phenotypes. Hematoxylin ＆ eosin staining was used to examine the histopathological change of cornea in one mouse with ENU-induced corneal opacity. To map the mutant gene, I （B6xD2）F1 ~B6 ] N2 mutant mice were bred, and the genome of the N2 mice was scanned by microsatellite markers distributed equally on the mouse chromosome. The microsatellite linked to the mutant gene was determined by the log odds score. This experimental procedure was approved by Ethic Committee about Experimental Animal Care and Use of Yangzhou University. Results The founder mouse, which was the progeny of an ENU-treated B6 male mouse and an untreated B6 female mouse,had a corneal opacity phenotype. After mating the mutant with B6 mice, 19 of 59 descendants appeared corneal opacity phenotype. Thickening of corneal stroma,neoangiogenesis, infiltration of inflammatory cells and proliferation of fibroblasts were exhibited in cloudy cornea in ENU-induced mutated mice under the optical microscope. After linkage analysis between microsatellite markers and the mutant gene,the mutant gene was linked to D2Mi307 ,which was located at 63.42 cM. Three cases of 26 N2 mice underwent recombination with the LOD 3.79. The mutant gene associated with the cornea phenotype was located on chromosome 2. Conclusions This study map the mutant gene associated with the cornea phenotype onchromosome 2. The strain might be used as a mouse model for heritable human corneal opacity.