BIGH3^(R555W)突变转基因小鼠模型的建立

Establishment of BIGH3^(R555W)-mutation transgenic mice

目的建立BIGH3^(R555W)突变转基因角膜营养不良小鼠模型。方法构建以磷酸甘油酸激酶(phosphoglycerate kinase,PGK)为启动子的BIGH3(M)-IRES-EGFP重组质粒载体,通过原核显微注射方法将线性化、纯化后的外源质粒注射入C57小鼠受精卵中,胚胎移植入假孕受体母鼠输卵管内,获得子代小鼠。用PCR方法检测子代鼠尾基因组DNA,通过RT-PCR及Western印迹法检测BIGH3^(R555W)基因表达。结果 8只假孕小鼠共移植注射后的胚胎84枚,出生35只子代鼠,经PCR检测得到4只转基因阳性小鼠。RT-PCR和Western印迹法检测结果显示,BIGH3^(R555W)在转基因小鼠角膜表达量明显增多。结论通过显微注射法使外源基因BIGH3^(R555W)突变体在小鼠基因组中整合,成功建立了BIGH3^(R555W)突变转基因小鼠模型,该模型可为今后进一步研究角膜营养不良疾病的发病机制提供依据。

Objective To establish an animal model of BIGH3R555wmutation. Methods The full- length human BIGH3 with R555w mutation was inserted into the IRES-EGFP vector under the control of phosphoglycerate kinase （Pgk） promoter. The recombinant transgenic fragment was constructed, linearized and purified, then microinjected into fertilized mouse eggs. These eggs were transplanted into pseudopregant mice. The genotypes of transgenic founders were identified by PCR. The expression of mutant BIGI-13R555w was confirmed by RT-PCR and Western blotting. Results Transgenic C57 mice were obtained by microinjection. PCR results showed 4 out of 35 mice were integrated. The mutant BIGH3R555w was overexpressed in the cornea of transgenic mice compared to wild-type mice as demonstrated by RT-PCR and Western blotting analysis. Conclusion BIGH3P555w mutation transgenic mouse model has been successfully established by pronuclear microinjection, the transgenic mice would provide a animal model for study of pathogenesis of corneal dystrophy.