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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 a BIGH3 ~ (R555W) mutant transgenic corneal dystrophy mouse model. Methods The recombinant plasmids of BIGH3 (M) -IRES-EGFP with phosphoglycerate kinase (PGK) as promoter were used to construct linearized and purified exogenous plasmids into C57 mice by pronuclear microinjection Fertilized eggs, the embryos were implanted into the fallopian tube of the female fetus of pseudopregnant recipients to obtain offspring mice. The tail genomic DNA of offspring rats was detected by PCR and the expression of BIGH3 ~ (R555W) gene was detected by RT-PCR and Western blot. Results Totally 84 embryos were implanted in 8 pseudopregnant mice and 35 offspring were born. Four transgenic mice were obtained by PCR. The results of RT-PCR and Western blotting showed that the expression of BIGH3 ~ (R555W) in the cornea of transgenic mice was significantly increased. Conclusion The BIGH3 ~ (R555W) mutant transgenic mouse model was successfully established by microinjection of the BIGH3 ~ (R555W) mutant in the mouse genome. This model can be used to further investigate the pathogenesis of corneal dystrophy Pathogenesis provide the basis.