目的:构建CDHR2基因条件性敲除小鼠模型,为研究CDHR2基因的生物学功能提供条件.方法:构建CDHR2基因条件打靶载体,电转入小鼠胚胎干细胞(ES细胞),用G418和GANC筛选阳性细胞克隆.胚胎注射阳性ES细胞入小鼠囊胚,获得嵌合体小鼠.嵌合体小鼠与Cre小鼠交配获得条件性敲除小鼠.分别通过PCR方法和免疫组织化学方法对CDHR2的敲除结果进行验证.结果:成功构建打靶载体,并获得6个正确同源重组的ES细胞阳性克隆.阳性ES细胞克隆注射入C57BL/6J小鼠的囊胚中,获得5只嵌合鼠.嵌合鼠再与Flp小鼠交配,获得6只阳性F1代去Neo小鼠.去Neo小鼠与Cre小鼠杂交,获得CDHR2基因肠道特异性敲除小鼠.免疫组织化学检测表明,阳性小鼠肠道组织中的CDHR2基因被特异性敲除,而肾脏组织CDHR2基因的表达没有受到影响.结论:成功构建CDHR2基因肠道特异性敲除小鼠,为进一步研究CDHR2基因的作用奠定了基础.%Objective:To construct a CDHR2 conditional knockout mouse model, and to provide conditions for the study on biological function of CDHR2 gene.Methods:The conditional CDHR2 targeting vector was constructed and transfected into mouse embryonic stem (ES) cells by electroporation.The positive ES cells screened by G418 and GANC were microinjected into the blastocysts of C57BL/6J mice.The chimeric mice were obtained and then mated with Cre mice to obtain conditional CDHR2 knockout mice.The phenotype was analyzed by PCR and immunohistochemistry, respectively.Results:The conditional CDHR2 targeting vector was successfully constructed, with six positive clones of ES cells being obtained.The positive clones of ES cells were microinjected into blastocysts of C57BL/6J mice with 5 chimeric mice being obtained.The chimeric mice were mated with Flp mice, and 6 positive F1 generation mice without Neo gene were obtained.Finally, such mice were hybridized with Cre mice to obtain intestine-specific CDHR2 knockout mice.Immunohistochemistry assay showed that the CDHR2 gene in intestinal tracts of the positive mice was specifically knocked out.In contrast, the expression of CDHR2 in kidney tissue was not affected.Conclusions:The successful construction of intestine-specific CDHR2 knockout mice has laid the foundation for provides a basis for further functional study on CDHR2 gene.
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