瞬时性受体电位通道香草酸受体亚型6基因敲除小鼠模型的建立

Generation of transient receptor potential vanilloid 6 gene knockout mouse model

目的建立瞬时性受体电位通道香草酸受体亚型6(Trpv6)基因敲除小鼠模型,为在体研究Trpv6的生物学功能及其与骨代谢关系奠定基础。方法从Ensembl数据库中获得小鼠Trpv6基因组序列。设计基因敲除策略,构建基因敲除载体pBR322-MK-Trpv6。以电穿孔方法将基因敲除载体导入胚胎多能干细胞(ES细胞),用G418和Ganciclovoir进行正负筛选,获得双抗性克隆。PCR鉴定出正确同源重组的ES细胞克隆。将正确同源重组的ES细胞注射到C57BL/6J小鼠的囊胚中,获得嵌合体小鼠。挑选嵌合率在50%的雄鼠与C57BL/6J小鼠交配,获得的灰鼠经PCR鉴定为杂合子小鼠。杂合子小鼠交配后获得纯合子小鼠。结果成功构建了打靶载体pBR322-MK-Trpv6。电穿孔后,共获得24个正确同源重组的克隆,同源重组效率为25%。同源重组的克隆经显微注射后,共获得4只嵌合率大于50%的雄鼠。嵌合鼠与C57BL/6J小鼠交配,获得57只来源于ES细胞的灰鼠,PCR鉴定证实其中17只为杂合子小鼠,阳性率为29.8%。杂合子小鼠交配获得纯合子小鼠。经蛋白质印迹分析证实纯合子小鼠无Trpv6蛋白的表达。结论成功建立了Trpv6基因敲除小鼠模型,其中纯合子小鼠未出现胚胎致死现象。

Objective To create transient receptor potential vanilloid 6 （Trpv6） gene knockout mouse model, so as to pave a way for further research of its biological function and its role in bone metabolism in vivo. Methods Mouse genomic DNA sequence of Trpv6 gene was obtained from Ensembl database. Trpv6 gene knockout vector （pBR322-MK-Trpv6） was constructed. Trpv6 knockout vector was transferred into the embryonic stem （ES） cells by electroporation and screening of both G418 and Ganciclovoir resistant clones were performed routinely. The homologous recombined ES cell clones were identified by PCR. The correct homologously recombined ES cells were microinjected into C57BL/6J mouse blastocysts to obtain chimera mouse. Male mice with a chimera rate of 50% were mated with C57BL/6J female mice; the offsprings with gray fur were obtained, which were identified as heterozygote mice by PCR. Heterozygote mice were intercrossed to generate homozygote mice. Results Targeting vector pBR322-MK-Trpv6 were successfully constructed. A total of 24 correct homologously recombined clones were gained after electroporation. The efficiency of homologous recombination was 25%. Four male mice with a chimera rate of more than 50% were acquired after homologously recombined clones through microinjection. After the chimera mice were mated with C57BL/6J mice, 57 grey-fur mice originated from ES cell were gained, including 17 （29.8%） with heterozygous genotype. Heterozygote mice were intercrossed to generate homozygote mice. Western blotting analysisshowed no Trpv6 protein expression in homozygote mice. Conclusion We have successfully established Trpv6 gene knockout mouse model, and there is no embryonic lethality in homologous mutant mice.