Generation of transient receptor potential vanilloid 6 gene knockout mouse model

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 analysis showed 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.