X-盒结合蛋白1条件性基因敲除小鼠模型的建立

Establishment of conditional knockout mouse model of Xbp1 gene

目的建立可进行条件性敲除X-盒结合蛋白1(Xbp1)基因的flox杂合子小鼠。方法通过ET-clone的方法构建基于cre-loxp系统和FLP-frt系统的条件性基因打靶载体。载体线性化后电转JM8A3 ES细胞,经G418和Ganc筛选获得抗性ES细胞克隆。经长片段PCR鉴定获得正确同源重组的阳性克隆。阳性ES细胞经克隆扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合鼠,筛选出高比例嵌合小鼠与野生型C57BL/6J小鼠交配后获得阳性F1代小鼠,并分别进行PCR和测序鉴定。结果成功构建打靶载体,共获得144个抗性ES细胞,克隆经长片段PCR鉴定,共获得4个正确同源重组的阳性克隆。获得4只阳性F1代小鼠,经测序鉴定正确。Xbp1基因flox杂合子小鼠表型无明显异常。结论成功构建了条件性敲除Xbp1基因的flox杂合子小鼠,为未来研究器官或组织特异性的Xbp1生物学作用奠定了基础。

Objective To establish the flox-heterozygous mouse model in which the X-box-binding protein 1(Xbp1)gene was conditionally knocked out.Methods The conditional gene-targeting vector,based on the cre-loxp system and the FLP-frt system,was constructed with the ET-clone method.The vectors were linearized and electroporated into the JM8A3 embryonic stem(ES)cells.The resistant ES cell clones were obtained through G418 and Ganc screening.The positive clones with correct homologousrecombination were obtained through identification with the long-range PCR method.Positive ES cells were clonally expanded,and injected into the blastocysts of C57 BL/6 J mice to obtain the chimeric mice.The high-rate chimeric mice were screened,and mated with the wild-type C57 BL/6 J mice to obtain the positive F1 mice,which were identified by PCR and sequencing.Results The targeting vectors were successfully constructed.A total of 144 resistant ES cell clones were obtained and identified by long-range PCR.Four positive homologous recombination clones were obtained.Four positive F1 mice were obtained and confirmed by sequencing.There was no obvious abnormality in the phenotypes of the Xbp1 gene flox-heterozygous mice.Conclusion The conditional knockout flox-heterozygous mouse model of Xbp1 gene was successfully established,which laid a foundation for further studies of the organ or tissue-specific biological effects of Xbp1 gene.