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目的建立人HBV、HBx定向敲入p53位点的大鼠胚胎干细胞株,为建立相关动物模型奠定基础。方法通过PCR方法扩增出人p53基因上下游同源臂、HBV全基因组序列、HBV的X蛋白编码基因(HBx),将其插入本实验室自主构建的基因打靶通用载体pKO中,分别构建pKO-gHBV及pKO-X打靶载体。载体经SalⅠ酶切线性化后,电转入状态良好的大鼠胚胎干细胞株中,经3轮药物筛选,获得单细胞克隆。通过PCR技术筛选获得阳性细胞克隆,并进行支原体污染鉴定和核型分析。结果成功构建pKO-gHBV及pKO-X打靶载体;电转大鼠胚胎干细胞株,经3轮药物筛选,分别挑取若干细胞克隆,其中2个pKO-X细胞克隆和1个pKO-gHBV细胞克隆经PCR鉴定、支原体检测和核型分析确定结果正确。结论成功建立了人HBV、HBx定向敲入p53位点的大鼠胚胎干细胞株,为后续建立动物模型奠定了基础。
OBJECTIVE: To establish a rat embryonic stem cell line targeting HBV and HBx targeting p53, which will lay the foundation for the establishment of related animal models. Methods The upstream and downstream homologous arms of human p53 gene, whole genome sequence of HBV and the gene encoding HBV X protein (HBx) were amplified by PCR and inserted into the gene targeting universal vector pKO independently constructed in our laboratory to construct pKO -gHBV and pKO-X targeting vector. The vector was linearized by Sal I digestion and electroporated into a rat embryonic stem cell line of good condition. After three rounds of drug screening, the single cell clone was obtained. The positive cell clones were screened by PCR and the mycoplasma contamination and karyotype analysis were performed. Results After successful construction of pKO-gHBV and pKO-X targeting vector, electroporation rat embryonic stem cell lines were selected after three rounds of drug screening, and several cell clones were picked out. Two pKO-X cell clones and one pKO-gHBV cell clone PCR identification, mycoplasma detection and karyotype analysis confirm the result is correct. Conclusion The rat embryonic stem cell line with human HBV and HBx targeted to p53 site was established successfully, which laid the foundation for the subsequent establishment of animal model.