人HBV、HBx定向敲入p53位点大鼠胚胎干细胞株的建立

Establishment of human HBV/HBx-Knockin-p53 rat embryonic stem cell line

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 human HBV/HBx-knockin-p53 rat embryonic stem （ES） cell line, so as to lay a foundation for establishment of animal models. Methods First we constructed the targeting vector pKO-gHBV or pKO-X with HBV whole genome or X and p53 homologue arms by PCR and connection with common gene targeted vector pKO; then after linearized with Sal I the vector was transferred into rat ES cells through electroporation. And 2i ES culture medium containing puromycin was used for a three-cycle selection of puromycin resistant clones. Then PCR was used to screen the obtained positive clones; mycoplasma examination and karyotypic analysis were used to confirm targeted ES cell clones. Results We successfully obtained two targeted vectors： pKO-gHBV and pKO-X then after three-cycle drug-selection we obtained 2 pKO-X clones and 1 pKO-gHBV clone, which were confirmed by PCR, mycoplasma examination and karyotypic analysis. Conclusion We have successfully established human HBV/HBx-knockin-p53 rat ES cells, paving a way for future establishment of HBV/HBx- knockin-p53 rat model.