| 摘要: |
| 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。 |
| 关键词: 乙型肝炎病毒 HBx基因 p53基因 胚胎干细胞 |
| DOI：10.3724/SP.J.1008.2012.00945 |
| 投稿时间：2012-05-16修订日期：2012-06-29 |
| 基金项目:国家杰出青年科学基金(30925037), 国家重点基础研究发展计划(“973”计划，2010CB529900-G). |
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| Establishment of human HBV/HBx-Knockin-p53 rat embryonic stem cell line |
| WANG Yan1,YE Zhen-long2,JIN Hua-jun1,LIU Tao1,LI Lin-fang1,QIAN Qi-jun1,2* |
(1. Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital,Second Military Medical University, Shanghai 200438, China;
2. Xinyuan Institute of Medicine and Biotechnology, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China
*Corresponding author.) |
| Abstract: |
| ObjectiveTo establish human HBV/HBx-knockin-p53 rat embryonic stem (ES) cell line, so as to lay a foundation for establishment of animal models. MethodsFirst 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 SalⅠ 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. ResultsWe 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. ConclusionWe have successfully established human HBV/HBx-knockin-p53 rat ES cells, paving a way for future establishment of HBV/HBx-knockin-p53 rat model. |
| Key words: hepatitis B virus HBx gene p53 gene embryonic stem cells |
