| 应用改良体细胞基因敲入技术内源标记跨膜丝氨酸蛋白酶RHBDDl的研究 |
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| 引用本文： | 宋伟,李尚泽,张慧慧,缪时英,王琳芳,张晓东,杜润蕾.应用改良体细胞基因敲入技术内源标记跨膜丝氨酸蛋白酶RHBDDl的研究[J].医学研究通讯,2011(11):21-24. |
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| 作者姓名： | 宋伟 李尚泽 张慧慧 缪时英 王琳芳 张晓东 杜润蕾 |
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| 作者单位： | [1]北京中国医学科学院基础医学研究所医学分子生物学国家重点实验室／北京协和医学院清华大学医学部基础学院,100005 [2]武汉大学生命科学学院,430072 |
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| 基金项目： | 国家重大研究计划基金资助项目（2011CB944302）;国家重点实验室专项基金资助项目（2060204）;中国医学科学院基础医学研究所院（所）长基金项目（2009PYl0） |
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| 摘 要： | 目的应用改良的体细胞基因敲入技术将标签序列靶向敲人目的基因。方法设计引物并构建RHBDDI的打靶载体，经过腺相关病毒包装、感染、新霉素筛选和鉴定等步骤获得含有新霉素抗性的阳性单克隆细胞株，最后通过ere病毒感染和筛选获得RHBDDI内源敲人FLAG和SBP双标签的HCTI16结直肠癌细胞株。结果经过包装病毒感染与筛选验证后得到6个打靶阳性克隆；进一步经ere病毒感染与筛选后得到2株去除新霉素抗性标记的阳性细胞克隆；通过Western blotting实验证明RHBDDl-FLAG-SBP融合蛋白的表达。结论通过改良的体细胞基因敲入技术成功构建跨膜丝氨酸蛋白酶RHBDDl内源敲入FLAG和SBP双标签的HCTll6结直肠癌细胞株。
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| 关 键 词： | 基因敲入 RHBDDl 同源重组 |
Study of Epitope Tagging of Intramembrane Serine Protease RHBDD1 by Improved Somatic Cell Knock - in Approach. |
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| Song Wei,Li Shangze,Zhang Huihui,Miao Shiying,Wang Linfang,Zhang Xiaodong,Du Runlei.Study of Epitope Tagging of Intramembrane Serine Protease RHBDD1 by Improved Somatic Cell Knock - in Approach.[J].Bulletin of Medical Research,2011(11):21-24. |
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| Authors: | Song Wei Li Shangze Zhang Huihui Miao Shiying Wang Linfang Zhang Xiaodong Du Runlei |
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| Institution: | , National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China |
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| Abstract: | Objective To apply improved somatic cell knock - in methods for endogenous tagging of target gene. Methods We designed primers and constructed targeting vector of RHBDD1. After adeno - associated virus （AAV） packaging, targeting, screening and verification, positive cell clones with neomycin resistance were obtained. Finally, we excised neomycin marker with adenovirus expressing ere- recombinase to get HCTll6 cell line expressing FLAG and SBP tagged RHBDD1. Results Six positive clones were identified through AAV infection, screening and verification. Two positive clones which neomycin marker was excised were obtained by cre adenovi- rus infection and screening. The expression of RHBDD1 - FLAG - SBP fusion protein was verified by Western blotting analysis. Conclusion We successfully constructed colorectal cancer cell line expressing FLAG and SBP tagged RHBDDlendogenously by improved somatic cell knock -in approach. |
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| Keywords: | Knock - in RHBDD1 Hoialologous recombination |
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