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RHBDD1的研究

Study of Epitope Tagging of Intramembrane Serine Protease RHBDD1 by Improved Somatic Cell Knock-in Approach

目的应用改良的体细胞基因敲入技术将标签序列靶向敲入目的基因。方法设计引物并构建RHBDD1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cre病毒感染和筛选获得RHBDD1内源敲入FLAG和SBP双标签的HCT116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cre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RHBDD1-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RHBDD1内源敲入FLAG和SBP双标签的HCT116结直肠癌细胞株。

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.