利用CRISPR/Cas系统构建在XRCC6内源基因插入Flag标签序列的HeLa细胞系  

Flag-Tag Inserted at the Endogenous Gene of XRCC6 by CRISPR/Cas System in He La Cells

作　　者：李瑞花[1,2] 付汉江[1] 沈远[1] 钟一然 朱捷[1] 郑晓飞[1,2] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,放射生物学北京市重点实验室,北京100850 [2]安徽医科大学研究生学院,安徽合肥230032

出　　处：《生物技术通讯》2016年第1期7-11,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2012AA022501);国家重点基础研究发展计划(2013CB910801);国家自然科学基金(31270836,31370760)

摘　　要：目的:利用CRISPR/Cas系统在XRCC6基因5′端插入Flag标签序列,筛选XRCC6-Flag稳定表达的宫颈癌细胞(He La)株。方法:根据Gen Bank中XRCC6基因序列,设计XRCC6基因敲入的g RNA序列,构建入p Cas-Guide载体中,获得p Cas-XRCC6载体;设计XRCC6基因的同源臂序列,利用搭桥PCR方法将同源臂和Flag标签序列作为模板进行扩增,得到供体DNA片段,并将其克隆到p Back Zero-T表达载体上,获得p XRCC6-Donor载体;将上述2个载体共转染He La细胞,采用PCR、Western印迹等方法检测和筛选Flag标签序列插入XRCC6基因5′端的细胞株。结果:构建了p Cas-XRCC6和p XRCC6-Donor载体,筛选获得3株XRCC6-Flag稳定表达细胞株。结论:构建了XRCC6-Flag稳定细胞株,为研究XRCC6基因及其蛋白产物的生物学功能奠定了基础。Objective： To obtain the HeLa cell lines of stable expression XRCC6-FIag by inserting Flag tag at the 5＇ terminus of XRCC6 in the genome by CRISPR/Cas9 system. Methods： According to the sequence of XRCC6 in the GenBank, gRNA sequences were designed and inserted into the pCas-Gnide vectors, and pCas- XRCC6 vectors were obtained; the sequences of homologous arms of XRCC6 gene were designed. Donor DNA were amplified by PCR with homologous arms and Flag sequences as templates. Then donor DNA were cloned in- to pBackZero-T vectors and pXRCC6-Donor vectors were obtained; the two vectors were co-transfected into HeLa cells, PCR and Western blot were used to analyze the Flag tag which were inserted into 5＇ terminus of XRCC6 in the genome. Results： pCas-XRCC6 vectors and pXRCC6-Donor vectors were sucessfully constructed, and three XRCC6-Flag stable cell lines were obtained. Conclusion： XRCC6-Flag stable cell lines were generated successful- ly, which contributed to study the function of XRCC6 gene and its protein.

关 键 词：CRISPR/Cas系统 XRCC6基因 Flag标签 

分 类 号：Q784[生物学—分子生物学]