利用CRISPR/Cas9技术构建DOC-1R基因敲除的HEK-293稳转细胞系  

Establishment of DOC-1R knockout HEK-293 stable cell line by CRISPR/Cas9 technology

作　　者：王世全 郭莹莹 沈飞 高锦兰[1] 邢雪莎[1] 王述森[1] 罗阳[1] 刘琦[1] WANG Shi-quan;GUO Ying-ying;SHEN Fei;GAO Jin-lan;XING Xue-sha;WANG Shu-sen;LUO Yang;LIU Qi(The Research Center for Medical Genomics,the Key Laboratory of Medical Cell Biology,Ministry of Education of China,School of Life Science,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学医学基因组学教研室,教育部医学细胞生物学重点实验室,生命科学学院,辽宁沈阳110122

出　　处：《解剖科学进展》2020年第3期339-342,共4页Progress of Anatomical Sciences

基　　金：国家自然科学基金(81571440,81400851);辽宁省高等学校基本科研项目(LZDK201703)。

摘　　要：目的利用CRISPR/Cas9技术敲除人胚肾(human embryonic kidney cell,HEK-293)细胞中DOC-1R,构建DOC-1R敲除的HEK-293稳转细胞系,用于进一步讨论DOC-1R的生物学功能。方法根据CRISPR/Cas9设计原则,设计向导RNA(single-guide RNA,sgRNA),构建表达载体,对sgRNA测序并转染包装HEK-293T收集上清液测定病毒滴度。前期用Cas9-puro慢病毒感染HEK-293,用已制备的DOC-1R慢病毒感染稳转Cas9的HEK-293,72 h后显微镜下观察HEK-293表达红色荧光蛋白,并用Western blot检测HEK-293中DOC-1R的表达以确定沉默效果。结果测序显示插入的sgRNA序列正确,表达载体成功构建,显微镜下观察到,90%以上HEK-293表达红色荧光蛋白,HEK-293中DOC-1R蛋白表达减少,确定了DOC-1R敲除效果最明显的序列为GCCTACCTATGCTGGCAGCA。结论利用CRISPR/Cas9技术成功构建DOC-1R基因敲除的细胞系,为进一步研究该基因的功能奠定了基础。Objective To construct HEK-293 cell line stably silencing DOC-1R expression by CRISPR/Cas9 genomic editing technology for further investigation of the biological function of DOC-1R.Methods According to the design principle of CRISPR/Cas9,the small guide RNAs were designed and the lentiviral expression vectors were constructed by recombinant technology.After sequencing,lentivirus particles was produced from HEK-293T followed by the determination of virus titer.Furthermore,lentivirus particles carring sgRNAs of DOC-1R were infected HEK-293 cells which have been infected with Cas9 lentivirus particles based on the protocol of manufacture previously.72 hours positioninfection,red fluorescent protein was observed under fluorescent microscope,and the expression of DOC-1R in HEK-293 cells was detected by Western blot to determine the silencing effect.Results DNA sequencing demonstrated that the sequences of inserted sgRNAs were correct and the recombinant vectors were successfully constructed.Under the microscope,more than 90%HEK-293 cells expressed red fluorescent protein after being infected by Lenti-DOC-1R-sgRNAs lentivirus.The expression level of DOC-1R protein in HEK-293 cells was decreased significantly,and the most efficient target for silencing the expression of DOC-1R was GCCTACCTATGCTGGCAGCA.Conclusion The DOC-1R knockout cell line was successfully constructed using CRISPR/Cas9 technology,which wound provide experimental basis for the further study of the DOC-1R biological function.

关 键 词：DOC-1R 基因敲除 CRISPR/Cas9技术 慢病毒 

分 类 号：Q782[生物学—分子生物学]