广西医学2018，Vol.40Issue(8)：934-938,943,6.DOI:10.11675/j.issn.0253-4304.2018.08.19
过表达和敲除沉默信息调节蛋白6质粒的构建及稳定肺癌A549细胞株的筛选
Plasmid construction of overexpression and knock-out of silent information regulator protein 6 and screening of stable lung cancer A549 cell lines
摘要
Abstract
Objective To develop the overexpression and knockout of silent information regulator protein 6(SIRT6) gene plasmids, and to screen the overexpression and knockout of stable lung cancer A549 cell lines.Methods The SIRT6 gene sequence was amplified using PCR technique,then ligated to the lentivirus plasmid CD513B via intermediate plasmid pSK,and accepted lentivirus packaging after identification.The A549 cells infected with the supernatant containing lentivirus particles were collected,then were treated with puromycin to screen out the stable cell lines of SIRT6 gene overexpressed,and Western blot was used to detect the expression level of SIRT6 protein. SIRT6 gene knockout was performed using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated 9(Cas9) gene editing technology,and the knockout target sites were identified as the exon 1 and the exon 2 of SIRT6 gene,then the SIRT6-small guide RNA(sgRNA) of the exon 1 and the exon 2 was constructed. A549 cells were transfected after SIRT6-sgRNA was ligated to plasmid CD513B of Cas9,and were treated with puromycin to screen out the successfully transfected cells.The SIRT6 knockout A549 cell lines were screened by infinite dilution method,and the expression of SIRT6 protein was detected by Western blot.Results The fluorescence efficiency of lentivirus-infected A549 cells was nearly 100%.Western blot analysis showed that overexpression of SIRT6 protein in A549 cells of SIRT6 gene resulted in high protein expression.DNA sequencing revealed that there was a frameshift mutation in the knockout SIRT6 A549 cell line,and no SIRT6 protein expression was observed by Western blot.Conclusion Overexpression and knockout of SIRT6 gene plasmids are successfully constructed,in which stable lung cancer A549 cell lines are screened out.关键词
沉默信息调节蛋白6/基因过表达/基因敲除/肺癌A549细胞Key words
Silent information regulator protein 6/Gene overexpression/Gene knockout/Lung cancer A549 cells分类
生物科学引用本文复制引用
吴爱鲁,梁世雄,宋晓,付佳美,周子洋,宋晓婷,赵志鹏,张春燕..过表达和敲除沉默信息调节蛋白6质粒的构建及稳定肺癌A549细胞株的筛选[J].广西医学,2018,40(8):934-938,943,6.基金项目
国家自然科学基金(81060173) （81060173）
上海市科学技术委员会科研计划(15411962400) （15411962400）
广西医疗卫生适宜技术研究与开发课题(S201417-01) （S201417-01）
西藏自治区高校青年教师创新支持计划(QCZ2016-34) （QCZ2016-34）
