| 摘要: |
| ［摘要］ 目的 利用CRISPR/Cas9技术敲除SiHa细胞中的卵泡抑素样蛋白1（FSTL1）基因，构建FSTL1基因敲除的SiHa细胞稳定株。方法 构建sgRNA-FSTL1重组质粒并包装成慢病毒。收集病毒液并感染SiHa细胞，使用嘌呤霉素筛选FSTL1基因敲除的SiHa细胞稳定株；采用RT-qPCR和Western blot检测SiHa细胞稳定株FSTL1 mRNA和蛋白的表达情况。结果 FSTL1-sgRNA慢病毒构建成功并筛选出FSTL1基因敲除的SiHa细胞稳定株；FSTL1的mRNA和蛋白表达水平在SiHa细胞稳定株中的表达量均比对照组低,差异有统计学意义（P<0.05）。结论 利用CRISPR/Cas9技术成功构建FSTL1基因敲除的SiHa细胞稳定株。 |
| 关键词: CRISPR/Cas9 卵泡抑素样蛋白1 基因敲除 SiHa细胞 |
| DOI：10.3969/j.issn.1674-3806.2019.08.06 |
| 分类号:R 331 |
| 基金项目:国家自然科学基金资助项目（编号：81660434） |
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| Construction of FSTL1 gene knockout SiHa cell stable strain using CRISPR/Cas9 technology |
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ZHANG Hong, HU Xiao-xia, LUO Ruo-yu, et al.
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Department of Gynaecology, Renmin Hospital of Wuhan University, Hubei 430000, China
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| Abstract: |
| ［Abstract］ Objective To knock out follistatin-like protein 1(FSTL1) gene in SiHa cells using CRISPR/Cas9 technology and to construct SiHa cell stable strain with knocking out FSTL1 gene. Methods The sgRNA-FSTL1 recombinant plasmid was constructed and was packaged into lentivirus. The virus solution was collected and infected with SiHa cells, and SiHa cell stable strain with knocking out FSTL1 gene was screened by puromycin. The expressions of FSTL1 mRNA and protein in SiHa cell stable strain were detected by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot. Results FSTL1-sgRNA lentivirus was successfully constructed and the SiHa cell stable strain with knocking out FSTL1 gene was successfully screened. The expressions of FSTL1 mRNA and protein in the SiHa cell stable strain group were significantly lower than those in the control group（P<0.05）. Conclusion SiHa cell stable strain with knocking out FSTL1 gene is successfully constructed using CRISPR/Cas9 technology. |
| Key words: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9（CRISPR/Cas9） Follistatin-like protein 1(FSTL1) Gene knockout SiHa cell |
