山东医药2017，Vol.57Issue(42)：1-4,4.DOI:10.3969/j.issn.1002-266X.2017.42.001
过表达和敲除SLUG基因卵巢癌SKOV3细胞稳定株的建立
Establishment of ovarian cancer stable SKOV3 cell lines with overexpression and knockout of SLUG protein
摘要
Abstract
Objective To establish the ovarian cancer stable SKOV3 cell lines with overexpression and knockout of SLUG protein by lentiviral vector and modified CRISPR/Cas9 gene editing system,respectively. Methods ①The HA-SLUG gene cloned from plasmid pCMV-Myc-SLUG was inserted into lentiviral vector pLVX-IRES-Hyg. The recombinant plasmid pLVX-HA-SLUG was used to infect SKOV3 cells (experimental group). The lentivirus of pLVX-IRES-Hyg empty vector was prepared as the negative control group under the same conditions. The expression of SLUG protein was detected by Western blotting. ② A pair of sgRNAs specifically binding to SLUG gene was inserted into the vector PX462. The pair of recombinant plasmids was transfected into SKOV3 cells by Lipofectamine method. We screened the stable cells,and se-lected monoclonal cells as the experimental group. The expression of SLUG protein was detected by Western blotting. Meanwhile,and the blank control group without any treatment was set up. Results ①PCR was performed on the extracted monoclonal bacteria,and the positive ones were extracted and identified by PCR and double digestion. The results showed that the recombinant plasmid was successfully constructed. The expression levels of SLUG protein in the negative control group and the experimental group were 0. 92 ± 0. 02 and 3. 14 ± 0. 04,respectively. The expression of SLUG protein in the experimental group was higher than that in the negative control group (P<0. 01). ②The four positive monoclonal cells were measured. The relative expression levels of SLUG protein were 0. 07 ± 0. 01,0. 06 ± 0. 01,0. 21 ± 0. 02 and 0. 20 ±0. 01,respectively,and that in the blank control group was 0. 56 ± 0. 03. The relative expression of SLUG protein in the experimental group was lower than that in the blank control group(all P<0.01).Conclusion Two stable SKOV3 cell lines with SLUG protein overexpression and knockout were constructed successfully,which provided the essential cell mod-els for further study about the role of SLUG protein in human ovarian cancer.关键词
SLUG基因/慢病毒载体/SKOV3细胞/卵巢癌/细胞模型Key words
SLUG gene/lentiviral vector/SKOV3 cells/ovarian carcinoma/cell model分类
医药卫生引用本文复制引用
雷静,赵然,高茹,辛灵彪,刘欣..过表达和敲除SLUG基因卵巢癌SKOV3细胞稳定株的建立[J].山东医药,2017,57(42):1-4,4.基金项目
天津市自然科学基金资助项目(17JCQNJC12600). （17JCQNJC12600）
