摘要
目的利用慢病毒载体和改良的CRISPR/Cas9基因编辑系统分别构建过表达和敲除SLUG基因的卵巢癌SKOV3细胞稳定株。方法 (1)以质粒p CMV-Myc-SLUG为模板扩增出HA-SLUG基因,将其连接到慢病毒表达质粒p LVX-IRES-Hyg中。将此重组表达载体p LVX-HA-SLUG通过病毒感染法感染SKOV3细胞(实验组),以相同条件制备p LVX-IRES-Hyg空载体的慢病毒作为阴性对照,Western blotting法检测两组SLUG蛋白表达。(2)将一对能特异结合SLUG基因的sgRNA分别连接到载体PX462中,获得的一对重组质粒通过脂质体法共转染SKOV3细胞,筛选稳定株,挑取单克隆细胞(实验组),用Western blotting法检测SLUG蛋白表达,另设空白对照组,不加任何处理。结果 (1)对挑取的单克隆菌液进行PCR扩增,阳性者提取质粒进行PCR和双酶切,两者验证正确后进行DNA测序鉴定,结果证实重组质粒构建成功。阴性对照组与实验组SLUG蛋白相对表达量分别为0.92±0.02和3.14±0.04,实验组SLUG蛋白表达高于阴性对照组(P<0.01)。(2)挑取4个单克隆细胞进行测定,SLUG蛋白的相对表达量分别为0.07±0.01、0.06±0.01、0.21±0.02、0.20±0.01,空白对照组为0.56±0.03。实验组SLUG蛋白相对表达量低于空白对照组(P均<0.01)。结论过表达和稳定敲除SLUG基因的SKOV3细胞株构建成功,为进一步探讨SLUG在卵巢癌中的作用提供了细胞模型。
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（1） The HASLUG gene cloned from plasmid p CMV-Myc-SLUG was inserted into lentiviral vector p LVX-IRES-Hyg.The recombinant plasmid p LVX-HA-SLUG was used to infect SKOV3 cells（experimental group）.The lentivirus of p LVX-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 selected 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（1）PCR 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（P0.01）.（2）The 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 P0.01）.Conclusion Two stable SKOV3 cell lines with SLUG protein overexpression and knockout were constructed successfully,which provided the essential cell models for further study about the role of SLUG protein in human ovarian cancer.
出处
《山东医药》
CAS
北大核心
2017年第42期1-4,共4页
Shandong Medical Journal
基金
天津市自然科学基金资助项目(17JCQNJC12600)
