过表达和敲除SLUG基因卵巢癌SKOV3细胞稳定株的建立
发布时间：2018-07-16 21:51
【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(P0.01)。(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。
[Abstract]:Objective to construct a stable cell line of ovarian cancer SKOV3 with lentivirus vector and modified CRISPRP / Cas9 gene editing system. Methods (1) HA-SLUG gene was amplified from plasmid pCMV-Myc-SLUG and ligated into lentivirus expression plasmid pLVX-IRES-Hyg. The recombinant expression vector pLVX-HA-SLUG was infected with SKOV3 cells by viral infection. The lentivirus containing pLVX-IRES-Hyg empty vector was used as negative control to detect the expression of sKOV3 by Western blotting. (2) A pair of lentiviruses capable of binding specifically to SKOV3 cells. The sgRNA of the SLUG gene was ligated into the vector PX462, respectively. A pair of recombinant plasmids were co-transfected into SKOV3 cells by liposome method. The stable cell lines were screened, and monoclonal cells (experimental group) were selected. The expression of sig protein was detected by Western blotting assay. A blank control group was set up without any treatment. Results (1) PCR amplification was carried out on the selected monoclonal bacteria, and the plasmid was extracted by PCR and double enzyme digestion. The results showed that the recombinant plasmid was successfully constructed after the two were verified correctly and identified by DNA sequencing. The relative expression levels of SLUG protein were 0.92 卤0.02,3.14 卤0.04 in the negative control group and 3.14 卤0.04 in the experimental group, respectively. The relative expression levels of the SLUG protein in the experimental group were higher than those in the negative control group (P0.01). (2) and the four monoclonal cells were 0.07 卤0.01-0.06 卤0.01 卤0.020.21 卤0.020.20 卤0.01, respectively, and 0.56 卤0.03in the blank control group. The relative expression of SLUG protein in the experimental group was lower than that in the blank control group (P 0.01). Conclusion the SKOV3 cell line with overexpression and stable knockout of SLUG gene was successfully constructed, which provides a cell model for further study of the role of SLUG in ovarian cancer.
【作者单位】： 天津医科大学;
【基金】：天津市自然科学基金资助项目(17JCQNJC12600)
【分类号】：R737.31
，
本文编号：2127776
[Abstract]:Objective to construct a stable cell line of ovarian cancer SKOV3 with lentivirus vector and modified CRISPRP / Cas9 gene editing system. Methods (1) HA-SLUG gene was amplified from plasmid pCMV-Myc-SLUG and ligated into lentivirus expression plasmid pLVX-IRES-Hyg. The recombinant expression vector pLVX-HA-SLUG was infected with SKOV3 cells by viral infection. The lentivirus containing pLVX-IRES-Hyg empty vector was used as negative control to detect the expression of sKOV3 by Western blotting. (2) A pair of lentiviruses capable of binding specifically to SKOV3 cells. The sgRNA of the SLUG gene was ligated into the vector PX462, respectively. A pair of recombinant plasmids were co-transfected into SKOV3 cells by liposome method. The stable cell lines were screened, and monoclonal cells (experimental group) were selected. The expression of sig protein was detected by Western blotting assay. A blank control group was set up without any treatment. Results (1) PCR amplification was carried out on the selected monoclonal bacteria, and the plasmid was extracted by PCR and double enzyme digestion. The results showed that the recombinant plasmid was successfully constructed after the two were verified correctly and identified by DNA sequencing. The relative expression levels of SLUG protein were 0.92 卤0.02,3.14 卤0.04 in the negative control group and 3.14 卤0.04 in the experimental group, respectively. The relative expression levels of the SLUG protein in the experimental group were higher than those in the negative control group (P0.01). (2) and the four monoclonal cells were 0.07 卤0.01-0.06 卤0.01 卤0.020.21 卤0.020.20 卤0.01, respectively, and 0.56 卤0.03in the blank control group. The relative expression of SLUG protein in the experimental group was lower than that in the blank control group (P 0.01). Conclusion the SKOV3 cell line with overexpression and stable knockout of SLUG gene was successfully constructed, which provides a cell model for further study of the role of SLUG in ovarian cancer.
【作者单位】： 天津医科大学;
【基金】：天津市自然科学基金资助项目(17JCQNJC12600)
【分类号】：R737.31
，
本文编号：2127776
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