【摘要】：目的利用慢病毒載體和改良的CRISPR/Cas9基因編輯系統(tǒng)分別構(gòu)建過表達(dá)和敲除SLUG基因的卵巢癌SKOV3細(xì)胞穩(wěn)定株。方法 (1)以質(zhì)粒p CMV-Myc-SLUG為模板擴(kuò)增出HA-SLUG基因,將其連接到慢病毒表達(dá)質(zhì)粒p LVX-IRES-Hyg中。將此重組表達(dá)載體p LVX-HA-SLUG通過病毒感染法感染SKOV3細(xì)胞(實(shí)驗(yàn)組),以相同條件制備p LVX-IRES-Hyg空載體的慢病毒作為陰性對照,Western blotting法檢測兩組SLUG蛋白表達(dá)。(2)將一對能特異結(jié)合SLUG基因的sgRNA分別連接到載體PX462中,獲得的一對重組質(zhì)粒通過脂質(zhì)體法共轉(zhuǎn)染SKOV3細(xì)胞,篩選穩(wěn)定株,挑取單克隆細(xì)胞(實(shí)驗(yàn)組),用Western blotting法檢測SLUG蛋白表達(dá),另設(shè)空白對照組,不加任何處理。結(jié)果 (1)對挑取的單克隆菌液進(jìn)行PCR擴(kuò)增,陽性者提取質(zhì)粒進(jìn)行PCR和雙酶切,兩者驗(yàn)證正確后進(jìn)行DNA測序鑒定,結(jié)果證實(shí)重組質(zhì)粒構(gòu)建成功。陰性對照組與實(shí)驗(yàn)組SLUG蛋白相對表達(dá)量分別為0.92±0.02和3.14±0.04,實(shí)驗(yàn)組SLUG蛋白表達(dá)高于陰性對照組(P0.01)。(2)挑取4個(gè)單克隆細(xì)胞進(jìn)行測定,SLUG蛋白的相對表達(dá)量分別為0.07±0.01、0.06±0.01、0.21±0.02、0.20±0.01,空白對照組為0.56±0.03。實(shí)驗(yàn)組SLUG蛋白相對表達(dá)量低于空白對照組(P均0.01)。結(jié)論過表達(dá)和穩(wěn)定敲除SLUG基因的SKOV3細(xì)胞株構(gòu)建成功,為進(jìn)一步探討SLUG在卵巢癌中的作用提供了細(xì)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 鹵0.02,3.14 鹵0.04 in the negative control group and 3.14 鹵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 鹵0.01-0.06 鹵0.01 鹵0.020.21 鹵0.020.20 鹵0.01, respectively, and 0.56 鹵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.
【作者單位】： 天津醫(yī)科大學(xué);
【基金】：天津市自然科學(xué)基金資助項(xiàng)目(17JCQNJC12600)
【分類號】：R737.31
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本文編號：2127776