慢病毒介导的靶向干扰MEX3A基因膀胱癌稳定细胞株的建立

发布时间：2018-05-09 14:18

本文选题：MEXA基因 + 膀胱癌　；参考：《中国医科大学学报》2017年12期

【摘要】：目的构建针对MEX3A基因的shRNA慢病毒载体,建立稳定干扰MEX3A基因表达的膀胱癌细胞株。方法采用实时PCR检测膀胱癌细胞株中MEX3A基因的表达;应用GV115质粒构建重组靶向MEX3A基因的shRNA慢病毒载体,鉴定及测序合格后,与包装载体共转染293T细胞产生慢病毒颗粒,病毒滴度测定后转染膀胱癌细胞,经抗生素筛选建立稳定表达si RNA的细胞株,实时PCR检测敲减效率。结果 MEX3A基因在膀胱癌5637和T24细胞中均有表达,且5637的表达量高于T24;鉴定及测序结果显示成功构建MEX3A-shRNA慢病毒载体,包装后病毒滴度较高,实时PCR结果表明慢病毒转染5637细胞后能稳定抑制MEX3A基因的表达。结论应用慢病毒介导的RNA干扰技术可成功建立稳定抑制MEX3A基因表达的细胞株。
[Abstract]:Objective to construct a shRNA lentivirus vector targeting MEX3A gene and to establish bladder cancer cell line which interferes with the expression of MEX3A gene. Methods the expression of MEX3A gene in bladder cancer cell line was detected by real-time PCR, and the recombinant shRNA lentivirus vector targeting MEX3A gene was constructed by using GV115 plasmid. After qualified identification and sequencing, lentivirus particles were co-transfected into 293T cells with packaging vector. After transfection into bladder cancer cells by titer assay, stable expression of si RNA was established by antibiotic screening, and knockout efficiency was detected by real time PCR. Results MEX3A gene was expressed in 5637 and T24 cells of bladder cancer, and the expression of 5637 was higher than that of T24.The identification and sequencing showed that MEX3A-shRNA lentivirus vector was successfully constructed, and the titer of MEX3A-shRNA lentivirus was higher after packaging. The results of real-time PCR showed that lentivirus transfected 5637 cells could stably inhibit the expression of MEX3A gene. Conclusion Lentivirus-mediated RNA interference technique can be successfully used to establish a cell line that stably inhibits the expression of MEX3A gene.
【作者单位】：中国医科大学附属盛京医院超声科;
【基金】：国家自然科学基金(81371552)
【分类号】：R737.14

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