应用Gateway技术构建pLenti6-Akt shRNA真核表达载体

目的 应用Gateway技术构建靶向人Akt基因的shRNA真核表达载体,转染肝癌细胞株HepG2,验证该载体能否下调Akt基因的mRNA水平.方法 以Akt基因为靶点设计两条具有短发卡结构的shRNA序列,经退火成互补双链后克隆入pENTR/U6入门表达载体(pENTR/U6-Akt1,pENTR/U6-Akt2);转化E.coli TOP10菌株,挑取阳性菌落进行菌落PCR和测序鉴定;利用Gateway技术进行LR位点特异性重组,将shRNA导入目的 表达载体pLenti6/Block-it DEST Vector,再行测序鉴定(pLenti6-Akt1,pLenti6-Akt2);将重组质粒转染肝癌细胞株HepG2,提取RNA,RT-PCR检测重组质粒对mRNA水平的抑制效果.结果 经菌落PCR和DNA测序鉴定:pENTR/U6-Akts入门载体和pLenti6-Akts目的 表达载体中插入片段的序列正确,RT-PCR结果表明将shRNA分别转染HepG2细胞后,Akt的mRNA表达受到明显抑制,其中1shRNA比2#shRNA抑制效果明显.结论 利用Gateway技术成功构建了靶向人Akt基因的shRNA表达载体,为以Akt基因为靶点的肝癌基因治疗方案奠定了实验基础.

Construction of pLenti6-Akt shRNA recombinant expression vector using Gateway technology

Objective To construct the expressing vector of small hairpin RNA(shRNA) targeting human Akt gene and detect its effectiveness of gene silencing in HepG2 cells(human hepatoma carcinoma cell line).Methods Complementary DNA oligonucleotides for shRNA expression,targeting Akt gene,were designed and chemically synthesized.The top and bottom strand oligos were annealed to generate a double-stranded oligonucleotide(ds oligo).The ds oligos was first cloned into pENTR/U6 entry expression vector(named pENTR/U6-Akt1,pENTR/U6-Akt2),and then cloned into pLenti6/Block-iT DEST Vector by LR recombination reaction(named pLenti6-Akt1,pLenti6-Akt2).In each step,PCR and sequencing analysis were performed to verify the constructs.After the verified plasmids were transfected into HepG2 cells,RT-PCR was performed to determine the mRNA level of Akt gene.Results PCR and sequencing analysis demonstrated that shRNA targeting Akt gene had been inserted at the expected site and the insertion sequence was perfectly corrected.The RT-PCR results showed that Akt expression in HepG2 was knockdown at mRNA level.The 1#shRNA had a better effect than 2#shRNA.Conclusion The shRNA expression vector targeting Akt gene was successfully constructed,which would facilitate further studies of Akt function and its application in gene therapy for hepatoma carcinoma.