摘要
目的采用RNA干扰(RNA interference,RNA i)技术,通过构建人O6-甲基鸟嘌呤-DNA甲基转移酶(O6-meth-ylguan ine-DNA methyltransferase gene,MGMT)基因的特异性siRNA(small interfere RNA)真核表达载体,体外观察对人MGMT(hMGMT)基因的沉默作用。方法将合成的发卡样特异性hMGMT RNA干扰寡核苷酸序列插入真核表达载体并转染体外HelaS3细胞株,以半定量RT-PCR法检测hMGMT mRNA的表达水平,以MTT法检测转染后HelaS3细胞对BCNU的敏感性变化。结果成功构建MGMT siRNA的真核表达载体;所构建的载体能够特异性降低hMGMT mRNA的表达水平,转染后HelaS3细胞对BCNU的敏感性增加。结论该RNA干扰真核表达载体可以特异性干扰hMGMT基因的表达。
Objective To construct the small interfering RNA (siRNA) eukaryotic expression vector specific to human MGMT gene ( pRNATin-H1.2/Neo MGMT siRNA) to observe its silencing effect on MGMT gene in vitro. Methods The pRNATin-H1.2/Neo MGMT siRNA expression vector was constructed by gene recombination, then transfected into the cultured HelaS3 cells. Inhibitory effect of siRNAs was detected by semiquantitative RT-PCR. Results The pRNATin-H1.2/Neo MGMT siRNA expression vector was successfully constructed. Cells transfected with pRNATin-H1.2/Neo MGMT siRNA could obviously inhibit the expression level of MGMT gene. Conclusion The pRNATin-H1.2/Neo MGMT siRNA expression vector could inhibit the MGMT gene expression.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第11期1138-1140,共3页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30300289)~~