RNA干扰技术抑制内源性绿色荧光蛋白的表达  

Inhibition of Intrinsic Expression of GFP by RNA Interferencing

作　　者：陈维贤[1] 黄爱龙[1] 闫歌[1] 唐霓[1] 张娟[1] 陈娟[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所感染性疾病重点实验室,重庆400016

出　　处：《生物技术通报》2005年第6期65-68,87,共5页Biotechnology Bulletin

基　　金：国家自然科学基金(30400374)

摘　　要：目的建立稳定表达绿色荧光蛋白(GFP)的细胞株;构建短发夹RNA(shRNA)表达质粒并观察其对内源性GFP的抑制作用。方法转染pEGFP-N1至HepG2细胞,利用G418筛选获得稳定表达GFP的细胞株(HepG2.GFP);设计合成针对GFP基因的siRNA对应的DNA片段,插入转录载体pTZU6+1,构建shRNA表达载体pSHGFP,转染HepG2.GFP,荧光显微镜观察细胞荧光强度,以western blot检测GFP蛋白水平,以RT-PCR检测mRNA水平。结果利用PCR方法从HepG2.GFP细胞基因组DNA中检测到GFP基因;pSHGFP能够显著抑制该细胞中GFP的表达。结论GFP基因成功整合至HepG2细胞基因组中,pSHGFP能够显著抑制内源性GFP的表达,该系统能够用于RNA干扰机制等研究中。Objectives To construct a cell strain expressing GFP stably and construct the plasmid containing short hairpin RNA of GFP to suppress the intrinsic expression of GFP. Methods transfected the HepG2 cells with pEGFP-N1 and then screening with G418 to get a cell strain（HepG2. GFP） expressing GFP stably; The plasmid of pSHGFP was constructed by inserting the reverse repeated motif of GFP into pTZU6 ＋1. Transfected the HepG2. GFP with pSHGFP and then the fluorescence was observed by fluorescent microscope and the protein and mRNA level of GFP was detected by wetern blot and RT-PCR,respectively. Results The GFP gene was detected in the genome DNA of HepG2. GFP by PCR; pSHGFP suppressed the intrinsic expression of GFP significantly. Conclusion A cell strain expressing GFP stably was constructed successfully and the expression of GFP can be suppressed by the short hairpin RNA efficiently.

关 键 词：RNA干扰 绿色荧光蛋白 短发夹状RNA 

分 类 号：Q78[生物学—分子生物学]