RNA干扰技术抑制内源性绿色荧光蛋白的表达

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。

Inhibition of Intrinsic Expression of GFP by RNA Interferencing

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.