shRNA表达载体特异性抑制人脑胶质瘤细胞BT325绿色荧光蛋白基因的表达

Specific inhibition of green fluorescence protein by small hairpin RNA (shRNA) in human glioma BT325 cell lines

目的探讨绿色荧光蛋白(GFP)特异性发夹结构RNA (shRNA)对人类胶质瘤BT325细胞系GFP基因表达的抑制作用。方法设计并合成针对GFP基因的shRNA序列,与报告基因质粒pEGFP-N1共转染,选择最佳转染比例,荧光显微镜下观察GFP表达的效率和对GFP的抑制效应。结果质粒pSIREN-RetroQ-RNAi经双酶切得到6 kb和100 bp条带,结合测序验证,与实验设计的shRNA长度相符。质粒DNA与XP-1转染比例为1∶2.5时GFP表达最强。BT325细胞干扰48 h时GFP表达显著减少。结论BT325细胞系中存在RNA干扰现象,且干扰效果特异、稳定;本实验为研究基因功能提供了新的策略。

Objective To investigate whether the green fluorescence protein (GFP) specific short hairpin RNA (shRNA) can inhibit the expression of GEP gene in human glioma BT325 cell lines. Methods An oligo sequence of shRNA was designed and constructed for GFP gene, and co-transfected with report gene pEGFP-N1 to BT325 cell. After confirming the optimal ratio of transfection, expression of GFP and depressing effect of shRNA on GFP were observed under fluorescence microscope. Results The plasmid carrying GFP gene was cut into 6 kb and 100 bp by restriction endonuclease analysis, which was proved to be the same as original construction. The highest expression of GFP was seen when the ratio of transfection for plasmid DNA and XP-1 was 1︰2.5. The expression of GFP was obviously inhibited by GFP shRNA after 48 h transfection. Conclusions RNA interference exists in BT325 cell line. shRNA could induce specific and stable inhibition of GFP expression, which will provide a new strategy for researching gene function in cancer cell lines.