短双链RNA对鸡胚盘细胞外源绿荧光蛋白基因表达的影响

Inhibition of green fluorescent protein gene expression in chicken blastoderm cells by siRNA

RNA干扰 (RNAinterference,RNAi)作为一种特异性沉默基因表达的方法 ,正在成为研究基因功能、胚胎发育及病毒性疾病治疗的重要工具。为了了解RNA干扰在禽类中的作用情况 ,实验将体外转录合成的绿荧光蛋白短双链干扰RNA (siGFP)和 3 磷酸甘油醛脱氢酶短双链干扰RNA (siGAPDH )分别同绿荧光蛋白(Greenfluorescentprotein ,GFP)表达载体 (pEGFP C1Vector)用脂质体转染试剂LipofectamineTM2 0 0 0共转染鸡胚盘细胞 ,并于转染后 36h在荧光显微镜下观察转染和干扰效果。对细胞绿荧光蛋白表达率的方差分析结果显示 ,不同处理组间差异达极显著水准 ,其中GFP组和GFP +siGAPDH组均同GFP +siGFP组差异极显著 ,GFP组同GFP +siGAPDH组差异不显著。实验结果说明 ,siGFP能特异、有效地敲低细胞绿荧光蛋白的表达。同线虫、真菌、拟南芥、水螅、锥虫、涡虫、果蝇、斑马鱼、小鼠等其它生物体一样 ,鸡胚盘细胞中也存在短双链干扰RNA (siRNA)

RNA interference (RNAi) has developed into a powerful tool for studying gene function, embryonic development and virosis therapy which can be capable of silenced the expression of special genes. In order to identify the mechanism of RNAi in the birds, The small interference RNA of GFP (siGFP) and GAPDH (siGAPDH) that synthesized in vitro transcription were cotransfected into the chicken blastoderm cells separately with the green fluorescent protein (GFP) expression vector (pEGFP-C1 Vector) by the transfection reagent (Lipofectamine TM 2000), then the numbers of fluorescent cell were measured after 36 h. The result of variance analysis showed that there were significant differences among the groups(P<0.01), and the result of multiple comparison showed that there were significant differences between the groups of GFP and GFP+ siGFP(P<0.01), GFP+ siGAPDH and GFP+siGFP(P<0.01), but no significant difference between GFP and GFP+ siGAPDH (P>0.05). The results indicate that the siGFP can knock-down the expression of GFP but the siGAPDH could not, which means there exists RNAi mechanism in chicken as well as in wireworm, epiphyte, cysticercoid, hydra, trypanosomiasis, turbination, drosophila, zebra, mice, and so on.