RNAi对小鼠胚胎成纤维细胞中MC1R基因表达的影响

Effect of RNAi on MC1R Expression in Mouse Embryo Fibroblasts

旨在研究RNAi对小鼠胚胎成纤维细胞(MEF)黑素皮质素受体1(Melanocortin-1receptor,MC1R)基因表达的影响;以MC1R基因作为影响哺乳动物毛色性状的候选基因,通过RNA干扰(RNAi)技术抑制小鼠胚胎成纤维细胞中MC1R基因表达。合成3对针对小鼠MEF中MC1R基因的瞬时干扰siRNA,采用反向转染法将siRNA转染入MEF,取转染siRNA 48h后的小鼠胚胎成纤维细胞提取总RNA和总蛋白,并通过QRT-PCR技术和Western bolt检测MC1RmRNA和其蛋白的相对表达量;结果显示,siRNA1组和siRNA2组的MC1R mRNA相对表达量和MC1R蛋白表达量显著低于空白对照组(P<0.05),但siRNA3组表达不显著;本试验利用反向转染方法,成功地对体外培养的小鼠胚胎成纤维细胞进行siRNA转染。MC1R基因与蛋白的抑制情况相一致,说明siRNA1和siRNA2对MC1R的抑制效果确实有效,并筛选出有显著干扰效果的siRNA,为进一步研究MC1R在毛色发育过程中的调控机理提供科学依据。

To study the effect of RNAi on MC1Rexpression in mouse embryo fibroblasts(MEF). MC1Rgene was taken as candidate gene affecting the formation of mammal's hair color.The aim of the study was to inhibit the expression of MC1Rby RNA interference in the MEF.Three pairs of specific siRNA for MC1Rgene were designed for RNA interference and transferred into the MEF by reverse transfection.After 48h,the total RNA and protein were extracted from the MEF,and the relative expression of MC1RmRNA and protein was detected by using QRT-PCR technology and Western bolt.The results showed that the relative expression of MC1R mRNA and MC1Rprotein in the siRNA1and siRNA2groups were significantly lower(P<0.05)than those in the blank control group,but the expression of siRNA3group was unconspicuous.These tests were successfully inhibited the expression of MC1Rin the MEF by RNA interference(reverse transfection)with LipofectamineTM RNAi-Mate.The expression trendency of MC1Rgene and protein are consistent.The results showed that the inhibition effect of siRNA1and siRNA2 groups were effective,the effective siRNA were screened from three specific siRNAs,can be served as a scientific foundation for the further study of regulation mechanism of MC1Rin the process of coat development.