RNAi(RNA interference, RNAi)是继基因打靶技术后的一种高效的研究基因功能的方法。细胞学实验和小鼠模型的研究结果表明, Pol Ⅱ型启动子可以实现组织特异的RNA干扰, 从而为鉴定基因在特定组织中的功能及作用机理提供了一个强有力的研究方法。为了能将这种方法用于转基因绵羊生产, 探讨基因与绵羊毛囊发育的关系及其作用机制等, 文章利用Pol Ⅱ型CMV启动子和毛囊组织特异表达的人角蛋白14 (K14)基因的启动子驱动eGFP-shRNA 融合转录本的生成, 从而实现敲低目的基因的表达。体外基因表达沉默效率分析(pEGFP- C1-shRNA和psiCHECK-BMP4双质粒共转染Hela细胞)结果表明, 6个干扰序列均能有效地抑制 BMP4基因的表达, 抑制效率达到 60% 以上; 体内表达沉默分析(只转染 pEGFP-K14-shRNA 质粒转染小鼠皮肤细胞系JB6-C41)的实验结果与体外分析结果相似, 除3#序列外, 其余干扰序列对BMP4基因的抑制效率都在60% 以上, 其中5#序列的效率达到80%以上。siRNA诱导的目标基因沉默中mRNA和蛋白水平的下降显著正相关。结果表明, 设计构建的由Pol Ⅱ型启动子K14驱动eGFP-shRNA融合转录本的形成, 从而实现RNAi的研究方法是可行的, 利用这种方法可以实现在特定细胞中敲低目的基因的表达水平。为在大家畜特别是绵羊中应用RNAi的方法分析目的基因在毛囊发育、对不同类型毛囊生长发育的诱导和调节等作用机理的研究提供一个参考方法。
RNA interference is an efficient method for exploring gene function. Accumulating evidence suggests that RNA Pol II promoters can direct cell- or tissue-specific gene silencing. A eGFP-shRNA fusion construct transcribed from an RNA Pol II promoter (K14 promoter) was used to induce gene-specific shRNA silencing of BMP4 gene expression. Re-combinant vectors (pEGFP-C1-shRNA, psiCHECK-BMP4, and pEGFP-K14-shRNA) were constructed. Vectors pEGFP-C1-shRNA and psiCHECK-BMP4 were cotransfected into Hela cells (in vitro) and shRNA-induced inhibi-tion efficiency was tested by a luciferase assay. The results showed that all the six interference sequences inhibited the ex-pression of BMP4 with high efficiency (>60%), and the interference sequence 5# showed the highest efficiency. For in vivo screening of JB6-C41 cells transfected with vector pEGFP-K14-shRNA, the inhibition efficiency was assayed by quantitative RT-PCR and Western blotting analyses. The results showed that the mRNA and protein products of the exoge-nous BMP4 gene were efficiently and specifically inhibited. The efficiency of gene silencing was greater than 60%, except for sequence 3#. The declines in mRNA and protein expression levels were significantly correlated during gene si-lence by the shRNA. This system may be adapted for in vivo shRNA expression and gene silencing. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in the analysis of the mechanisms of hair follicle development in sheep.
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