摘要
角蛋白是一个多基因家族,不同的角蛋白表达呈现表达的组织和时空特异性。因此,利用不同的角蛋白启动子可以实现外源基因在特定组织和特定发育时期的表达。为了探讨某一基因在皮肤和毛囊生长发育及在疾病发生中的作用机制,常选择角蛋白启动子实现外源基因在皮肤和毛囊组织里特异表达。
为制备在皮肤和毛囊组织里特异表达外源基因的转基因动物,首先需要选择组织特异表达的启动子。为此,本实验从人血中提取基因组,克隆得到在皮肤和毛囊组织中特异表达的人K14和K5启动子。瞬时转染体外培养不同类型的细胞,利用萤光素酶检测系统,分析这两个启动子的启动活性及组织特异性。结果表明,两个启动子在选择的细胞系中都有表达,但以小鼠皮肤来源的细胞系里的活性最强。其中K14启动子在小鼠皮肤细胞系中活性远高于其它细胞系(P<0.01),K5启动子的活性也比较高(P<0.05)。为充分利用角蛋白启动子,明确其表达特点,使之更好地应用于外源基因的定位表达提供一定的参考依据。
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,PolⅡ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,本研究利用PolⅡ型CMV启动子和毛囊组织特异表达的人角蛋白14(K14)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-Cl-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PolⅡ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。
RNAi是一种行之有效的基因沉默的新方法,越来越广泛地应用于基因功能的研究、疾病的治疗以及新型疫苗的研制等领域。本研究通过原核显微注射干扰载体的方法制备转基因小鼠。选用皮肤组织特异表达的人源角蛋白14(K14)基因启动子(2000bp)作为表达载体启动子,成功地驱动融合表达载体EGFP-shRNA进行干扰片段前体的转录,进而生成成熟的干扰片段,靶向小鼠BMP4基因使其发生沉默。所得到的转基因小鼠及其杂交后代经PCR和Southern杂交鉴定,结果表明外源基因准确无误地整合到小鼠基因组。Northern杂交结果证明小干扰RNA在皮肤组织中有较高水平的表达,在肺和肠组织中有较低水平的表达。研究结果表明：利用PolⅡ型(K14)启动子驱动shRNA融合转录本的表达,在特定组织高表达siRNA,从而达到抑制特定组织目的基因表达的技术路线是可行的。在取E18.5的转基因小鼠皮肤组织做组织切片并用BMP4抗体杂交后发现,阳性小鼠表达BMP4小干扰RNA的皮肤局部出现毛囊增多的现象,初步确定该基因受到抑制后,毛囊在胚胎期的发生被激活,从而产生更多的毛囊。
我们的研究结果表明,利用polⅡ型启动子K14融合表达mRNA的方法实现体内小干扰RNA的表达是可行的,为利用K14启动子进行毛囊相关基因干扰研究积累了基础数据,也为制备组织特异抑制基因表达的转基因大家畜提供了一个参考方法。
Keratin promoter 5, and 14 are the best available promoter for expression genes in specific tissue. This review summarized the expression characteristics of these promoters and application in transgenic animal models.In this paper, the activities of promoter K14 and K5 were analyzed by luciferase reporter in different cell lines. The results showed that two promoters were both express in all cell lines, but the activity was the highest in skin cell lines. The activity of K14 promoter in skin cell line was higher than others cell lines(P<0.01), and K5 promoter is same too.
Keratins are a family of cytoskeleton component proteins of epithelial cells. Because keratin generally are expressed in a differentiation-specific and spatiotemporal manner, so keratin promoter regions have proven invaluable for targeting transgene expression to specific compartments within epithelia.
RNA interference is an efficient method for exploring gene function. Accumulating evidence suggests that RNA PolⅡpromoters can direct cell- or tissue-specific gene silencing. A GFP-shRNA fusion construct transcribed from an RNA PolⅡpromoter (K14 promoter) was used to induce gene-specific shRNA silencing of BMP4 gene expression. Recombinant vectors (pEGFP-Cl-shRNA, psiCHECK-BMP4 and pEGFP-K14-shRNA) were constructed. Vectors pEGFP-Cl-shRNA and psiCHECK-BMP4 were cotransfected into Hela cells (in vitro) and shRNA-induced inhibition efficiency was tested by a luciferase assay. The results showed that six interference sequences all inhibited the expression of BMP4 with high efficiency (>60%), with interference sequence 5# showing 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. The results showed that the mRNA and protein products of the exogenous 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 silence 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.
Small interfering RNAs are short, double-stranded RNA molecules that can target mRNAs with complementary sequences for degradation. As a method for silencing of gene expression, RNAi is more simple and convenient compared with gene targeting. RNAi has been applied universally in research on gene function, treatment of disease and vaccine production. Accumulating evidence suggests that the Pol II promoter can be applied to RNAi. In this research, the human keratin 14 (K14) gene promoter was used to drive eGFP-shRNA fusion expression in order to interfere with BMP4 expression in skin. Transgenic mice were produced by the method of pronuclear microinjection and identified by PCR screening and Southern blotting. PCR and Southern blotting experiments showed that the foreign gene was integrated in the genome of the transgenic mice. Northern blotting detected siRNA expression in lungs, intestines and skin, with highest siRNA expression level found in skin. Our results demonstrate that in vivo siRNA expression driven by PolⅡpromoter fusion constructs is feasible. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression for the analysis of gene function in skin and also for skin appendage development in livestock. In addition, it provides further data advancing progress of the use of polⅡpromoters in combination with RNAi.
Our results demonstrate that the fusion construct transcribed from an PolⅡpromoter to induce gene-specific RNAi was feasible.
引文
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