| 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 Polâ…¡promoters can direct cell- or tissue-specific gene silencing. A GFP-shRNA fusion construct transcribed from an RNA Polâ…¡promoter (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 Polâ…¡promoter 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 polâ…¡promoters in combination with RNAi.Our results demonstrate that the fusion construct transcribed from an Polâ…¡promoter to induce gene-specific RNAi was feasible.
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