The Application Of RNAi To The Acetyltransferase P300 Gene Silencing In The Mammalian Cells

The dsRNAs synthesized in vitro or formed in vivo trigger the sequenced-specificmRNA destruction and the suppression of corresponding gene. Because the effect of RNAi ison the level of mRNA, RNAi is a kind of PTGS (post-transcription gene silencing). In recentyears, RNAi has been found in organisms from epiphytes to plants, and from invertebrates tomammalian animals. Compared with gene knockout, RNAi is simple, time saving andeffective, RNAi has now been widely used in studies of gene function. With the breakthroughof RNAi technology in mammalian cells, more and more researchers begin to apply the RNAiin the studies of Functional genomics and gene therapy. It is now clear that the acetylation modification of core histone tails is involved in themodulation of chromatin structure and function that leads to the activation/suppression ofgene expression. p300 is an important HAT and has implicated in the regulation of geneexpression, including many cytokine genes. The state of histone acetylation is related withgene expression. This study demonstrates that p300 stimulated the activation of IL-12 p40 promoter.Moreover, p300 was shown to be able to work synergistically with the transcription factorp65 to enhance the activation of p40 promoter and this effect could be impaired by HDACs.In order to further analyse the roles of p300 in the transcriptional regulation, we apply RNAiassay. Firstly, we constructed two plasmids of siRNA. But it seemed that neither was effective.Then we design two sequences of dsDNAs according to the p300 mRNA. The dsDNAs weretranscribed by T7 polymerase and digested into siRNAs by RNase III. Then the siRNAs weretransfected into 293T cells. The RT-PCR assay implied that the suppression of p300expression triggered by one of the two dsDNAs is obvious. This result contributes to theresearch of p300's function in the transcription control.