Construction Of SiRNA Plasmids Of Several AD Associated Genes And Their Identification

RNAi technology is a powerful experimental tool in laboratory. Using of homologous double-stranded RNA (dsRNA)-induced sequence-specific silencing of target genes, it can rapidly block gene activity. SiRNA which plays a central role in the RNA degradation is the guiding elements for the degeneration of specific mRNA. Nucleotide adopted in this study contains a small hairpin-line design of the two chains of I1PP2A, P53 and CREB corresponds to the template DNA sequence. After the annealing, phosphorylation, connecting to psuppress plasmid, we constructed the recombinant plasmids psuppress-siI1, psuppress-siP53, psuppress-siCREB. Through digestion and sequencing we identified the validity of the reorganization of the product. And then we transfected these plasmids to the HEK293 cells with lipofectAMINE2000 reagent and extracted the protein after a certain period time of culturing. Then western blot was used to detect the protein levels of I1PP2A, P53 and CREB. Results: we successfully constructed the recombinant plasmids identified by restriction enzyme digestion and sequencing. They can be significantly decreased by 75.1%, 90.5% or 72.1% of I1PP2A, P53 or CREB protein level compared to the non-transfected group and negative group, respectively. Conclusion: recombinant plasmids psuppress-siI1, psuppress-siP53, psuppress-siCREB can significantly suppress the protein level of relative genes in HEK293 cells. It provided the experimental tools to further study the function of their protein in the pathogenesis of AD.