Construction and Functional Identification of pSUPER Vector of p21 RNAi

Objective: To construct a pSUPER RNAi vector that can inhibit p21 gene expression and identify its function. Methods: A pair of 60bp oligonucleotides coding for short hairpin RNA and targeting p21 gene of rat were chemically synthesized and annealed and inserted into pSUPER plasmids digested with Bgl Ⅱ and Hind Ⅲ to construct the recombinant pSUPER RNAi plasmid (pSU-PER-p21). Recombinant pSUPER-p21 plasmid was identified by enzyme digestion and sequencing analysis. After transfection of pSU-PER-p21 into primary cultured cortical neurons of rat, western blotting is used to determine the p21 protein expression of neurons treated with kainate(KA). Results: Recombinant pSUPER-p21 vector was identified correct by enzyme digestion and sequencing analysis. Western blotting showed pSUPER-p21 vector can inhibit the p21 protein up-regulation of primary cultured cortical neurons induced by KA. Conclusion: The pSUPER RNAi vector targeting p21 is successfully constructed and it can specifically inhibit p21 gene expression.