The construction and application of siRNA-DNA expression vector

Objective To construct DNA expression vector of siRNA,and to study the target gene expression inhibition by RNAi in mammalian cells.Methods The hairpin structure of siRNA transcript template targeting EGFP gene was first synthesized.Then, both PTZU6+1 empty vector and siRNA transcript template were digested with SalI/XbaI. Two digested fragments were ligated with T_4 DNA ligase. The recombinant vector was then transfected into hapartocarcinoma SMMC-7721 cells and the expression alteration of EGFP was detected.Results The recombination plasmid was amplified in the E. coli. JM109. The identification of redigesting with HindⅢ /EcoRI and sequencing showed that the reconstructive plasmid contained the correct and full nucleotide sequence of EGFP-siRNA transcript template. The mRNA and protein of EGFP gene were inhibited by RNAi in hapartocarcinoma SMMC-7721 cells.Conclusion The DNA expression siRNA vector was constructed successfully and could be used to suppress target gene.