Construction and identification of siRNA expression vectors of adaptor protein 2 gene

Objective To construct μ2 subunit of adaptor protein 2(AP2-μ2) small interfering RNA(siRNA) and expression vectors,and detect their silencing effects.Methods AP2-μ2 mRNA targeted hairpin siRNAs were devised and three oligonucleotide strands of DNA fragments encoding the above siRNAs were synthesized.After annealing of the complementary strands,the DNA fragments were cloned into pSilencer 3.1 neo H1 plasmid,followed by amplification in E.coli and DNA sequencing.The transfected cells were divided into five groups:control,scramble plasmid,AP2 plasmid-1,AP2 plasmid-2 and AP2 plasmid-3.Three groups of recombinant plasmids and the negative control siRNA plasmid(scramble plasmid) were transfected into rat cardiac cell line H9C2 by liposome transfection method,and the expression of AP2-μ2 mRNA was examined by RT-PCR.Results The synthesis of six single-stranded oligonucleotide showed multiple bands by gel electrophoresis.After annealing respectively,the product by gel electrophoresis showed a single band.After double-stranded DNA templates were connected with plasmid,they digested by restriction enzyme of BamHⅠ and Hind Ⅲ,the 4300 bp and 66 bp fragments were seen by gel electrophoresis.DNA sequencing analysis showed that the connecting sequence of nucleic acid sequences was same as the designed,indicating three AP2-μ2 siRNA vectors were successfully constructed.RT-PCR results showed that in AP2 plasmid-1 group,the AP2-μ2 gene expression level in transfected H9C2 cells reduced by 96% compared with negative control group.Conclusion Three kinds of AP2-μ2 siRNA vectors are successfully constructed,and AP2 plasmid-1 transfected cells can significantly inhibit the AP2-μ2 gene expression.