Establishment and identification of ShRNA expression vectors of recombinant targeting gene EGFR

Objective To establish and identify the plasmid expression vector encoding short hairpin RNA(shRNA) targeting epidermal growth factor receptor(EGFR) of human gene.Methods ①Four short hairpin RNAs(shRNA) were designed according to the homo sapiens EGFR mRNA ID,and then recombinant shRNA was reconstructed,with PGPU6/GFP/Neo plasmids as vectors,respectively.The plasmids were transferred into E.coli DH5α for plasmid extraction and the subsequent identification by enzyme digesting and sequencing.②Four recombinant plasmids were transfected into Colo-16 cells with lipofectamine 2000 and were screened for positive clones by G418.Results ①The results of enzyme-digestion sequencing confirmed that four plasmids containing shRNA were successfully constructed.②Four recombinant plasmids were successfully transfected into Colo-16 cells and the cell clones with stable expression were obtained.Conclusion With RNAi technology,the recombinant shRNA plasmids targeting EGFR were successfully established and transfected into Colo-16 cells.