Construction and identification of hif-1? shRNA recombinant plasmids / 吉林大学学报(医学版)

ABSTRACT

Objective To construct the expression vectors of pSilencer3.1-hif-1? and identify the inhibition of hif-1? in Hela299 cells after transfection with the combinative plasmids.Methods The interfering sequences of hif-1? were designed according to the sequence of hif-1? of GenBank.Three recombinant plasmids of pSilencer3.1-hif-1? were constructed by DNA ligase.Trizol reagent was used to extract the whole RNA of cells and RT-PCR assay was applied to detect the expression of hif-1? mRNA.Lysis assay was used to extract the protein from cells and Western blotting was adopted to observe the expression of HIF-1? protein.Results The vectors were identified to be right after sequencing.The mRNA level was decreased 24 h after transfection with three vectors of pSilencer-hif-1?,and the ratios of hif-1? /GAPDH in control,group transfecting with pSilencer-sihif-1?-1,group transfecting with pSilencer-sihif-1?-2,group transfecting with pSilencer-sihif-1?-3 were 0.55,0.13,0.33,and 0.08,respectively(P