Construction Of Human Transforming Growth Factor Beta 1 RNA Interference Eukaryotic Expression Vector

OBJECTIVETo construct eukaryotic expression vectors of short hairpin RNA (shRNA) targetinghuman transforming growth factor beta 1 (TGF-β1) gene for the study of RNAinterference (RNAi) therapy for fibrotic diseases.METHODSThe cDNA sequence of human TGF-β1 was retrieved from Genebank. Three pairs ofsmall interferencing RNAs (siRNA) targeted against human TGF-β1 were designedaccording to the Tuschl principal, and corresponding negative control siRNAs werealso designed. Six pairs of hairpin oligonucleotides coding the shRNA correspond tothe siRNAs were designed and synthesized. After annealing, the oligonucleotidesform six double-stranded DNA inserts with sticky ends that are compatible with theends of the lined psiSTRIKE neomycin vector, and then the DNA inserts were ligatedinto the psiSTRIKE neomycin vector by sticky ligation. The ligation product weretransformed into E. coli DH5α, recombinant plasmids were extracted, the presence ofinserts were confirmed by PstⅠdigestion, and the sequences were identified by DNAsequence analysis.RESULTSThree siRNAs targeted against human TGF-β1 and corresponding negative controlsiRNAs were successfully designed. Six pairs of hairpin oligonucleotides coding theshRNA correspond to the siRNAs were successfully designed and synthesized. Therecombinant vectors were digested into 958bp and 3655bp fragments by PstⅠdigestion. The results of DNA sequence analysis showed the sequence of DNA inserts were coincident with design.CONCLUSIONThree siRNA eukaryotic expression vectors targeted against human TGF-β1 andcorresponding negative control siRNA eukaryotic expression vectors weresuccessfully constructed, which will be helpful to the study RNAi therapy for f?broticdiseases.