Construction of conditionally replicative adenovirus expressing short interference RNA targeting the β-actin and GAPDH genes with hTERT promoter regulation and control

Abstract

Objective To construct conditionally replicative adenovirus (CRAds) expressing short interference RNA (siRNA) targeting the β-actin and GAPDH genes with hTERT promoter regulation and control. Methods β-actin and GAPDH siRNA template DNA sequences were inserted into pGPH1/GFP/Neo plasmid, respectively. Two pairs of specific primers were designed and used to get the expression cassettes of β-actin-siRNA and GAPDH-siRNA which were inserted into pZHTERT and pZD55 plasmid to construct the recombinant plasmid pZHTERT-β-actin and pZD55-GAPDH, respectively. The pZHTERT-β-actin plasmid was cut with Kpn Ⅰ and Pvu Ⅰ to get the expression cassette of β-actin which was cloned in-to pZD55-GAPDH to form pTD-GAPDH-β-actin plasmid. The plasmid pTD-GAPDH-β-actin was transfect-ed into 293 cells together with plasmid pBHGE3 to obtain the recombinant CRAds,TD-GAPDH-β-actin. The viral plaques appeared 9-12 days after infection. The extracted DNA of recombinant adenoviruses was verified by PCR. Viruses were plaque purified, propagated on HEK293 cells and purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. Results The conditionally replicative adenovirus expressing siRNA targeting the GAPDH and β-actin genes with hTERT promoter regulation and control had been constructed successfully. Viral PFU titers were 2 × 1011 PFU/ml. Conclusion The conditionally replicative adenovirus will lay a good foundation for fur-ther study on cancer gene therapy. Key words: GAPDH; Promoter; Conditionally rephcative adenovirus; RNA interference