Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting osteopontin gene

Abstract

Objective To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting osteopontin (OPN) gene (OPN-siRNA),and to screen for OPN-siRNA that may be most effective in gene-silencing. Methods Two pairs of oligonucleotides for short hairpin expression targeting OPN mRNA (shRNA1 and shRNA2) were inserted into pGenesil-1 vector. Recombinant expression vector was identified by enzyme cutting and sequencing analysis. OPN shRNAs were transfected into rat blood vessel smooth muscle cells (VSMC) via LipofectamineTM 2000. The transfected cells were visualized by using inverted fluorescent microscope. Forty-eight hours later,the VSMCs transfected by optimal recombined plasmid were selected by culturing in G418. Nude cells and cells transfected by PGH were used as control.The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. Results VSMCs were stably transfected by pshRNA1-OPN in more than 50% of total cells. The expression levels of OPN mRNA and proteins were 0.16±0.04 and 0.30±0.09 in shRNA1 transfected VSMCs,0.23±0.06 and 0.44±0.06 in shRNA2 transfected VSMCs,which were significantly different between the two groups and with compared normal cells (P 0.05). Conclusions The plasmid expression vectors coding for shRNA targeting OPN mRNA are constructed successfully. shRNA 1 appears to be most effective for gene-silencing. Key words: RNA interference; Osteopontin; Plasmids; Small hairpin RNA; Vessel smooth muscle cells