Construction of Omi/HtrA2 specific small hairpin RNA expression plasmid and its role in a hypoxia-reoxygenation model
Rongshan Li · Shanxi Yike Daxue xuebao · 2008
Objective To construct and identify two Omi/HtrA2 specific small hairpin RNA(shRNA)expression plasmids,and to explore its influence on Omi/HtrA2 expression in a hypoxia-reoxygenation model.Methods Two pairs of Omi/HtrA2 specific oligonucleotides were designed and synthesized.These oligonucleotides were annealed to form double strand DNA fragments,and then these fragments were cloned into Pgenesil-1 plasmid,respectively.The recombinant Omi/HtrA2-shRNA expression constructs were confirmed by restriction enzymes digestion and by sequencing.Rat renal epithelial cell NRK-52E hypoxia-reoxygenation model was established.The recombined plasmids were transected into NRK-52E with liposome-mediated method.Expression of Omi/HtrA2 in transferred cells was detected by Western blot.Results The Omi/HtrA2-shRNA expression plasmids,named PGP1 and PGP2,were successfully constructed and identified by restriction enzyme and sequence analysis.Expression of Omi/HtrA2 in cells transfected with Omi/HtrA2-shRNA was much lower than that transfected with Pgenesil-1 plasmid.Conclusion Two Omi/HtrA2-shRNA expression plasmids are successfully constructed and they could effectively inhibit Omi/HtrA2 expression in epithelial cells of hypoxia-reoxygenation rats.