Construction of LRIG1 Specific RNAi Expressing Vector and Screening of Stably Transfected Cell Clone

Objective To construct eukaryotic expression vector encoding RNA interference sequences specific for LRIG1 gene,to screen the stably transfected cell clone,and to observe its effect on the expression of LRIG1.Methods Two shRNAs sequences based on the sequence of LRIG1 mRNA in the GenBank were designed and one scrambled shRNA sequence was regarded as negative control.The synthesized sequences were inserted into shRNA expression vector pGenesil2 and sequenced.The shRNA vectors were transfected into GL15 by Metafectene.The stably transfected cell clones were obtained after being screened with G4l8.Western Blotting was performed to examine the inhibitory effect at the protein level.Results The recombinant plasmids containing shRNA were analyzed by restriction endonuclease analysis and DNA sequencing.The screening concentration of G418 to GL15 cell was 600mg/L.LRIG1 expression was significantly down-regulated by siRNA as validated by Western Bloting.Conclusion RNA interfering (RNAi) mediated by the shRNA expression vector could significantly down-regulate the expression of LRIG1 in glioma cell line GL15.The stably transfected cell clone was obtained for further study.