Construction of LRIG3 specific short-hairpin RNA expressing vector and screening of stably transfected cell clone

Objective To construct eukaryotic expression vectors of RNA interference specific for LRIG3 gene,and to screen the stably transfected cell clone.Methods Genomic sequences of LRIG3 gene was retrieved from Genbank and cDNA was designed encoding shRNA (small hairpin RNAs) for LRIG3. The cDNA was synthesized and inserted into plasmid pGenesil2. Recombinant vectors were then transformed into competent E. coli. The positive clones were selected and recombinant plasmids were extracted. The plasmids were digested with Sal I and loaded in agarose gel electrophoresis. The three shRNA vectors were transfected into GL15 by Metafectene. The stably transfected cell clones were obtained after being screened with G4l8. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were performed to examine the inhibitory effect at the RNA level and protein level.Results The stably transfected pGenesil2-LRIG3-shRNA cell clones was significantly down-regulated by siRNA as validated by RT-PCR and Western blotting. Conclusion RNA interfering (RNAi) mediated by the shRNA expression vector can significantly down-regulate the expression of LRIG3 in glioma cell line GL15. The stable transfected cell clone is obtained for further study.