Construction and identification of RNAi expressing vector specific for leucine-rich repeats and immunoglobulin-like domains 1 gene and selection of stably transfected cell clone / 中国组织工程研究

ABSTRACT

BACKGROUND: Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) gene showed low expression in glioma cells. LRIG1 gene overexpression significantly enhanced LRIG1 mRNA and protein expression and inhibited its biological behavior. However, very few researchs are reported from the stand-point of inhibition of LRIG1 gene expression. OBJECTIVE: To construct specific RNA interference plasmids for LRIG1, establish stably transfected human glioma GL15 cell line, and observe its effect on expression of target gene LRIG1. METHODS: Designed and synthesized two shRNAs (named LRIG1-shRNA1 and LRIG1-shRNA2) specific for LRIG1 mRNA according to the GenBank, and one scrambled shRNA sequence as negative control, named pGenesil2-negative shRNA. The shRNA was inserted into pGenesil2 vector and sequenced. The recombinant vectors were transformed into E. coli. Picked up the positive clones and extracted the plasmids, which were transfected into GL15 cells by Metafectine. G418 was applied to select the stably transfected cell clones. Western Blotting was performed to examine the LRIG1 protein level.RESULTS AND CONCLUSION: The recombinant plasmids which contain shRNA were analyzed by restriction endonuclease digestion and DNA sequence, and it was proved that the fragment was inserted into the expected sites. Compared with the negative control group, the level of LRIG1 protein expression in pGenesil2-LRIG1-shRNA1(LRIG11) transfected cells and in pGenesil2-LRIG1-shRNA2(LRIG12) transfected cells was decreased by 47.9% (P 0.05). The results confirmed that RNAi expressing vector specific for LRIG1 gene (pGenesil2-LRIG1-shRNA1) was successfully constructed, and the stable cell clones transfected with the shRNA expression vector showed inhibition of the expression of LRIG1 in glioma cell line GL15.