Construction of siRNA adenovirus vector targeting rat glucocorticoid-induced leucine zipper and infection to rat L6 myoblasts

XIANG Fang-hua; XIA Yan; YANG Shi-hong; XIONG Jian; CHEN Wen-ge; YUAN Lin; YANG Zhao-hui

Abstract：Objective To establish siRNA adenovirus vectors targeting rat glucocorticoid-induced leucine zipper ( GILZ) , and infect rat L6 myoblasts cultured in vitro, in order to provide experimental basis for further investigation of regulatory effects of GILZ on skeletal muscle metabolism in sepsis.Methods (1) Three specific siRNA sequences targe-ting rat GILZ mRNA and one control sequence were designed and cloned.Double stranded oligo DNAs were inserted into vector GV119 which had been double-digested by AgeⅠand EcoRⅠto construct the shuttle plasmid.After confirmation by sequencing, the shuttle plasmid and the adenovirus backbone plasmid pBHG lox△E1,3 Cre were co-transfected into HEK293 cells.The recombinant adenoviruses named Ad-siRNA-1-GILZ, Ad-siRNA-2-GILZ and Ad-siRNA-3-GILZ were acquired, propagated and purified, before detection of the recombinant adenovirus titer.(2)0.1, 1.0, 10.0 μL recombinant adenovirus were infected to L6 myoblasts in different media ( complete medium, Enhanced Infection Solution, complete medium with 5μg/mL Polybrene, Enhanced Infection Solution with 5 μg/mL Polybrene) .Expression of green fluorescent protein and morphological changes of L6 myoblasts were observed microscopically to identify the best condition for infection.(3) The L6 myoblasts were infected by negative control adenovirus (negative control group), Ad-siRNA-1-GILZ ( GroupⅠ) , Ad-siRNA-2-GILZ ( GroupⅡ) and Ad-siRNA-3-GILZ ( GroupⅢ) .Ex-pression of GILZ mRNA was determined via real-time PCR.Results (1) DNA sequencing and PCR results demonstra-ted that recombinant adenovirus was successfully constructed, and the titer of recombinant adenovirus was 2.0 ×1010 PFU/mL.(2) The best infection effect was demonstrated in Enhanced Infection Solution with 10μL recombinant adenovirus, in which the infection efficiency of L6 myoblasts was up to 80%.(3) Cell necrosis was not observed in the L6 myoblasts in-fected with the recombinant adenovirus, and there were large amounts of green fluorescent protein expressed in L6 myo-blasts.(4) Real-time PCR showed that there was no difference in GILZ mRNA expression between GroupsⅡ,Ⅲand the negative control group (P>0.05).While expression of GILZ mRNA in GroupⅠwas significantly decreased compared with the negative control group(P<0.01), with a silencing efficiency of 59.8%.Conclusion The siRNA adenovirus vectors targeting rat GILZ are successfully constructed, which markedly down-regulates the expression of GILZ in vitro. The study provides experimental basis for investigating the regulatory effects of GILZ on protein metabolism of skeletal mus-cles in sepsis.