Construction Of Adeno-associated Virus Vector Of Ciliary Neurotrophic Factor Gene

Objective To construct the recombinant adeno-associated virus (rAAV)vector of CNTF gene.Methods 1.The CNTF gene was obtained by gene splicing. Then the CNTF genefragment was directional cloned into plasmid pAAV-MCS ,to construct therecombinant pAAV-CNTF plasmid.2.Co-transfect AAV-293 cells with threeplasmids(pAAV-CNTF,pAAV -RC and pHelper)by lipofectamine2000.Meanwhile,pAAV —LacZ control plasmid was co-transfected together with pAAV —RC andpHelper into the AAV-293 cells in parallel with the recombinant plasmidcontaining the CNTF gene in the same transfectional condition. The AAV-293cells containing pAAV —LacZ-derived virus can be stained by In Situ β-Galactosidase Staining Kit(X-gal)to confirm that the AAV-293 transfectionwas successful.Harvest the recombinant AAV particles containing LacZ.Diluteviral stocks over a 10-fold series from 10-2 to 10-5 and infect HT-1080 cellswith different folds of diluted viral stocks.HT-1080 cells containing pAAV—LacZ-derived virus was stained by In Situ β-Galactosidase Staining Kitto estimate the viral titer of harvested AAV particles.Results 1.Successfully construct the recombinant AAV expressing system ofciliary neurotrophic factor gene.2.The viral titer of recombinant AAVmeasured by staining of In Situ β-Galactosidase Staining Kit is 7.0×107particles/ml.Conclusion Successful construction of AAV expression system for CNTF genewill provide a foundation for investigating the effects of CNTF on theregeneration of the optic nerve .