Construction And Identification Of Adenovirus Vector Encoding Mouse PGC1A Gene

ObjectiveTo construct an adenovirus vector encoding mouse PGC1αgene, for further study on its function and application in antiobesity gene therapy.MethodsThe PGC1αgene sequence was amplified by RT-PCR from mouse muscle and then cloned into shuttle plasmid pAdTrack-CMV, which was then linearized by PmeI and transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdeasy-1 to obtain adenovirus plasmid through homologous recombination. The adenovirus plasmid was digested with PacI and transfected into HEK293 cells to form adenovirus particle。The successfully packaged virus was upscaled through 3 to 5 circles and then its titer was determined. PGC1αexpression was confirmed by western blotting and RT-PCR.ResultsRestrictive endonuclease digestion, PCR and sequencing showed that the favorite gene was correctly cloned into the shuttle plasmid and the recombinant virus plasmid was obtained through homologous recombination.The packaging cells showed typical CPE, the virus titer was about 3×1010TCID50/ml.The effective expression of PGC1αmediated by the recombinant vector was verified by western blotting and RT-PCR. ConclusionThe adenovirus vector encoding mouse PGC1αgene was successfully constructed.