Construction and identification of the recombinant Adenovirus vector for Dmrt7 gene

To study the construction of recombinant Adenovirus vector containing Dmrt7 gene and GFP,the experiment adopted the plasmid pCMV5-Dmrt7 which was double-digested,purified and pluged into the pShuttle-CMV vector with IRES-GFP fragment.After sequenced,the recombinant Adenovirus vector Ad-Dmrt7 was constructed with Dmrt7 and GFP through homologous recombination technique in bacteria using Adeasy-1 system.After linearized by Pme Ⅰ,the vector was transfected into HEK 293 cell and then the viral titer was checked by GFP.The results indicated that the recombinant Adenovirus vector digested by restrictive endonuclease PacⅠwas constructed successfully.It could be cut into two fragments of 23 kb and 2.9 kb.At 24 hours after transduction mediated by liposome,the fluorescence was observed in the 293 cell.The viral titer was 0.95×108 pfu/mL,which was checked by GFP.This revealed that the recombinant Adenovirus vector containing human Dmrt7 gene and GFP gene was constructed successfully.