A rapid generation of adenovirus vector with a genetic modification in hexon protein.
The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy.
Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an 710062, PR China.
Bingyan
B
Di
Qinwen
Q
Mao
Junli
J
Zhao
Xing
X
Li
Dongyang
D
Wang
Haibin
H
Xia
D000256:Adenoviridae / Q000235:genetics*; D036022:Capsid Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015183:Restriction Mapping
D036022:Capsid Proteins; C485184:enhanced green fluorescent protein; C018315:hexon capsid protein, Adenovirus; D049452:Green Fluorescent Proteins