[The construction of a general adeno-associated virus vector].
The general adeno-associated virus(AAV) vector pACR-Neo was constructed by substituting AAV's construct gene with cassette that was composed of CMV-IE promoter, multiple cloning sites and SV40-polyA. After transfecting pACR-Neo into recombinant AAV's packaging cell line AE1201, which was infected by Adenovirus 5 before transfection, we got rAAV/ACR-Neo at the titer of 4.2 x 10(5) CFU/ml. Using rAAV/ACR-Neo infected host cell A549, the recombinant AAV could transfer reporter gene Neo into host cell and mediate the integration of viral genome into host cell's chromosome DNA. By extracting chromosome DNA of host cell A549 that had been infected by rAAV/ACR-Neo, and digestion respectively with restriction endonuclease PvuII and HindIII, each of which has only one cutting site within rAAV/ACR-Neo's genome. We did Southern blot to check the hybridization of endonuclease digested chromosome to digoxin labelled Neo gene probe. After analyzing the experimental results, we found that rAAV/ACR-Neo's genome had integrate into host cell's chromosome compared to wild type AAV's site-specific integration. Through this work, we constructed a general AAV vector successfully. It lays foundation for research on AAV vector and application on gene therapy.
Yao
E
E
National Lab of Molecular Virology and Genetic Engineering, Beijing 100052.
Feng
Z
Z
Zheng
Y
Y
D000256:Adenoviridae / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D014162:Transfection; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant