Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2001-05

AUTHORS

H-G Zhang, Y M Wang, J F Xie, X Liang, H-C Hsu, X Zhang, J Douglas, D T Curiel, J D Mountz

ABSTRACT

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap. More... »

PAGES

704-712

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/sj.gt.3301454

DOI

http://dx.doi.org/10.1038/sj.gt.3301454

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1044982995

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/11406765


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