Document Detail


Rep/Cap gene amplification and high-yield production of AAV in an A549 cell line expressing Rep/Cap.
MedLine Citation:
PMID:  11991756     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cell lines stably expressing rep/cap are important tools for studying adeno-associated virus (AAV) biology and producing AAV vectors. Several rep/cap cell lines have been isolated, each of which is based on HeLa cells. Infection of these cell lines with adenovirus for production of AAV vector is associated with substantial amplification of the rep/cap gene. Concerns over the presence of human papilloma viral (HPV) sequences in HeLa cells may limit use of such lines for production of clinical-grade vectors. Here we describe a non-HeLa-derived rep/cap cell line called K209, generated by stable transfection of A549 cells with a plasmid construct containing the P5 rep/cap cassette from AAV2. Infection of K209 cells with adenovirus leads to a 1000-fold amplification of the rep/cap gene with high-yield production of AAV vectors. The multiplicity of infection (MOI) of adenovirus that led to maximum amplification of the rep/cap gene and high-level production of AAV is 10 times higher in the HeLa-based cell line than that required in K209 cells. Our data suggest that papilloma-derived gene products present in HeLa cells are not required for high-yield production of AAV vectors.
Authors:
Guang-ping Gao; Fengmin Lu; Julio C Sanmiguel; Phoi T Tran; Zahra Abbas; Kimberly S Lynd; Jon Marsh; Nancy B Spinner; James M Wilson
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Molecular therapy : the journal of the American Society of Gene Therapy     Volume:  5     ISSN:  1525-0016     ISO Abbreviation:  Mol. Ther.     Publication Date:  2002 May 
Date Detail:
Created Date:  2002-05-06     Completed Date:  2002-09-12     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  100890581     Medline TA:  Mol Ther     Country:  United States    
Other Details:
Languages:  eng     Pagination:  644-9     Citation Subset:  IM    
Affiliation:
Institute for Human Gene Therapy, Department of Molecular, the Wistar Institute, Philadelphia, Pennsylvania, 19104-4268, USA.
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MeSH Terms
Descriptor/Qualifier:
Blotting, Western
Cells, Cultured / cytology,  virology
DNA Helicases / genetics*,  metabolism
DNA Primers / chemistry
DNA, Recombinant / genetics,  metabolism
DNA, Viral / genetics*
DNA-Binding Proteins / genetics,  metabolism*
Dependovirus / genetics*,  metabolism
Fluorescent Antibody Technique
Gene Amplification*
Genetic Vectors
Humans
In Situ Hybridization, Fluorescence
Polymerase Chain Reaction
Trans-Activators / genetics*,  metabolism
Transfection
Viral Proteins / genetics,  metabolism*
Virus Replication
Grant Support
ID/Acronym/Agency:
P01 HL59407/HL/NHLBI NIH HHS; P30 DK47757/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/DNA Primers; 0/DNA, Recombinant; 0/DNA, Viral; 0/DNA-Binding Proteins; 0/Trans-Activators; 0/Viral Proteins; 0/replication initiator protein; 137750-19-7/rep proteins, Adeno-associated virus 2; EC 3.6.1.-/DNA Helicases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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