Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative, real-time PCR.
Recombinant AAV vectors are produced by transient transfection of mammalian cells. The virus is usually purified from a combination of lysed cells and spent culture medium by HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated single-stranded viral DNA (i.e. DNA-containing virions) in cell lysates and the spent culture medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility of this assay, we produced recombinant AAV in HeLa cells and five different types of 293 cells. We used primers to the EGFP transgene to detect the production of a recombinant AAV. We assayed the cell lysates and media by both our quantitative PCR assay and a functional transduction assay. The quantitative PCR assay data correlated well with the transduction assay data. Because this assay only requires standard PCR primers and SYBR Green I dye to detect the amplification of the PCR template, it will readily adapt to any target DNA sequence within the recombinant AAV genome. The recombinant AAV vector does not need to express a reporter gene, such as EGFP or beta-galactosidase in order to assay the amount of virus produced.
Mayginnes
John P
JP
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, United States.
Reed
Sharon E
SE
Berg
Heath G
HG
Staley
Elizabeth M
EM
Pintel
David J
DJ
Tullis
Gregory E
GE
D052160:Benzothiazoles; D002460:Cell Line; D003470:Culture Media; D004247:DNA / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D003959:Diamines; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D011995:Recombination, Genetic; D013194:Staining and Labeling; D013223:Statistics as Topic; D014161:Transduction, Genetic; D014771:Virion / Q000302:isolation & purification*; D014773:Virology / Q000379:methods
D052160:Benzothiazoles; D003470:Culture Media; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C098022:SYBR Green I; D004247:DNA; D003850:Deoxyribonuclease I