Development of a competitive PCR method for physical titration of recombinant EBV vector in a helper-dependent packaging system.
Epstein-Barr virus (EBV) is a gamma-herpesvirus with B lymphotropism and a double-stranded DNA genome of 172 kb that is episomally maintained in permissive cells during latency. EBV-based vectors containing minimal cis elements for replication, amplification, and helper-dependent packaging in a producer cell line HH514 have been developed to deliver therapeutic/suicide transgenes as infectious viral particles (miniEBV) to EBV-transformed B lymphoblastoid cells or B lymphoma cells. A quantitative, competitive PCR-based assay was developed to determine the relative packaging efficiencies of miniEBV and helper P3HR1 coproduced in HH514 cells. This provides a rapid and accurate quantitation of the physical titer of the virus preparation, which helps preserve the biological titer of the virus preparation and increase the efficiency of transgene delivery by miniEBV infection. In addition, it provides a sensitive and accurate way to evaluate future development of a helper-free packaging system by detecting any possible helper virus contamination.
Wang
J
J
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295, USA. jianlongnc@genetics.med.harvard.edu
Vos
J M
JM
D017931:DNA Primers / Q000737:chemistry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006378:Helper Viruses / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D017186:Titrimetry / Q000379:methods*; D014407:Tumor Cells, Cultured; D014771:Virion; D014779:Virus Replication
D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins