This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwon, Y. J.
Right arrow Articles by Yu, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwon, Y. J.
Right arrow Articles by Yu, H.

 Previous Article  |  Next Article 

Journal of Virology, May 2003, p. 5712-5720, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5712-5720.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Determination of Infectious Retrovirus Concentration from Colony-Forming Assay with Quantitative Analysis

Young Jik Kwon,1 Gene Hung,2,{dagger} W. French Anderson,2 Ching-An Peng,1,{ddagger}* and Hong Yu3*

Department of Chemical Engineering,1 Gene Therapy Laboratories,2 Department of Surgery, University of Southern California, Los Angeles, California 900893

Received 20 November 2002/ Accepted 28 February 2003

The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ± 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

* Corresponding author. Present address for Hong Yu: Vascular Biology Institute, Department of Surgery, University of Miami School of Medicine, P.O. Box 019132 (R104), Miami, FL 33101. Phone: (305) 243-6477. Fax: (305) 243-2810. E-mail: hyu{at}med.miami.edu.

{dagger} Present address: Arena Pharmaceuticals, Inc., San Diego, CA 92121.

{ddagger} Mailing address for Ching-An Peng: Department of Chemical Engineering, University of Southern California, 925 Bloom Walk, HED 208, Los Angeles, CA 90089-1211. Phone: (213) 740-2067. Fax: (213) 740-8053. E-mail: capeng{at}usc.edu

Journal of Virology, May 2003, p. 5712-5720, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5712-5720.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Thomas, J. A., Ott, D. E., Gorelick, R. J. (2007). Efficiency of Human Immunodeficiency Virus Type 1 Postentry Infection Processes: Evidence against Disproportionate Numbers of Defective Virions. J. Virol. 81: 4367-4370 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»