The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MO
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BioMed Central
Open Access
Research
The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events Bing Zhang1, Pat Metharom1, Howard Jullie1, Kay AO Ellem2, Geoff Cleghorn3, Malcolm J West1 and Ming Q Wei*1 Address: 1Department of Medicine, University of Queensland, Prince Charles Hospital, Brisbane, AUSTRALIA, 2Queensland Institute of Medical Research, Brisbane, AUSTRALIA and 3Department of Paediatrics and Child Health, Royal Children's Hospital, Brisbane, AUSTRALIA Email: Bing Zhang - [email protected]; Pat Metharom - [email protected]; Howard Jullie - [email protected]; Kay AO Ellem - [email protected]; Geoff Cleghorn - [email protected]; Malcolm J West - [email protected]; Ming Q Wei* - [email protected] * Corresponding author
Published: 04 August 2004 Genetic Vaccines and Therapy 2004, 2:6
doi:10.1186/1479-0556-2-6
Received: 24 October 2003 Accepted: 04 August 2004
This article is available from: http://www.gvt-journal.com/content/2/1/6 © 2004 Zhang et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background: Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. Methods: Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact and adsorption periods were studied. MOI between 0–32 was assessed on commonly used cell lines as well as a new cell line. Results: We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene
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