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Journal of Biomedicine and Biotechnology
Volume 2009, Article ID 901079, 7 pages
http://dx.doi.org/10.1155/2009/901079
Methodology Report

Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors

1Institute of Biotechnology, Northwest A&F University, Yangling, Shaanxi 712100, China
2Department of Physiology and Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78245, USA

Received 2 October 2008; Revised 8 February 2009; Accepted 11 March 2009

Academic Editor: Gerald Schumann

Copyright © 2009 Yuehong Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of 4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at , increased the coinfection rate to 10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to 25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or 50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.