Improved coinfection with amphotropic pseudotyped retroviral vectors.
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 approximately 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 15 degrees C, increased the coinfection rate to approximately 10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to approximately 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.
Wu
Yuehong
Y
Institute of Biotechnology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Melton
David W
DW
Zhang
Yong
Y
Hornsby
Peter J
PJ
D016475:3T3 Cells; D000818:Animals; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000821:virology; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D012190:Retroviridae / Q000145:classification* / Q000235:genetics / Q000502:physiology*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein