High-efficiency retroviral infection of primary myoblasts.
In the past, it has been hard to introduce genes into primary myoblasts without selection, as they have been very difficult to transfect or infect. We describe conditions under which mouse primary skeletal muscle myoblasts can be infected with retroviral vectors at high efficiency. Infection can be greatly increased by minimizing the time during which cells are exposed to virus, adding a minimal centrifugation step, and supplementing the infection cocktail to mimic more closely primary myoblast growth medium. Under these conditions, one round of exposure to virus results in an infection efficiency of up to 80%, whereas 4-5 rounds of infection over a two day period reproducibly yield an infection efficiency of > 99%. These methods greatly enhance the potential for studying genetically engineered primary myoblasts from any mouse strain, transgenic or knockout, and may have useful application to other primary cell types that are refractory to transfection or infection.
Springer
M L
ML
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford University Medical Center, California 94305-5332, USA.
Blau
H M
HM
D000818:Animals; D001053:Apolipoproteins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology* / Q000821:virology*; D012190:Retroviridae / Q000235:genetics / Q000502:physiology*; D012692:Serial Passage; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication
D001053:Apolipoproteins