Preparation of a specific retrovirus producer cell line.
Establishing a cell line that produces high levels of a specific retrovirus construct involves several steps. First, the retroviral construct must be stably introduced into an appropriate packaging cell line. This can be accomplished either directly by transfection or by transfection to produce a transient virus stock followed by cross-infection of this stock into a separate packaging line; procedures for both approaches are described in this unit. After stable lines are produced, they must be characterized to identify lines that produce high titers of virus with an appropriate structure. A drug selection protocol (the most common method for determining virus titer) is provided along with a sample calculation of BAG virus titer. Sometimes, a short and direct method of estimating virus titer is available when the virus encodes a histochemically detectable gene such as lacZ. For this case, an Xgal staining protocol is described. Similarly, a protocol is provided to estimate the levels of virus being produced by a given cell line by quantitating any specific product of the virus (e.g., RNA) present within the producer line.
Cepko
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Havard Medical School, Boston, Massachusetts, USA.
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003470:Culture Media; D004279:DNA, Viral / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics* / Q000254:growth & development; D014162:Transfection / Q000379:methods; D014779:Virus Replication / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D003470:Culture Media; D004279:DNA, Viral; D012367:RNA, Viral; D001616:beta-Galactosidase