Application of the polymerase chain reaction in determination of recombinant retrovirus titers as fifty percent endpoints.
Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. One of the most laborious routine assays in the application of retroviral-mediated gene transfer is the determination of viral titers of vector producer cell lines. Traditionally, determination of virus titer involves the testing of culture medium from individual packaging cell lines for the ability to transfer drug resistance to susceptible cells - a process that can easily take up to 14 days. It is generally agreed that this method is cumbersome. We sought to develop PCR-based protocols that would significantly simplify and shorten this procedure. Using PCR and primers specific for the Neoregion of the MLV-derived vector LeGSN, we determined 1. the proviral integration in target cells, and 2. the viral nucleic acid (RNA or DNA) content of the vector stock. Results were compared with those using the conventional method. We found that these specific PCR-based procedures were indeed useful for rapid determination of viral titers as well as for quick screening for high-titer vector-producing cell clones and successful transduction of target cells.
Husemoen
L L
LL
Laboratory for Infectious Diseases, Hvidovre Hospital, Denmark.
Gram
G J
GJ
Hansen
J E
JE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D009052:Leukemia Virus, Murine / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014161:Transduction, Genetic; D016662:Virus Integration
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral