Rapid retrovirus titration using competitive polymerase chain reaction.
A quantitative polymerase chain reaction (PCR) procedure has been developed for rapid retrovirus titration. This procedure, which is based on the simultaneous amplification of the sample with known amounts of a competitor DNA fragment (competitive PCR), was used for the quantification of viral RNA genomes in retrovirus-producing cell clone supernatants and of proviral DNA molecules formed at 24 h after infection of different reference cell lines. The results obtained from the analysis of several samples indicated that proviral DNA quantification is in complete agreement with the number of selectable colonies in a standard colony assay. Conversely, the number of viral RNA genomes in the producer cell clone supernatants is a poor predictor of the actual efficiency of infection. Repeated competitive PCR experiments for provirus copy number determination at different times after transduction indicated that the number of proviral DNA molecules remains stable over time, suggesting stable integration into the host genome. The developed procedure is rapid and simple, is applicable to retroviral constructs not containing a selectable gene and can be used to directly measure the efficiency of infection of any target cell type, thus overcoming the problem of the dependency of retroviral titer determination on the rate of expression of a selectable gene and on the efficiency of colony formation of a reference cell line.
Tafuro
S
S
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Zentilin
L
L
Falaschi
A
A
Giacca
M
M
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive; D004279:DNA, Viral / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification*; D017186:Titrimetry / Q000379:methods
D004279:DNA, Viral