One step screening of retroviral producer clones by real time quantitativePCR

Background Recombinant retroviruses are obtained from either stably or tran siently transfected retrovirus producer cells. In the case of stably produc ing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre produci ng clones is time consuming and expensive. Methods We have taken advantage of retroviral endogenous reverse transcript ion to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman (R) PCR technology, which, by using fluorescence me asurement at each cycle of amplification, allows PCR product quantification . Fluorescence results from specific degradation of a probe oligonucleotide by the Tag polymerase 3'-5' exonuclease activity. Primers and probe sequen ces were chosen to anneal to the viral strong stop species, which is the fi rst DNA molecule synthesised during reverse transcription. The protocol con sists of a single real time PCR, using as template filtered viral supernata nt without any other pre-treatment. Results We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 20 0 GFP-expressing retroviral-producing clones either by FAGS analysis of inf ected cells or by using the quantitative PCR. We confirm that the Taqman (R ) protocol allows the detection of virus in supernatant and selection of hi gh titre clones. Furthermore, we can determine infectious titre by quantita tive PCR on genomic DNA from infected cells, using an additional set of pri mers and probe to albumin to normalise for the genomic copy number. Conclusion We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre re troviral producer clones. Copyright (C) 1999 John Wiley & Sons, Ltd.