One step screening of retroviral producer clones by real time quantitative PCR

✍ Scribed by Greg J. Towers; Daniel Stockholm; Valérie Labrousse-Najburg; Frederique Carlier; Olivier Danos; Jean-Christophe Pagès

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 170 KB
• Volume: 1
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/(sici)1521-2254(199909/10)1:5<352::aid-jgm57>3.0.co;2-i

✦ Synopsis

Background Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing 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 producing clones is time consuming and expensive.

Methods

We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman 1 PCR technology, which, by using ¯uorescence measurement at each cycle of ampli®cation, allows PCR product quanti®cation. Fluorescence results from speci®c degradation of a probe oligonucleotide by the Taq polymerase 3k±5k exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the ®rst DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template ®ltered viral supernatant 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 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We con®rm that the Taqman 1 protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers 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 retroviral producer clones.