Nucleic Acids Research Advance Access published online on August 18, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn523
Methods Online |
Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs
1Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, 2Cancer and Inflammation Program, National Cancer Institute-Frederick, Frederick, MD 21702, USA and 3Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK
*To whom correspondence should be addressed. Tel: +1 301 435 1906; Fax: +1 301 402 2170; Email: pams{at}mail.nih.gov
Received March 31, 2008. Revised July 28, 2008. Accepted July 31, 2008.
We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination.