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Nucleic Acids Research Advance Access published online on August 18, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn523
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs

Julio Gómez-Rodríguez1, Valance Washington2, Jun Cheng1, Amalia Dutra1, Evgenia Pak1, Pentao Liu3, Daniel W. McVicar2 and Pamela L. Schwartzberg1,*

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.


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[Abstract] [Full Text] [PDF]


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