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

Nucleic Acids Research
Julio Gómez-RodríguezPamela L Schwartzberg

Abstract

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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Citations

Dec 3, 2009·Genome Research·Hyung Joo LeeJin-Soo Kim
Jun 27, 2012·Journal of Neuroscience Methods·Jorge H Limón-PachecoMagda Giordano
Jun 12, 2013·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Joseph E DohertyMatthew H Wilson

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Methods Mentioned

BETA
PCR
electrophoresis
transfection

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