title: Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs.
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.
authors: Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Julio
authors: J
authors: Gómez-Rodríguez
authors: Valance
authors: V
authors: Washington
authors: Jun
authors: J
authors: Cheng
authors: Amalia
authors: A
authors: Dutra
authors: Evgenia
authors: E
authors: Pak
authors: Pentao
authors: P
authors: Liu
authors: Daniel W
authors: DW
authors: McVicar
authors: Pamela L
authors: PL
authors: Schwartzberg
mesh_terms: D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D018390:Gene Targeting / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic