Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs.
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.
Gómez-Rodríguez
Julio
J
Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Washington
Valance
V
Cheng
Jun
J
Dutra
Amalia
A
Pak
Evgenia
E
Liu
Pentao
P
McVicar
Daniel W
DW
Schwartzberg
Pamela L
PL
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