Gómez-Rodríguez Julio, Washington Valance, Cheng Jun, Dutra Amalia, Pak Evgenia, Liu Pentao, McVicar Daniel W, Schwartzberg Pamela L
Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2008 Oct;36(18):e117. doi: 10.1093/nar/gkn523. Epub 2008 Aug 18.
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.
我们在此评估实时定量聚合酶链反应（q-PCR）作为一种筛选方法的应用，该方法用于筛选在哺乳动物细胞中由传统基因靶向构建体或基于细菌人工染色体（BAC）的完整构建体产生的同源重组体。利用不同位点的基因靶向事件，我们表明q-PCR是一种用于筛选传统基因靶向的高度灵敏且准确的方法，它可以减少需要通过Southern印迹进行后续筛选的克隆数量。我们进一步将q-PCR与荧光原位杂交（FISH）进行比较，以检测使用基于全长BAC的构建体进行的基因靶向事件，这些构建体旨在将突变引入一个基因或同时引入两个相邻基因。我们发现，尽管通过FISH评估时基于BAC的构建体似乎具有较高的同源重组率，但FISH筛选容易出现假阳性，而q-PCR可以检测到这些假阳性。我们的结果证明了q-PCR作为基因靶向筛选工具的实用性，并进一步突出了使用基于完整BAC的构建体进行同源重组时的潜在问题。
