Nucleic Acids Research, 2008, 1–9 doi:10.1093/nar/gkn523 Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs Julio Go ´mez-Rodrı´guez 1 , Valance Washington 2 , Jun Cheng 1 , Amalia Dutra 1 , Evgenia Pak 1 , Pentao Liu 3 , Daniel W. McVicar 2 and Pamela L. Schwartzberg 1, * 1 Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, 2 Cancer and Inflammation Program, National Cancer Institute-Frederick, Frederick, MD 21702, USA and 3 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK Received March 31, 2008; Revised July 28, 2008; Accepted July 31, 2008 ABSTRACT We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homol- ogous 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 com- pared 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. INTRODUCTION Homologous recombination in mouse embryonic stem cells has provided a powerful tool for the generation of gene-targeted mice carrying mutations of defined loci (1,2). However, since homologous recombination can be a relatively infrequent event in mammalian cells (3,4), detec- tion of homologous recombination events can require the screening of large numbers of clones. The gold standard for screening is Southern blot analyses, which both iden- tifies correctly targeted clones and permits analysis of the structure of the gene-targeted event, including verification of single copy insertion of the construct. However, Southern analyses can be time-consuming, particularly in cases of loci with low-frequency targeting where hun- dreds of cell lines may need to be screened. The use of PCR-based strategies, most of which utilize one primer outside of the targeting constructs in conjunc- tion with a primer present on the selectable marker, has permitted more rapid screening for homologous recombi- nants (5–8). Other PCR strategies, including screening for loss of plasmid sequences from the targeting construct, have also been used (9). However, conventional PCR pro- vides limited information on the number and structure of inserts. Moreover, conventional PCR-based strategies are prone to false negatives resulting from problems with long-range PCR or low amounts of DNA. Conversely, false positives can arise due to annealing of products initiated by the different primers, giving rise to products consistent with appropriate targeting but that actually derive from clones containing random insertions. Thus, PCR-based screens cannot replace the information gained through Southern analyses, although this techni- que can reduce the numbers of clones to be evaluated in greater detail. Recent data have suggested that whole BAC-based gene-targeting vectors can provide an alternative method allowing for more rapid generation of homologous recom- binants (10–12). However, screening for homologous recombination after introduction of such vectors can be complicated by the long length of homologous arms used, which most often preclude screening by either Southern analyses or PCR strategies. To bypass these assay pro- blems, several alternatives have been utilized. These include: (i) the use of one short-length arm on the target- ing construct to permit screening by Southern analyses using an outside probe (10); (ii) the use of fluorescent *To whom correspondence should be addressed. Tel: +1 301 435 1906; Fax: +1 301 402 2170; Email: pams@mail.nih.gov ß 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. Nucleic Acids Research Advance Access published August 18, 2008 by guest on May 24, 2016 http://nar.oxfordjournals.org/ Downloaded from