PCR-Based Strategy for Genotyping Mice and ES Cells Harboring LoxP Sites

Increasingly, the Cre-loxP system is being used to generate both null and conditional mutations in mice by homologous recombination in embryonic stem (ES) cells (1,3–5). The advantage of the Cre-loxP approach in generating mutations in mice, is that the gene of interest can be inactivated in any organ at a given time during the mouse’s life, thus bypassing developmental and/or pleiotropic effects inherent in the classical knock-out approach. Furthermore, this strategy allows for the generation of null alleles as well as “floxed” alleles (genomic DNA flanked by loxP sites) by using just one construct for gene targeting, since one parental homologous recombinant ES clone can yield subclones with both null and floxed alleles, following transient transfection with a Cre-recombinase expressing plasmid. Genotype analysis of mutant mice, generated by homologous recombination is done using genomic DNA for either Southern analysis or by use of the polymerase chain reaction (PCR). PCR is preferred since it rapidly yields the required results and allows for a larger throughput of samples. For classical knock-out techniques, where a large marker gene such as a neomycinresistance gene or the lacZ gene (or both) is either inserted into or replaces an exon(s) (2), PCR analysis requires the use of three primers (see below). In our experience, amplifying mouse genomic DNA is not straightforward, because determining the correct conditions with respect to annealing temperature, magnesium concentration, quality and amount of template, are all critical factors in allowing for the development of a robust assay. The more primers used for this type of assay, the more difficult the task of determining the optimal PCR conditions. We have been using the Cre-loxP system to generate both null and conditional mutations in a number of genes, including the following transcription factors: cAMP responsive element binding protein (CREB), glucocorticoid receptor (GR) and hepatocyte nuclear factor 4γ (HNF4γ). Here, we describe the PCR strategy we use in analyzing the genotypes of our floxed mice, using only two primers, making the development of assays simpler, quicker and more robust. During our analysis of CREB and HNF4γ ES cell clones, we realized that this approach also allowed rapid screening for multicopy insertions of the targeting construct in putative homologous recombined ES clones. For the classical knock-out approach, analysis of mouse genotypes by PCR requires use of three primers, one common to both mutant and wild-type (WT) alleles and one specific to each (Figure 1A; primers 1, 2wt and 3mut) allowing for the amplification of relatively short products in each case. The amplification yields two different sized products, one mutant specific (null) and one WT specific. Use of only two primers on either side of the selection cassette in this case is problematic, because the size of the reporter gene(s) is relatively large (>1 kb), making routine amplification of large genomic fragments in multiple heterogeneous samples difficult. Secondly, the large size difference, hence amplification rate difference, between the mutant and WT products in this case would further complicate the determination of suitable PCR conditions. For floxed mice, genotyping by PCR is possible using only two primers flanking one loxP site (Figure 1B; primers 1 and 2). This allows for the amplification of two different sized products depending on the presence or absence of the loxP site. The size difference between the floxed allele and WT allele is due both to the extra nucleotides that comprise the loxP sequence (34 bp) and the surrounding restriction sites used for constructing the targeting construct. Because the size difference is relatively small, differential amplification of one product over the other is avoided. Moreover, by designing primers outside of both loxP