Robust method for distinguishing heterozygous from homozygous transgenic alleles by multiplex ligation-dependent probe assay

Transgenic mouse alleles continue to be used heavily in biomedical research (1). Transgenes insert into the genome at random sites, typically in a tandem array of 1 to 20 copies. Both Southern blot analysis and real-time PCR can be used for determining the zygosity of transgenes, but each have practical and technical limitations (2–4). Here we describe a robust, easily implemented method for determination of zygosity of transgene alleles, which gives a clear, reproducible distinction between 1 versus 2 alleles. The method uses the multiplex ligation-dependent probe assay (MLPA) with a competitor oligonucleotide. MLPA is a widely used method for assessing the relative copy number of multiple genomic sequences in a DNA sample (5). During MLPA, an oligonucleotide ligation reaction is performed, followed by PCR using a fluorescein-conjugated primer, such that the amount of PCR product generated for each genomic sequence is directly proportional to the number of input copies (Figure 1A, left). For this MLPA assay, genomic DNA samples were isolated from mouse tail or toe snips using the Puregene® Tail DNA method (Gentra, Minneapolis, MN, USA) and adjusted to approximately 50 ng/μL. Mice bearing four different Cre transgene alleles were studied: SynICre (6), TetOpCre (7), Nestin-Cre, and Wnt-Cre, as well as two other transgene alleles: the Sleeping Beauty transposon T2/Onc (8) and enhanced green fluorescent protein (EGFP) (7). For each of the three transgenes analyzed, two MLPA probe sets were designed (see Supplementary Table S1 available online at www.BioTechniques. com) to have amplification products of size approximately 120 bp and approximately 170 bp. Three control probes elsewhere in the mouse genome were used, with amplification products ranging in size from 108 to 136 bp. Each probe set was composed of a 5′ and a 3′ half-probe, each containing unique target specific sequence, stuffer sequence, and universal primer Robust method for distinguishing heterozygous from homozygous transgenic alleles by multiplex ligation-dependent probe assay