Abstract
The ability to rapidly and unequivocally distinguish heterozygous from homozygous transgenic mice is an integral part of any breeding strategy. Here we describe a quick and simple protocol for determining the zygosity of transgenic mice at multiple loci in a single reaction. This involved the development of a multiplex ligation-dependent probe amplification (MLPA) probe mix to simultaneously measure common transgenic alleles such as Cre recombinase (Cre), neomycin (Neo), β-galactosidase (LacZ) and enhanced green fluorescent protein (eGFP), as well as loci specific to the X and Y chromosome to allow sexing. Each reaction required as little as 100 ng of genomic DNA isolated from a tail biopsy using a simple procedure. Normalization against autosomal control loci resulted in 100% call accuracy, with no ambiguous results. This probe mix can be easily implemented in any laboratory with access to a PCR machine and a DNA sequencer, and can be rapidly adapted to genotype any additional loci of interest.
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Acknowledgments
We would like to thank Anna Piper and Christine Hall for animal husbandry. This work was supported by the National Health and Medical Research Council Australia (334314 to AS, 491293 and 546478 to SW).
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Notini, A.J., Li, R., Western, P.S. et al. Rapid and reliable determination of transgene zygosity in mice by multiplex ligation-dependent probe amplification. Transgenic Res 18, 987–991 (2009). https://doi.org/10.1007/s11248-009-9284-x
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DOI: https://doi.org/10.1007/s11248-009-9284-x