title: Rapid and reliable determination of transgene zygosity in mice by multiplex ligation-dependent probe amplification.
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), beta-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.
authors.affiliation: Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Melbourne, VIC, Australia. Amanda.notini@mcri.edu.au
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Notini
authors.forename: Ruili
authors.initials: R
authors.lastname: Li
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Western
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Sinclair
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: White
mesh_terms: D000818:Animals; D005838:Genotype; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D019076:Transgenes