Abstract
Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.
Highlights
Transgenic mice are invaluable for studying gene function and modeling human disease
Zygosity assessment of transgenic mice is a common task for laboratories engaged in transgenic mouse research
We investigated Fluorescence in situ Hybridization (FISH) as a method for determining transgene zygosity status based on the evaluation of hybridization signals
Summary
Transgenic mice are invaluable for studying gene function and modeling human disease. If coexistence of three loci is required, the expected yield of triple transgenic mice from a hemizygous cross will be 12.5%, meaning that the large majority of the offspring from each cross will need to be weaned, genotyped, and eventually eliminated. In such situations, the use of homozygous mice ensures that animals with the desired genotype can be rapidly attained while preventing the generation of animals with unproductive genotypes
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