期刊
PLOS ONE
卷 7, 期 5, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0037881
关键词
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资金
- ERC Advanced Investigator award [250356]
- University of Zurich
- Dr. Max Husmann Foundation Zurich
- Swiss National Science Foundation [3100A0-111917]
- Center of Transgenesis Expertise of the National Center of Competence in Research Neural Plasticity and Repair
- Novartis Research Foundation
- European Union programs Prion Research Projects Community (PRIORITY) [222887]
- Seventh Framework Programs [24098, 278611]
- European Research Council (ERC) [250356] Funding Source: European Research Council (ERC)
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.
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