zyfish a simple, rapid and reliable zygosity assay for transgenic micezyfish一个简单、快速、可靠的转基因老鼠接合性试验.pdfVIP

ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice Donal McHugh, Tracy O’Connor, Juliane Bremer, Adriano Aguzzi* Institute of Neuropathology, Department of Pathology, University Hospital of Zurich, Schmelzbergstrasse, Switzerland 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. Citation: McHugh D, O’Connor T, Bremer J, Aguzzi A (2012) ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice. PLoS ONE 7(5): e37881. doi:10.1371/journal.pone.0037881 Editor: Ya-Ping Tang, Louisiana State University Health Sciences Center, United States of America Received December 8, 2011; Accepted April 26, 2012; Published May 29, 2012 Copyright: 2012 McHugh et al. This is an open-access article distributed under t