Priyadarshini Raman1, Vladimir Grachtchouk1, Robert H Lyons2, Ronald J Koenig1. 1. 1 Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan , Ann Arbor, Michigan. 2. 2 Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan.
Abstract
BACKGROUND: It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task. METHODS: A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions. RESULTS: The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ∼1.5 and 0.9 Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products. CONCLUSIONS: Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.
BACKGROUND: It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task. METHODS: A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions. RESULTS: The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ∼1.5 and 0.9 Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products. CONCLUSIONS: Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.
Authors: Melissa E Dobson; Ericka Diallo-Krou; Vladimir Grachtchouk; Jingcheng Yu; Lesley A Colby; John E Wilkinson; Thomas J Giordano; Ronald J Koenig Journal: Endocrinology Date: 2011-09-27 Impact factor: 4.736
Authors: Yun Ji; Natalie Abrams; Wei Zhu; Eddie Salinas; Zhiya Yu; Douglas C Palmer; Parthav Jailwala; Zulmarie Franco; Rahul Roychoudhuri; Eric Stahlberg; Luca Gattinoni; Nicholas P Restifo Journal: PLoS One Date: 2014-05-14 Impact factor: 3.240