Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter–Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method

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.