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

Abstract

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. 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. 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. 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.