Abstract
Random integration transgenesis has been foundational to modern mouse genetics, generating thousands of lines that continue to serve as important tools in biomedical research. Yet for many of these models, the genomic integration site and associated genomic alterations remain unknown, complicating zygosity determination and potentially obscuring disruption of endogenous loci. Mice expressing inducible CreERT2 under rat Nes regulatory elements are widely used for in vivo lineage tracing of neural stem and progenitor cells, yet the genomic integration site of the Tg(Nes-cre/ERT2)KEisc/J transgene has remained undefined. Here, we used PacBio HiFi whole-genome sequencing to resolve its genomic locus and molecular architecture. We reconstructed the complete 13.7-kb transgene and mapped its integration to chromosome 6 within intron 1 of Kcnd2, where insertion was accompanied by deletion of 17.4 kb of endogenous genomic DNA. Identification of the integration junctions further enabled development of an allele-specific PCR assay for direct zygosity determination. Beyond resolving the molecular architecture of a widely used CreERT2 mouse line, this study demonstrates the utility of long-read HiFi sequencing for revisiting legacy transgenic models, uncovering otherwise hidden genomic alterations while enabling precise molecular genotyping.