Long-read PacBio HiFi sequencing resolves the genomic integration site and structure of the Nestin-CreERT2KEisc/J transgene

Random integration transgenesis has been a cornerstone of modern mouse genetics, resulting in thousands of lines that remain crucial tools in biomedical research. However, for a significant portion of these models, the precise genomic integration site and associated alterations remain unknown, making zygosity determination challenging and potentially leading to the unnoticed disruption of endogenous loci.

Mice expressing inducible CreERT2 under rat Nes regulatory elements are commonly utilized for in vivo lineage tracing of neural stem and progenitor cells. Nevertheless, the genomic integration site of the Tg(Nes-cre/ERT2)KEisc/J transgene has remained unclear.

In a groundbreaking study, researchers employed PacBio HiFi whole-genome sequencing to definitively resolve the genomic locus and molecular architecture of this transgene. They reconstructed the full 13.7-kb transgene and pinpointed its integration to intron 1 of Kcnd2 on chromosome 6. This insertion was found to have removed 17.4 kb of endogenous genomic DNA.

By identifying the integration junctions, the team was able to create an allele-specific PCR assay, facilitating direct zygosity determination. This research not only clarified the molecular structure of a widely used CreERT2 mouse line but also showcased the potential of long-read HiFi sequencing in reassessing older transgenic models. The study revealed hidden genomic changes that were previously undetected, and it paved the way for precise molecular genotyping.