Amanda DuBose didn’t set out to come up with a new technical solution to characterizing transgenic DNA in animal models. A postdoctoral fellow in Francis Collins’ lab at the National Human Genome Research Institute, DuBose was using mice to study the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) when she and her colleagues realized that they were hindered by their inability to determine which mice were homozygous for the transgene containing the mutated gene of interest. This was crucial information: mice that are homozygous for the transgene with the HGPS-causing variant have a more severe phenotype with an earlier onset, making them more useful for research than the heterozygotes.
The scientists tried a number of tools — including qPCR, FISH, and Southern blots — but none of the assays could quickly and reliably report the number of gene copies in a mouse. So the team resorted to what DuBose calls “an invention of necessity” and made a custom microarray to identify the site of transgene integration and develop a genotyping assay that could deliver the results they needed.
The results of that work were published in Nucleic Acids Research in a paper entitled “Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene.” Lead author DuBose and her colleagues analyzed regions of the transgenic mouse genome next to BAC sequence by using arrays to capture the DNA, size selecting with the Pippin Prep, and sequencing on the Illumina HiSeq.
DuBose, Collins, and the other authors found that the BAC had broken more than expected when it was injected into the mouse; there were four mouse-human junctions. “I was surprised by how rearranged it was,” DuBose says, noting that the junctions within the BAC were quite unexpected. “We know it’s not unique to our model. I have a feeling that all of the transgenic models have crazy rearrangements — it’s just that nobody goes in to look this closely.”
Ultimately, DuBose used the sequence data to design a PCR assay with three primers crossing one of the BAC-mouse junctions. That assay is now a standard tool that can be used to determine gene copy number in the model mice: results indicate whether a mouse is homozygous mutant, heterozygous mutant, or wild type.
The lab has now returned to its HGPS research, but DuBose says that the technological approach to characterizing a transgene and developing a PCR genotyping assay can be used by anyone working on transgenic models or genome sequence that has foreign DNA in it. She notes that the array her team used for targeted capture is no longer available, so the technique would have to be adapted for liquid capture.