A protocol for locating and counting transgenic sequences from laboratory animals using a map-then-capture (MapCap) sequencing workflow: procedure and application of results.

Abstract

Transgenic rodent models for human diseases have been widely used over the past 50 years and are a mainstay of many biomedical research programs. Oftentimes the sequence of the transgenic segment of DNA is carefully designed but incorporation of this DNA into the host genome is less well understood. Structural variation and insertional mutagenesis may occur at transgenic insertion sites. Here, we present a robust workflow including identification of the transgene locus via selective Illumina sequencing followed by Cas9-mediated target DNA enrichment of the locus, which successfully identified beginning and end sites of a large transgenic insertion into a murine model for human amylin-induced type II diabetes. Enriched sequences were mapped via Oxford Nanopore sequencing. Although the insertion was too long for a single mapped genetic sequence to encompass, the method provided multiple insights relevant to the animal model: a minimum number of forward- and reverse-facing transcript copies as well as characterization of an inversion point within the insertion site. The insertion start point containing both murine and human DNA was used to identify and separate animals hemizygous for the transgenic insertion from homozygous animals. This identification could be performed early in the rodent life cycle prior to maturation (i.e. breeding age), thus allowing for management of colony phenotypes and eliminating the need to "genotype by phenotype" later on (onset of amylin-induced type II diabetes does not occur until ~8-10 weeks of age for this model). We further confirmed our homozygous diabetic mice function the same as colonies established in other labs and present full antibody and fluorescent-staining protocols (available in SI). Lastly, we note that, due to our genotyping, a novel animal was identified within our colony: non-diabetic homozygous mice. Indeed, only 37% of homozygous mice bred in our colony became diabetic. AUTHOR SUMMARY (for broad audience)Transgenic rodent models are important to studying human diseases. When creating a new rodent model, one may insert new DNA into a well-characterized background genome. However, it is oftentimes not known where the new DNA was incorporated, how many times it was incorporated, or if any coding sequences or regulatory elements within native DNA were disrupted. Here, we have developed a method to characterize transgenic animals, and have applied it to a popular model for studying human amylin-induced type II diabetes.