Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

Abstract

A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was 10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

Expression dynamics and relations with nearby genes of rat transposable elements across 11 organs, 4 developmental stages and both sexes

“…Rats and mice have been the most widely used models in biomedical research and drug development for many years [ 1 – 3 ]. However, a shift has taken place that mice rapidly overtake rats as the major model of choice [ 4 ].…”

Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

“…Because producing transgenic mice have traditionally been less difficult than rats, a large body of literature has accumulated demonstrating the ease with which the genomes of mice can be manipulated using these tools to, for example, reprogram somatic cells in into iPS cells, induce random mutagenesis, conduct precise genome editing through oligo replacement, and knock out gene function via indels 33 37 39 49 50 . Although much smaller in number relative to mouse models, transgenic rats have been generated using these tools as well 42 51 52 53 54 55 .…”

A piggyBac transposon‐ and gateway‐enhanced system for efficient BAC transgenesis

“…3) can be readily applied to the engineering of any chosen BAC clone for mouse transgenesis. Moreover, as piggyBac efficiently transposes in a wide variety of vertebrate species, including zebrafish (Suster et al, 2011), rat (Furushima et al, 2012), pig , goat (Bai et al, 2012), and chicken , we suggest that this system may be adaptable for transgenesis in other organisms.…”