Transposon‐mediated transgenesis, transgenic rescue, and tissue‐specific gene expression in rodents and rabbits

Abstract

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

“…The SB100X-mediated protocol was optimized by carefully titrating the relative amounts of transposase and transposon to obtain optimal rates of transgenesis to generate founders, and was extensively evaluated for efficacy, toxicity, mosaicism, germline transmission, insertion site preferences, transgene copy number and silencing. Genotyping of numerous transgenic lines produced by SB-transposition demonstrated single-copy integrations of the transposon as expected from the cut-and-paste integration reaction catalyzed by the SB-transposase 30 . The numbers of integrations per genome was shown to be dependent on and hence controllable by the concentration of both components of the transposon system in the injection cocktail 30 .…”

“…Genotyping of numerous transgenic lines produced by SB-transposition demonstrated single-copy integrations of the transposon as expected from the cut-and-paste integration reaction catalyzed by the SB-transposase 30 . The numbers of integrations per genome was shown to be dependent on and hence controllable by the concentration of both components of the transposon system in the injection cocktail 30 . One of the most important aspects of using this transposon-mediated transgenic protocol is that no major mosaicism was observed, and transgene expression was maintained for several generations in all species tested.…”

“…This feature makes transposons easily controllable DNA delivery vectors that can be used for versatile applications, including germline gene transfer. A hyperactive variant of the SB transposase, called SB100X, was recently developed by in vitro evolution 29 , and shown to support efficient germline transgenesis in mice [29][30][31] , rats 30,31 , rabbits 30 and pigs 32,33 . The SB100X-mediated protocol was optimized by carefully titrating the relative amounts of transposase and transposon to obtain optimal rates of transgenesis to generate founders, and was extensively evaluated for efficacy, toxicity, mosaicism, germline transmission, insertion site preferences, transgene copy number and silencing.…”

“…Furthermore, unlike retroviral vectors [34][35][36][37] , SB100X transposase-catalyzed transgene integration does not seem to trigger transcriptional silencing 24,30 . Therefore, the application of the Sleeping Beauty transposon system described here can significantly enhance the rabbit genomic toolbox.…”

“…1) into the pronucleus of a zygote, with frequencies of transgenic founders (per born live pup) of 15 % or higher 30 . The protocol consists of the following major stages: Transgenesis with Sleeping Beauty in rabbits (Steps 27-49).…”