Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Abstract

2The pig has emerged as an important large animal model in biomedical and pharmaceutical research.We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer, and offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing as well as the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in one day; generation of germline-transgenic lines by using this protocol takes approximately one year.

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…The SB reprogramming transposon carried the murine cDNAs of Oct4, Sox2, Klf4, and cMyc, each separated by sequences coding for self-cleaving 2A peptide. The SB transposase (SB100X) helper plasmid has been described before Ivics et al, 2014;Kues et al, 2013;Talluri et al, 2014).…”

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

“…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.…”

Transgenic bovine as bioreactors: Challenges and perspectives

“…21 Studies using transposon-mediated transgenesis resulted in a higher efficiency of transgene expression than lentiviruses, and mosaicism in the newborns has not been reported as has been done for lentiviruses. 23,24 These features make the transposon system an attractive methodology for the transgenesis in the bovine. However, the efficacy in cattle must be proven principally using promoters driving the recombinant protein expression in milk.…”

“…21 Transposon plasmids have also been developed for use in transgenic procedures, 22 and the generation of transgenic pigs and rabbits using this system was successful. 23,24 Evaluation of the vectors constructed Prior to the generation of transgenic animals as bioreactors, it is important to evaluate the vector construction of the transgene for functionality of the promoter, its ability to respond to hormonal induction and its capacity to express the recombinant protein of interest. The evaluation of recombinant protein production in milk of transgenic cattle involves a long interval from birth until the first lactation cycle, thus it is essential to estimate the efficiency of the vector in the production of the recombinant protein.…”

“…Transposon plasmids were cytoplasmically microinjected to introduce DNA into the fertilized oocytes of pigs, and high rates (40 to 60%) of transgenic founders per animals born were achieved. 23 In rabbits, transposon plasmids were co-injected into the pronuclei of fertilized oocytes along with synthetic transposase mRNA, resulting in efficiencies of transgenesis of 15% or higher. 24 Transposon-mediated transgenesis offers comparable efficacies to lentiviral approaches but without the toxicity and biosafety concerns of working with lentiviral vectors.…”