Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

Abstract

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

“…This study also reported that shRNA mediated by the Sleeping Beauty transposon achieved an increased integration rate over random integration of anti- VP1 shRNA. Furthermore, Sleeping Beauty transposase and Tn5 transposase were cytoplasmically injected into sheep zygotes to integrate transposon containing recombinant human factor IX (rhFIX) driven by the BLG promoter (Bevacqua et al, 2017). No transgenic lambs have been obtained from Tn5 transposase injection, while injection of Sleeping Beauty transposase resulted in two lambs that carried the transgene 2/7 (29%) (Bevacqua et al, 2017).…”

Gene Therapy with the Sleeping Beauty Transposon System

“…The most hyperactive SB transposase version currently available, SB100X, displays a $100fold hyperactivity compared with the originally resurrected transposase [50]. The SB100X transposase enables highly efficient germline transgenesis in relevant mammalian models, including mice, rats, rabbits, pigs, sheep, and cattle [51][52][53][54][55][56]. Moreover, the use of the SB100X system yielded robust gene transfer efficiencies into human HSCs [50,57], mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells (iPSCs) [58], and T cells [59], which are relevant targets for regenerative medicine and gene-and cell-based therapies of complex genetic diseases.…”

Cytoplasmic Microinjection of <i>piggyBac</i> Transposase mRNA and Transposon Vectors for Efficient <i>In Vitro</i> Production of Transgenic Porcine Parthenotes

“…Previous studies have successfully conducted transposon-based gene delivery to embryos using SB and Tol2 (a transposon derived from fishes) via CPI of a combination of a plasmid DNA (encoding Trans) and a transposon DNA (carrying GOI) to generate Tg animals from bovines [50], pigs [51][52][53], sheep [50], and mice [54]. For example, Garrels et al [51] co-injected a plasmid DNA coding for SB-based hyperactive Trans called SB100X and a transposon plasmid DNA into the cytoplasm of porcine zygotes and obtained Tg fetuses and piglets with high efficiencies (7.5%; 9/120 of transferred embryos).…”