355 COMPARISON OF Tn5 AND SLEEPING BEAUTY SYSTEMS IN BOVINE EMBRYOS AND IN OVINE OFFSPRING

Abstract

Current techniques for the production of transgenic domestic animals remain inefficient. Only recently, DNA transposons resulted in improved efficiencies for mouse and pig transgenesis. In this work, we evaluated Tn5 and Sleeping Beauty systems for transgenesis in bovine and ovine species. First, both transposon systems were assessed in vitro in bovine embryos employing transposons carrying fluorescent reporter genes. In vitro-produced bovine zygotes were microinjected with either 1) a complex of Tn5:egfp transposon (20 ng μL^–1) (protein: transgene with mosaic ends recognised by Tn5, in Mg+2 free medium), or 2) two plasmids carrying Sleeping Beauty 100X (pSB100X, 5 ng μL^–1) and pT2/Venus transposon (10 ng μL^–1). In vitro results for Tn5 transgenesis in bovine showed that blastocysts, Day 4 egfp embryos and egfp blastocysts rates for the group injected with Tn5:egfp did not differ from the group injected with the egfp transposon alone (73/145, 50%; 86/145, 59%; and 65/145, 45% v. 65/129, 50%; 87/129, 67%; and 57/129, 44%, respectively). For SB transgenesis, blastocysts, D4 Venus embryos, and Venus blastocysts rates did not differ between co-injection of pSB100X and pT2/Venus or injection with pT2/Venus alone (46/99, 46.5%; 64/99, 64.6%; and 33/99, 33.3% v. 41/83, 49.4%; 52/83, 62.7%; and 26/83, 31.3%, respectively). However, Venus intensity in blastocysts was markedly higher for the group co-injected with pSB100X and pT2/Venus respective to pT2/Venus alone. Both systems were assessed in vivo for the production of transgenic lambs employing a functional transposon (hrFIX, recombinant human factor IX driven by a Beta-lactoglobulin promoter). Laparoscopic artificial insemination of donor sheep was performed, and presumptive zygotes were flushed from the oviducts. The microinjections were done identically as described for the bovine embryos. A total of 24 presumptive zygotes were recovered and injected with the Tn5:hrFIX complex. Then, 21 zygotes were transferred to 5 synchronized ewes; one pregnancy of siblings was obtained, and one animal was born. Genomic DNA from skin, placenta, and blood was genotyped by PCR, but the hrFIX gene could not be detected. For the SB approach, 64 presumptive zygotes were recovered from 4 superovulated ewes, microinjected with the SB plasmids, and 21 of them were transferred to 7 oestrous synchronized recipients. The remaining zygotes were cultured in vitro and blastocysts (n = 7) were vitrified. Currently, 3 donor ewes are pregnant, one with siblings (4 total fetuses). Deliveries are expected by the end of August of this year. Our results indicate that both Tn5 and SB systems are capable of resulting in the production of transgene expressing embryos, and the presence of the transposases does not affect embryo viability. However, phenotyping of blastocyst stages does not seem to be predictive for stable transgene integration. The in vivo results will help to better address the suitability of Tn5 and SB approaches for the production of transgenic sheep.