Evaluation of TN5 mediated transgenesis in vivo in ovine

Transposable elements are DNA segments with the unique ability to move within the genome. In particular, transposons can be applied as vectors for gene transfer in vertebrates, and have already proven efficient to produce transgenic mice and pigs. Among them, the Tn5 transposition system allows the use of the commercial Tn5 transposase protein to generate in vitro the transposome complex with the transgen. Previously, we demonstrated that simple Tn5 cytoplasmic injection was efficient to produce high rates of transgene expressing embryos in vitro and confirmed integration in embryos by southern blot. In this work, Tn5 transposition mediated transgenesis was evaluated for the first time to produce transgenic ovine capable to express human recombinant factor IX (hrFIX) in milk. With this aim, adult Hampshire Down donor sheep were superestimulated (n=4) by insertion of intravaginal sponges for 14 days and during days 12-14, were injected with decreasing doses of FSH im every 12 h, administered twice daily for up to 12 h before sponge removal. On day 14, sponges were removed and a single dose of eCG (200 IU) was applied. The estrus was detected by males, and the ewes were inseminated by laparoscopy with frozen/thawed semen. Afterwards (16 h post insemination), presumptive zygotes were collected from the oviducts and cytoplasmically microinjected with the complex Tn5: hrFIXtransposon (20 ng/ul; protein: transgene with mosaic ends recognized by the transposon), in Mg+2 free medium. Immediately after microinjection, zygotes were transferred into receptor ewes oviducts (synchronized as donors, without the addition of FSH). As a result, a total of 28 presumptive zygotes were recovered and microinjected with the complex Tn5:hrFIX. A total of 12 microinjected presumptive zygotes were transferred to 5 donor ewes. It was obtained 1 pregnancy of siblings, but only one animal was born. The tissues and blood of the newborn were analyzed by nested PCR and it was determined that the animal was not transgenic. It is necessary to repeat the experience in order to determine the efficiency of Tn5 transposon transgenesis technique in ovine.