Efficient production of transgenic lambs by cytoplasmic injection with Sleeping Beauty transposition plasmids.

BEVACQUA R; FERNANDEZ-MARTIN, R; GIBBONS A; CANEL N; LANGE F; TABOGA O; KUES W; S FERRARIS; SALAMONE D

Congreso; X Transgenic Animal Research Conference, TARC (2015); 2015

Almost twenty years ago, the molecular reconstruction of Sleeping Beauty transposonfrom salmonid genomes (Ivics et al, 1997) expanded the utility of transposon-basedtechnologies to vertebrates. More recently, Venus transgenic pigs and cattle wereproduced by simple cytoplasmic injection of the hyperactive SB100X system (Garrelset al. 2011; Garrels et al. 2015). The objective of this work was to produce transgeniclambs coding for human recombinant Factor IX (hrFIX) in the milk by cytoplasmicinjection with plasmids coding for Sleeping Beauty transposition system. To this aim,12 donor sheep were superovulated and subjected to laparoscopic artificial inseminationby standard procedures. Zygotes were collected from the oviducts and cytoplasmicallymicroinjected with plasmids coding for Sleeping Beauty 100X transposase (5 ng/ul) andfor BLGhrFIX transposon (10 ng/ul). After microinjection, zygotes were transferredinto the oviduct of surrogate ewes. In total, 107 zygotes were microinjected, and 33 ofthem were transferred to 11 surrogate sheep. A total of 4 (36.4%) pregnancies wereestablished, and 5 (45.5%) lambs were born. Three of the newborns were transgenic(60%). Two of them were highly mosaic, showing signals of the transgene only in liverand placenta (this could be checked only in one of the animals, as the other placentacould not be recovered); while the third one showed signals of the transgene in most ofthe tissues analyzed (skin, kidney, placenta, muscle and cells derived from testis andumbilical cord). No signals of the transposase SB100X plasmid were detected in thetissues of the animal, indicating that the plasmid was not integrated. The lamb died twodays after its birth of cardiopulmonary arrest. Southern blot results showed bandscompatible to three integrations of the transgene. These results are clear proof thatSleeping Beauty transgenesis by simple cytoplasmic injection of plasmids can be usedto produce transgenic domestic species of pharmaceutical interest with high efficiencyand simplicity.