321 PRECISION GENETIC ENGINEERING OF THE PIG GENOME AND SKIN TRANSPLANTATION WITHIN A SYNGENIC CLONE COHORT CARRYING DIFFERENT VITAL REPORTER TRANSPOSONS

Abstract

Farm animals have a long-standing tradition as models in transplantation medicine. The implementation of techniques for precision genetic engineering techniques in pigs will allow to assessing safety and efficacy of novel cell therapies in this large animal model (Garrels et al. 2011 PLoS One 6, e235731). Recently, we generated a syngenic clone cohort of Sleeping Beauty (SB) transposon transgenic pigs by active transgenesis. Eight syngenic pigs carried a monomeric transposon with a Venus cDNA, and 4 animals carried a transposon with an mCherry cDNA; both groups of animals had the same genetic background and differed only in regard to the reporter transgene. In addition, half siblings resulting from conventional breeding were used as control animals (heterologous). To assess immunocompatibility within the clone cohort, skin transplantation between mCherry pigs and Venus pigs was performed. The donors (2 mCherry pigs) and the recipients (3 Venus pigs) were anesthetized and 25-mm² patches of skin were transplanted under sterile conditions. As control, skin patches of half siblings (heterologous) were transplanted to the Venus recipients. Each recipient was transplanted with 6 small skin patches. The fluorescence reporters Venus and mCherry allowed for unambiguous tracking of cell origin, potential cell fusion, or cell migration in the transplants. Skin patches, which were transplanted between animals of the clone cohort, were accepted within 2 to 3 weeks (n = 14), whereas heterologous skin transplants (n = 4) were rejected. Some of the skin transplants were recovered for fluorescence microscopic analysis after 3 to 6 weeks. Fluorescence microscopy allowed unequivocal discrimination between host cells and cells derived from the transplant in the “wound” zone. Major advantages of this approach are the identical genetic background, omission of an immunosuppressive regime, and direct visual discrimination of transplanted and host cells by different fluorophore reporters. Importantly, no signs of transgene silencing were found for the monomeric SB transposon constructs. These data show that precision genetic engineering is possible in the domestic pig, and that the mCherry and Venus reporters are suitable for long-term cell tracking in transplantation situations. This approach is promising for the development of a preclinical model for novel cell therapies.