High throughput production of multi-transgenic pig for xenotranplantation research

Due to the increasing demand of organ for transplantation and the shortage of human donors several areas of research, including regenerative medicine and xenotransplantation, are currently explored to provide different clinical solutions to a varieties of pathological conditions. While regenerative medicine will relay on stem cell auto-regeneration and/or cell transplantation in partially compromised tissues and organs, xenotransplantation would provide ready to use tissues or organs for more severe pathologies or organ failures. Genetic engineering of porcine genome lies at the heart of xenotransplantation research since the pig is currently considered, on the risk/benefit ratio, the most appropriate species. The stepsinvolved in the creation of candidate animals for xenotransplantation research include the selection of a somatic cell line (usually fibroblasts) that is engineered using current technologies, either for the inactivation or for the insertion of candidate genes, followed by somatic cell nuclear transfer (SCNT) procedure to generate live animals. The founder animals obtained can be suitable directly for xenotransplantation and/or their cells can be further engineered. In our laboratory we have been working primarily with a male GAL-KO miniature pig cell line (provided by D. Sachs, MGH, Boston USA) with the aim of overexpressing under an ubiquitous promoter (pCGACGS) several transgenes controlling complement mediated lysis and inflammation (hCD55, hCD39), and coagulation (hEPCR, hTM). To speed up the process we cotransfected by nucleofection two transgenes at the same time, one of which carried the selectable marker (hCD55HygromycinR ? hCD39; hEPCRPuromycinR ? hTM). Forty-eight hours later the cells were subjected to drug selection for 15 days to isolate resistant cell clones that were expanded in duplicates for SCNT and phenotypic characterisation by Western blot and immunocitochemistry to assess the presence of the protein and the uniform expression of the transgenes in all morphologically normal cells. At this point the clones expressing single or the double transgenes were selected for nuclear transfer. Zona-free enucleated oocytes were fused with fibroblasts, activated and cultured in vitro up to the blastocyst stage for 6 days. Blastocysts were subsequently transferred to synchronised recipient sows on day 5 after ovulation. Pregnant animals were allowed to go to term and farrowing was induced with prostaglandin, if it did not occur by day 118 of gestation, or by caesarian section. After nucleofection of 106 cells with CD55-CD39 we obtained 60 colonies of which 12 did not express the transgenes, 15 expressed one and 21 expressed both transgenes while 12 were composed of negative and positive cells (variegated). After nucleofection of 106 cells with EPCRhTM we obtained 12 colonies of which 3 did not express the transgenes, 7 expressed one and 2 expressed both transgenes however possessed some negative for EPCR cells. ICC analyses matched the data of WB and gave possibility to see the quality of the cells and the degree of uniform expression (absence of variegation) and possible contamination of colonies by negative cells. For each recipient sow we implanted pools of 86 ± 12 (range 59–109) embryos coming from 4 cell colonies with high expression either of CD55 alone or both CD55-CD39 to optimise pregnancy rates and embryo survival. We transferred embryos to 27 sows and obtained 6 out of 10 pregnancies for the single and 7 out of 16 for the double transgene, but one of the single and three with double transgenic embryos were lost by day 40. Forty-nine piglets were obtained of which 28 were alive at birth and 20 were alive after 1 week. The development to term of the single transgenic CD55-embryos was higher in comparison with double transgenic embryos (6 piglets/sow vs. 3.25 piglet/sow). The higher abortion rate and small number of piglets per litter suggest the detrimental effect of ubiquitous high CD39 expression in the donor cells. The WB analysis of the umbilical cord of all animals and of some organs of the stillborn confirmed the expression in all animals obtained with some minor differences in the levels of the proteins. After first PCR screenings, Southern Blot (SB) analysis done on CD55+ piglets live at birth confirmed that starting from 8 selected fibroblasts colonies (used in two different pools) we obtained 6 different founder animals of which 5 were CD55+ positive. The same strategy was used also on CD55+ -CD39+ piglets live at birth identifying 3 different founders starting from 4 double transgenic colonies. In conclusion, we implemented a system that, by combining thorough analysis of the transfected donor fibroblast cell clone for uniformity and high level of protein expression and pooling embryos of different clonal origin, can efficiently and reliably generate founder animals in sufficient number to be used directly for xenotransplantation. Moreover particularly interesting phenotypes can be re-cloned for breeding purposes