Isolation and characterization of porcine Gal KO fibroblasts expressing hCD55-hCD39 and hEPCR-hTPA

Xenotransplantation will benefit from genetic modification of the pig genome to reduce immunogenicity; the first obstacle in pig to human xenotransplanation was represented by hyperacute rejection (HAR), which has been overcome by genetic ablation of a1,3 galactosyltransferase and by expression of the hDAF. The second obstacle is represented by acute vascular rejection (AVR) that is characterised by vascular thrombosis, blood extravasation and edema. Expression of molecules involved in the coagulation cascade (ePCR and tPA) and in inflammatory-apoptotic events (CD39) are potential strategies to bypass AVR. The aim of this work is the production of transgenic cell lines to use in SCNT for the production of pigs for xenotransplantation studies. Two neonatal pig Gal KO fibroblasts lines cultured in DMEM/M199 1:1 + 10% FCS + 5ng/ml bFGF were co-transfected by nucleofection with ubiquitous expression vectors pMG5’3’MARHyg2272-CXhDAF and pCXhCD39-3'MAR or pMG5’3’MARPuro5171-CXhEPCR and pJC13I-CXhTPA. After nucleofection, cells were plated in Petri dishes and selected for eight days with Hygromycin: 150 µg/ml or Puromycin: 1 µg/ml. Drug resistant colonies were isolated and expanded for transgene expression analysis. We used immunohistochemistry (IHC) to detect the expression of the protein. For hDAF we used IA10 (BD Pharmingen) and for hCD39 BU61(Ancell). For hEPCR and hTPA we screened a battery of antibodies without detecting a clear specific signal. Therefore we used RT-PCR to detect the presence of the transcripts. Cells from DAF-CD39 colonies were serum starved for 24h before being fused to enucleated oocytes. Following electric activation, embryos were grown in vitro to the blastocyst stage. Thirty-nine colonies derived from a cell line transfected with hDAF-hCD39 were analysed by IHC: 48,7 % of the colonies expressed both hCD55-hCD39; 28,2 % expressed only CD55; 15,3 % expressed only CD39; 5,1 % showed mosaic expression. IHC findings were confirmed also by RT PCR. We analysed nine selected colonies from hEPCR-hTPA by RT PCR; six colonies (66.6 %) showed the presence of both transcripts, two colonies (22.2 %) showed only TPA transcript. Gal KO cell colonies co-expressing DAF-CD39 were used in SCNT experiments obtaining 32.4% compacted blastocyst development (n=1446). We obtained 40.7 % (n=2583) blastocyst development using only Gal KO cells. In this experiment was demonstrated that this system is very efficient to produce DAF-CD39 cloned pig blastocysts using GAL KO cells. Moreover, Gal KO TPA/EPCR cell lines were established for the first time. This study was supported by EU grant n_ LSHB-CT-2006-037377 and Fondazione Banca Popolare di Cremona