Double transgenic Gal(-/-) piglets over-expressing hCD39

Over-expression of human CD39 in transgenic pigs is a potential strategy to bypass acute vascular rejection in xenotransplantation. The aim of this work is the production of transgenic cloned pigs using a Gal -/- CD55/CD39 cell line. A neonatal pig Gal -/- fibroblast line cultured in DMEM/M199 1:1 + 10% FCS + 5 ng/ml bFGF was co-transfected by nucleofection with two ubiquitous expression vectors, the first carrying hCD55 under Elongation Factor promoter and a HygroR cassette; the second carrying hCD39 under pCAGGS promoter and a 3¢ MAR region. After nucleofection, cells were plated in Petri dishes and selected with Hygromycin B for 8 days. Drug resistant colonies were isolated and expanded for transgene expression analysis. We used immunohistochemistry (IHC) to detect the expression of the proteins. For hCD55 we used IA10 and for hCD39 BU61. Cells co-expressing CD55–CD39 were serum starved for 24 h before being fused to enucleated oocytes. Following electric activation, embryos were grown in vitro up to compact morula/blastocyst and all (n=144) were transplanted in two synchronized sows. PAEC and fibroblasts derived from delivered piglets were analysed with FACS, using the following antibodies: BRIC110, IH4, 2G2, and MEM-118 for hCD55 and TU66 for hCD39 detection respectively. Using a double transgenic CD55/CD39 Gal -/- colony in a Somatic Cell Nuclear Transfer (SCNT) experiment we have obtained 35.4% compacted morula/blastocyst development. One of two sows resulted in a pregnancy. At day 117 of gestation, this sow was induced to farrowing and delivered two stillborn piglets that were probably too immature and died from respiratory failure. Nevertheless, IHC analysis performed on PAEC and fibroblasts derived from these piglets showed strong expression of CD55– CD39, as in the original colony. FACS analysis confirmed robust human CD39 expression but showed a very low level of hCD55 expression. Two Gal -/- CD55/CD39 piglets were obtained. Over-expression of hCD39 seems to be compatible with normal pig fetus development. Efficency of SCNT using the double transgenic Gal -/- line was slightly lower than that observed using the Gal -/- line itself, from which we obtained 40.7% (n=2583) blastocyst development in previous experiments. This cell line will be used for generation of other cloned piglets and for in vitro test analysis