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Background: Somatic cell nuclear transfer is the method of choice to generate transgenic large animals. Success largely depends on a high expression level of the target protein. Selection of cell clones with the desired expression level is of paramount importance before nuclear transfer. In this work we compare different methods that can be used on a small number of cells and their predictive value. Methods: Two expression vectors for CD55 and CD39 were separately transfected to PK15 cell line. Following selection, five of the best growing clones resulting from each transfection were expanded and subjected to RT-PCR and Immunohistochemistry (IHC) analyses. For IHC analyses the mAB IA10 (BD-Pharmingen) – for CD55 – and the mAB BU61 (Ancell) – for CD39 – were used. The same antibodies were also used in Western blot (WB) analyses performed on samples subjected to non-reducing SDS–PAGE and electroblotted on PVDF membrane. The presence of the target transcripts was confirmed by Northern blot (NB) analyses using DIGlabeled probes (Roche). The proteins expression was also analysed by FACS conducted on chosen clones. Fibroblasts and PAECs deriving from one CD55–CD39 stillborn cloned piglet were subjected to IHC, NB and FACS analyses. Results: Three out of five (#24, #2 and #15) PK15–CD55 clones were positive to RT-PCR but only clone #24 was positive to IHC. Clone #24 was further analysed by NB, WB and FACS that confirmed the high expression level. Clone #2 revealed a low expression level by FACS not detected by IHC. All five PK15–CD39 clones were positive to IHC and RT-PCR analysis. Clone #10 was further analysed and confirmed positive by NB and WB. The IHC, NB and FACS data obtained on fibroblasts and PAECs of cloned piglet confirmed the donor cell lines CD39 expression detected by IHC. This was not the case with CD55 expression since the positivity detected by IHC was not confirmed with FACS and NB analyses. Conclusion: IHC is the method of choice when few cells for each

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 seem