431 GENERATION OF CLONED CD55-CD39 TRANSGENIC α1,3-GALACTOSYLTRANSFERASE DEPLETED (GAL-/-) PIGLETS

Abstract

Success in xenotransplantation relies on engineering of the pig genome to express human transgenes, such as CD55/CD39, that can control coagulation and inflammation to prolong the graft survival of a 1,3-galactosyltransferase depleted (Gal-/-) pig organs in nonhuman primates and then able to bypass the hyperacute rejection. The aim of our work was to produce Gal-/- piglets overexpressing CD55/CD39. In experiment (Exp.) 1 exploiting 2 ubiquitous expression vectors (pCAGGS-CD55 and pCAGGS-CD39), we transfected immortalized porcine kidney cells (PK15) with CD55 and CD39 using Nucleofector (Amaxa Biosystems, Cologne, Germany) and selected 5 cell colonies each (PK15-CD55 and PK15-CD39) that were expanded and analyzed by RT-PCR, immunohistochemistry (IHC) and Western blot (WB). The monoclonal antibodies IA10 for hCD55 and BU61 for hCD39 were used. Transgenic transcription was confirmed by Northern blot (NB) using digoxigenin-labeled probes. In Exp. 2, a neonatal pig Gal-/- fibroblast line was co-transfected by Nucleofector using 2 ubiquitous expression vectors (hEF-CD55 and pCAGGS-CD39) for the expression of CD55 and CD39. Colonies were analyzed by RT-PCR and IHC only, because of the limited number of cells available. Cells from one colony with a high level of CD55/CD39 expression according to IHC were used for nuclear transfer into enucleated oocytes. Day 5 compact morula/blastocyst (n = 144) were transplanted in 2 synchronized sows. Porcine aortic endothelial cells (PAEC) and fibroblasts derived from 2 stillborn piglets were analysed with IHC, NB, and WB. The expression level of transgenes from both experiments was compared with human umbilical vein endothelial cells (HUVEC) by fluorescence-activated cell sorting (FACS), using IA10, BRIC110, IH4, 2G2, and MEM-118 antibodies for hCD55 and TU66 for hCD39. In Exp. 1, RT-PCR showed CD55 mRNA expression in 3 out of 5 (2, 15, 24) PK15-CD55 colonies. A high level of CD55 expression was confirmed only in colony 24 by IHC, NB, WB, and FACS. Low expression level in colony 2 revealed by FACS was not detected by IHC, indicating that FACS analysis is more accurate to quantify the level of expression. All PK15-CD39 colonies were positive according to RT-PCR and IHC. Only one colony PK15-CD39 was further analyzed by NB and WB and confirmed positive. In Exp. 2, IHC, NB, WB, and FACS analyses of fibroblasts and PAEC derived from both cloned piglets confirmed the high level of CD39 expression detected by IHC in donor cells used for nuclear transfer. However, strong CD55 expression detected by IHC was not confirmed by NB analyses and, by FACS, was lower than in HUVEC cells. In conclusion, we produced cloned CD55-CD39 transgenic Gal-/- piglets with a high level of CD39 expression but the expression level of CD55 was lower than in HUVEC cells. We found that although IHC is the method of choice in preliminary screening, it is not sufficiently quantitative when only a few cells for each clone are available. Thus, IHC needs to be complemented with additional methods (e.g. WB, FACS, real-time RT-PCR) to obtain complete evaluation of the expression pattern of transgenes before nuclear transfer experiments.

This study was supported by EU grant no. LSHB-CT-2006-037377 and Fondazione Banca Popolare di Cremona.