327 PRODUCTION OF TRIPLE TRANSGENIC hHO-1/GGTA-1–/–/hCD55 TRANSGENIC PIGS USING SLEEPING BEAUTY TRANSPOSITION AND ZINC-FINGER NUCLEASES
Authors: Hauschild-Quintern J. , Petersen B. , Herrmann D. , Lucas-Hahn A. , Petkov S. , Schwinzer R. , Niemann H. 
Published: December 2012

Advances in xenotransplantation (pig-to-baboon or human transplantation) require multi-transgenic pigs with a homozygous knockout (KO) of the α1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) to control the hyperacute rejection response. To achieve prolonged survival of the porcine xenograft, transgenic expression of additional immune modulatory genes on the GGTA-1–/– background is considered the solution of choice. The acute vascular rejection (AVR) is primarily caused by endothelial cell activation and leads to rejection of GGTA-1–/– pig organs. Here, we set out to produce triple transgenic pigs with high expression of the human complement regulatory gene hCD55 (human decay acceleration factor, hDAF) and the anti-apoptotic and anti-inflammatory gene human heme oxygenase-1 (hHO-1), respectively. Porcine ear fibroblasts carrying a hHO-1 transgene (Petersen et al. 2011 Xenotransplantation 18, 355–368) were transfected with zinc-finger nucleases (ZFN) targeting the GGTA-1 gene, leading to a biallelic KO of the GGTA-1 gene in ~1% of the transfected cells (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010). Somatic cell nuclear transfer (SCNT) was accomplished with cells from double transgenic cell line (hHO-1/GGTA-1–/–), and one pregnancy was terminated to obtain fetal cell cultures. Fluorescence-activated cell sorting (FACS) analysis revealed that all 11 fetuses (C6F1-F11) were lacking Gal-epitopes. One pure fetal cell culture (C6F5) was used for co-transfection with the newly designed SB-CAGGS-hDAF-Puror transposon, based on the Sleeping Beauty transposon plasmid pT2/HB, together with the SB transposase 100X plasmid (both kindly provided by Dr. Zoltan Ivics). After 14 days of selection with puromycin, cell colonies were picked and expanded to obtain cells for analysis and as a backup for SCNT. The PCR amplification with hDAF-specific primers revealed that ~50% of picked colonies had integrated the transgene. Two hDAF-positive colonies were pooled and used for SCNT. One pregnancy was sacrificed and 4 hHO-1/GGTA-1–/–/hCD55 fetuses were obtained for real-time PCR analysis and subsequent use in SCNT. Real-time PCR showed elevated hCD55 expression representing a 0.9-fold (0.7- to 1.0-fold) expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), while FACS analysis confirmed absence of Gal-epitopes in all 4 fetuses. Binding of human-specific anti-CD55 antibodies showed high expression of hCD55 by FACS measurement. Triple transgenic fetuses C8F2 and C8F4 showed higher expression of hCD55 by real-time PCR and by FACS analysis compared with fetuses C8F1 and C8F3. Fetuses C8F3 and C8F4 were used for re-cloning experiments to produce live offspring, which will further be characterised and used in transplantation experiments. This work underlines the importance of new genomic technologies to further improve the efficiency of the generation of transgenic pigs suitable for xenotransplantation.