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BACKGROUND: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. METHODS: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. RESULTS: The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. CONCLUSION: Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.

Original publication

DOI

10.1111/xen.12382

Type

Journal article

Journal

Xenotransplantation

Publication Date

03/2018

Volume

25

Keywords

ROSA26 , CD55, heme oxygenase 1, transgene stacking, xenotransplantation, Animals, Animals, Genetically Modified, CD55 Antigens, CD59 Antigens, Fibroblasts, Genetic Loci, Heme Oxygenase-1, Humans, Insulin-Secreting Cells, Promoter Regions, Genetic, Swine, Transgenes, Transplantation, Heterologous