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.
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Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.
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Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
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Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
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Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.
Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants.
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