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- Title
Strong xenoprotective function by single‐copy transgenes placed sequentially at a permissive locus.
- Authors
Rieblinger, Beate; Fischer, Konrad; Kind, Alexander; Saller, Benedikt S.; Baars, Wiebke; Schuster, Marion; Wolf‐van Buerck, Lelia; Schäffler, Andrea; Flisikowska, Tatiana; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Flisikowski, Krzysztof; Wolf, Eckhard; Seissler, Jochen; Schwinzer, Reinhard; Schnieke, Angelika
- Abstract
Abstract: 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 <italic>ROSA26</italic> 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 <italic>ROSA26</italic> 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 <italic>ROSA26</italic>.
- Subjects
PANCREATIC beta cells; TRANSGENES; IMMUNOHISTOCHEMISTRY; IMMUNOFLUORESCENCE; FLOW cytometry
- Publication
Xenotransplantation, 2018, Vol 25, Issue 2, p1
- ISSN
0908-665X
- Publication type
Article
- DOI
10.1111/xen.12382