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.
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 humanCD55 transgene adjacent to a humanheme 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.
Authors: Tatiana Flisikowska; Jerome Egli; Angelika Schnieke; Antonio Iglesias; Krzysztof Flisikowski; Marlene Stumbaum; Erich Küng; Martin Ebeling; Roland Schmucki; Guy Georges; Thomas Singer; Mayuko Kurome; Barbara Kessler; Valeri Zakhartchenko; Eckhard Wolf; Felix Weber Journal: Nat Biomed Eng Date: 2022-09-22 Impact factor: 29.234