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Enabling Methods/Technologies in Transplantation

Saturday September 25, 2021 - 15:50 to 17:05

Room: General Session

315.5 Strategies for precise placement of xenoprotective transgenes

Beate Rieblinger, Germany

Chair of Livestock Biotechnology
Technische Universität München


Strategies for precise placement of xenoprotective transgenes

Beate Rieblinger1, Konrad Fischer1, Agnieszka Bak1, Andrea Fischer1, Eckhard Wolf2, Angelika Schnieke1.

1Chair of Livestock Biotechnology, School of Life Sciences, Technical University of Munich, Freising, Germany; 2Chair of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-Universität München, Oberschleissheim, Germany

Introduction: A clinically effective xenodonor pig requires a range of genetic modifications. Xenoprotective transgenes are best placed at a permissive site within the porcine genome which supports high expression even from a single-copy transgene, and allows subsequent addition of further xenoprotective genes. Combining all transgenes at a single site avoids segregation, simplifies breeding of the xenodonor animals and ensures a ready supply of porcine organs and tissues for transplantation.

Methods: We used Bxb1-integrase-mediated and CRISPR/Cas9-assisted approaches to place transgene constructs either at the porcine ROSA26 locus or adjacent to a previously introduced multi-transgene construct at 6q22.

Results: We previously showed that the porcine ROSA26 locus could be targeted efficiently supporting abundant ubiquitous transgene expression and allows consecutive addition of further transgenes. Here, we placed a multifunctional integrase (MIN) tag at this site that serves as a genetic entry site for the Bxb1 integrase and generated a transgenic animal. Functionality was shown via Bxb1-mediated integration of an AttB-eGFP construct and this system can now be used for placement of a battery of xenoprotective transgenes. Moreover, we adopted a CRISPR/Cas9-assisted approach, called CRISPlace, for highly efficient transgenes introduction at the ROSA26 or 6q22 locus in a recombination-independent manner. Clones with transgene integration at the desired site were generated with high efficiencies and used for somatic cell nuclear transfer to generate transgenic animals.

Conclusion: We demonstrated effective strategies for the generation of transgenic xenodonor pigs. Controlled transgene addition at the porcine ROSA26 or 6q22 locus provides a suitable strategy for the generation of optimised xenodonor pigs.


DFG TRR 127: Biology of xenogeneic cell, tissue and organ transplantation - from bench to bedside.

Presentations by Beate Rieblinger