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.