Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus

Area of Science:

• Genetics
• Xenotransplantation
• Molecular Biology

Background:

• Consolidating multiple xenoprotective transgenes at a single genetic locus is crucial.
• This approach prevents gene segregation during breeding and streamlines the generation of genetically modified donor animals.

Purpose of the Study:

• To develop a method for efficiently grouping xenoprotective transgenes at a specific locus in pigs.
• To assess the expression and function of integrated transgenes in a porcine model.

Main Methods:

• Utilized transgene stacking to insert human CD55 and heme oxygenase 1 genes adjacent to each other at the porcine ROSA26 locus.
• Generated transgenic pigs via nuclear transfer and analyzed transgene expression using PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry.
• Evaluated cellular resistance to complement-mediated lysis and caspase 3/7 activation in vitro.

Main Results:

• Efficient retargeting of the ROSA26 locus and successful generation of transgenic animals.
• Abundant expression of transgenes detected in all analyzed organs, including pancreatic beta cells, at both RNA and protein levels.
• Transgenic porcine kidney fibroblasts demonstrated significant protection against complement-mediated lysis and reduced caspase 3/7 activation.

Conclusions:

• Step-by-step transgene placement at the porcine ROSA26 locus is an effective strategy.
• This method allows for the grouping of highly expressed, single-copy xenoprotective transgenes.
• The approach simplifies the production of donor animals for xenotransplantation research.