Assembling multiple xenoprotective transgenes in pigs.

This review gives a brief overview of the genetic modifications necessary for grafted porcine tissues and organs to overcome rejection in human recipients. It then focuses on the problem of generating and breeding herds of donor pigs carrying modified endogenous genes and multiple xenoprotective transgenes. A xenodonor pig optimised for human clinical use could well require the addition of ten or more xenoprotective transgenes. It is impractical to produce the required combination of transgene by cross-breeding animals bearing individual transgenes at unlinked genetic loci, because independent segregation means that huge numbers of pigs would be required to produce relatively few donor animals. A better approach is to colocate groups of transgenes at a single genomic locus. We outline current methods to assemble transgene arrays and consider their pros and cons. These include polycistronic expression systems, in vitro recombination of large DNA fragments in PAC and BAC vectors, transposon vectors, classical gene targeting by homologous recombination at permissive loci such as ROSA26, targeted transgene placement aided by gene editing systems such as CRISPR/Cas9, and transgene placement by site-specific recombination such as Min-tagging using the Bxb1recombinase.