GalSafe pigs were generated through rDNA-mediated homologous recombination using porcine fetal fibroblasts followed by somatic cell nuclear transfer into enucleated oocytes. Hemizygous GalSafe lineage progenitors were bred to produce homozygous GalSafe animals.

The vector pPL657 was used to transfer the recombinant DNA (rDNA) consisting of DNA sequences homologous to the targeted region of the porcine galactosyltransferase alpha 1,3 gene (GGTA1) flanking a neomycin phosphotransferase gene (nptII) as selectable marker.

Disruption of the GGTA1 gene results in undetectable endogenous galactose-alpha-1,3-galactose sugar residues on the cell surfaces of these pigs. Hence, these alpha-gal sugars are undetectable on biological derivatives such as tissues and organs. This has implications for people who suffer from alpha-gal syndrome (AGS), an allergy to red meat from food producing mammals and other products containing mammalian based materials, including cosmetics and medicines. GalSafe pigs are intended to be used as sources of food or human therapeutics including excipients, devices, drugs, or biological products.

Molecular characterization indicated that the rDNA construct was stably integrated in both alleles of the targeted location in the GGTA1 gene. Southern blot analysis indicates that no vector backbone was integrated in the genome of the GalSafe pig. Both the genotype and phenotype of GalSafe pigs were conserved across multiple generations.

Sources:

1. Freedom of Information Summary (FOI), Original New Animal Drug Application (NADA 141-542): pPL657 rDNA Construct in Domestic Pigs. Date of Approval: December 14, 2020.

2. Dai et al. (2002), Targeted disruption of the α-1,3-galactoysltransferase gene in cloned pigs. Nature 20:251–255.