Targeted knock-in of human immune-regulatory genes into the porcine GGTA1 exon 4 reveals divergent expression on red blood cell membranes

The decline in blood donations has become a global challenge, emphasizing the urgent need for alternatives to meet the growing demand. One potential solution is the development of transgenic pigs whose blood can overcome immune rejection and can therefore be used in xenotransfusion. We generated two types of transgenic pigs, one expressing human CD46 ( hCD46 ) and human thrombomodulin ( hTBM ) and the other expressing human CD59 ( hCD59 ) and human CD47 ( hCD47 ). These genes were inserted into the exon 4 of glycoprotein alpha-galactosyltransferase 1 ( GGTA 1 ) locus to eliminate galactose-α-1,3-galactose (α-gal) , while simultaneously enabling the expression of human immune-regulatory genes. Among these, hCD59 and hCD47 were detected on the membranes of porcine red blood cells (pRBCs) of the transgenic pigs, whereas hCD46 and hTBM were not. However, all four inserted genes were expressed in other tissues. Notably, although hCD46 and hTBM mRNA were transcribed in erythroid cells, their proteins were absent from the pRBC membrane, suggesting that they were lost or degraded during erythropoiesis. This study shows that expression patterns of transgenes may be unique for different proteins in RBCs, and a greater understanding of post-translational modifications and membrane localization is needed to enable better expression of human immune-regulatory genes.