Introduction:  The continuous decline in blood donations has become a critical global issue, emphasizing the urgent need for alternative solutions to meet the growing global demand. One promising approach is the development of transgenic pigs capable of overcoming immune rejection, enabling their blood to be used in therapeutic applications. To this end, we aim to generate transgenic pigs by eliminating genes responsible for xenoantigen production and inserting human genes that regulate immune rejection, thereby enabling the development of xenogeneic porcine red blood cell (pRBC) products.
Methods:  Two types of transgenic cells were generated by inserting hCD46-2A-hTBM (TG1) or hCD59-2A-hCD47 (TG2) vectors into exon 4 of the GGTA1 gene, which encodes alpha-1,3-galactosyltransferase, via homologous recombination. Subsequently, cloned transgenic pigs were generated via somatic cell nuclear transfer based on these cells. Blood samples were collected from the resulting pigs, and FACS analysis was performed to verify the proper insertion and expression of the inserted human genes.
Results:  Cloned transgenic pigs were produced using transgenic cell lines carrying the TG1 and TG2 constructs. In peripheral blood mononuclear cells (PBMCs), all transgenes were successfully expressed. However, in pRBCs, hCD46 and hTBM were not detected, whereas the other inserted genes, hCD59 and hCD47, showed clear expression. Notably, hCD46 and hTBM were expressed in PBMCs and other tissues, indicating that their lack of expression is specific to red blood cells. Further analysis at the mRNA level revealed that hCD46 and hTBM transcripts were present in pRBCs.
Conclusion:  In summary, to develop xenogeneic RBC products, we successfully produced transgenic pigs with the human immune regulatory genes hCD46 and hTBM or hCD59 and hCD47 inserted into the GGTA1 exon 4 locus. While the inserted genes were properly expressed in PBMCs and other tissues, hCD46 and hTBM were not expressed on the membrane of erythrocytes, in contrast to hCD59 and hCD47. Although mRNA transcription occurs in erythrocyte precursor cells, challenges seem to arise during post-transcriptional or subsequent processes. Further research is needed to clarify the precise mechanisms involved.