One of the major challenges in organ transplantation is reducing the risk of rejection, which is especially critical in xenotransplants. Studies have identified key molecules targeted by pre-formed antibodies in recipients of pig xenotransplants. These xenoantigens include cell surface glycans such as alpha-Gal, SDa, and Neu5Gc. Pigs lacking these xenoantigens were produced by somatic cell nuclear transfer (SCNT) using fibroblasts that were edited to remove the genes responsible for these molecules, followed by implantation of knockout embryos into synchronized surrogates. Eliminating these glycans reduced hyperacute rejection and enabled the identification of additional antigenic molecules, particularly Swine Leukocyte Antigens (SLAs), the pig orthologs of Human Leukocyte Antigens (HLAs). Some patients awaiting organ transplants have pre-formed antibodies against SLAs, which can jeopardize transplant safety and recipient survival. Therefore, we aimed to characterize the SLAs present in pig fibroblasts used as donors for SCNT. This would allow us to express these specific SLAs on the surface of lymphoblastoid cells lacking HLAs and assess immunoreactivity. Our results will be essential to determine if a patient is more suitable to receive an organ from a particular clonal group of our edited donor pigs.

First, SLA genes from fibroblasts of five different pigs were amplified by PCR from isolated genomic DNA. The PCR products were cloned using the pGEMT system and sequenced by Sanger sequencing. In parallel, SLA mRNA was amplified by RT-PCR from isolated RNA and sequenced. Finally, sequences were assembled and aligned to compare them and determine their identity.

Amplification of a region including SLA exons 2 and 3 from genomic DNA yielded two bands: one 495 base pairs (bp) long and another 545 bp long. Amplification of a larger region including exons 2 to 4 yielded a single 1,345 bp band. BLAST alignments confirmed these sequences as SLAs. Our analysis showed that the main difference between the 495 bp and 545 bp products lies in the intron sequence. Notably, the 495 bp product showed low similarity to both the 1,345 bp product and the transcripts, suggesting it may be a pseudogene. The 545 bp and 1,345 bp sequences matched each other well and aligned with the transcripts, confirming amplification of SLA class I genes. Finally, the SLA sequences from the five pigs grouped into three distinct SLA clusters. In conclusion, three different SLA sequences should be considered when immunologically testing if a patient is more likely to receive an organ from one pig group or the others.