Background: The Hanganutziu-Deicher (H-D) antigen, NeuGc, is widely distributed in mammalian species including monkeys and apes, but is not found in humans and birds. After the knock out of alpha-1,3galactosyltransfease (alpha-Gal),the H-D antigen became a major antigen of the "non-Gal antigen". The expression of NeuGc is controlled by the activity of cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase. Several strategies for reducing the level of this antigen in pig cells are under consideration and include the knockdown and knockout of the CMP-NeuAc gene. Findings reported in our previous study indicated that it was possible that the over expression of N-acetylglucosaminyltransferase-III (GnT-III) reduced the levels of H-D antigen, because GnT-III does not act exclusively on alpha-Gal. GnT-III acts only on an N-linked sugar of a glycoprotein, but it clearly has antigenicity, including the H-D antigen. In this study, we report on an attempt to reduce the expression levels of H-D antigens by RNA interference (siRNA) for pig CMP-N-acetylneuraminic acid hydoxylase, as the first step in producing H-D knockdown and knockout pigs. Methods: Although several groups have reported on the pig CMP-NeuAc gene, we cloned and characterized it, by comparing it to the mouse CMP-NeuAc gene. Several synthetic siRNAs targeting the gene were then designed according to published guidelines :four siRNAs(21 mer) and three siRNAs(25 mer:S). Real-time PCR (RT) primers were also inserted stradding intron 15. The PECs and fibroblasts from wild-type and an α-Gal KO pig were used to check the changes in antigenicity for the human natural antibody by knocking down the H-D antigen. For the next experiment, plasmids with siRNA for the stable clones were prepared, pSXGH: the polymerase-III H1-RNA gene promoter with pCXHL-GFP, a green florescence protein subcloned into the betaactin promoter with the CMV enhancer and hygromycin resistance, pSilencer 1.0-U6: U6-RNA gene promoter and piGENE: tRNA promoter. Results: I. Analysis of the gene. The ATG is located in exon 4, which corresponds to the mouse gene, and the stop codon in exon 17. While exon 18 was identified in the 3 'site of the gene, in the case of the 5' site of the gene, exon 3 was also identified but exons 1 and 2 are still being investigated. II. Inhibition of H-D antigen expression on PEC by synthetic siRNA. 1. Single suppression < siRNA #1 (exon 1): 27-56%, siRNA #2 (exon 2): 32-48%, siRNA #4 (exon 8): 42-42%, siRNA #5 (exon 11): 43-83%, siRNA S#2 (exon 8-9): 25-89%, siRNA S#6 (exon 11): 25-52%, siRNA S#8 (exon 12-13): 41-77%>. 2. Dowble suppression < siRNA #1+#5 : 46-94%, siRNA #5+S#8: 44-66%, siRNA #1+S#8 : 42-100%, siRNA #1+S#2: 52-97% >. 3. Triple suppression < siRNA #1+#5+S#8 : 26-94%>. Conclusion: We analyzed the pig CMP-NeuAc gene and successfully downregulated the expression of H-D antigen from the PECs by siRNA. The results represent useful information for future clinical xenotransplantation studies.
