Anemic Pathological Features Development In GGTA1/CMAH Double Knockout Piglets

Background Cystidine monophosphate-N-acetylneuraminic acid hydroxylase(CMAH)and glycoprotein alphal 3-galactosyltransferase(GGTA1)are encoded by CMAH and GGTA1 gene,and these two genes were mutated in human evolution,resulting in absence of two antigens,the galactosyl-alpha 1,3-galactose(αGal)epitope and N-glycolylneuraminic acid(Neu5Gc).Recently,with great breakthroughs in gene engineering technologies,various animal models of GGTA1 and CMAH knockout(KO)have been generated for the sake of xenotransplantation as a potential solution to donor organ shortage crisis worldwide.GGTA1 and CMAH are key glycosylation enzymes that mediate different metabolisms of glycosylation,and the function and role of these two enzymes and its products,α-Gal and Neu5Gc,in animals are not fully understood.Objectives Thus far,however,the studies on the generation of GGTA1 and CAMH knockout animals have had a focus on application of these animal models into xenotransplantation,and the after-effects of GGTA1 and CMAH KO in animal models have not studied,yet.Here in the present study,therefore,we investigated the effects of GGTA1 and CMAH knockout in GGTA1/CMAH double knockout(DKO)pigs.Methods Blood type of pigs for fibroblast cell donor was checked by a clinical blood typing kit and PCR methods.GGTA1 and CMAH genes were knockouted by Crispr/Cas9 system,and these cells were used for somatic cell nuclear transfer.Gene knockout was confirmed through gene sequencing,immunofluorescent and flow cytometry.Hematological profile of GGTA1/CMAH DKO piglets was analyzed by complete blood count.Histological observation of bone marrow,liver and spleen was performed by Wright-Giemsa,and hematoxylin and eosin staining.For the compositional and structural changes of sialylated N-glycans on the membrane of erythrocytes of GGTA1/CMAH DKO piglets,lectin blotting assay and mass spectrometry were performed.Major findingsIn the first part of the experiment,we produced 62(9 stillborn)blood type A GGTA1/CMAH DKO piglets,and 17(3 stillborn)wild type(WT)piglets through 2 round of somatic cell nuclear transfer(SCNT)and Crispr/Cas9 system.In the first SCNT,one fetus(F3)was confirmed as homozygous mutant carrying lbp deletion in the exon 8 of GGTA1,and used for the establishment of GGTA1 knockout(KO)cell line.Using GGTA1 KO cell line,in the second SCNT,various mutations in the exon 6 of CMAH gene were confirmed among 9 fetuses(F3-1,2,3,4,5,6,7,8,9).Using a fetus of F3-6,the fibroblast cell line of GGTA1/CMAH double KO(DKO)were generated,and using this cell line,2817 embryos carrying the somatic cells of GGTA1/CMAH were transferred to 15 recipient sows.As a control,580 embryos carrying WT cells were transferred to 3 surrogate pigs.Gene knockouts in GGTA1 and CMAH genes were confirmed with sequencing.The absence of α-Gal and Neu5Gc in DKO piglets was confirmed through immunofluorescent staining and flow cytometric analysis.In the second part of the experiment,we observed that DKO piglets developed common clinical signs such as weakness(100%),dyspnea(90%)and constipation(65%)compared to WT piglets within 2 weeks after born.Subsequently,compared to WT,GGTA1/CMAH DKO revealed a significant decrease(P<0.05)in RBC(1012/L),HCT(%)and HGB(g/L),and increase in WBC(109/L),LYM(109/L),MON(109/L)and MCV(fL)(P<0.05).DKO piglets showed a more than 10%of giant multinucleated cells in bone marrow and a significant decrease in the levels of serum immunoglobulins,including IgG-n,IgA and IgM(P<0.05).DKO piglets also showed swollen liver and spleen,and exhibited raised deposition of hemosiderin and severe bleeding.The observed findings indicated that the pathological features developed in DKO piglets were similar to symptoms of anemia.In the third part of the experiment,we analyzed glycosialylation changes in the membrane of erythrocytes of DKO piglets through lectin blotting and mass spectrometry(MS),as a possible etiology of the observed pathological features.The lectin blotting assay revealed the small difference of α2,6-sialylated glycans(Siaα2,6-Gal/GalNAc)between two groups and the expression of α2,3-sialylated glycans(Siaα2,3-Galβ1-4GlcNAc)in A-DKO RBC membrane glycoproteins with approximately 55～100 kDa.DKO piglets showed about 83%normalized intensity of NeuAc-sialylated complex/hybrid glycans as a major glycan of erythrocyte membrane,while WT piglets displayed NeuGc-sialylated complex/hybrid(57%normalized intensity)as a major glycan of erythrocyte membrane.MS analysis showed that A-DKO increased di-sialylated N-glycans and supported the lectin assay,showing theα2,3-sialylation linkages on the RBC membrane glycoproteins with 5 5～100 kDa extracted from lectin blotting.With these results,we demonstrated that the composition and structure of sialylation on the membrane of erythrocytes of DKO piglets were remarkably altered compared to that of WT piglets,which might be associated with the observed pathological features.ConclusionIn conclusions,the findings indicate that GGTA1 and CMAH knockout in pigs displayed pathological features similar to symptoms of hemolytic anemia,and the altered glycosylation patterns in erythrocyte membrane of DKO piglets could be implicated in the observed pathologies.This study will contribute to better understanding of the after-effects of GGTA1/CMAH knockout in animal models and will give a more comprehensive view on the generation of GGTA1/CMAH knockout animal models.