Antigenicity of tissues and organs from GGTA1/CMAH/β4GalNT2 triple gene knockout pigs

Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigens[Gal,Neu5 Gc,and Sd(a)]present on pig cells and tissues.Triple gene(GGTAI,CMAH,and β4 GalNT2)knockout(TKO)pigs were produced in our laboratory by CRISPR-Cas9 targeting.To investigate the antigenicity reduction in the TKO pigs,the expression levels of these three xenoantigens in the cornea,heart,liver,spleen,lung,kidney,and pancreas tissues were examined.The level of human IgG/IgM binding to those tissues was also investigated,with wildtype pig tissues as control.The results showed that aGal,Neu5 Gc,and Sd(a)were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs.Compared to wildtype pigs,the liver,spleen,and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding,whereas corneas,heart,lung,and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding.These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.

Overexpressing dominant-negative FGFR2-IIIb impedes lung branching morphogenesis in pigs

Genetic studies with mouse models have shown that fibroblast growth factor receptor 2-Ⅲb（FGFR2-Ⅲb）plays crucial roles in lung development and differentiation. To evaluate the effect of FGFR2-Ⅲb in pig lung development, we employed somatic cell nuclear transfer（SCNT） technology to generate transgenic pig fetuses overexpressing the transmembrane（dn FGFR2-Ⅲb-Tm） and soluble（dn FGFR2-Ⅲb-HFc） forms of the dominant-negative human FGFR2-Ⅲb driven by the human surfactant protein C（SP-C） promoter,which was specifically expressed in lung epithelia. Eight dn FGFR2-Ⅲb-Tm transgenic and twelve dn FGFR2-Ⅲb-HFc transgenic pig fetuses were collected from three and two recipient sows, respectively.Repression of FGFR2-Ⅲb in lung epithelia resulted in smaller lobes and retardation of alveolarization in both forms of dn FGFR2-Ⅲb transgenic fetuses. Moreover, the dn FGFR2-Ⅲb-HFc transgenic ones showed more deterioration in lung development. Our results demonstrate that disruption of FGFR2-Ⅲb signaling in the epithelium impedes normal branching and alveolarization in pig lungs, which is less severe than the results observed in transgenic mice. The dn FGFR2-Ⅲb transgenic pig is a good model for the studies of blastocyst complementation as well as the mechanisms of lung development and organogenesis.