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.

Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting

Unbalanced brain serotonin(5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2(TPH2). In the present study, the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas) system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer(SCNT) and 10 Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth.However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

Induction of diabetes in cynomolgus monkey with one shot of analytical grade streptozotocin

Backgrounds:Streptozotocin(STZ)-induced diabetic monkey is a wide used preclinical animal model for the investigation of diabetes such as islet transplantation and development of diabetic drugs.There are serious side effects of this method,including nausea,emesis,weight loss,liver damage,renal failure,and metabolic acidosis.In order to reduce the side effects,diabetic monkeys were induced using clinicalgrade STZ.However,clinical-grade STZ is not available in China.Here,we establised a method by using 100 mg/kg analytical-grade STZ to induce complete diabetes in cynomolgus monkey without generating adverse effects to liver and renal.Methods:Three cynomolgus monkeys were used in this study.100 mg/kg STZ dissolved in normal saline and infused through vein line in 5 minutes after indwelling catheter in the carotid artery and jugular vein.After the STZ administration,blood glucose levels were examined every 1 or 2 hours in the first 48 hours.Then,blood glucose levels were examined twice per day during the first week after the STZ injection.Insulin and C-peptide levels were measured by ELISA.Blood chemistry of hepatic and renal function tests were performed.Insulin and glucagon expression in the islet of diabetic monkey and normal monkey were examined by immunohistochemistry assays.Results:The stimulated C-peptide level(Intravenous glucose tolerance test)which is less than 0.5 ng/mL,the triphasic blood glucose response and the destroyed β cell suggested the complete induction of diabetes model.No apparent adverse effects were observed including no signs of vomiting and toxicity after STZ injection.Conclusion:In summary,we established a safe and reproducible STZ-induced diabetic cynomolgus monkey model for islet transplantation which will be used to develop novel approaches for the treatment of diabetes.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.

Erratum to“A Bama miniature pig model of monoallelic TSC1 mutation for human tuberous sclerosis complex”[Journal of Genetics and Genomics(2020)47,735–742]

This erratum clarifies information in the article“A Bama miniature pig model of monoallelic TSC1 mutation for human tuberous sclerosis complex”by Li et al.(2020).In the“Acknowledgment”section,the statement that“This work was supported by grants from the National Natural Science Foundation of China(31701283,81970164)”should say“This work was supported by grants from the National Natural Science Foundation of China(81874144,81970164)”.

A Bama miniature pig model of monoallelic TSC1 mutation for human tuberous sclerosis complex

Tuberous sclerosis complex(TSC)is a dominant genetic neurocutaneous syndrome characterized by multiple organ hamartomas.Although rodent models bearing a germline mutation in either TSC1 or TSC2 gene have been generated,they do not develop pathogenic lesions matching those seen in patients with TSC because of the significant differences between mice and humans,highlighting the need for an improved large animal model of TSC.Here,we successfully generate monoallelic TSC1-modified Bama miniature pigs using the CRISPR/Cas9 system along with somatic cell nuclear transfer(SCNT)technology.The expression of phosphorylated target ribosomal protein S6 is significantly enhanced in the piglets,indicating that disruption of a TSC1 allele activate the mechanistic target of rapamycin(mTOR)signaling pathway.Notably,differing from the mouse TSC models reported previously,the TSC1^(+/−)Bama miniature pig developed cardiac rhabdomyoma and subependymal nodules,resembling the major clinical features that occur in patients with TSC.These TSC1^(+/−)Bama miniature pigs could serve as valuable large animal models for further elucidation of the pathogenesis of TSC and the development of therapeutic strategies for TSC disease.

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.