Dear Editor: Glucose-dependent insulinotropic polypeptide (GIP) and proglucagon product glucagon-like peptide-1 (GLP- 1) and their corresponding receptors promote secretion of glucose-dependent insulin and may b...Dear Editor: Glucose-dependent insulinotropic polypeptide (GIP) and proglucagon product glucagon-like peptide-1 (GLP- 1) and their corresponding receptors promote secretion of glucose-dependent insulin and may be responsible for up to 70% of postprandial insulin secretions.展开更多
Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRIS...Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic lgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the lgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.展开更多
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 develo...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.展开更多
Using a data set from our laboratory, we assessed the effects of several factors on pig cloning ef?ciency. The results demonstrated that cells at high con?uence( > 90%) used as donor cell resulted in higher pregnan...Using a data set from our laboratory, we assessed the effects of several factors on pig cloning ef?ciency. The results demonstrated that cells at high con?uence( > 90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning ef?ciency(number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to79% con?uence and 80% to 89% con?uence. Cells with four, ?ve and six passages compromised the pregnancy and delivery rates compared with ?rst passage cells. The number of blastocysts transferred by somatic cell nuclear transfer(SCNT) did not signi?cantly affect the cloning ef?ciency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a signi?cantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig ?broblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT ef?ciency in pigs.展开更多
文摘Dear Editor: Glucose-dependent insulinotropic polypeptide (GIP) and proglucagon product glucagon-like peptide-1 (GLP- 1) and their corresponding receptors promote secretion of glucose-dependent insulin and may be responsible for up to 70% of postprandial insulin secretions.
基金supported by a grant from the Jiangsu Key Laboratory of Xenotransplantation (BM2012116)
文摘Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic lgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the lgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.
基金supported by grants from the National Natural Science Foundation of China(Nos.81570402 and 31701283)the National Key R&D Program of China(2017YFC1103701 and 2017YFC1103702)+3 种基金the Jiangsu Key Laboratory of Xenotransplantation(BM2012116)the Sanming Project of Medicine in Shenzhen(SZSM201412020)the Fund for High Level Medical Discipline Construction of Shenzhen(2016031638)the Shenzhen Foundation of Science and Technology(JCYJ20160229204849975 and GCZX2015043017281705)
文摘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.
基金supported by the National Natural Sciences Foundation of China (30871408 and 31371487)partially supported by grants from the Sanming Project of Medicine in Shenzhen+1 种基金the Fund for High Level Medical Discipline Construction of Shenzhen (2016031638)the Shenzhen Foundation of Science and Technology (JCYJ20160229 204849975 and GCZX2015043017281705)
文摘Using a data set from our laboratory, we assessed the effects of several factors on pig cloning ef?ciency. The results demonstrated that cells at high con?uence( > 90%) used as donor cell resulted in higher pregnancy rate, delivery rate and overall cloning ef?ciency(number of live offspring born per reconstructed embryo transferred to recipients) compared with the cells at 60% to79% con?uence and 80% to 89% con?uence. Cells with four, ?ve and six passages compromised the pregnancy and delivery rates compared with ?rst passage cells. The number of blastocysts transferred by somatic cell nuclear transfer(SCNT) did not signi?cantly affect the cloning ef?ciency, but transfer of blastocyst derived from in vitro culture 5 d after SCNT achieved a signi?cantly higher pregnancy rate compared with one to two cell SCNT embryos from overnight culture. The highest pregnancy rate, delivery rate and the largest litter size were obtained when Bama Miniature pig ?broblasts were used as donor cells and Landrace/Yorkshire hybrid gilts were used as recipients. Recipients treated with chemicals for estrus synchronization had higher pregnancy rates compared with untreated recipients. Our data might be helpful for improving SCNT ef?ciency in pigs.