Background: Liver has important immune function during fetal development and after birth.However,the effect of maternal malnutrition on immune function of the fetal liver is rarely reported.In this study,twelve pregna...Background: Liver has important immune function during fetal development and after birth.However,the effect of maternal malnutrition on immune function of the fetal liver is rarely reported.In this study,twelve pregnant goats(Xiangdong black goat,at d 45 of gestation) were assigned to the control group(fed 100% of nutritional requirements) and the restriction group(fed 60% of the intake of the control group) during gestation from d 55 to100.Fetal goats were harvested at d 100 of gestation and immune indexes and amino acid profiles of the umbilical cord blood and liver Toll-like receptors(TLRs) signaling pathways were measured.Results: Maternal body weight in the restriction group was lower than the control group(P < 0.05).Maternal feed intake restriction decreased(P < 0.05) heart weight,heart index,alkaline phosphatase and serum amyloid protein A in the umbilical cord blood(UCB).Moreover,only histidine was decreased in the restricted group(P = 0.084),and there were no differences in other amino acids contents in the UCB between the two groups(P > 0.05).The TLR2 and TLR4 mRNA expression in the fetal liver in the restriction group was greater(P < 0.05) than that in the control group.Furthermore,the mRNA expression levels of myeloid differentiation primary response 88(MyD88),TNF receptor associated factor 6,nuclear factor kappa B subunit 1,NFKB inhibitor alpha,IFN-β,TGF-β,TNF-α and IL-1β in the restricted group were upregulated(P < 0.05),and the expression of TLR3(P = 0.099) tended to be higher in the restricted group.However,protein levels of TLR2,TLR4,IκBα,phosphorylated IκBα,phosphorylated IκBα/total IκBα,TRIF and MyD88 were not affected(P > 0.05) by maternal intake restriction.Conclusions: These results revealed that the restriction of maternal feed intake influenced the development of heart and hepatic protein synthesis at the acute phase of fetal goats and upregulated the mRNA expression of genes involved in MyD88-dependent signaling pathways and of target cytokines.展开更多
This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effe...This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.展开更多
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
基金jointly supported by the National Natural Science Foundation of China(Grant No.31760678,31730092)Youth Innovation Team Project of ISA,CAS(2017QNCXTD_ZCS)
文摘Background: Liver has important immune function during fetal development and after birth.However,the effect of maternal malnutrition on immune function of the fetal liver is rarely reported.In this study,twelve pregnant goats(Xiangdong black goat,at d 45 of gestation) were assigned to the control group(fed 100% of nutritional requirements) and the restriction group(fed 60% of the intake of the control group) during gestation from d 55 to100.Fetal goats were harvested at d 100 of gestation and immune indexes and amino acid profiles of the umbilical cord blood and liver Toll-like receptors(TLRs) signaling pathways were measured.Results: Maternal body weight in the restriction group was lower than the control group(P < 0.05).Maternal feed intake restriction decreased(P < 0.05) heart weight,heart index,alkaline phosphatase and serum amyloid protein A in the umbilical cord blood(UCB).Moreover,only histidine was decreased in the restricted group(P = 0.084),and there were no differences in other amino acids contents in the UCB between the two groups(P > 0.05).The TLR2 and TLR4 mRNA expression in the fetal liver in the restriction group was greater(P < 0.05) than that in the control group.Furthermore,the mRNA expression levels of myeloid differentiation primary response 88(MyD88),TNF receptor associated factor 6,nuclear factor kappa B subunit 1,NFKB inhibitor alpha,IFN-β,TGF-β,TNF-α and IL-1β in the restricted group were upregulated(P < 0.05),and the expression of TLR3(P = 0.099) tended to be higher in the restricted group.However,protein levels of TLR2,TLR4,IκBα,phosphorylated IκBα,phosphorylated IκBα/total IκBα,TRIF and MyD88 were not affected(P > 0.05) by maternal intake restriction.Conclusions: These results revealed that the restriction of maternal feed intake influenced the development of heart and hepatic protein synthesis at the acute phase of fetal goats and upregulated the mRNA expression of genes involved in MyD88-dependent signaling pathways and of target cytokines.
基金supported by the National Transgenic Project of China (2016ZX08010001-002)the National Natural Science Foundation of China (81471001)+1 种基金the Inner Mongolia Science and Technology Program, China (201502073)the National 863 Prgram of China (2009AA10Z111)
文摘This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
