This study aims to evaluate the effect of serum concentration, synchronization time, and confluence degree on the synchronisation efficiency of goat fibroblast cycle. The results indicated that there was no difference...This study aims to evaluate the effect of serum concentration, synchronization time, and confluence degree on the synchronisation efficiency of goat fibroblast cycle. The results indicated that there was no difference in the percentage of nucleated fibroblasts in the G0/G1 stage between serum concentrations of 0.3% and 0.4% (83.89% and 82.69%, respectively, P > 0.05) as well as between serum concentrations of 0.2% and 0.5% (76.95% and 75.46%, respectively, P > 0.05). The percentage of nucleated fibroblasts in the G0/G1 stage was highest at the concentration of 0.3% and lowest in the control group (83.89% vs. 62.67%, P 0.05). The beneficial effect of high confluence was confirmed by the large percentage of nucleated fibroblasts at the G0/G1 stage. The 60% confluency was significantly lower than the 80% and 100% confluency (73.44%, 86.63%, and 87.17%, respectively, P < 0.05). The results indicate that the goat fibroblast cycle synchronization is the most effective at the serum concentration of 0.3%, 72 hours of synchronization and 100% confluency.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
文摘This study aims to evaluate the effect of serum concentration, synchronization time, and confluence degree on the synchronisation efficiency of goat fibroblast cycle. The results indicated that there was no difference in the percentage of nucleated fibroblasts in the G0/G1 stage between serum concentrations of 0.3% and 0.4% (83.89% and 82.69%, respectively, P > 0.05) as well as between serum concentrations of 0.2% and 0.5% (76.95% and 75.46%, respectively, P > 0.05). The percentage of nucleated fibroblasts in the G0/G1 stage was highest at the concentration of 0.3% and lowest in the control group (83.89% vs. 62.67%, P 0.05). The beneficial effect of high confluence was confirmed by the large percentage of nucleated fibroblasts at the G0/G1 stage. The 60% confluency was significantly lower than the 80% and 100% confluency (73.44%, 86.63%, and 87.17%, respectively, P < 0.05). The results indicate that the goat fibroblast cycle synchronization is the most effective at the serum concentration of 0.3%, 72 hours of synchronization and 100% confluency.
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.
