The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Randall S Prather - One of the best experts on this subject based on the ideXlab platform.
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oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Mingtao Zhao, Lee D Spate, Eric M Walters, Melissa Samuel, Clifton N Murphy, Kristin M Whitworth, Chad Ogorman, Kevin D Wells, Rocio Melissa Rivera, Randall S PratherAbstract:Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low Cloning Efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the c...
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66 dna methylome analysis in nuclear transfer donor cells and its relationship to Cloning Efficiency in swine
Reproduction Fertility and Development, 2011Co-Authors: J J Whyte, Eric M Walters, S C Isom, William G Spollen, Sean M Blake, Randall S PratherAbstract:The success of Cloning by somatic cell nuclear transfer is unpredictable due in part to abnormal patterns of DNA methylation and histone modifications in the donor genome. The state of these epigenetic markers appears to regulate the timing and degree of embryonic gene expression by altering chromatin structure. In this study, we sought to determine the pattern of DNA methylation in genetically distinct clonal populations of swine fetal fibroblast donor cells that have previously been demonstrated to have either consistently high or consistently low success at producing healthy cloned pigs. To accomplish this goal, we combined methylated DNA immunoprecipitation using antibodies to 5-methylcytosine with Illumina high throughput sequencing (MeDIP-seq) to detect broad regions of methylated and unmethylated DNA in the 2 populations of donor cells (termed high success v. low success). Size-selected immunoprecipitated DNA samples each had matching input controls. Model-based analysis of ChIP-Seq revealed a total of 272 598 and 261 339 genomic regions enriched for methylated cytosine in high v. low success donor cells, respectively. The majority of these enriched regions (peaks) in high and low success samples coincided in genomic position, but 12.9% of the peaks were unique in high success cells, while 20.4% were unique in low success cells. When intersected with 38 778 predicted CpG islands in the swine genome, 3129 of the total MeDIP peaks (1.1%) overlapped for high success cells as compared to 1836 peaks (0.7%) for the low success cells. Of these intersecting MeDIP-CpG peaks for both groups, 1505 peaks were located in unique locations for high success cells and 205 were unique for low success cells. The chromosomal location of the CpG intersections differed between each cell populations, with the greatest number of overlaps on chromosomes 1 and 3 for high and low success cells, respectively, and the least number of overlaps on chromosomes 9 and 13, respectively. Of the total MeDIP peaks, 6632 (2.4%) from the high success cell population overlapped with predicted swine genes (UCSC Genome Database/Ensembl) compared to 4041 (1.5%) from low success donor cells. Less similar was the percentage of the predicted genes in these methylated regions that were unique to each cell group: 2875 (43.4%) for high success cells v. 338 (8.3%) for low success cells. These results highlight differences in the degree of DNA methylation and the pattern of methylated genes in NT donor cells that produce healthy clones v. those that do not. Such comprehensive genomic analyses of epigenetic markers may provide predictive data to screen for optimal NT donor cells, thereby improving Cloning Efficiency. This work was supported by the National Institutes of Health NIH R01 RR013438 and Food for the 21st Century at the University of Missouri.
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significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer
Biology of Reproduction, 2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the Cloning Efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
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method of oocyte activation affects Cloning Efficiency in pigs
Molecular Reproduction and Development, 2009Co-Authors: Kristin M Whitworth, Lee D Spate, August Rieke, David Wax, Jeffrey J Whyte, Gaurishankar Manandhar, Miriam Sutovsky, Jonathan A Green, Peter Sutovsky, Randall S PratherAbstract:The following experiments compared the Efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken β-actin/rabbit β-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca2+ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. Mol. Reprod. Dev. 76: 490–500, 2009. © 2008 Wiley-Liss, Inc.
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title significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer short title hdac inhibitor improves somatic Cloning Efficiency summary sentence hdac inhibit
2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the Cloning Efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM 6-(1,3-Dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from zero to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
Jianguo Zhao - One of the best experts on this subject based on the ideXlab platform.
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repsox increases porcine Cloning Efficiency by improving pluripotency of donor nuclei
Cellular Reprogramming, 2019Co-Authors: Jianguo Zhao, Jiaojiao Huang, Guosong QinAbstract:Abstract Accumulating evidence suggests that a low pluripotency of donor nuclei might lead to abnormal development of cloned embryos and underlie the inEfficiency of mammalian somatic cell nuclear ...
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bix 01294 increases pig Cloning Efficiency by improving epigenetic reprogramming of somatic cell nuclei
Reproduction, 2016Co-Authors: Jiaojiao Huang, Hongyong Zhang, Jing Yao, Guosong Qin, Feng Wang, Xianlong Wang, Ailing Luo, Qiantao Zheng, Chunwei Cao, Jianguo ZhaoAbstract:Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (Cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.
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significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer
Biology of Reproduction, 2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the Cloning Efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
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title significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer short title hdac inhibitor improves somatic Cloning Efficiency summary sentence hdac inhibit
2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the Cloning Efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM 6-(1,3-Dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from zero to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
Eric M Walters - One of the best experts on this subject based on the ideXlab platform.
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oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Jiude Mao, Lee D Spate, Eric M Walters, Melissa Samuel, Clifton N Murphy, Kristin M Whitworth, Mingtao Zhao, Chad Ogorman, Kiho Lee, Kevin D WellsAbstract:Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low Cloning Efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve Cloning Efficiency.
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oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Mingtao Zhao, Lee D Spate, Eric M Walters, Melissa Samuel, Clifton N Murphy, Kristin M Whitworth, Chad Ogorman, Kevin D Wells, Rocio Melissa Rivera, Randall S PratherAbstract:Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low Cloning Efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the c...
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66 dna methylome analysis in nuclear transfer donor cells and its relationship to Cloning Efficiency in swine
Reproduction Fertility and Development, 2011Co-Authors: J J Whyte, Eric M Walters, S C Isom, William G Spollen, Sean M Blake, Randall S PratherAbstract:The success of Cloning by somatic cell nuclear transfer is unpredictable due in part to abnormal patterns of DNA methylation and histone modifications in the donor genome. The state of these epigenetic markers appears to regulate the timing and degree of embryonic gene expression by altering chromatin structure. In this study, we sought to determine the pattern of DNA methylation in genetically distinct clonal populations of swine fetal fibroblast donor cells that have previously been demonstrated to have either consistently high or consistently low success at producing healthy cloned pigs. To accomplish this goal, we combined methylated DNA immunoprecipitation using antibodies to 5-methylcytosine with Illumina high throughput sequencing (MeDIP-seq) to detect broad regions of methylated and unmethylated DNA in the 2 populations of donor cells (termed high success v. low success). Size-selected immunoprecipitated DNA samples each had matching input controls. Model-based analysis of ChIP-Seq revealed a total of 272 598 and 261 339 genomic regions enriched for methylated cytosine in high v. low success donor cells, respectively. The majority of these enriched regions (peaks) in high and low success samples coincided in genomic position, but 12.9% of the peaks were unique in high success cells, while 20.4% were unique in low success cells. When intersected with 38 778 predicted CpG islands in the swine genome, 3129 of the total MeDIP peaks (1.1%) overlapped for high success cells as compared to 1836 peaks (0.7%) for the low success cells. Of these intersecting MeDIP-CpG peaks for both groups, 1505 peaks were located in unique locations for high success cells and 205 were unique for low success cells. The chromosomal location of the CpG intersections differed between each cell populations, with the greatest number of overlaps on chromosomes 1 and 3 for high and low success cells, respectively, and the least number of overlaps on chromosomes 9 and 13, respectively. Of the total MeDIP peaks, 6632 (2.4%) from the high success cell population overlapped with predicted swine genes (UCSC Genome Database/Ensembl) compared to 4041 (1.5%) from low success donor cells. Less similar was the percentage of the predicted genes in these methylated regions that were unique to each cell group: 2875 (43.4%) for high success cells v. 338 (8.3%) for low success cells. These results highlight differences in the degree of DNA methylation and the pattern of methylated genes in NT donor cells that produce healthy clones v. those that do not. Such comprehensive genomic analyses of epigenetic markers may provide predictive data to screen for optimal NT donor cells, thereby improving Cloning Efficiency. This work was supported by the National Institutes of Health NIH R01 RR013438 and Food for the 21st Century at the University of Missouri.
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significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer
Biology of Reproduction, 2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the Cloning Efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
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title significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer short title hdac inhibitor improves somatic Cloning Efficiency summary sentence hdac inhibit
2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the Cloning Efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM 6-(1,3-Dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from zero to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
D F Salamone - One of the best experts on this subject based on the ideXlab platform.
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20 aggregation of yak heterospecific somatic cell nuclear transfer embryos improves Cloning Efficiency
Reproduction Fertility and Development, 2020Co-Authors: Yauri M Felipe, Duque M Rodriguez, A De Stefano, D F SalamoneAbstract:Cloning endangered species has the limitation that generally the number of available oocytes is limited. Reprogramming the nuclei heterospecifically using an enucleated oocyte from a different species is an alternative. Aggregation of SCNT (somatic cell nuclear transfer) embryos from the same specie results in improved embryo development. However, after aggregation of heterospecific SCNT embryos from different genera, no effects were observed (Moro et al. 2015 Reproduction 50, 1-10). The objective of this study was to evaluate the influence of aggregation of yak (Bos grunniens) embryos produced by heterospecific SCNT using enucleated oocytes from an animal from the same genus Bos taurus. As control homospecific SCNT of Bos taurus, parthenogenic zone-free embryos and IVF embryos were used. Cumulus-oocyte complexes were recovered from bovine slaughterhouse ovaries by follicular aspiration. The cumulus-oocyte complexes were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10 μg mL−1 FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic for 22 h, at 6.5% CO2 in humidified air and 38.5°C. After denudation, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation. Staining was performed with Hoechst 33342 to observe MII. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with the donor cell followed by electrofusion. All reconstituted embryos were activated using ionomcine. This was followed by a treatment with 6-dimethylaminopurine for 3 h. Zona-free reconstituted cloned embryos were cultured in the wells of the well system, placing one (1×) or two (2×) per microwell, in synthetic oviductal fluid medium. The experimental groups were parthenogenic zone free; IVF; reconstituted embryos bull fibroblast-enucleated oocyte from cow (BC1×); reconstituted embryos yak fibroblast-enucleated oocyte from cow (YC1×); and reconstituted embryos aggregated yak fibroblast-enucleated oocyte from cow (YC2×). In all experimental groups, cleavage of at least one embryo in the wells and blastocyst formation at Day 7 were assessed. The effect of cloned embryo aggregation on blastocyst rates was analysed using Fisher exact tests (GraphPad Prisma 8), and results are shown on Table 1. Results demonstrated that aggregation of two SCNT heterospecific embryos increased the blastocyst formation rate of yak (P < 0.05). In conclusion aggregation in yak heterospecific SCNT embryos from species of the same genus (Bos) can improve development to blastocyst. Table 1.Aggregation of yak heterospecific somatic cell nuclear transfer embryos Experimental group1 No. of embryos No. of embryos-wells2 Cleavage (%) Blastocyst (%) PZF 68 68 66 (97.06%)a 17 (25.00%)acd IVF 89 - 81 (91.01%)ab 39 (43.82%)b BC1× 45 45 41 (91.11%)b 6 (13.33%)cd YC1× 101 101 77 (76.24%)c 14 (13.86%)c YC2× 134 67 61 (91.04%)ab 21 (31.34%)ab a-dDifferent superscripts in the same column indicate significant difference (Fisher's exact test, P < 0.05). 1PZF, parthenogenetic zone free; IFV, IVF fecundation; BC1×, clone of bovine; YC1×, clone of yak-bovine; YC2×, clone of yak-bovine added. 2Wells used with embryos.
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embryo aggregation in pig improves Cloning Efficiency and embryo quality
PLOS ONE, 2016Co-Authors: C Buemo, A Gambini, L N Moro, M I Hiriart, R Fernandezmartin, Philippe Collas, D F SalamoneAbstract:In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.
Lee D Spate - One of the best experts on this subject based on the ideXlab platform.
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oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Jiude Mao, Lee D Spate, Eric M Walters, Melissa Samuel, Clifton N Murphy, Kristin M Whitworth, Mingtao Zhao, Chad Ogorman, Kiho Lee, Kevin D WellsAbstract:Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low Cloning Efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve Cloning Efficiency.
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oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming
Cellular Reprogramming, 2015Co-Authors: Mingtao Zhao, Lee D Spate, Eric M Walters, Melissa Samuel, Clifton N Murphy, Kristin M Whitworth, Chad Ogorman, Kevin D Wells, Rocio Melissa Rivera, Randall S PratherAbstract:Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low Cloning Efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the c...
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significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer
Biology of Reproduction, 2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the Cloning Efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
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method of oocyte activation affects Cloning Efficiency in pigs
Molecular Reproduction and Development, 2009Co-Authors: Kristin M Whitworth, Lee D Spate, August Rieke, David Wax, Jeffrey J Whyte, Gaurishankar Manandhar, Miriam Sutovsky, Jonathan A Green, Peter Sutovsky, Randall S PratherAbstract:The following experiments compared the Efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken β-actin/rabbit β-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca2+ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. Mol. Reprod. Dev. 76: 490–500, 2009. © 2008 Wiley-Liss, Inc.
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title significant improvement in Cloning Efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer short title hdac inhibitor improves somatic Cloning Efficiency summary sentence hdac inhibit
2009Co-Authors: Jianguo Zhao, Jason W Ross, Yanhong Hao, Lee D Spate, Eric M Walters, Melissa Samuel, August Rieke, Clifton N Murphy, Randall S PratherAbstract:The NIH miniature pig was developed specifically for xenotransplantation and has been extensively used as a large animal model in many other biomedical experiments. However the Cloning Efficiency of this pig is very low (less than 0.2%) and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Here we report that treatment with 500 nM 6-(1,3-Dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDACi, significantly enhanced the development SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) (21% vs. 9%, P < 0.05) were used as donors compared to the untreated group. Scriptaid treatment resulted in 8 pregnancies from 10 embryo transfers (ET) and 14 healthy NIH miniature pigs from 8 litters while no viable piglets (only 3 mummies) were obtained from 9 ETs in the untreated group. Thus scriptaid dramatically increased the Cloning Efficiency when using inbred genetics from zero to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in IVF embryos (from 37% to 26%, P < 0.05). In conclusion, the extreme low Cloning Efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.