Trichostatin A does not correct specific errors of somatic cell nuclear transfer on the transcriptomic level; highlighing the non-random nature of oocyte-mediated reprogramming errors.
Ontology highlight
ABSTRACT:
ORGANISM(S): Bos taurus
PROVIDER: GSE57247 | GEO | 2014/05/03
SECONDARY ACCESSION(S): PRJNA246052
REPOSITORIES: GEO
Similar Datasets
Project description:Bovine cloned and IVF blastocysts at day 7 were collected and used for comparative transcriptomics. A potent histone deacetylase inhibitor, Trichostatin A (TSA), was used to understand the effect of assisted epigenetic modification on the transcriptional profile of SCNT blastocysts and to identify specific genes or categories of genes affected in the developing embryos.
2014-05-03 | E-GEOD-57247 | biostudies-arrayexpress
Project description:The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected.TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592?=?4.3 %) and TSA-NT (1907?=?5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos.The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.
| S-EPMC4698792 | biostudies-literature
Project description:Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain M-bM-^@M-^\unclonableM-bM-^@M-^] for unknown reasons. Here we examined whether neurons from adult mice could be cloned, using a combination of two epigenetic approaches. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark - dimethylated histone H3 lysine 9 (H3K9me2) - and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (per embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrated for the first time that adult neurons could be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos. Comparative gene expression analyses using blastocysts of cloned embryos were performed by microarray. Cloned embryos were produced with three different types of donor cells (neonatal Sertoli cells, CA1 pyramidal cells and Purkinje cells) and all cloned embryos were treated with Trichostatin A (TSA). Each embryos were cultured for 96 h and blastocysts derived from each donor cell types were subjected to gene expression microarray. For comparison of gene expression, the data sets of control sex- and genotype-matched embryos produced by in vitro fertilization and SCNT-derived blastocysts from cumulus cells treated with TSA from our previous paper (Inoue K. et al. Science 2010) were also used.
2015-02-03 | E-GEOD-65497 | biostudies-arrayexpress
Project description:Somatic cell nuclear transfer (SCNT) in mammals has been mostly unsuccessful. In this study, we measured and compared transcription profiles of cloned and fertilized embryos using RNA sequencing. Before zygotic genome activation (ZGA), fewer genes were activated in the cloned embryos than in vitro fertilization (IVF), leading to insufficient activation of protein transport and degradation functions. In blastocysts, terms of “embryo development” and “adherens junction” represented major differences for repressed genes in SCNT compared to IVF. Vitamin C (Vc) was found to relax donor cell chromatin and promote cloned embryo development. RNA sequencing indicated that Vc treatment restored some abnormal genes and processes in the cloned embryos. Processes of autophagy, RNA-editing and cell junction were not fully established, but they were improved by Vc treatment. Overexpression of ZNF641, one of the completely restored genes by Vc treatment, improved the cloned embryos’ potential. Finally, Vc treatment rescued some long non-coding RNAs that were aberrantly expressed in the cloned embryos. Collectively, many important genes and biological processes were abnormal in the cloned embryos, some of which can be improved by Vc treatment. This investigation provides several practical aspects that could be useful for improving cloning efficiency and its future applications.
2020-12-12 | GSE123705 | GEO
Project description:Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF) and artificial insemination (AI) at or near term development was performed in order to better understand why SCNT and IVF often result in placental defects, hydrops and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF and AI on gene expression, taking into account the effects of parturition, sex of the fetus, breed of dam, breed of fetus and disease status of the offspring. The differential expression of 21 physiologically important genes was confirmed using quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentae were collected at term or preterm (whether the collection was due to disease or to obtain matching case-controls) followed by placentome source (AI, IVF or SCNT). Gene expression in SCNT placentomes was dramatically different from AI (N=336 genes; 276 >2-fold) and from IVF (N=733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, gene expression, posttranslational modification, molecular transport and connective tissue development. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS. Keywords: gene expression; development; SCNT; cloning; nuclear transfer; IVF; AI
2007-12-21 | GSE8923 | GEO
Project description:Somatic cell nuclear transfer (SCNT) into an enucleated oocyte can reprogram a differentiated nucleus to a totipotent state. However, the time course of transcriptional reprogramming has not been determined. To monitor the inactivation of somatic genes after SCNT in the bovine model system, we determined transcript levels of 159 genes highly expressed in fetal fibroblast nuclear donor cells, but not in metaphase II recipient oocytes. For 18 of these genes significantly higher transcript levels were found in four-cell SCNT embryos compared with four-cell embryos derived by in vitro fertilization (IVF), indicating incomplete silencing of somatic genes at this stage. RNA sequencing revealed that nearly 600 genes with no transcripts in oocytes were activated in eight-cell IVF embryos, in agreement with major embryonic genome activation (EGA). De novo transcription in SCNT embryos was delayed (16 genes before the 16-cell stage, 300 and >800 genes at the 16-cell and blastocyst stages). Intron-containing primary transcripts as another option to detect embryonic gene transcription revealed activation of >2,200 genes in IVF embryos at the eight-cell stage, while only 828 genes were activated in SCNT embryos. At the 16-cell stage, a higher number of activated genes was found in SCNT (1,917) than in IVF embryos (738). Self-organizing tree algorithm clustering confirmed that in SCNT embryos transcription of genes that are normally activated during major EGA is delayed. Our study provides detailed insight into the timing of genome activation in cloned embryos that is substantially different from IVF embryos in spite of similar developmental kinetics.
2021-05-31 | GSE125724 | GEO
Project description:Transcriptomic analysis of 5 IVF-derived bovine blastocysts using Illumina sequencing technology
2013-02-03 | GSE44023 | GEO
Project description:Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain “unclonable” for unknown reasons. Here we examined whether neurons from adult mice could be cloned, using a combination of two epigenetic approaches. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark - dimethylated histone H3 lysine 9 (H3K9me2) - and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (per embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrated for the first time that adult neurons could be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.
2015-02-03 | GSE65497 | GEO
Project description:Transcriptomic analysis of 5 IVF-derived bovine blastocysts using Illumina sequencing technology RNA was extracted from individual D7 bovine blastocysts (Arcturus Picopure), cDNA was synthesized and amplified (Nugen Ovation) and indexed libraries were created for sequencing (Nugen Encore)
2013-02-03 | E-GEOD-44023 | biostudies-arrayexpress
Project description:The exchange of the oocyte’s genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cell types affected in degenerative human diseases. Such cells, carrying the patient’s genome, might be useful for cell replacement. Here we report that the development of human oocytes activated after genome exchange invariably arrests at the late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, they efficiently develop to the blastocyst stage. Human stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies the removal of the oocyte genome as the primary cause of developmental failure after genome exchange. Future work should focus on the critical elements that are associated with the human oocyte genome. Stem cells were derived by reprogramming of skin cells using oocytes ('nuclear transfer') or defined factors (iPS cells), or from IVF blastocysts
2011-10-05 | E-GEOD-27507 | biostudies-arrayexpress