Global gene expression profiles of NT, AI and IVF bovine blastocysts, examination of nuclear reprogramming
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ABSTRACT:
ORGANISM(S): Bos taurus
PROVIDER: GSE3568 | GEO | 2005/11/30
SECONDARY ACCESSION(S): PRJNA93643
REPOSITORIES: GEO
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Project description:Nuclear reprogramming reinstates totipotency or pluripotency in somatic cells by changing their genetic transcription. This technology is widely used in medicine, animal husbandry and other industries. However, certain deficiencies, such as the poor developmental ability of reprogrammed nuclear transfer (NT) embryos and the low birth rate (less than 5%) of cloned animals, severely restrict the promotion of this technology. Multiple nuclear reprogramming barriers likely exist in the process of somatic nuclear reprogramming. Furthermore, the NT embryos obtained via this technology have transcriptional defects in embryonic development. To address these problems, in this study, two types of NT embryos were constructed using the same genetic background in mice. We detected the full transcriptome of all stages of NT embryos using single-cell RNA-seq and obtained the following results: 1) NT embryos have translation initiation defects during the minor zygotic genome activation (ZGA) period; NT embryos display RNA processing and RNA modification defects during the major ZGA period, and their protein kinase activity in protein phosphorylation exhibits defects during blastocyst formation; and 2) 2003 constant genes cannot be reprogrammed in cumulus cells (CCs) and mouse embryo fibroblast cells (MEFs); of these constant genes, 399/583 reprogramming barrier genes (RBGs) continue to mis-transcribe from the pronuclear stage to the blastula stage, and the same 136 RBGs are found in both the CCs and MEFs. The main functions of these RBGs is to positively regulate certain factors. In conclusion, our study offers full transcriptome blueprints of all stages of CC donor NT embryos, MEF donor NT embryos and in vivo embryos and reveals new transcriptional defects and reprogramming barriers in nuclear reprogramming. These findings provide insight for further mechanism studies investigating somatic nuclear reprogramming.
2018-08-11 | GSE113164 | GEO
Project description:Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming both for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA into oocytes in somatic cell nuclear transfer (SCNT) embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity. Transcriptome sequencing of 4-cell NT embryos, Luciferase 4-cell SCNT embryos, 4-cell NT embryos_H3.3KD, 4-cell NT embryos_H3.3KD+H3.3mRNA, H3.3 KD + H3.2 mRNA SCNT embryos
2014-06-02 | E-GEOD-56762 | biostudies-arrayexpress
Project description:Genome wide comparison of gene expression between EpiSC lines derived from fertilized (FT) embryos and somatic cell nuclear transfer (NT) embryos. EpiSC lines were derived from fertilized and somatic cell nuclear transfer embryos and cultured until 15 to 20 passages. RNA was then extracted in order to compare transcriptomic profiles.
2010-02-05 | GSE17402 | GEO
Project description:We aimed to identify a reprogramming factor in mammalian oocytes. DJ-1 is one candidate gene of the factor. Inhibition of DJ-1 function in nuclear transfer embryos affected developmental abilities. The downstream effect of this DJ-1 inhibition was examined using microarrays. Nuclear transfer (NT) embryos (23-26 embryos per each sample) were collected at 28 h after nuclear transfer when many of the embryos had just reached the 2-cell stage. Total RNA was extracted and hybridized on the Affymetrix GeneChip Porcine Genome Array. Global transcripts were compared among NT embryos injected with anti-DJ1 antibody (αDJ1-NT_1), NT embryos injected with IgG (IgG-NT_1), non-injected NT embryos (NT_1) and donor cells (DonorCell_1). Biologically different samples of αDJ1-NT (αDJ1-NT_2) and IgG-NT (IgG-NT_2) were prepared and the microarray analysis was repeated. In both trials, NT embryos injected with IgG (IgG-NT_1 or IgG-NT_2) were used as controls.
2011-04-07 | E-GEOD-18104 | biostudies-arrayexpress
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. Duplicate cDNA libraries of two IVF-ESCs, three sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast line were sequenced using Illumina HiSeq 2000. The sequence reads were mapped to hg19 reference genome and hits that passed quality filters were analyzed for differential expression.
2014-07-08 | E-GEOD-53094 | biostudies-arrayexpress
Project description:Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient because of aberrant genomic reprogramming. In addition to random reprogramming errors, we hypothesized the presence of specific errors as evidenced by common anomalies among clones. We found that Xist, which normally inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa normalized global gene expression and produced about a 10-fold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically downregulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning, which provide promising targets for breakthroughs in SCNT cloning technology. Gene expression were measured in mouse in vitro fertilized and somatic cell cloned blastocysts. More than three biological replicates were performed in each group using defferent nuclear donor cells.
2010-09-16 | E-GEOD-23181 | biostudies-arrayexpress
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. 16 matched samples, two IVF-ESCs, five sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, the parental fibroblast line, and the sperm and oocyte donor were genotyped using the Illumina Omni5, which interrogates 4.3 million SNPs across the human genome. Additionally, matched samples from a patient with Leigh syndrome, a NT-ESC line, three iPSC lines, and the parental fibroblast line were genotyped using the Illumina Omni5.
2014-07-08 | E-GEOD-53141 | biostudies-arrayexpress
Project description:Somatic cell nuclear transfer (SCNT) enables gaining of totipotency by reprogramming nuclei of terminally differentiated donor cell. Recent studies have clearly demonstrated that intervention of epigenetic networks can significantly elevate both in vitro and in vivo development potential of NT embryos. Specifically, trichostatin A (TSA), a kind of histone deacetylase inhibitors (HDACi), was proved to functionally works during cloning in various mammal systems. However, how it modulates histone acylation lacks careful illustration. Here, we systematically evaluate the effect and limitation of TSA during SCNT embryo development by generating genome-wide H3K9ac maps. In addition, a Dux-dependent 2-cell (2C) activation deficiency is observed in SCNT embryos as compared with their natural fertilized counterparts. Strikingly, a refined Dux supplement can successfully assist SCNT embryos in overcoming the 2C activation defect and further promotes the overall cloning efficiency. Together, our study for the first time reveals the regulation mechanism of histone marker H3K9ac in SCNT and provides the new insight of Dux during embryogenesis.
2020-10-15 | GSE143523 | GEO
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies.
2014-07-08 | GSE53141 | GEO
Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies.
2014-07-08 | GSE53094 | GEO