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Reprogramming is achieved within a single cell cycle after mouse nuclear transfer

ABSTRACT: Although nuclear transfer allows the reprogramming of somatic cells to totipotency, little is known concerning the kinetics by which it takes place or the minimum requirements for its success. Here, we demonstrate that reprogramming can be achieved within a few hours and a single cell-cycle as long as two key constraints on reprogramming are satisfied. First, the recipient cell chromosomes must be removed during mitosis. Second, the nuclear envelope of the donor cell must be broken down and its chromosomes condensed, allowing an embryonic nucleus to be constructed around the incoming chromosomes. If these requirements are not met, then reprogramming fails and embryonic development arrests. These results point to a central role for processes intimately linked to cell division in mediating efficient transitions between transcriptional programs.

ORGANISM(S): Mus musculus

PROVIDER: GSE28811 | GEO | 2011/10/04

SECONDARY ACCESSION(S): PRJNA138795

REPOSITORIES: GEO

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Reprogramming is achieved within a single cell cycle after mouse nuclear transfer

Project description:Although nuclear transfer allows the reprogramming of somatic cells to totipotency, little is known concerning the kinetics by which it takes place or the minimum requirements for its success. Here, we demonstrate that reprogramming can be achieved within a few hours and a single cell-cycle as long as two key constraints on reprogramming are satisfied. First, the recipient cell chromosomes must be removed during mitosis. Second, the nuclear envelope of the donor cell must be broken down and its chromosomes condensed, allowing an embryonic nucleus to be constructed around the incoming chromosomes. If these requirements are not met, then reprogramming fails and embryonic development arrests. These results point to a central role for processes intimately linked to cell division in mediating efficient transitions between transcriptional programs. tail tip skin fibroblasts were transferred into mitotic mouse zygotes, blastomeres or oocytes.

2011-10-03 | E-GEOD-28811 | biostudies-arrayexpress

Transcriptional failures arrest development after human nuclear transfer

2011-10-04 | GSE24631 | GEO

Mapping of mitotic gene conversion tracts associated with repair of a defined double-strand break (DSB) using SNP microarray

Project description:Mitotic recombination between homologous chromosomes can lead to loss-of-heterozygosity (LOH), which is an important contributor to human disease. In the current study, a defined double-strand break (DSB) on chromosome IV was used to initiate LOH in a yeast strain with sequence-diverged chromosomes. Associated gene conversion tracts, which reflect the repair of mismatches formed when diverged chromosomes exchange single strands, were mapped using microarrays. LOH events reflected two broken chromosomes, one of which was repaired as a crossover and the other as a noncrossover.

2017-04-29 | GSE98349 | GEO

Transcriptional failures arrest development after human nuclear transfer

Project description:Reprogramming occurs after nuclear transfer into zygotes whose genomes have been removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase. Our results suggest that there is a previously unappreciated barrier to successful human nuclear transfer, and that future studies should focus on the requirements for somatic genome activation. 1-3 embryos were used for analysis. RNA amplification was done using two or three rounds of T7-mediated RNA amplification using the Illumina Total Prep RNA Amplification kit. Somatic cells 1-000 and 1-011 required only one round of RNA amplification because starting amounts of RNA were 100-500ng, while embryonic samples were amplified from single cells or embryos.

2011-10-03 | E-GEOD-24631 | biostudies-arrayexpress

Mus musculus

Project description:Cell type-specific requirements for iPSC reprogramming

| PRJNA256932 | ENA

First round of DNA replication is essential for transcriptional reprogramming of somatic nuclei in mouse oocytes

Project description:Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

2016-12-17 | GSE53497 | GEO

Impeding Xist expression from the active X chromosome improves mouse somatic cell cloning

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

Gene expression in pluripotent stem cells derived after somatic cell genome transfer into human oocytes

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

Characterization of chromosomal architecture in Arabidopsis thaliana by Chromosome Conformation Capture

Project description:Background: The packaging of long chromatin fibres in the nucleus poses a major challenge, as it must fulfil both physical and functional requirements. Until recently, insight into the chromosomal architecture of plants was mainly provided by cytogenetic studies. Complementary to these analyses, chromosome conformation technologies promise to refine and improve our view on chromosomal architecture and to provide a more generalised description of nuclear organization. Results: Employing circular chromosome conformation capture (4C), this study describes chromosomal architecture in Arabidopsis nuclei from a genome-wide perspective. Surprisingly, the linear organisation of chromosomes is reflected in the genome-wide interactome. In addition, we studied the interplay of the interactome and epigenetic marks and report that the heterochromatic knob on the short arm of chromosome 4 (hk4s) maintained a pericentromere-like interaction profile and interactome despite its euchromatic surrounding. Conclusion: Despite the extreme condensation that is necessary to pack the chromosomes into the nucleus, the Arabidopsis genome appears to be packed in a predictive manner, according to the following criteria: (i) heterochromatin and euchromatin represent two distinct interactomes, (ii) interactions between chromosomes correlates with the linear position on the chromosome arm, and (iii) distal chromosome regions have a higher potential to interact with other chromosomes. This study includes circular chromosome conformation capture (4C) sequencing information of 13 samples, present in two batches, each present in duplicates (A and B). The individual 4C sequencing information can be retrieved by the 4C primer sequence, given in the 4C primer information file.

2013-11-01 | E-GEOD-50181 | biostudies-arrayexpress

Incoming human papillomavirus 16 genome is lost in PML protein-deficient HACAT keratinocytes.

Project description:Human papillomaviruses (HPVs) target PML nuclear bodies during infectious entry and PML protein is important for efficient transcription of incoming viral genome.We used shRNA to knockdown PML protein in HaCaT keratinocytes to further investigate the role of PML protein in HPV entry. We used microarrays to compare the transcriptome of PML protein-deficient with vector control-transduced HaCaT cells.

2016-11-17 | GSE89937 | GEO

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