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Olga Østrup - One of the best experts on this subject based on the ideXlab platform.
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long term effect on in vitro cloning efficiency after treatment of somatic cells with xenopus Egg Extract in the pig
Reproduction Fertility and Development, 2013Co-Authors: Ying Liu, Olga Østrup, Poul Hyttel, Dan A. Klaerke, Gabor Vajta, P M Kragh, Mette Schmidt, Stig Purup, H CallesenAbstract:In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus Egg Extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus Egg Extract on donor cells was maintained for several cell passages. The Extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7–22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus Egg Extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.
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increased blastocyst formation of cloned porcine embryos produced with donor cells pre treated with xenopus Egg Extract and or digitonin
Zygote, 2012Co-Authors: Ying Liu, Olga Østrup, Gabor Vajta, P M Kragh, Stig Purup, Lin Lin, P Hyttel, H CallesenAbstract:Pre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and Extract from Xenopus laevis Eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in Egg Extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, Extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus Egg Extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without Extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.
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Remodeling of ribosomal genes in somatic cells by Xenopus Egg Extract
Biochemical and biophysical research communications, 2011Co-Authors: Olga Østrup, Poul Hyttel, Dan A. Klaerke, Philippe CollasAbstract:Extracts from Xenopus Eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of Extract treatment and is distinct from a stress response. Egg Extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by Extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus Egg Extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.
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379 treatment of 293t cells with xenopus Egg Extract induces not stress related epigenetic remodeling of ribosomal genes
Reproduction Fertility and Development, 2010Co-Authors: Olga Østrup, Poul Hyttel, D Klœrke, Philippe CollasAbstract:Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis Egg Extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis Egg Extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with Egg Extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or Extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between Extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in Extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, Extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity. The research was supported by Carlsberg Foundation 2008-01-0105.
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68 effect of xenopus Egg Extract treatment of donor cells on porcine somatic cell nuclear transfer
Reproduction Fertility and Development, 2010Co-Authors: Y Liu, Olga Østrup, P M Kragh, Stig Purup, G Vajta, Lin Lin, H CallesenAbstract:Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevis Egg Extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to Egg Extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mM CaCl2 for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO Extract-treated cells (SETC) and digitonin Extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that Extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and Extract treatment. Table 1.Effect of different permeabilization agents prior to SCNT
H Callesen - One of the best experts on this subject based on the ideXlab platform.
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long term effect on in vitro cloning efficiency after treatment of somatic cells with xenopus Egg Extract in the pig
Reproduction Fertility and Development, 2013Co-Authors: Ying Liu, Olga Østrup, Poul Hyttel, Dan A. Klaerke, Gabor Vajta, P M Kragh, Mette Schmidt, Stig Purup, H CallesenAbstract:In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus Egg Extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus Egg Extract on donor cells was maintained for several cell passages. The Extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7–22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus Egg Extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.
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increased blastocyst formation of cloned porcine embryos produced with donor cells pre treated with xenopus Egg Extract and or digitonin
Zygote, 2012Co-Authors: Ying Liu, Olga Østrup, Gabor Vajta, P M Kragh, Stig Purup, Lin Lin, P Hyttel, H CallesenAbstract:Pre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and Extract from Xenopus laevis Eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in Egg Extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, Extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus Egg Extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without Extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.
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21 effect of second time xenopus Egg Extract treatment on colony formation and cloned blastocyst formation in pig
Reproduction Fertility and Development, 2012Co-Authors: Y Liu, Gabor Vajta, P M Kragh, Stig Purup, O Ostrup, H CallesenAbstract:Extract from Xenopus Eggs can induce reprogramming in somatic cells. In our previous study, cell colony formation was induced during culture of porcine fetal fibroblasts after a single treatment with Xenopus Egg Extract and culture for several passages and using these long-term cultured cells for cloning increased the resulting blastocyst rate (Liu et al. 2011 Reprod. Fertil. Dev. 23, 130). However, both colony number and cloned blastocyst rate decreased after Passage 15 and no colonies formed after Passage 18. Therefore, in this study we investigated the effect of a second Extract treatment on colony formation and cloned blastocyst formation. Extract-treated (ExT) porcine fetal fibroblasts at Passage 13 (P13) grown on poly-L-lysine-coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated in Extract at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445). The treated cells were split onto 2 coverslips on Day 7 after the second Extract treatment (2ExT), defined as Passage 1 (2ExT P1, comparable with ExT P14). New subcultures were made every 7 to 8 days when 70 to 80% clusters became colonies (i.e. 2ExT P8). Colony cells from both ExT (P14 and P16) and 2ExT (P1, P3 and P6) were used for handmade cloning and nontreated cells were used as control (Day 0). Blastocyst rates were analysed by chi-square test and colony numbers were analysed by 1-way ANOVA (SAS version 9.2). Colony numbers and cloned blastocyst rates on Day 6 are summarised in Table 1. Colonies continued to form in treated cells from 2ExT P1 to P8. The colony number maintained at a high level (60 to 80) from 2ExT P4 to P8 and it was significantly higher than that of ExT cells at the comparable passage numbers. No colonies formed in control cells. When using 2ExT colony cells at P3 and P6 for cloning, the blastocyst rates increased compared with controls and they were also higher than in the ExT group. Cloned blastocyst rates were not different between 2ExT P1 and ExT P14 groups. In conclusion, a second Extract treatment can induce colony formation and increase cloned blastocyst rates, indicating that this repeated Extract treatment again could activate the Extract-treated cells to an activity level similar to that achieved after the first treatment. Table 1.Summary of colony number and cloned blastocyst rate with ExT and 2ExT colony cells
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68 effect of xenopus Egg Extract treatment of donor cells on porcine somatic cell nuclear transfer
Reproduction Fertility and Development, 2010Co-Authors: Y Liu, Olga Østrup, P M Kragh, Stig Purup, G Vajta, Lin Lin, H CallesenAbstract:Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevis Egg Extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to Egg Extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mM CaCl2 for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO Extract-treated cells (SETC) and digitonin Extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that Extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and Extract treatment. Table 1.Effect of different permeabilization agents prior to SCNT
Philippe Collas - One of the best experts on this subject based on the ideXlab platform.
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Remodeling of ribosomal genes in somatic cells by Xenopus Egg Extract
Biochemical and biophysical research communications, 2011Co-Authors: Olga Østrup, Poul Hyttel, Dan A. Klaerke, Philippe CollasAbstract:Extracts from Xenopus Eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of Extract treatment and is distinct from a stress response. Egg Extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by Extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus Egg Extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.
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379 treatment of 293t cells with xenopus Egg Extract induces not stress related epigenetic remodeling of ribosomal genes
Reproduction Fertility and Development, 2010Co-Authors: Olga Østrup, Poul Hyttel, D Klœrke, Philippe CollasAbstract:Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis Egg Extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis Egg Extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with Egg Extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or Extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between Extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in Extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, Extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity. The research was supported by Carlsberg Foundation 2008-01-0105.
Timothy J Mitchison - One of the best experts on this subject based on the ideXlab platform.
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Assembly of Spindles and Asters in Xenopus Egg Extracts.
Cold Spring Harbor protocols, 2018Co-Authors: Christine M. Field, Timothy J MitchisonAbstract:Here, we provide methods for assembly of mitotic spindles and interphase asters in Xenopus laevis Egg Extract, and compare them to spindles and asters in the Egg and zygote. Classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (CSF) Extract and inducing entry into interphase Extract to promote nucleus formation and DNA replication. Interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of CSF Extract. Kinetochores assemble in this reaction and these spindles can segregate chromosomes. CSF spindles are made by addition of sperm nuclei to CSF Extract. They are less physiological and lack functional kinetochores but suffice for some applications. Large interphase asters are prepared by addition of artificial centrosomes or sperm nuclei to actin-intact Egg Extract. These asters grow rapidly to hundreds of microns in radius by branching microtubule nucleation at the periphery, so the aster as a whole is a network of short, dynamic microtubules. They resemble the sperm aster after fertilization, and the asters that grow out of the poles of the mitotic spindle at anaphase. When interphase asters grow into each other they interact and assemble aster interaction zones at their shared boundary. These zones consist of a line (in Extract) or disc (in zygotes) of antiparallel microtubule bundles coated with cytokinesis midzone proteins. Interaction zones block interpenetration of microtubules from the two asters, and signal to the cortex to induce cleavage furrows. Their reconstitution in Extract allows dissection of the biophysics of spatially regulated cytokinesis signaling.
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purification and fluorescent labeling of tubulin from xenopus laevis Egg Extracts
Methods of Molecular Biology, 2016Co-Authors: Aaron C Groen, Timothy J MitchisonAbstract:For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or Extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the Eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog Egg tubulin incorporates into meiotic spindles assembled in Egg Extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus Egg Extract experiments. Frog Egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.
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the kinesin eg5 drives poleward microtubule flux in xenopus laevis Egg Extract spindles
Journal of Cell Biology, 2004Co-Authors: David T Miyamoto, Zachary E Perlman, Kendra S Burbank, Aaron C Groen, Timothy J MitchisonAbstract:Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis Egg Extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus Extract spindles.
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microtubule plus end dynamics in xenopus Egg Extract spindles
Molecular Biology of the Cell, 2004Co-Authors: Jennifer S Tirnauer, Timothy J Mitchison, E D SalmonAbstract:Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus Egg Extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at ∼11 μm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization (∼30 μm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by α-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.
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identification of xmap215 as a microtubule destabilizing factor in xenopus Egg Extract by biochemical purification
Journal of Cell Biology, 2003Co-Authors: Mimi Shirasuhiza, Peg Coughlin, Timothy J MitchisonAbstract:Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus Egg Extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus Extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in Extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.
Poul Hyttel - One of the best experts on this subject based on the ideXlab platform.
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long term effect on in vitro cloning efficiency after treatment of somatic cells with xenopus Egg Extract in the pig
Reproduction Fertility and Development, 2013Co-Authors: Ying Liu, Olga Østrup, Poul Hyttel, Dan A. Klaerke, Gabor Vajta, P M Kragh, Mette Schmidt, Stig Purup, H CallesenAbstract:In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus Egg Extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus Egg Extract on donor cells was maintained for several cell passages. The Extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7–22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus Egg Extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.
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Remodeling of ribosomal genes in somatic cells by Xenopus Egg Extract
Biochemical and biophysical research communications, 2011Co-Authors: Olga Østrup, Poul Hyttel, Dan A. Klaerke, Philippe CollasAbstract:Extracts from Xenopus Eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of Extract treatment and is distinct from a stress response. Egg Extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by Extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus Egg Extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.
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379 treatment of 293t cells with xenopus Egg Extract induces not stress related epigenetic remodeling of ribosomal genes
Reproduction Fertility and Development, 2010Co-Authors: Olga Østrup, Poul Hyttel, D Klœrke, Philippe CollasAbstract:Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis Egg Extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis Egg Extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with Egg Extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or Extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between Extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in Extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, Extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity. The research was supported by Carlsberg Foundation 2008-01-0105.