Experiment 2: Pou5f1 and Sox2 overexpression experiments protein synthesis inhibition
Ontology highlight
ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Wolfgang Driever
PROVIDER: E-GEOD-41107 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-12-31 | E-GEOD-45013 | biostudies-arrayexpress
Project description:Overexpression experiments with or without protein synthesis inhibition were performed to investigate transcriptional targets of Klf2a, Klf2b and FoxD3 in the blastula stage embryos (4,7 hours postfertilization) of wild type or MZspg backgroumd Experiments were performed in biological duplicates. Cy3 and Cy5 channels were splitted in Genedata Analyst software, quantile normalised and used for independent comparisons.
2013-12-31 | E-GEOD-45010 | biostudies-arrayexpress
Project description:Morpholino injections were performed to investigate transcriptional targets of Klf4, FoxD3 and FoxD5 in the blastula stage embryos (4,7 hours postfertilization) of wild type zebrafish embryos. Experiments were performed in biological duplicates or triplicates. Cy3 and Cy5 channels were splitted in Genedata Analyst software, quantile normalised and used for independent comparisons.
2013-12-31 | E-GEOD-45012 | biostudies-arrayexpress
Project description:Morpholino injections were performed to investigate transcriptional targets of Klf2a, and Klf2b in the blastula stage embryos (4,7 hours postfertilization) of wild type zebrafish embryos. Experiments were performed in biological duplicates. Cy3 and Cy5 channels were splitted in Genedata Analyst software, quantile normalised and used for independent comparisons.
2013-12-31 | E-GEOD-45011 | biostudies-arrayexpress
Project description:Overexpression experiments with or without protein synthesis inhibition were performed to investigate transcriptional targets of Klf4, FoxD3, and Nanog-like in the blastula stage embryos (4.7 hours postfertilization) of wild type or MZspg background. Experiments were performed in biological triplicates. Cy3 and Cy5 channels were splitted in Genedata Expressionist and Analyst software, quantile normalised and used for independent comparisons.
2013-12-31 | E-GEOD-44982 | biostudies-arrayexpress
Project description:The MTF-1 transcription factor is considered to be a master regulator of zinc homoestasis. We previously characterized a constitutively nuclear, dominant-negative zebrafish MTF-1/eGFP fusion protein (dnMTF-1). In this study, in vitro transcribed dnMTF-1 mRNA was microinjected into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. The role of MTF-1 signaling in zebrafish embryos was examined by injection of in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. A Narishige IM-300 microinjector was used to microinject 1-2 cell embryos with ~100 pg of dnMTF-1 IVT mRNA (~2.1 nL microinjection volume with a concentration 50 ng/M-BM-5L IVT mRNA), or ~100 pg of enhanced green fluorescent protein (eGFP) IVT mRNA. Immediately after microinjection, embryos from each treatment were divided into 4 replicates of 30 pooled embryos and held in 25 mL of 0.3X DanieauM-bM-^@M-^Ys at 28.5M-BM-0C under a 14 hour light/10 hour dark cycle. eGFP positive embryos (dnMTF-1 and eGFP) were collected at 28 and 36 hpf and flash frozen in liquid nitrogen for future RNA isolation and microarray hybridization. Total RNA was prepared using the RNA STAT60 protocol (Tel-Test, Inc., TX). RNA was quantified using a Nanodrop 2000C spectrophotometer and assessed for quality using an Agilent 2100 Bioanalyzer Lab-on-chip system. Only samples with RNA integrity number (RIN) between 9.8-10 were used for microarray analysis. For each RNA sample, a single microarray was hybridized with 750 ng Cy3 labeled cDNA using AgilentM-bM-^@M-^Ys standard conditions for single-color microarrays. The samples were hybridized to a the Agilent 4x44K feature zebrafish microarray using the Agilent In situ Hybridization Kit Plus by the Genome Technology Core at the Whitehead Institute for Biomedical Research (Cambridge, MA). Post-hybridization, microarray slides were washed as per the Agilent In situ Hybridization Kit Plus and scanned with an Agilent DNA Microarray Scanner. Extraction of raw microarray results was performed using AgilentM-bM-^@M-^Ys Feature Extraction software with background detrending (spatial and multiplicative). The data were normalized using a non-linear scaling method based on rank invariant probes. Cy3 signal saturated probes and probes not above background in all replicated were then removed prior to statistical analysis. Statistical tests were performed using the MeV microarray analysis suite. All data were log transformed and median centered for each probe. A two-factor ANOVA was performed for time (28 vs. 36 hpf), treatment (dnMTF-1 vs. eGPF), and their interaction with p-value based on 1000 permutations of the data and alpha of 0.01. For post-hoc analyses to find biologically relevant results, the probes significant for treatment and time-treatment interaction were secondarily examined using Rank Product (RP) analysis. Rank product analysis was performed to provide lists of probes up- and down-regulated between GFP control and MTF-1 for each time point. For each test, a two-class unpaired RP analysis was performed using 1000 permutation of the data with a false discovery rate (FDR) not exceeding 5%.
2014-04-25 | E-GEOD-51298 | biostudies-arrayexpress
Project description:Genetic control of pluripotent mammalian ES cells is determined by a transcriptional network, with a "central core" of transcription factors, Pou5f1, Sox2 and Nanog. Zebrafish homologues of the "core pluripotency factors" Pou5f1, SoxB1 and Nanog-like are also crucially involved in early development. However, the degree of functional similarity of the network between mammals and non-mammals is a matter of debate. To identify the components of Pou5f1-dependent transcriptional networks, we determined the genomic binding sites for Pou5f1 and Sox2 in late blastula stage zebrafish embryos using ChIP-seq. We found that Sox2 and Pou5f1 are co-binding to the regulatory regions of Sox2, Pou5f1, and Nanog-like, as well as to multiple orthologues of mammalian plutipotency network components. Deep sequencing was performed using the Illumina GAIIx on DNA samples obtained from Sox2 ChIP, Pou5f1-Flag ChIP and Input Control. Pou5f1 was analysed in technical duplicates to obtain higher sequencing depth.
2013-08-10 | E-GEOD-39780 | biostudies-arrayexpress
Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg. 43 samples were analyzed Fib, Human fibroblast F134, 2 replicates CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates
2014-09-12 | E-GEOD-55107 | biostudies-arrayexpress
Project description:We have employed the Zebrafish gene expression microarray (MZH_Zebrafish_16k_v1.0) as a discovery platform to analyze the trancriptome of 108hpf (hours post fertilization) embryos exposed from the 96hpf to 108hpf to 100µM of diethylmaleate (DEM). Four two-color microarray studies for measuring expression levels of zebrafish embryos treated with 100µM of diethylmaleate (DEM) were performed. 8 samples were analyzed in total. Four samples were treated with 0.1% DMSO as a control; four samples were treated with 100 µM of DEM and 0.1% DMSO.
2011-09-23 | E-GEOD-32267 | biostudies-arrayexpress
Project description:In zebrafish, ovulated oocytes contain both cortisol deposited from the maternal circulation and maternal mRNA for the glucocorticoid receptor (gr mRNA), which is spread as granular structures throughout the central ooplasm. At the 1-cell stage (0.2 hpf), this transcript is relocated by streamers in the blastodisc area and equally partitioned among blastomeres. At 15 hpf, it is replaced by the zygotic transcript. Morpholino knockdown was applied to block translation (grATG1MO or MO2-nr3c1 and grATG2MO or MO3-nr3c1) of both maternal and zygotic gr transcripts, while a missplicing morpholino (grmismMO or MO4-nr3c1) was used to block post-transcriptionally the zygotic transcript alone. MO2-nr3c1 and MO3-nr3c1 (but not MO4-nr3c1) treatment produced craniofacial and caudal malformations in 1-dpf embryos and 5-dpf larvae, which were also affected by pericardial oedema, persistent yolk sac, reduced subintestinal veins, altered neurogenesis and uninflated swim bladder. Such effects were rescued with trout gr2 mRNA. Pangenomic microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal GR protein deficiency in 5-hpf embryos. Similar alterations were found at 10 hpf. These effects were confirmed by real-time PCR of 2 up- (casp8, grp1 and igf2a) and 1 down-regulated transcripts (mcm6) evaluated at 4, 8 and 12 hpf. As the contents of transcripts were modified already at 4 hpf, it seems that the lack of GR affects both ways the molecular machinery for the degradation of maternal mRNAs. These results indicate that the maternal gr transcript participates in the maternal programming of zebrafish development. MO2-nr3c1 morphants were compared with MO2-nr3c1-5m morphants at 5 hpf and 10 hpf. MO2-nr3c1 morphants were compared with wild type (WT) at 5 hpf and 10 hpf. MO2-nr3c1 is a morpholino selected to knockdown translation of gr mRNA. MO2-nr3c1-5m is a specific control morpholino.
2011-11-16 | E-GEOD-25517 | biostudies-arrayexpress