Collection of mouse ES cell lines engineered for the forced induction of transcription factors
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Minoru Ko
PROVIDER: E-GEOD-31381 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Here we report the generation of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7 - 10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phonotypes. These cell lines and microarray data provide the building blocks for a variety of future biomedical research applications as a community resource. This set of data uses Universal Mouse Reference on the Cy5 channel. ES cells (passage 25) were cultured in the standard LIF+ medium with Dox+ on a gelatin-coated dish through the experiments. Cells from each cell line were split into 6 wells and the media was changed 24 hr after cell plating: 3 wells with Dox+ medium, and 3 wells with Dox- medium to induce transgenic TFs. Dox was removed via washing 3 times with PBS at 3 hour intervals. Total RNA was isolated by TRIzol (Invitrogen) after 48 hr, and two replications were used for real time qPCR and for microarray hybridization. RNA samples were labeled using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). For most TFs, we hybridized a Cy3-CTP labeled sample from Dox- medium together with a Cy5-CTP labeled sample from Dox+ medium. But for 7 TFs, we labeled samples from Dox- and Dox+ with Cy3, and hybridized them independently with a Cy5-labeled reference target, which is a mixture of Stratagene Universal Mouse Reference RNA and MC1 cells RNA (this method requires a double number of arrays). Analysis showed that both methods produce results of comparable quality.
2011-11-10 | E-GEOD-30917 | biostudies-arrayexpress
Project description:The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, due to a poor understanding of the initial reprogramming mechanism, it is difficult to control the efficiency and quality of the iPSCs. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Unlike other factors, Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process through the recapitulation of the early embryonic program. V6.5 ES cells derived from an F1 hybrid strain (C57BL/6 x 129/Sv) were purchased from Thermo Scientific Open Biosystems. ES cells were cultured at 37C in 5% CO2 in a complete ES medium: DMEM, 15% FBS, 1000 U/ml LIF (ESGRO, Chemicon), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM GlutaMAX, 0.1 mM -mercaptoethanol, penicillin, and streptomycin. V6.5 ES cells (5x105 cells) in suspension were cotransfected with 2 g of a linearized pCAG-Zscan4-ERT2 vector and 0.4 g of a PL452 vector (Liu et al., 2003) (a neomycin-resistant gene driven by a PGK promoter) using the Effectene (QIAGEN) according to the manufacturer s protocol, and plated in 100 mm cell culture dishes. After selecting with G418 for 8 days, resulting ES cell colonies were picked, expanded, and frozen. Subsequently, an ES-ZERT cell clone was selected based on the results of genotyping, qPCR, and puromycin-resistance. ES-ZERT cells (10-15 cells) were injected into 2N blastocysts and then transferred to E2.5 recipient females. After genotyping the pups, ZERT chimeric mice carrying pCAG-Zscan4-ERT2 DNA were established.
2012-01-06 | E-GEOD-31423 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-08-20 | E-GEOD-50863 | biostudies-arrayexpress
Project description:Millions of sweat glands required to maintain body temperature develop from embryonic ectoderm by a poorly defined mechanism. We present evidence for temporal cascade regulation of sweat gland development by Wnt, Eda and Shh pathways. The first stage, sweat gland induction, failed completely when Wnt/β-catenin signaling was blocked in skin epithelium, accompanied by sharp downregulation of Wnt, Eda and Shh pathway genes. In a meta-layer of regulation, Wnt antagonist Dkk4 appeared to operate on sweat gland induction in a negative feedback loop: Dkk4 was itself sharply downregulated in β-catenin-ablated mice, whereas its over-expression repressed Wnt target genes and significantly reduced gland numbers. Eda signaling succeeded Wnt, and activated downstream Shh pathway. Thus, in absence of Eda, Wnt pathway was still active and initial sweat gland pre-germs were seen but failed to develop germs, and dwonstream Shh pathway was repressed. When both Wnt and Eda were intact but Shh was ablated, early stage induction and subsequent duct formation occurred normally, but the final stage formation of secretory coil failed. Thus, sweat gland development shows a relay of regulatory steps, initiated by Wnt/β-catenin -- itself modulated by Dkk4 -- with subsequent tandem action of Eda and Shh pathways. Compared expression changes between wild type and skin specific beta-catenin knockout footpads at 3 developmental time points, E15.5, E16.5 and E17.5. Data were duplicated with 2 sets of samples.
2014-08-20 | E-GEOD-50859 | biostudies-arrayexpress
Project description:Millions of sweat glands required to maintain body temperature develop from embryonic ectoderm by a poorly defined mechanism. We present evidence for temporal cascade regulation of sweat gland development by Wnt, Eda and Shh pathways. The first stage, sweat gland induction, failed completely when Wnt/?-catenin signaling was blocked in skin epithelium, accompanied by sharp downregulation of Wnt, Eda and Shh pathway genes. In a meta-layer of regulation, Wnt antagonist Dkk4 appeared to operate on sweat gland induction in a negative feedback loop: Dkk4 was itself sharply downregulated in ?-catenin-ablated mice, whereas its over-expression repressed Wnt target genes and significantly reduced gland numbers. Eda signaling succeeded Wnt, and activated downstream Shh pathway. Thus, in absence of Eda, Wnt pathway was still active and initial sweat gland pre-germs were seen but failed to develop germs, and dwonstream Shh pathway was repressed. When both Wnt and Eda were intact but Shh was ablated, early stage induction and subsequent duct formation occurred normally, but the final stage formation of secretory coil failed. Thus, sweat gland development shows a relay of regulatory steps, initiated by Wnt/?-catenin -- itself modulated by Dkk4 -- with subsequent tandem action of Eda and Shh pathways. Compared expression changes between WT and skin specific Dkk4 transgenic footpads at 2 developmental time points, E15.5 and E16.5. Data were duplicated with 2 sets of samples.
2014-08-20 | E-GEOD-50862 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE28436: Zscan4 initiates direct reprogramming of somatic cells by transiently reactivating early embryonic genes Part A GSE31423: Zscan4 initiates direct reprogramming of somatic cells by transiently reactivating early embryonic genes Part B Refer to individual Series
2012-01-06 | E-GEOD-31435 | biostudies-arrayexpress
Project description:K and Cl channels play critical role for sweat secretion, however, individual K or Cl channels and their functions are largely unknown. By comparing expreession profilings between wild-type and Eda mutant Tabby footpads we identified 4 K and 2 Cl channels highly expressed in mouse eccrine sweat glands. Total RNAs isolated from microdissected wild-type and Tabby footpads were profiled with Agilent 44K NIA mouse microarrays.
2015-12-09 | E-GEOD-73715 | biostudies-arrayexpress
Project description:Here we report the generation of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7 - 10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phonotypes. These cell lines and microarray data provide the building blocks for a variety of future biomedical research applications as a community resource. This set of data does not use Universal Mouse Reference on Cy5 channel. ES cells (passage 25) were cultured in the standard LIF+ medium with Dox+ on a gelatin-coated dish through the experiments. Cells from each cell line were split into 6 wells and the media was changed 24 hr after cell plating: 3 wells with Dox+ medium, and 3 wells with Dox- medium to induce transgenic TFs. Dox was removed via washing 3 times with PBS at 3 hours intervals. Total RNA was isolated by TRIzol (Invitrogen) after 48 hr, and two replications were used for real time qPCR (see primers in Supplementary Table S2) and for microarray hybridization. RNA samples were labeled with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent). For most TFs, we hybridized Cy3-CTP labeled sample from Dox- medium together with Cy5-CTP labeled sample from Dox+ medium. But for 7 TFs we labeled samples from Dox- and Dox+ with Cy3, and hybridized them independently with Cy5-labeled reference target, which is a mixture of Stratagene Universal Mouse Reference RNA and MC1 cells RNA (this method requires a double number of arrays). Analysis showed that both methods produce results of comparable quality. The arrays are for 2 channel agilent 44K. We used treated two channels are two separate arrays, and normalized them with an quantile method described in the data processing sections. So we used one supplemental file for two samples (Cy3 and Cy5, or ch1 and ch2). Supplementary 'processed data' file linked below.
2011-11-10 | E-GEOD-31374 | biostudies-arrayexpress
Project description:The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, due to a poor understanding of the initial reprogramming mechanism, it is difficult to control the efficiency and quality of the iPSCs. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Unlike other factors, Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process through the recapitulation of the early embryonic program. Secondary MEFs were isolated from E13.5 embryos, which were harvested by tetraploid complementation. Secondary MEFs were plated on gelatin-coated 6-well plates at a density of 1x105 cells/well in the complete ES medium. After 24 h incubation, secondary MEFs were fed with complete ES medium with or without Dox (1.5 ?g/ml) and with or without 200 nM Tmx. Culture medium was changed every day. Withdrawal of drugs (Dox or Tmx) was always followed by 1x washing by PBS before changing culture medium. We cotransfected MEFs with PB-Tet-MKOS and either a PB-Tet-Zscan4 or PB-Tet-DsRed (control). These cells were subsequently cultured in the standard iPSC generation condition (Dox+). Unexpectedly, we found that MEFs transfected with a PB-Tet-Zscan4 show consistently higher efficiency of iPSC generation than MEFs transfected with a PB-Tet-DsRed (control). All selected iPSCs expressed pluripotent marker genes according to RT-PCR analysis and were positive for ALP, Nanog, and SSEA-1. These iPSCs also formed embryoid bodies in hanging drops without LIF and could be differentiated in vitro to cells of all three germ layers. We are currently studying whether the presence of Zscan4 has the beneficial effects on iPSCs in terms of genome stability.
2012-01-06 | E-GEOD-28436 | biostudies-arrayexpress
Project description:To decipher the structure and behaviors of the transcription factor (TF) network, we created 50 permanent mouse ES cell lines, in each of which one of the 50 transcription factors tagged with FLAG, is inserted into the doxycycline (dox)-repressible ROSA26 locus. We have obtained time series data for 10 of the transcripts at 24h,48h and 72hr. Expression profiling reveals Cdx2 as the most potent inducer of transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. Immunoprecipitation (IP) with a FLAG-antibody in Cdx2-induced ES cells, identifies NuRD in CDX2-associated protein complexes; and chromatin-IP-sequencing identifies CDX2-binding sites predominantly in genes up-regulated by CDX2. Compendium analyses of Cdx2- and the other TF-inducible ES cells suggest a central role of the POU5F1/SOX2/NANOG protein complex in a swift-acting control mechanism to down-regulate a common set of genes at the beginning of multi-lineage ES cell differentiations. These ES cell lines will be a valuable resource to study biological networks in ES cells and mice. Keywords: dose response design,genetic modification design,individual genetic characteristic design,reference design,replicate design,time series design MC1 mouse ES cells derived from 129S6/SvEvTac were cultured in DMEM with 15% FBS and LIF on feeder cells. Cells were electroporated with linearlized pMWROSATcH and selected by hygromycine B. Knock-in for ROSA-TET locus in ES[MC1R(20)] cells was confirmed by southern blotting. For exchange vectors, PCR amplified ORFs were subcloned into pZhcSfi that was modified to express His6-FLAG tagged protein and puromycin resistant gene. ES[MC1R(20)] cells (passage 17) cultured on feeder cells were co-transfected with sequence verified exchange vector and pCAGGS-Cre and selected by puromycin in the presence of doxycycline. Isolated clones were tested for Venus expression, hygromycin B susceptibility, transgene RNA expression, genotyping for Cre mediated integration, karyotyping, western blotting using anti-FLAG antibody (Sigma-Aldrich) and mycoplasma contamination. ES cells (passage 25) were plated onto gelatin-coated dish. Doxycycline was removed through washing 3 times with PBS at 3 hours intervals. Total RNA was isolated by TRIzol (Invitrogen) at time points indicated in the sample names. Cy3-CTP labeled sample targets were prepared with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cells RNA.
2010-05-23 | E-GEOD-16148 | biostudies-arrayexpress