Unknown,Transcriptomics,Genomics,Proteomics

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Global comparison of mouse ES cell lines derived from blastocysts and preblastocysts.

ABSTRACT: Standard mouse ES cell derivation protocols use blastocysts at 3.5 dpc, but the efficiency of successful line derivation is low in commonly-used inbred strains, and much lower in other mouse strains. By using 2-cell zona pellucida-free embryos, culturing them on embryonic feeder cells, and passaging the outgrowths in several different media formulations, we have dramatically improved mouse ES cell line derivation efficiency. Mouse ES cell lines derived from blastocyst outgrowths using standard procedures were compared to preblastocyst (2-cell outgrowth)-derived lines, each derived in three different media formulations, using the genome-scale MEEBO gene expression oligo probe set. The overall goal of this microarray study is to determine the overall similarity between cell lines derived using the two methods, in terms of gene expression, as well as to identify important differences. Additionally, we hope to identify effects of the media formulation on gene expression in the cell lines. Seven-condition experiment (two cell types, three derivation conditions, plus one control cell line). 2 biological replicates for each cell line, independently grown and harvested. One dye-swapped pair of arrays per replicate, one probe replicate per array.

ORGANISM(S): Mus musculus

SUBMITTER: Mark Carter

PROVIDER: E-GEOD-17560 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:CcpA is a global regulator of transcription in Gram-positive bacteria that controls gene expression in response to carbohydrate availability. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrate that CcpA does indeed play a major role in carbohydrate catabolite repression. Using DNA microarrays, the expression of at least 170 genes differed in CcpA-deficient cells compared to the parental strains when cells are grown in the presence of glucose, but only 90 genes showed altered expression when cells were cultivated in the poorly-repressing substrate galactose. Interestingly, 90 genes were differently expressed in wild-type S. mutans in response to cultivation in glucose versus galactose, but the expression of over 500 genes was altered when growth in glucose and galactose were compared in the CcpA-deficient strain. The results support a major role for CcpA in regulation of gene expression, but reveal that a substantial CcpA-independent network exists in S. mutans to regulate gene expression in response to carbohydrate source. A reference RNA that had been isolated from 2 liters of UA159 cells grown in BHI broth to OD600 = 0.5 was used in every experiment. Our experimental conditions consisted of S. mutans UA159 and TW1 grown in TV supplemented with 0.5% glucose or 0.5% galactose and collected at the mid-exponential phase of growth (OD600 = 0.5). All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy3)-dUTP, while reference cDNA was coupled with indodicarbocyanine (Cy5)-dUTP (Amersham Biosciences, Piscataway, NJ). Four individual Cy3-labeled cDNA samples originating from four different cultures of UA159 or TW1, grown in each experimental condition, were hybridized to the arrays along with Cy5-labeled reference cDNA, generating a total of 16 slides. Hybridizations were carried out in a Maui 4-chamber hybridization system (BioMicro Systems, Salt Lake City, Utah) for 17 h at 42ºC. The slides were then washed according to TIGR protocols and scanned using a GenePix Scanner (Axon Instruments Inc., Union City, CA). After the slides were scanned, single-channel images were loaded into TIGR Spotfinder software and overlayed. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, then the intensity values of each spot were measured. Data was normalized using TIGR Microarray data analysis software (MIDAS), by using LOWESS and Iterative Log Mean Centering with default settings, followed by In-Slide Replicate Analysis. Statistical analysis was carried out using BRB Array Tools with a cutoff P value of 0.001 for class prediction and class comparison.

Project description:Clostridium difficile is a gram-positive, spore-forming enteric anaerobe which can infect humans and a wide variety of animal species. Recently, the incidence and severity of human C. difficile infection has markedly increased. In this study, we evaluated the genomic content of 73 C. difficile strains isolated from humans, horses, cattle, and pigs by comparative genomic hybridization with microarrays containing coding sequences from C. difficile strains 630 and QCD-32g58. The sequenced genome of C. difficile strain 630 was used as a reference to define a candidate core genome of C. difficile and to explore correlations between host origins and genetic diversity. Approximately 16% of the genes in strain 630 were highly conserved among all strains, representing the core complement of functional genes defining C. difficile. Absent or divergent genes in the tested strains were distributed across the entire C. difficile 630 genome and across all the predicted functional categories. Interestingly, certain genes were conserved among strains from a specific host species, but divergent in isolates with other host origins. This information provides insight into the genomic changes which might contribute to host adaptation. Due to a high degree of divergence among C. difficile strains, a core gene list from this study offers the first step toward the construction of diagnostic arrays for C. difficile.investigated by determining changes in transcript profiles when aerobic steady-state cultures were depleted of air. Dye-swap experiments with genomic DNA of tested and reference strains 8383cy3.gpr- Cy3 – test, Cy5 – reference 8384cy3.gpr- Cy3 – test, Cy5 – reference 8384cy5.gpr- Cy3 – reference, Cy5 – test 8385cy3.gpr- Cy3 – test, Cy5 – reference 8385cy5.gpr- Cy3 – reference, Cy5 – test 8386cy3.gpr- Cy3 – test, Cy5 – reference 8525cy3.gpr- Cy3 – test, Cy5 – reference 8525cy5.gpr- Cy3 – reference, Cy5 – test 8527cy5.gpr- Cy3 – reference, Cy5 – test 8529cy5.gpr- Cy3 – reference, Cy5 – test 8531cy3.gpr- Cy3 – test, Cy5 – reference 8531cy5.gpr- Cy3 – reference, Cy5 – test 8533cy3.gpr- Cy3 – test, Cy5 – reference 8533cy5.gpr- Cy3 – reference, Cy5 – test 8596cy3.gpr- Cy3 – test, Cy5 – reference 8596cy5.gpr- Cy3 – reference, Cy5 – test 8694cy3.gpr- Cy3 – test, Cy5 – reference 8694cy5.gpr- Cy3 – reference, Cy5 – test 2 GPR files per Sample record except for 4 Sample records (GSM480414, GSM480417, GSM480419, and GSM480420) which have 1 GPR file each 4 GPR file for those Sample records are lost

Project description:Prostaglandin E2 (PGE2) is involved in several inflammatory conditions including periodontitis. The aim of this study was to investigate the global gene expression profile of tumor necrosis factor alpha (TNFalpha) stimulated human gingival fibroblasts, focusing on signal pathways related to PGE2 production and the new PGE2-synthesizing enzymes, prostaglandin E synthases (PGES). The expression of microsomal prostaglandin E synthase-1 (mPGES-1) as well as the upstream cyclooxygenase-2 (COX-2) was up-regulated by TNFalpha, accompanied by increased PGE2 production. In contrast, the expression of microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNFalpha. Using microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, we identified differentially expressed genes in response to TNFalpha treatment. Enrichment analysis of microarray data identified two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappaB (NF-kappaB). We used specific inhibitors and phosphorylation analysis to confirm their role in PGE2 regulation. Both JNK and NF-kappaB inhibitors reduced the TNFalpha-stimulated up-regulation of mPGES-1 and COX-2 as well as subsequent PGE2 production. The novel finding that TNFalpha-stimulated mPGES-1 is regulated by JNK suggests this kinase as a potential future target for treatment strategies in inflammatory disorders, including periodontitis. Keywords: Time course, gene expression, factorial design. Three human gingival fibroblast cell lines were established from gingival biopsies obtained from 3 healthy patients, 3 to 12 years of age, with no clinical signs of periodontal disease. Cells were incubated with or without TNF-alpha (20 ng/ml) for 1, 3 or 6h. After incubation for 1, 3 or 6 h in the two conditions, the cells were immediately frozen in liquid nitrogen and then stored at -70°C for subsequent isolation of total RNA. The experimental design of the microarray study was set up as a time-course factorial design, to best observe the TNF-alpha induced gene expression changes over time. C++ program (G.F. Glonek, P.J. Solomon, Factorial and time course designs for cDNA microarray experiments, Biostatistics 5 (2004) 89-111) was used to determine the exact layout of the design in order to estimate the interaction effect between treatment and time, i.e. genes that are differentially expressed over time, with optimal statistical efficiency. Cyanine 5 and cyanine 3 were used for labeling. For each cell line 12 hybridizations were performed in a time-course factorial design. In total, 36 hybridizations were performed.

Project description:Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism. RNA extraction. S. mutans strains were grown in 50 ml of BHI broth and harvested at an OD600 of [congruent with]0.5 by centrifugation at 3,800 × g at 4°C for 5 min. The pellets were quickly resuspended and treated with RNAprotect (QIAGEN, Inc., CA) by using the procedures recommended by the supplier. The treated cells were collected by centrifugation at 3,800 × g at 4°C for 10 min and stored at −80°C. RNA extractions were performed by using hot phenol as previously described (33). To remove all DNA, the purified RNAs were treated with DNaseI (Ambion, Inc., Austin, TX) and retrieved with the RNeasy purification kit (QIAGEN, Inc., CA). To prepare a reference RNA, S. mutans UA159 was grown in 3 liters of BHI until mid-exponential phase (OD600 [congruent with] 0.5) and total RNA was extracted as described above. The purified RNA was then aliquoted and stored at −80°C until use. cDNA synthesis, Cy dye coupling and array hybridization. Array analysis was performed by using the whole-genome S. mutans microarrays that were obtained from PFGRC at TIGR. The S. mutans genome array consisted of 70-mer oligonucleotides representing 1,960 open reading frames from strain UA159. The full 70-mer complement was printed four times on the surface of the slides. Experiments, including cDNA synthesis, Cy dye coupling, hybridization, and washing, were performed by using protocols from the PFGRC with minor modifications. Briefly, cDNA synthesis was carried out in a total volume of 45 μl by mixing 10 μg total RNA with 3 μg random hexamers (Invitrogen Life Technologies, CA), 9 μl 5× first-strand buffer, 4.5 μl 0.1 M dithiothreitol, 2 μl 12.5 mM dNTP/aa-UTP with a 1.5- to-1 ratio of aa-dUTP to dTTP, and 3 μl Superscript III reverse transcriptase. This mixture was incubated at 42°C for 16 h. Following the completion of cDNA synthesis, RNA was hydrolyzed with NaOH and the aminoallyl-labeled cDNA samples were purified by using the QIAGEN QIAquick PCR purification kit, followed by drying in a speed vacuum. The samples were then resuspended in 5 μl 0.1 M NaCO3 and incubated with 5 μl of Cy dye (resuspended as recommended by the manufacturer) for 2 h in the dark. For all experiments, the reference RNA samples were coupled to Cy5 and both wild-type and the BrpA-deficient mutant RNA samples were coupled to Cy3. Uncoupled Cy dyes were removed by the QIAquick PCR purification kit, and the coupled samples were eluted in 100 μl elution buffer. Each Cy3-labeled experimental sample was mixed with a Cy5-labeled reference sample, and the mixtures were allowed to dry in a speed vacuum. To hybridize, the samples were resuspended in 60 μl hybridization buffer and heated at 90°C for 10 min. Following a quick centrifugation, the Cy dye-coupled probes were applied to microarray slides and incubated at 42°C in a water bath for 17 h. Slides were then washed and scanned by using an Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA). Array data normalization and statistical analysis. Data were collected from at least four separate array slides, which contained four copies of the genome, with RNA isolated from four independent experiments. 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Project description:In this report, codY mutant strains were constructed and used to demonstrate the relationship of (p)ppGpp synthesized by RelP and RelQ with the activation of CodY. In addition, because CodY has not been studied in S. mutans, we used microarrays to demonstrate that this protein function as a global regulator of gene expression in S. mutans. Keywords: stress response, gene knockout analysis For microarray analysis, cells were grown in complete FMC to an optical density at 600 nm (OD600) of 0.3 collected by centrifugation, quick frozen in a dry-ice/ethanol bath, and stored at - 80â??C for RNA isolation. RNA was isolated from exponential phase (OD600 0.3), as described previously. All samples were digested with DNase I (Ambion, Austin, TX) and purified using the RNeasy mini kit column (Qiagen, Inc., Chatsworth, CA). RNA concentrations were determined spectrophotometrically in triplicate and one Âµg of RNA was run in a formaldehyde gel to verify RNA quality. Reverse transcription (RT) reaction was performed with 10 mg of RNA following the protocol provided by TIGR with minor modifications. Transcriptome analysis was performed using the S. mutans UA159 microarrays provided by The Institute for Genomic Research (TIGR). The microarrays consisted of 1,948 70-mer oligonucleotides representing 1,960 open reading frames printed four times on the surface of each microarray slide. Additional details regarding the arrays are available at http://pfgrc.tigr.org/descriptions/S_mutans.shtml. A reference RNA prepared from a large-scale culture of S. mutans UA159 cells that had been grown in BHI broth to an OD600 = 0.5 was used in every experiment. cDNA labeling was performed according to TIGR protocols with minor modifications as described elsewhere. Four cDNA samples originating from four independent cultures of UA159 and JLcodY strains were hybridized to the arrays along with the reference cDNA, generating a total of 16 slides. Hybridizations were performed in a Maui hybridization chamber (BioMicro Systems, Salt Lake City, UT). Additional details regarding array protocols are available at http://pfgrc.tigr.org/protocols/protocols.shtml. Data from all individual experiments were analyzed using Spotfinder software and normalized using Midas according to TIGR specifications (http://www.tigr.org/software/). Statistical analysis was carried out using BRB Arrays Tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) with a P-value of 0.001.

Dataset Information

Global comparison of mouse ES cell lines derived from blastocysts and preblastocysts.

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