Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Mus musculus MEFs wide type and knocked out gene Lsh to investigate DNA methylation status

ABSTRACT: MBD-affinity purification (MAP) was employed to investigate the DNA methylation status at promoters of mouse embryonic fibroblasts (MEFs), both wild type and Lsh knock-out cells.
MAP is conceptually identical to ChIP, using an affinity column rather than an antibody.

ORGANISM(S): Mus musculus

SUBMITTER: Jose de las Heras

PROVIDER: E-MEXP-2383 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages. We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis. We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation. Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.

Project description:Purification of Nucleosomal DNA T47D-MTVL breast cancer cells carrying one stably integrated copy of the luciferase reporter gene driven by the MMTV promoter (Truss et al., 1995) were routinely grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mL streptomycin. For DNA preparation, cells were plated in RPMI medium without phenol red supplemented with 10% dextran-coated charcoal-treated FBS (DCC/FBS), and 48 hr later medium was replaced by fresh medium without serum. After 1 day in serum-free conditions, cells were treated for 5 minutes at 37ºC with 2mL of buffer A (150mM sucrose, 15mM Tris pH 7.5, 60mM KCl, 15mM NaCl, 2mM CaCl2) containing 0,5 mg/mL of lysolecithin. Micrococcal nuclease digestion was performed by adding 2mL of buffer A, supplemented with 720U of MNase (Worthington) for 3 minutes at 37ºC. The reaction was stopped with 40mM EDTA. Cells were scrapped and lysated with 1% SDS, 10mM EDTA and 50mM Tris pH 8.1 buffer, during 10 minutes on ice. Samples were then treated with 0,1mg/mL RNAse A for 30 minutes at 37ºC and 0,1 mg/mL of proteinase K for 1 hour at 45ºC. DNA was purified by phenol/chloroform extractions and ethanol precipitation. The digested DNA was run on 1% agarose gel, and band corresponding to mononucleosomes was gel-extracted. Microarray hybridization Labelling was done using Bioprime genomic labeling kit (Invitrogen, cat.no. 18095-011 and 18095-012) following manufacturers instructions. Briefly, 500 ng isolated nucleosomal DNA or total genomic DNA partially digested with MNase was labeled with either dUTP-Cy5 or dUTP-Cy3 fluorescent mononucleotides (Amersham GE PA55322), respectively. 2 mg of the obtained labeled sample were co-hybridized to the Agilent customized oligo microarray in the presence of 50 mg Human Cot-1 DNA and Agilent oligo aCGH hybridization kit (cat.no. 5188-5220) with includes blocking agents for unspecific hybridization following manufacturers instructions. Hybridization was performed in a rotating oven at 65ºC for 40 hours. Arrays were washed and fluorescent images were acquired using G2565BA Microarray Scanner System (Agilent) with 100% laser power and 100% PMT settings. 16-bit TIFF images, one for each channel, were quantified using GenePix Pro 6.0 microarray analysis software (Axon Instruments Inc.). Microarray data was normalized applying a global loess algorithm using a smoothing factor of 0.2 (Yang et al. 2002) obtaining normalized log2[intensity Cy5]/[Intensity Cy3] mean values for each spot on the array.

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Transcription profiling of Mus musculus MEFs wide type and knocked out gene Lsh to investigate DNA methylation status

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