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Gene expression data from cultured mouse cortical (mCTX) neurons in different stimulation and knockdown conditions

ABSTRACT: Although the induction of C-FOS in the brain has been extensively studied for several decades to date there has been no attempt to identify the targets of C-FOS at a genome wide level, and it was not known how many genes C-FOS activates in a given cell. To identify potential C-FOS target genes, we performed microarray analysis on RNA obtained from mouse cortical (mCTX) neurons infected with lentivirus containing either a control shRNA (targeting firefly luciferase) or c-Fos shRNA that were subsequently depolarized with 0, 1, 3, or 6 hours of KCl.

ORGANISM(S): Mus musculus

PROVIDER: GSE60049 | GEO | 2014/09/07

SECONDARY ACCESSION(S): PRJNA257387

REPOSITORIES: GEO

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Gene expression data from cultured mouse cortical (mCTX) neurons in different stimulation and knockdown conditions

Project description:Although the induction of C-FOS in the brain has been extensively studied for several decades to date there has been no attempt to identify the targets of C-FOS at a genome wide level, and it was not known how many genes C-FOS activates in a given cell. To identify potential C-FOS target genes, we performed microarray analysis on RNA obtained from mouse cortical (mCTX) neurons infected with lentivirus containing either a control shRNA (targeting firefly luciferase) or c-Fos shRNA that were subsequently depolarized with 0, 1, 3, or 6 hours of KCl. Embryonic day 16.5 cortices from C57BL/6 mice were dissected, dissociated, plated, and subsequently grown in a cell culture incubator that maintained a temperature of 37 degrees C and a CO2 concentration of 5%. On DIV5, lentiviral supernatant containing either a lentivirus expressing a control shRNA targeting firefly luciferase or a lentivirus expressing shRNA targeting c-Fos was applied to cultures for 6hrs with 6ug/mL polybrene. At DIV7, mCTX neurons were silenced and stimulated with KCl for either 0, 1, 3, or 6hrs, and total RNA was purified using Trizol and the RNeasy mini kit from Qiagen. 10ug of labeled cDNA was subsequently hybridized to Affymetrix mouse genome 430 2.0 arrays with affymetrix processing.

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Project description:To identify the activity-induced gene expression programs in inhibitory and excitatory neurons, we analyzed RNA extracted from cultured E14 mouse MGE- and CTX-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. To identify the gene programs regulated in these cells by the activity-induced early-response transcription factor Npas4, we repeated the same experiment in the MGE- and CTX-cultures lacking Npas4 (Npas4-KO). Littermate mouse E14 MGE- or CTX-derived neurons (WT or KO for Npas4) were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and analyzed using Affymetrix GeneChip Mouse Expression Set 430 2.0 microarray platform.

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