Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis of gene expression in the paraventricular nucleus of Sim1-Cre specific DNMT3a deletion mice.

ABSTRACT: Analysis of gene expression in the paraventricular nucleus in the hypothalamus (PVH) of WT and Sim1-Cre specific DNMT3a deletion mice. Results provide important information about genes regulated by DNMT3a. For the gene expression profiling, total RNA was extracted from PVH of WT and Sim1-Cre specific DNMT3a deletion mice (C57BL6 background) at 6 weeks old using Qiagen RNeasy Lipid Tissue Mini Kit (Qiagen) and Phase Lock Gel (5 Prime Inc., Gaitherburg, MD). To make sure reproducibility and biological significance, triple hybridizations were performed for each genotype with the RNA from 3 indipendent PVH samples, each sample contains RNA from 5 animals. Genomics and Microarray Core Facility at UTSW (http://microarray.swmed.edu/) checked RNA quality with Bioanalyzer Chip and processed the samples for hybridization with Illumina Mouse-6 V2 BeadChip (Illumina Inc., San Diego, CA). We used GeneSpring GX (Agilent Technologies, Santa Clara, CA) for data analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Daisuke Kohno

PROVIDER: E-GEOD-42304 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).

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Genome-wide analysis of gene expression in the paraventricular nucleus of Sim1-Cre specific DNMT3a deletion mice.

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