Project description:Retinal histogenesis occurs over an extended period, which necessitates retinal progenitors to self-renew to sustain the temporal generation of diverse cell types. Embryonic day 18 retinal cells showed self-renewal properties such as clonal propagation without compromising multipotentiality when grown in the presence of endothelial cell conditioned medium and not EGF alone.
Project description:Retinal histogenesis occurs over an extended period, which necessitates retinal progenitors to self-renew to sustain the temporal generation of diverse cell types. Embryonic day 18 retinal cells showed self-renewal properties such as clonal propagation without compromising multipotentiality when grown in the presence of endothelial cell conditioned medium and not EGF alone. Timed pregnant Sprague Dawley (Embryonic day 18) rats were obtained from Charles River Laboratories. Embryos were harvested and enucleated. Retinae were dissected out and dissociated by trypsin-EDTA followed by manual trituration. The retinal cells obtained from 10 embryos were cultured in the presence of EGF (condition1) or EGF and Endothelial cell conditioned medium (condition 2) to obtain neurospheres. To get biological replicate for both conditions, another batch of embryos were pooled and subjected to neurosphere formation. Total RNA was extracted from both conditions using Qiagen RNeasy mini kit following manufacturer's protocol. The RNA was subjected to microarray analysis using Affymetrix Rat 230 2.0 platform by UNMC microarray Core facility. RMA analysis was performed using Genepattern software. Log2 transformed values of each condition was used to obtain fold change of specific genes.
Project description:To explore the gene expression prolife in the chroniclly hypoxic myocardium, 8 rats were divided randomly into normoxic (n=4) or chroniclly hypoxic (n=4) group, and were exposed to room air (21% O2) or continued hypoxia (10% O2) for 4 weeks. Heart tissues were collected and RNA sequencing was applied to detect the overall gene expression prolife. Genes with adjusted P-value ≤0.01 (corrected by Benjamini-Hochberg) and |log2_ratio|≥0.585 are identified as differentially expressed genes. RNA sequencing identified a total of 2014 gene with statistical significances, among which 1260 genes were significantlly increased and 754 genes were significantlly decreased. The results showed that gene expression profiling was perturbed in chronically hypoxic myocardium.
Project description:Oligodendrocytes undergo extensive changes as they differentiate from progenitors into myelinating cells. To better understand the; molecular mechanisms underlying this transformation, we performed a comparative analysis using gene expression profiling of A2B5+; oligodendrocyte progenitors and O4+ oligodendrocytes. Cells were sort-purified ex vivo from postnatal rat brain using flow cytometry. Using Affymetrix microarrays, 1707 transcripts were identified with a more than twofold increase in expression inO4+oligodendrocytes. Many genes required for oligodendrocyte differentiation were upregulated in O4+ oligodendrocytes, including numerous genes encoding; myelin proteins. Transcriptional changes included genes required for cell adhesion, actin cytoskeleton regulation, and fatty acid and; cholesterol biosynthesis. At the O4+ stage, there was an increase in expression of a novel proline-rich transmembrane protein (Prmp). Localized to the plasma membrane, Prmp displays adhesive properties that may be important for linking the extracellular matrix to the; actin cytoskeleton. Together, our results highlight the usefulness of this discovery-driven experimental strategy to identify genes relevant; to oligodendrocyte differentiation and myelination. Experiment Overall Design: Whole brain dissociates were prepared from one litter of 10 male postnatal day 7 rat pups for each of the 5 A2B5 bioligcal replicates and the 4 O4+ bioligical replicates. Total RNA was extracted from single A2B5+ and single O4+ cells sorted directly from postnatal day7 rat whole brain dissociates using flow cytometry.