Transcription profiling of mouse transformed glial progenitor cells

ABSTRACT: The goals of this research are three-fold: (i) One is to identify genetic changes associated with tumorgenesis that interfere with the ability of transformed glial cells to differentiate. This is connected with (ii) our analysis of the regulation of differentiation in normal glial progentor cells, as only by understanding this in normal cells can we understand what is occurring when differentiation fails in transformed versions of the same cells. (iii) The third is to identify the molecular basis for the greater resistance of transformed glial progenitor cells to chemotherapeutic agents as compared with normal glial progenitor cells. Thus, analysis of the populations provided will yield data crucial to two of our central areas of research, as awarded by NINDS in "Oligodendrocytes & precursors: toxicity of chemotherapy" (5R01NS044701), and "Low-level toxicant perturbation of neural cell function" (1R01ES012708). Our ability to work with essentially unlimited numbers of primary progenitor cells and transformed derivatives of these cells offers one of the few systems in which side-by-side comparison of normal and transformed cells at both the biochemical and molecular levels is possible. Using defined cell populations, expression profiles will be compared to reveal changes in cell type specific markers and regulatory factors associated with transformation. In addition, retention of markers despite transformation may enable identification of new markers useful in the diagnosis of glial tumors. Transformation of glial progenitor cells is associated both with loss of the proteins required for normal differentiation and with an inability to normally activate proteins that function in differentiation control. By side-by-side analysis of gene expression in normal and transformed glial progenitor cells, and analysis of the response to inducers of differentiation in each these populations, we will identify candidate regulators critical in the control of normal differentiation. Identification of these regulators will also provide clues as to overcome the blockade of differentiation that occurs in glial cancers (which represent the majority of adult brain tumors). RNA will be isolated from two separate preparations of glial progenitors. Preparation of purified populations has been described in multiple of our previous publications (Mayer, M. et al. (1994) Development 120, 142-153; Mayer-Pröschel, M. et al. (1997) Neuron 19, 773-785; Rao, M. and Mayer-Pröschel, M. (1997) Dev. Biology 188, 48-63). Total RNA from purified cell cultures will be extracted in a two step process. RNA will first be extracted by dounce homogenization in phenol/guanidine isothiocyonate solution (Trizol, Invitrogen, California) followed by isopropanol precipitation. Contaminating traces of DNA and protein will then be removed by silica-based affinity chromatography and Dnase treatment (NucleoSpin RNA Purification system, Clontech, California). Depending on the cell type, 5x105 cells will typically yield 5-15µg of high quality RNA (OD260/280=2.1 at pH7.5). In order to determine purity and molecular weight distribution, samples will be routinely tested on a Agilent Bioanalyzer. cDNA will be generated from total RNA and then be submitted to one round of amplification and labeling using the messageAmp system (Ambion). Probes are then hybridized to the Affymerix Rat 230 (A/B or 2.0) GeneChip expression array. Array data will be processed using the Micro Array Suite 5.0 (MAS, Affymetrix). .Cel files containing normalized, relative expression data for all genes tested will be used for downstream statistical analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Elizabeth Salomon 

PROVIDER: E-GEOD-9763 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress