Transcription profiling of MutaMouse epithelial lung cell line, MutaMouse lung tissue and a primary MutaMouse lung cell culture
ABSTRACT: In this study, we employ high-density oligonucleotide microarrays to characterize the MutaMouse FE1 cell line at various stages of cell growth, in primary MutaMouse lung epithelial cell cultures, and in whole lung. Global transcriptional analysis and real-time RT-PCR was applied to (1) further define the cellular origin of the FE1 cell line and its responses under different culture conditions (media and substratum), (2) provide insight into the transcriptional differences in cellular processes between FE1 cultures compared to whole lung tissues, more specifically in toxicological response, and (3) preliminarily examine FE1 culture response to exposure of benzo(a)pyrene compared to whole animals. Total RNA samples from 3 cell culture types (50% FE1, %100 FE1, and Primary lung) or MutaMouse lung were labeled with Cyanine 5-CTP, and universal reference total RNA (Stratagene, CA, USA) was labeled with Cyanine 3-CTP (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) using Agilent linear Amplification kits (Agilent Tech. Inc. Mississauga, ON, Canada) following the manufacturer's instruction. Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 5 ug total RNA. Cyanine-labeled cRNA targets were in vitro transcribed using T7 RNA polymerase. The synthesized cRNA was precipitated by LiCl and fragmented at 60 degrees C for 30 min with fragmentation solution. Cy5- sample cRNA and Cy3- reference cRNA were hybridized to Agilent mouse development microarrays (containing ~20,000 unique 60 mer oligonucleotides; Agilent Tech. Inc. Mississauga, ON, Canada) at 60 degrees C overnight with Agilent hybridization solution and washed according to manufacturer's instruction. Arrays were scanned on a VersArray ChipReader (BioRad Laboratories Ltd., Waterloo, Ontario, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, Inc. CA, USA). Present calls were determined as signals that were greater than the mean plus three times the standard deviation of the average of the negative control spots.
Project description:Identification of Hox gene downstream genes at embryonic stages 11 and 12<br><br>Functional diversification of body parts is dependent on the formation of specialized structures along the various body axes. In animals, region-specific morphogenesis along the anterior-posterior axis is controlled by a group of conserved transcription factors encoded by the Hox genes. Although it has long been assumed that Hox proteins carry out their function by regulating distinct sets of downstream genes, only a small number of such genes have been found, with very few having direct roles in controlling cellular behavior. We have quantitatively identified hundreds of Hox downstream genes in Drosophila by microarray analysis, and validated many of them by in situ hybridizations on loss- and gain-of-function mutants. One important finding is that Hox proteins, despite their similar DNA binding properties in vitro, have highly specific effects on the transcriptome in vivo, as expression of many downstream genes responds primarily to a single Hox protein. In addition, a large fraction of downstream genes encodes realizator functions, which directly affect morphogenetic processes, such as orientation and rate of cell divisions, cell-cell adhesion and communication, cell shape and migration, or cell death. Focusing on these realizators, we provide a framework for the morphogenesis of the maxillary segment. Since the genomic organization of Hox genes and the interaction of Hox proteins with specific cofactors are conserved in vertebrates and invertebrates, and similar classes of downstream genes are regulated by Hox proteins across the metazoan phylogeny, our findings represent a first step towards a mechanistic understanding of morphological diversification within a species as well as between species.
Project description:Biological comparison of gene expression profiles of adult male whole MutaMouse lung with its immortalized 100% confluent epithelial lung cell line counterpart. White, P.A.,et al. 2003. Development and characterization of an epithelial cell line from MutaMouse lung. Environ Mol Mutagen 42,3 pgs 166-184
Project description:Lymph node sections from 29 patients with StageIIIB and IIIC melanoma, with divergent clinical outcome including 16 poor and 13 good prognosis patients as defined by time to tumor progression (TTP), were profiled using MWG oligonucleotide arrays,.
Project description:A common reference experiment design was performed. mRNAs from 149 New Zealand colorectal cancer samples were labeled with Cy5 and a common reference RNA from 12 cell lines were labeled with Cy3. Data were log2 transformed and normalized. Class prediction was performed to identify genes capable of predicting future samples.
Project description:In order to study the physiological consequences of a high-copper diet on hepatic gene expression, 6 mM CuCl2 was added to the drinking water for a period of 1 month. After this period, livers of seven control mice and eight copper-treated mice were isolated and were subjected to microarray analysis and copper measurements. The hepatic gene expression profile of copper-treated mice was compared to non-treated mice using a pooled reference.
Project description:Nine time points for microarray analysis were chosen to study early and late transcriptional responses in copper metabolism upon copper overload in HepG2 cells. Samples of copper-treated cells were hybridized using non-treated samples as a reference.
Project description:Cross-platform target gene screening in colorectal cancer (CRC): we have compared 25 tumoural CRC biopsies against their normal counterpart in 30 hybridizations with a home-made cDNA array (CNIO oncochip) and 16 hybridisations with a custom oligoarray (Agilent Technologies).
Project description:Exponentially growing Sulfolobus acidocaldarius were treated with NaAc to generate replication runout and arrest in G2 phase. The cells were then resuspended in fresh acetate-free media which generates a synchronous population. Samples for investigation of gene expression change were taken during the synchronised populations progress through the cell cycle.