Gene expression profiling of bovine in vitro adipogenesis using a cDNA microarray

The gene expression profile of bovine bone marrow stromal cells undergoing adipogenesis was established using a custom cDNA microarray. Cells that were treated with adipogenic stimulants and those that were not were collected at each of the six time points, and gene expression differences between the treated and untreated samples within each time point were compared using a microarray. Statistical analyses revealed that 158 genes showed a minimum fold change of 2 in at least one of the five post-differentiation time points. These genes are involved in various cellular pathways and functions, including lipogenesis, glycolysis, cytoskeleton remodelling, extracellular matrix, transcription as well as various signalling pathways such as insulin, calcium and wingless signalling. The experiment also identified 17 differentially expressed (DE) microarray elements with no assigned function. Quantitative real-time PCR was employed to validate eight DE genes, and the PCR data were found to reproduce the microarray data for these eight genes. Subsequent gene ontology annotation was able to provide a global overview of the molecular function of DE genes during adipogenesis. This analysis was able to indicate the importance of different gene categories at various stages of adipogenic conversion, thereby providing further insights into the molecular changes during bovine adipogenesis.

[1]  S. Moore,et al.  Mapping of 12 bovine ribosomal protein genes using a bovine radiation hybrid panel. , 2001, Animal genetics.

[2]  B. Dalrymple,et al.  An interactive bovine in silico SNP database (IBISS) , 2004, Mammalian Genome.

[3]  J. Ntambi,et al.  The mechanism of inhibition of 3T3-L1 preadipocyte differentiation by prostaglandin F2alpha. , 1996, Endocrinology.

[4]  Y. H. Wang,et al.  Development and application of a bovine cDNA microarray for expression profiling of muscle and adipose tissue , 2004 .

[5]  A. Reverter,et al.  A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae. , 2004, Journal of animal science.

[6]  M. Pfaffl,et al.  Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species. , 2002, Domestic animal endocrinology.

[7]  T. Leff,et al.  c-Jun N-Terminal Kinase Phosphorylates Peroxisome Proliferator-Activated Receptor-γ1 and Negatively Regulates Its Transcriptional Activity. , 1999, Endocrinology.

[8]  W. Wilkison,et al.  Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous , 2001, Journal of cellular biochemistry.

[9]  A. Reverter,et al.  A mixture model-based cluster analysis of DNA microarray gene expression data on Brahman and Brahman composite steers fed high-, medium-, and low-quality diets. , 2003, Journal of animal science.

[10]  Ken W. Y. Cho,et al.  Microarray optimizations: increasing spot accuracy and automated identification of true microarray signals. , 2002, Nucleic acids research.

[11]  Radhakrishnan Nagarajan,et al.  Microarray analysis of differentiation-specific gene expression during 3T3-L1 adipogenesis. , 2004, Gene.

[12]  S. McWilliam,et al.  Transcriptional profiling of skeletal muscle tissue from two breeds of cattle , 2005, Mammalian Genome.

[13]  J. Clapham,et al.  The thiazolidinedione insulin sensitiser, BRL 49653, increases the expression of PPAR-gamma and aP2 in adipose tissue of high-fat-fed rats. , 1996, Biochemical and biophysical research communications.

[14]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[15]  D. Bernlohr,et al.  The Mammalian Fatty Acid-binding Protein Multigene Family: Molecular and Genetic Insights into Function , 2000, Trends in Endocrinology & Metabolism.

[16]  Z. Herceg,et al.  Genome-wide analysis of gene expression regulated by the HAT cofactor Trrap in conditional knockout cells. , 2003, Nucleic acids research.

[17]  J. Novakofski Adipogenesis: usefulness of in vitro and in vivo experimental models. , 2004, Journal of animal science.

[18]  B. Spiegelman,et al.  ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. , 1996, Genes & development.

[19]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[20]  Reiko Itoh,et al.  Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36,000 bovine ESTs. , 2001, Nucleic acids research.

[21]  J. Wood,et al.  Manipulating meat quality and composition , 1999, Proceedings of the Nutrition Society.

[22]  S. Moore,et al.  Global gene expression patterns spanning 3T3-L1 preadipocyte differentiation , 2004 .

[23]  N. Lee,et al.  A concise guide to cDNA microarray analysis. , 2000, BioTechniques.

[24]  W. Wilkison,et al.  Role of intracellular calcium in human adipocyte differentiation. , 2000, Physiological genomics.

[25]  M. Rajeevan,et al.  Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. , 2001, Methods.

[26]  A. Muise,et al.  A eukaryotic transcriptional repressor with carboxypeptidase activity. , 1995, Nature.

[27]  T. Braulke,et al.  Proteolysis of insulin-like growth factors (IGF) and IGF binding proteins by cathepsin D. , 1997, Endocrinology.

[28]  Ronald W. Davis,et al.  Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray , 1995, Science.

[29]  David S. Wishart,et al.  The CyberCell Database (CCDB): a comprehensive, self-updating, relational database to coordinate and facilitate in silico modeling of Escherichia coli , 2004, Nucleic Acids Res..

[30]  A. Reverter,et al.  Joint analysis of multiple cDNA microarray studies via multivariate mixed models applied to genetic improvement of beef cattle. , 2004, Journal of animal science.

[31]  A. Greenberg,et al.  The Short- and Long-Term Effects of Tumor Necrosis Factor-α and BRL 49653 on Peroxisome Proliferator-Activated Receptor (PPAR)γ2 Gene Expression and Other Adipocyte Genes , 1998 .

[32]  M. Ozbun,et al.  Variable expression of some "housekeeping" genes during human keratinocyte differentiation. , 2002, Analytical biochemistry.

[33]  E. Walters,et al.  β-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels , 2002, Thorax.

[34]  J. Ntambi,et al.  Role of Ca2+ in the early stages of murine adipocyte differentiation as evidenced by calcium mobilizing agents. , 1996, Differentiation; research in biological diversity.

[35]  Francesco Pinciroli,et al.  GFINDer: Genome Function INtegrated Discoverer through dynamic annotation, statistical analysis, and mining , 2004, Nucleic Acids Res..

[36]  P. J. Higgins,et al.  Control selection for RNA quantitation. , 2000, BioTechniques.

[37]  M. Lazar,et al.  Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[38]  David Peel,et al.  The EMMIX Algorithm for the Fitting of Normal and t-Components , 1999 .

[39]  Peter Adams,et al.  The EMMIX software for the fitting of mixtures of normal and t-components , 1999 .

[40]  D. Fergusson,et al.  Muscle metabolism in relation to genotypic and environmental influences on consumer defined quality of red meat , 2005 .

[41]  L. A. García-Cortés,et al.  VCE4.0 : a (Co)variance Component Package for Frequentists and Bayesians , 1998 .

[42]  G. Vassaux,et al.  Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent. , 1992, The Journal of biological chemistry.

[43]  W. Siegert,et al.  Guideline to reference gene selection for quantitative real-time PCR. , 2004, Biochemical and biophysical research communications.

[44]  O. Evanson,et al.  Sequential patterns of gene expression by bovine monocyte-derived macrophages associated with ingestion of mycobacterial organisms. , 2004, Microbial pathogenesis.

[45]  I. Yang,et al.  Concise Guide to cDNA Microarray Analysis – II , 2001 .

[46]  S. Perrey,et al.  Thiazolidinedione- and tumor necrosis factor alpha-induced downregulation of peroxisome proliferator-activated receptor gamma mRNA in differentiated 3T3-L1 adipocytes. , 2001, Metabolism: clinical and experimental.

[47]  Hinrich W. H. Göhlmann,et al.  Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures. , 2003, Nucleic acids research.

[48]  A. Greenberg,et al.  The short- and long-term effects of tumor necrosis factor-alpha and BRL 49653 on peroxisome proliferator-activated receptor (PPAR)gamma2 gene expression and other adipocyte genes. , 1998, Molecular endocrinology.

[49]  T. Hudson,et al.  Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. , 2002, Genome research.

[50]  X. Wang,et al.  Inhibition of adipogenesis by the stress‐induced protein CHOP (Gadd153). , 1995, The EMBO journal.

[51]  J. Warrington,et al.  Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. , 2001, Analytical biochemistry.

[52]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[53]  Andrea Didier,et al.  Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR. , 2003, Molecular and cellular probes.