ToxicogenomicsToxicogenomicsGenomic Sequences: More Genetic Information than Ever Before!More Genetic Information than Ever Before!Gene FunctionSequenceNature Reviews: Drug Discovery Nicholson et al. (2002)Contemporary Toxicology Experimental DesignCh i l f i l d l?MicroarrayAnalysisToxicologicalCharacterizationFractionation of targetand nontarget tissuesProteomic AnalysisChemical exposure of animal modelsBioinformaticsanalysis• acute toxicity• pathology• clinical chemistry•chronic toxicityand non-target tissues2D-PAGE / MS SELDI yIdentification ofcandidate•chronic toxicity• carcinogenicityCandidate proteinidentification (mass spec)Data Integrationcandidatebiomarkersidentification (mass spec)Analysis of serum forcandidate proteinsData IntegrationVerifyBiomarker Discovery, Key events,Mechanisms of actionVerifyCredit: M. Waters (NIEHS)Toxicogenomics is a new scientific field that elucidates how the entire genome is involved inbiological responses of organisms e xposed to environmental toxicants/stressorsDisease Mechanisms: application of gene expression profiling technologies to define the mechanistic underpinnings of environmentally related diseases; genetic and environmental components of disease, elucidation of disease pathways and networks, and development of disease models. Susceptibility: individual and population susceptibilities to exposure and disease as derived from genetic and environmental analysis and integration; identification of gene targets and factors mediating susceptibility and genderenvironmental analysis and integration; identification of gene targets and factors mediating susceptibility, and gender‐, strain‐ and species‐susceptibility.Comparative Genomics: comparative and integrated responses of organisms to environmental stimuli; cross‐species comparisons of biological responses to environmental factors at the gene, transcription, and protein level and their integration in model organisms; conserved biological components, pathways and responses to environmental factors; and computational tools to support comparative to xicogenomics.Predictive Toxicology: development and application of gene expression, proteomics and metabolomics technologies in predictive toxicology; development of model systems and research tools, and linkage of pr edictive responses to disease phenotype.Gene Expression (mRNA) Studies:p()• Pattern of genes expressed in a cell is characteristic of its current statecurrent state • Many differences in cell state or type are correlated with h i RNA l l fchanges in mRNA levels of many genes •Expression patterns of many previously uncharacterized pp ypygenes may provide clues to their possible function by comparison with how known genes act• Gene expression data can be combined with metabolic schemas to understand how pathways are changed under iditi(i hifti)varying conditions (i.e., mechanisms of action)Microarray TechniqueMicroarray TechniqueMicroarray: A substrate with bound capture probesCapture probe: An oligo/cDNA with gene (DNA sequence) of interestGeneric experimental steps:1. Fabrication:Ph t lith hAff t i(l )500 000 bPhotolithographyAffymetrix(one-color array)>500,000 probesPrinting Agilent (two-color arrays) ~240,000 probesSpotting In-house (two-color arrays) <24,000 probes2. Target Generation from a sample of interest:One color (biotin labeled cRNA, phycoerythrin-streptavidin detection) Two color (Cy3 and Cy5 cDNA labeling)3. Hybridization4. Analysis“Scanning” of arrayAmount of hybridized target is assessedStatistical interrogation of the dataNat. Biotech. 24, 1112-3 (2006)DNA sequences complementary to genes of i t t t d d l id t i tionMicroarray Techniqueinterest are generated and laid out in microscopic quantities on solid surfaces at defined positionsFabricad ncDNAs (from mRNA) from samples are labeled with fluorescent probes and eluted over the surface complementary DNA binds Target generation anhybridizationPresence of bound DNA is detected by fluorescence following laser excitationand data is interrogated using specialized alysisand data is interrogated using specialized computational approachesAnaNat. Biotech. 24, 1112-3 (2006)Microarray Experiments:Microarray Experiments:analysis of gene expressionanalysis of gene expressionanalysis of gene expressionanalysis of gene expression• Genome-wide (e.g., yeast)• Partial selection of known/unknown genes-Normal vs DiseaseExample: Analysis of expression patterns in cancer-Pattern of gene expression-networks-Novel gene association/discovery-Molecular ClassificationExample:Comparison of Breast TumorsExample:Comparison of Breast Tumors-Samples classified into subtypes -Genome-Wide AnalysisyExample: Genome-wide expression in S. cerevisiae-Discovery of yet unidentified molecular pathwaysFabrication of twoFabrication of two--color oligo/cDNA arrayscolor oligo/cDNA arraysNat Genet211519 (1999)Nat. Genet.21, 15-19 (1999) Oligos(20-70 bp)or cDNAs96-well plate384-well plateprinted on a glass slideOligo/cDNA Arrays MethodOligo/cDNA Arrays MethodTwo mRNA sources to be compared are labeled with fluorescent probes:probes:Cy3 (green) used for one sample (e.g., “control”)Cy5 (red)used for the other (e g“treatment”)Cy5 (red)used for the other (e.g., treatment )Probes are mixed and washed over the microarray (hybridization)Each probe is excited using a laser and its fluorescence (RandG)atEach probe is excited using a laser, and its fluorescence (Rand G) at each element detected with a scanning confocal microscopeThe ratio between the signals in two channels (R:G) is calculated for each array spotRatios of intensity of Cy5/Cy3 probes is a reliable measure of the abundance of specific mRNA’s in each sample compared to“control”abundance of specific mRNAs in each sample compared to controlTwoTwo--color oligo/cDNA arrayscolor oligo/cDNA arraysmRNA from Sample 1(e.g. control)mRNA from Sample 2(e.g., treated)Scan and quantitate gene expression levelsScan and quantitate gene expression levelsTwoTwo--color oligo/cDNA arrayscolor oligo/cDNA arrays150 m200 mTwo-color oligonucleotide (60-mer) commercial arraysDATAAffymetrix® GeneChip array formatAffymetrix® GeneChip array format• One-color chip (biotin labeled cRNA, phycoerythrin-streptavidin detection))• Oligonucleotide probes are synthesized in situ on the chip• Semiconductor photolithography technology is used to th i liiitlbtt12synthesize oligos in situ on a glass substrate 1 cm2•
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