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PGA DescriptionMicroarray Expression Profiling of Rodent Models of Human Disease
The Institute for Genomic Research
The Theme of our PGA: Examining Gene/Environment Interactions in Rodent Models of Human Disease Using cDNA Microarrays to link phenotype to genotype Disease phenotypes arise from complex interactions of organisms with their environments. While we have a long history of associating genes and gene defects with a large array of disease phenotypes, a growing body of data suggests that many disease phenotypes arise from the interactions of genes with their environments, including the genetic background in which the genes are expressed. Our goal is to begin an exploration of these interactions using rodent models of human disease and cDNA microarray assays to elucidate patterns of gene expression. Overview of our PGA As stated in RFA HL-99-024, The principal goal of the NHLBI Programs for Genomic Applications (PGAs) will be to link genes to function on a genomic scale in order to facilitate investigations in physiological and pathophysiological mechanisms underlying heart, lung, blood, and sleep function and disease. Together, researchers at The Institute for Genomic Research (TIGR), The Jackson Laboratories (TJL), Boston University (BU), Duke University (Duke), University of Pennsylvania (Penn) and Medical College of Wisconsin (MCW), have developed a broad-based response to this challenge in which we will develop a number of reagents, tools, and techniques that will allow us to provide links between physiologically relevant animal models of human disease and the genes that are differentially expressed in those phenotypes. The starting point for our proposed studies is the identification of rodent phenotypes that are of relevance to heart, lung, and blood diseases as well as sleep disorders. Those animals will be identified and characterized using heart, lung, kidney, vasculature, blood, and sleep responses to environmental stressors, including diet, exercise, hypoxia and pharmacological challenges. cDNA microarrays will then be used to assay patterns of gene expression in these animals with wild-type animals and their responses to identical stimuli as controls. We will present our data to the research community, integrating it with available mapping, sequencing, and physiological data.
Our PGA brings together biologists, statisticians, computer scientists, engineers, and physicists who are will lend their expertise to the achievement of our common goals. Our proposal builds on existing expertise at TIGR in the analysis of gene expression using cDNA microarrays, the mouse mutagenesis and phenotyping programs that have been developed by The Jackson Laboratories and their collaborators at Penn, Duke, and Boston Universities, and the efforts in rat genomics underway at the Medical College of Wisconsin, including the generation of phenotypically characterized consomic and congenic rat strains. Linking these programs are coordinated efforts in informatics that will both facilitate data exchange between consortium members and will make that data easily accessible to the wider research community. Underlying this is a commitment to continuing to develop and improve the reagents and assays a to provide a firm statistical basis for any inferences that our assays provide. Finally, we maintain a commitment to community service and will provide reagents, software, and data generated as part of this PGA to the wider research community.
As we are generating expression profiles, we will continue to develop and refine the laboratory resources necessary for microarray analysis, including the creation of a well maintained, sequence verified collection of reference cDNA clones, and the development of advanced statistical techniques for microarray data analysis. We will develop web-based presentations for our data and integrate it with the Gene Expression Database (GXD; <http://www.informatics.jax.org/userdocs/aboutGXD.shtml>) at TJL, the Rat Genome Database (RGD; <http://rgd.mcw.edu/>) at MCW, and the TIGR Gene Index Databases (<http://www.tigr.org/tdb/tgi.html>). Finally, we will provide educational and community outreach services to make both the techniques and reagents developed as part of this project accessible to the broader research community. This will include courses on microarray techniques and analysis and a visitorÌs program that will allow researchers not directly participating in this PGA to spend time at TIGR analyzing rodent models of HLBS disorders.
The PGA will be divided into seven operational components covering its various aspects:
Overall Specific Aims of our PGA The mission of our PGA will be to identify subsets of genes that are particularly relevant to the biology, diagnosis, management, treatment, and prevention of heart, lung, blood, and sleep related disorders and to prioritize the information for further focused study. We will achieve this through microarray analysis of patterns of gene expression in mouse and rat models of these disorders, with a focus on developing an understanding of how environmental and genetic factors combine to produce the disease phenotype. To meet this ambitious goal, we have assembled a diverse group of highly skilled research scientists with diverse but complementary talents. The research plan we will outline represents the efforts of those individuals to contribute their expertise to meeting what we believe will be a most rewarding challenge. The division of this proposal into Components has allowed the participating investigators to highlight their primary research interests and expertise. However, the division is, in many ways an artificial one. All of the participating investigators are committed to a collaborative effort to meet our overall objective and that the ultimate success of the PGA will rely on an integrated approach to large-scale, discovery-driven research. Our underlying hypothesis in this proposal is that through the careful design of our experiments, the thoughtful selection of rodent phenotypes, the integration of diverse data, and the learned analysis of experts in these disorders, we will not only recapitulate what is known about these diseases, but that we also discover new leads that will allow us to expand our understanding. With those cautions and caveats, we present the following outline for our PGA:
Specific Aim 1: To establish an Administration and Coordination Component. The goals of this Component will be to create an environment in which the diverse collection of research scientists participating in this PGA will be able to most efficiently work together and in which the PGA as a whole can adapt to experimental difficulties and advances, including those made by researchers outside of this specific PGA. Our plan to achieve these goals are outlined in the Specific Aims for this Component:
Specific Aim 2: To establish a Mouse cDNA Microarray Development and Analysis Component.
Specific Aim 3: To establish a Rat Gene Expression Anatomy Component. Microarray analysis of consomic rats offer an unprecedented means to link complex gene expression profiles to pathophysiological pathways. A consomic rat has a full-length chromosome from one inbred strain introgressed onto the background of another inbred strain. Hence, the contribution of genes on each chromosome can be assessed by phenotyping and expression profiling. Comparisons between the congenic strains will provide valuable insights into the genomic pathways (ÏclusteredÓ gene expression patterns) that differ between strains and how these differences might be connected to a particular pathogenic phenotype of the animal.
Specific Aim 4: To establish an Applications to Mouse Models Component. The theme of this application is a study of the interactions of genes with their environment to understand the generation of a disease phenotype. The mouse microarrays and the protocols and analysis tools that will be developed in the Component 3 represent an unprecedented reagent for the genome scale analysis of gene expression. However, to achieve the goal outlined in our theme, we will require mouse models that are well characterized by careful phenotypic analysis of their responses to various stresses. This component will screen mouse models of human heart, lung, blood, and sleep disorders and assist in developing experiments that will allow us to:
Specific Aim 5: To establish an Advanced Analysis Techniques Component. The members of this Component, who are trained biostatisticians and software developers, will work closely with the members of the other Components to refine experimental design and to develop novel approaches to data analysis. It is our hope that this will allow us to generate the most comprehensive and cost-effective data in the most efficient manner possible. To meet these objectives, this Component will undertake the following research plan:
Specific Aim 6: To establish a Database Integration Component. The unprecedented quantity of gene expression data that this PGA will generate for rodent models of HLBS disorders presents a number of challenges to effective analysis and interpretation. In order to fully understand the changes in expression that will be observed, we must integrate this data with phenotype, genotype, and information, including tissue distribution and time course expression data gleaned from previous studies. The Jackson Laboratories maintain two important resources, the Mouse Genome Database (MGD) and the Mouse Gene Expression Database (GXD). The objectives of this Component will be to integrate these with the Gene Expression Laboratory databases that have been developed for microarray analysis at TIGR in order to provide an integrated view of the data to participants in this PGA and to the community as a whole. This will be achieved through meeting the following goals:
Specific Aim 7: To establish an Education and Outreach Component. The advances developed as part of this PGA will provide researchers with the means to explore complex relationships between genes and the environment in HLBS disorders. We anticipate that there will be broad interest in learning the techniques and in gaining access to those resources. Consequently, the participants in this PGA are committed to a proactive community outreach program that goes beyond posting data on a website. We propose to both offer courses, training researchers in the applications of cDNA microarray technology and to create an environment where researchers with relevant biological models can visit TIGR to perform microarray assays and analyze data. Specifically:
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Description