CCBM: NHLBI Canine Microarray

Large-scale measurement of gene expression using complex mixtures of mRNAs from target tissues has the potential to provide a wealth of information regarding the mechanisms of adaptive and maladaptive responses to disease. However, the ability to study temporal changes in gene expression under carefully controlled conditions during disease progression is severely limited when working with human subjects. We have assembled a group of collaborating investigators from the departments of Medicine, Oncology, Surgery, Anesthesiology and Biomedical Engineering who have extensive experience in the study of the pathobiology of a number of diseases of the heart, lungs, kidneys and vasculature in humans and animal models. Most importantly, all share a long-standing interest in the use of canine models of human disease to study the natural history of disease and the effects of therapeutic interventions using serial tissue sampling; as a consequence there is a wealth of biological material that will serve as targets for expression profiling experiments.

In an effort to better understand the molecular mechanisms of adaptive and maladaptive responses to cardiovascular, pulmonary, vascular and renal disorders we propose to profile global changes in gene expression in canine models of disease that are well studied at our institution. The aims of this proposal are:

Aim 1. Generate and validate canine cDNA arrays for expression profiling. At present there are no canine oligonucleotide or cDNA arrays available from commercial or academic sources. Indeed the amount of canine DNA sequence information in the public domain is quite limited. We have made substantial investments in the first stages of this project by generating normalized cDNA libraries from pooled fetal, neonatal and adult canine tissues. Completion of Aim 1 will provide a unique resource which our local research community will use to study the molecular basis of a broad range of disease processes, and will contribute to a more comprehensive understanding of gene expression in a commonly used large mammalian model of human disease.

Aim 2. To provide computational and informatics support for the research activities of the proposed array facility including: Aim 2A. use of multi-gene alignment and clustering algorithms to generate and annotate a World Wide Web accessible canine Unigene database; Aim 2B : development of gene-specific parametric models of expression intensity, methods for model parameter estimation, and application of these models and methods to identification of statistically significant changes in gene expression under control versus test conditions; Aim 2C : development of statistical algorithms for identification of gene expression patterns from time series measurements, using the gene-specific models developed in Aim 2B. Aim 2D : to implement and maintain databases supporting tissue-specific and regional analyses of gene expression data.

Aim 3. To profile gene expression in the dog including: Aim 3A. The characterization of regional variations in gene expression in the normal canine heart. The heart is functionally heterogeneous and a large body of evidence demonstrates regional differences in mechanical, electrical, signaling and hormonal function in the heart. The molecular basis of this heterogeneity is not understood. We will utilize expression profiling to examine the differences in gene expression between the atria and ventricles, the right and left ventricles and regionally across the wall of the left ventricle. These experiments will serve the dual purpose of providing insight into the molecular mechanisms regional differences in transcriptomes in the heart and will serve as a series of validation experiments for the array data. Aim 3B. The characterization of spatiotemporal changes in gene expression during the development of models of canine cardiovascular, pulmonary and renal disease.

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