This project will examine the brain genomic response to hypoxia. Chronic hypoxia induces """"""""tolerance"""""""" to ischemia, chronic hypoxia protecting against a stroke whereas acute hypoxia does not. The identification of genes induced by chronic hypoxia would help delineate genes that produce tolerance. The genomic effects of hypoxia are also of interest since we have identified a transcription factor, HIF-1, that is induced in response to chronic but not acute hypoxia, and therefore is a candidate gene for conferring hypoxia induced tolerance. It would be important to identify target genes of HIF using genomic approaches. Lastly, the induction of genes related to acute hypoxia is of interest because acute hypoxia stimulates a """"""""stress response,"""""""" and could help in characterizing the genes that mediate physiological stress in contrast to psychic stress. These studies are designed to test several hypotheses: (1) short durations of hypoxia induce genes that are mainly related to stress; (2) chronic hypoxia induces genes that protect the brain against ischemia; and differences in gene expression in HIF-1a knockout mice compared to their normal littermates will identify candidate HIF-1a target genes.
The aims are to:
Aim #1 a. Examine the genomic response of 7d old and adult rat brain to acute hypoxia; (#lb); examine the genomic response of 7d old and adult rat brain to chronic hypoxia that produces tolerance to focal ischemia; and (#lc); and confirm that chronic hypoxia, but not acute hypoxia, produces hypoxia-induced tolerance to ischemia.
Aim #2. Perform Suppressive Subtractive Hybridization (SSH) by making cDNA from acutely hypoxic brain compared to chronically hypoxic brains of 7-day old rats. Clone SSH products to make subtracted libraries. PCR amplify the clone inserts and array the inserts. The microarrays are then screened with cDNA made from mRNA from the acute and chronically hypoxia brains. Genes that are confirmed to be induced by chronic hypoxia are sequenced.
Aim #3. Examine the genomic response to chronic hypoxia in HlF-1a knockout mice compared to wild type mice. The long-term goals are to identify the acute and chronic hypoxia responsive genes in the mammalian genome, to describe the mechanisms of induction, to identify those genes that mediate physiological responses to hypoxia via the carotid body, and to identify genes that mediate hypoxia induced tolerance to ischemia.
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