Leukotriene (LT)C4 synthase is the pivotal enzyme in the biosynthesis of LTC4, the parent compound of the receptor active cysteinyl leukotrienes. Thus, LTC4 synthase can regulate the biosynthesis of this potent lipid mediator, which contributes to the pathobiology of bronchial asthma and other inflammatory diseases through its metabolites, LTD4 and LTE4. The objectives of this project are to define the transcriptional regulation of the murine LTC4 synthase gene (Aim 1); to generate LTC4 synthase transgenic mice and the LTC4 synthase gene-disrupted mice (Aim 2); and to examine the in vivo responses of these gene manipulated mice to various inflammatory insults (Aim 3). To examine the transcriptional regulation of mouse LTC4 synthase gene, we will start with a previously identified 402-bp genomic fragment of mouse LTC4 synthase gene contining 352-bp of 5' flanking region. Promoter and enhancer elements will be defined by reporter contructs, mutagenic analysis, and by Dnase 1 hypersensitivity assays. Trans-acting factors will be characterized by gel shift and supershift assays. The major focus, however, is to assess the role of the cysteinyl leukotrienes in physiologic and pathobiologic processes by selective alterations of the terminal biosynthetic enzyme, LTC4 synthase. Transgenic mice will be created by pronuclear injection of a 5.5-kb genomic fragment containing the entire human LTC4 synthase gene with its native promoter. Mice will be screened for the transgene by PCR with human specific oligonucleotide primers and their gene dosage determined by Southern blot analysis. The gene-disrupted mice will be created with a targeting construct which contains a neomycin gene in place of exon 2 to exon 4 of mouse LTC4 synthase gene and a thymidine kinase gene downstream of the disrupted LTC4 synthase gene. Double resistence clones with homologous recombination will be used for blastocysts injection to create chimeric mice and subsequently gene-disrupted mice. The growth and development of these gene manipulated animals and the ability of their BMMC to express LTC4 synthase and to generate cysteinyl leukotrienes will be examined. Assessment of their inflammatory responses will include airway hyperreactivity to protein sensitization and aerosol challenge, arachidonic acid induced inflammation, systemic anaphylaxis, T. spiralis infection, and NSAID induced gastric ulcers.
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