Functional Characterization of the Mouse LTC4 Synthase Gene

白三烯（LT）C4合成酶是生物合成具有受体活性的半胱氨酰白三烯的母体化合物LTC 4的关键酶。 因此，LTC 4合酶可以调节这种有效脂质介质的生物合成，其通过其代谢产物LTD 4和LTE 4促进支气管哮喘和其他炎性疾病的病理生物学。 本项目的目的是确定小鼠LTC 4合酶基因的转录调控（目标1）;产生LTC 4合酶转基因小鼠和LTC 4合酶基因破坏小鼠（目标2）;并检查这些基因操作小鼠对各种炎症损伤的体内反应（目标3）。 为了研究小鼠LTC 4合酶基因的转录调控，我们将从先前鉴定的小鼠LTC 4合酶基因的402-bp的基因组片段开始，其包含352-bp的5'侧翼区。 启动子和增强子元件将通过报告构建体、致突变分析和DNA酶1超敏试验来确定。 反式作用因子将通过凝胶迁移和超迁移试验进行表征。 然而，主要的焦点是通过选择性改变末端生物合成酶LTC 4合酶来评估半胱氨酰白三烯在生理和病理过程中的作用。 将通过原核注射含有完整的人LTC 4合酶基因及其天然启动子的5.5-kb基因组片段来产生转基因小鼠。 通过使用人特异性寡核苷酸引物的PCR筛选小鼠的转基因，并通过Southern印迹分析确定其基因剂量。 将用靶向构建体产生基因破坏的小鼠，所述靶向构建体含有新霉素基因代替小鼠LTC 4合酶基因的外显子2至外显子4和破坏的LTC 4合酶基因下游的胸苷激酶基因。 具有同源重组的双抗性克隆将用于胚泡注射以产生嵌合小鼠和随后的基因破坏小鼠。 将检查这些基因操作动物的生长和发育以及它们的BMMC表达LTC 4合酶和产生半胱氨酰白三烯的能力。 炎症反应的评估将包括气道对蛋白质致敏和气溶胶激发的高反应性，花生四烯酸诱导的炎症，全身过敏反应，T。螺旋体感染和NSAID诱导的胃溃疡。

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.