HEX EXPRESSION AND REGULATION IN DEVELOPING LUNG

这项提议的研究目标是阐明分子 参与肺发育的机制。同源异型盒基因，编码 起序列特异性DNA结合蛋白作用的蛋白质有 在发育中的小鼠体内以特定的时空模式表达 已经被证明沿着前后轴线引导图案和 在指定区域身份方面。不同的同源异型盒基因十六进制 在发育中的肺中表达，但对其时间- 表达的空间模式或其调节。的具体目标 这个项目是：1.在正在开发的鼠标中识别特定的 表示十六进制的区域和细胞类型，并确定该模式如何 随着胎龄的增加，表达的变化；2.识别肺- 转基因小鼠Hex报告基因分析的特异性增强子； 以及3.鉴定相互作用的反式转录因子 Hex的肺特异性增强子(S)。要使用的方法 完成这些特定目的包括：原位杂交、分离 和用于十六进制的基因组克隆的限制图.鉴定 利用报告基因分析转基因小鼠的增强子元件； 肺组织特异性增强子元件的缺失鉴定 分析，包括使用一种新的穿梭载体来切除特定的 含有十六进制基因组序列的PI克隆的同源序列分析 重组；以及相互作用的反式作用因子的特征 肺特异性增强剂的体外DNA-蛋白质结合研究 包括鉴定可能的DNA的共识结合部位 结合蛋白及其与DNA结合的特异性检测 通过竞争、突变分析和超移位分析使用特定的 抗体。任何已识别的转录因子的能力 激活的十六进制肺增强剂将通过在组织中共转染法进行测试 培养细胞。最终，十六进制的潜在监管者将是异地 在转基因小鼠中表达，以测试这些元件的能力 在体内调节Hex肺的表达。实验室研究将是 由首席研究员Clifford W.Bogue博士在 与一个由两个赞助商指导的说教项目相结合， 弗兰克·H·拉德尔博士和哈里斯·C·雅各布斯博士以及一个咨询委员会 它将定期与私家侦探会面，提供指导和评估 进步。这项提议将使候选人能够进一步发展 在分子遗传学和发育领域的技术专长 生物学，以及加强他的基础知识和提炼他的 以智力科学的方法解决这些领域的问题。这个 本计划中提供的额外培训将为候选人做好准备 在肺生物学领域拥有独立而富有成效的职业生涯 发展。

The research goals of this proposal are to elucidate the molecular mechanisms involved in lung development. Homeobox genes, which encode proteins which function as sequence-specific DNA binding proteins, are expressed in specific temporo-spatial patterns in the developing mouse and have been shown to direct patterning along the anterior-posterior axis and in specifying regional identity. The divergent homeobox gene Hex is expressed in the developing lung but nothing is known about its temporo- spatial pattern of expression or its regulation. The specific aims of this project are: 1. to identify, in the developing mouse, the specific regions and cell types that express Hex and to determine how that pattern of expression changes with advancing gestational age; 2. to identify lung- specific enhancers for Hex by reporter gene analysis in transgenic mice; and 3. to characterize trans-acting transcription factors interacting with the lung-specific enhancer(s) of Hex. The methodology to be used to complete these specific aims includes: In situ hybridization, isolation and restriction mapping of genomic clones for Hex; identification of enhancer elements using reporter gene analysis in transgenic mice; characterization of lung-specific enhancer elements by deletional analysis, including the use of a novel shuttle vector to excise specific regions of PI clones containing Hex genomic sequence by homologous recombination; and characterization of trans-acting factors interacting with lung-specific enhancers by in vitro DNA-protein binding studies including identification of consensus binding sites for possible DNA binding proteins and examination of the specificity of DNA-protein binding by competition, mutational analysis and supershift assays using specific antibodies. The ability of any identified transcription factors to activate Hex lung enhancers will be tested by cotransfection in tissue culture cells. Ultimately, potential regulators of Hex will be ectopically expressed in transgenic mice to test the ability of these elements to regulate Hex lung expression in vivo. The laboratory research will be conducted by the principal investigator, Dr. Clifford W. Bogue, in conjunction with a didactic program under the guidance of two sponsors, Dr. Frank H. Ruddle and Dr. Harris C. Jacobs and an advisory committee that will meet regularly with the P.I. to offer guidance and to assess progress. This proposal will allow the candidate to further develop technical expertise in the areas of molecular genetics and developmental biology as well as enhancing his fundamental knowledge and refining his intellectual scientific approach to problems in these areas. The additional training provided in this proposal will prepare the candidate for an independent and productive career in the biology of lung development.