HEX EXPRESSION AND REGULATION IN DEVELOPING LUNG

本研究的目的是阐明 参与肺发育的机制。同源异型盒基因， 作为序列特异性DNA结合蛋白的蛋白质， 在发育小鼠中以特定的时空模式表达， 已经显示出引导图案沿着前后轴， 明确地区身份。趋异同源盒基因Hex 在发育中的肺中表达，但对它的时间， 表达的空间模式或其调节。 的具体目标 本项目是：1.在发育中的小鼠中， 表达Hex的区域和细胞类型， 随着胎龄的增加表达量也随之变化; 2.识别肺- 在转基因小鼠中通过报告基因分析Hex的特异性增强子; 和3.来表征与以下物质相互作用的反式作用转录因子： Hex的肺特异性增强子。将采用的方法 完成这些具体目标包括：原位杂交，分离 Hex基因组克隆的限制性酶切图谱; 在转基因小鼠中使用报告基因分析增强子元件; 肺特异性增强子元件的表征 分析，包括使用一种新的穿梭载体，以切除特定的 含有Hex基因组序列的PI克隆的同源区域 重组;和表征的反式作用因子相互作用 通过体外DNA-蛋白结合研究， 包括鉴定可能的DNA的共有结合位点 结合蛋白和DNA-蛋白结合特异性的检测 通过竞争、突变分析和使用特异性 抗体的任何已鉴定的转录因子的能力， 将通过在组织中共转染来测试激活的Hex肺增强剂 培养细胞。最终，Hex的潜在调节剂将异位 在转基因小鼠中表达，以测试这些元件 在体内调节Hex肺表达。实验室研究将 由首席研究员Clifford W.博格，在 在两个赞助商的指导下， 博士Frank H. Ruddle和Dr. Harris C.雅各布斯和一个顾问委员会 会定期和私家侦探见面以提供指导和评估 中求进工作总这一建议将允许候选人进一步发展 分子遗传学和发育领域的技术专业知识 生物学以及提高他的基础知识和完善他的 用科学的方法解决这些问题。的 本建议书中提供的额外培训将为候选人做好准备 在肺部生物学领域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.