EXPRESSION AND FUNCTION OF MAMMALIAN HOMEOBOX GENES

哺乳动物同源盒基因的表达和功能

• 批准号: 2198526
• 负责人: Robert E. Maxson
• 金额: $ 28.33万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2198526
• 关键词: Drosophilidae; Retroviridae; animal population genetics; athymic mouse; biochemical evolution; central nervous system disorders; complementary DNA; crosslink; cytogenetics; developmental genetics; diphtheria toxin; embryo /fetus; embryo /fetus protein; embryogenesis; epitope mapping; flow cytometry; fusion gene; gene expression; gene mutation; genetic enhancer element; genetic manipulation; genetic mapping; genetic promoter element; genetic regulation; genetic regulatory element; genetically modified animals; genotype; germ cells; homeobox genes; immunofluorescence technique; in situ hybridization; mammalian embryology; meiosis; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; protein sequence; tissue /cell culture

Valuable insight into normal and abnormal embryonic development may be gained from the study of genes and gene products that control these processes. Current knowledge on the structure, evolution and expression of mammalian homeobox genes identifies these as excellent candidates for loci that regulate mammalian development. We propose experiments that explore the regulation of the homeobox genes Hox 1.3 and Hox 5.1 and the function of Hox 1.3. 1. The cis-acting elements responsible for the spatial expression of Hox 1.3/lacZ and Hox 5.1/lacZ gene fusions in the central nervous system of transgenic mice will be defined. The cis-acting elements will be used as affinity tools in order to characterize the trans-acting factors involved. 2. Previous results suggest that the developing spinal cord may be subdivided into discrete rostrocaudal regions based on the activity of particular Hox genes. Therefore the brachial and cervical CNS cells from Hox 1.3/lacZ and Hox 5.1/lacZ mice will be purified using a protocol based on fluorescence activated cell sorting of lacZ+ cells. It will then be determined whether the brachial and cervical CNS domain express different combinations of active genes, including Hox genes. This analysis will be done with purified cells using immunoblots, RNase mapping and subtractive cDNA cloning. Genetic ablation of brachial cells will also be performed in transgenic mice expressing a Hox 1.3/diphtheria toxin A fusion gene. The resulting phenotype will be described. 3. Transgenic mice that express ectopically the Hox 1.3 coding region under the control of the ubiquitous GRP70 promoter or the specific Hox 5.1 promoter will be produced and analyzed with respect to several molecular and morphological parameters. 4. Vectors that allow for enrichment of ES cells containing a Hox 1.3 gene activated by homologous recombination will be used to isolate such cells. Chimeric mice will be produced from these ES cells and bred to generate mice heterozygous and homozygous for the inactivated Hox 1.3 gene.

对正常和异常胚胎发育的宝贵见解可能是 从控制这些的基因和基因产物的研究中获得 过程。 当前关于结构，演变和表达的知识 哺乳动物同源基因基因将其识别为基因座的出色候选者 调节哺乳动物的发展。 我们提出了探索的实验 同型基因HOX 1.3和HOX 5.1的调节以及功能 Hox 1.3 1。负责HOX空间表达的顺式作用元素 1.3/lacz和HOX 5.1/lacz基因融合在中枢神经系统中 将定义转基因小鼠。 顺式作用元素将用作 亲和力工具以表征所涉及的跨性别因素。 2。先前的结果表明，发育中的脊髓可能是 根据 特定的HOX基因。 因此，来自 HOX 1.3/LACZ和HOX 5.1/LACZ小鼠将使用基于协议的 在荧光激活的LAC+细胞的细胞分选中。 那将是 确定臂和宫颈CNS结构域是否表达不同 活性基因的组合，包括HOX基因。 该分析将是 使用免疫印迹，RNase映射和减法使用纯化的单元 cDNA克隆。 臂细胞的遗传消融也将在 表达HOX 1.3/白喉毒素融合基因的转基因小鼠。 这 将描述由此产生的表型。 3。在异位表达HOX 1.3编码区的转基因小鼠 在无处不在的GRP70启动子或特定HOX 5.1的控制下 将根据几个分子产生和分析启动子 和形态参数。 4。允许富集含有HOX 1.3基因的ES细胞的载体 通过同源重组激活将用于分离此类细胞。 嵌合小鼠将从这些ES细胞产生并繁殖以产生 无效的HOX 1.3基因的小鼠杂合和纯合子。