GENETIC ANALYSIS OF MESODERM INDUCTION IN MICE

小鼠中胚层诱导的遗传分析

• 批准号: 6386306
• 负责人: BERNADETTE C HOLDENER
• 金额: $ 27.18万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386306
• 关键词: biological signal transduction; cell cycle; cell differentiation; gene deletion mutation; gene expression; genetic mapping; genetic regulation; growth factor; in situ hybridization; laboratory mouse; mesoderm; molecular cloning; phenotype; transcription factor; transfection

DESCRIPTION (adapted from investigator's abstract): This is a revised application to study the function of the msd locus in mouse development. The msd mutant phenotype consists of a failure to form mesoderm and the locus is defined by a series of overlapping deletions within the albino complex. The broad goal of the proposed research is to understand the genetic regulation of mesoderm differentiation. The specific aims of the proposal are to 1) determine if the msd mutation disrupts growth factor signaling. 2) To assess whether msd has other roles during development besides its involvement in mesoderm formation, and 3) to clone the msd gene. For specific aim 1, marked wild type ES cells will be injected into msd mutant blastocysts to determine whether msd endoderm is able to produce a mesoderm inducing signal(s). In addition, the expression patterns of Nodal, BMP-4 and Bmpr will be examined in mutant msd embryos to determine if msd functions by blocking production or reception of known mesoderm inducing signals. In a related series of experiments, regulated ectopic expression of known downstream components of mesoderm signaling pathways such as Mad1 , T and Map kinase will be examined for their ability to bypass the mesoderm block in msd mutants. In section 2 additional characterization of the msd phenotype will include experiments to address the cell-cycle length during early gastrulation, in situ hybridization studies to determine if early markers of anteroposterior axis formation are expressed in msd mutant embryos, production of chimeric embryos containing mutant msd cells in a wildtype background, and isolation of new ENU induced mutations in the msd gene. In the third section of the grant, approaches to clone the msd region are described. They include the isolation of a contig spanning the msd region and identification of transcription units within the contig by cDNA selection. The msd gene or genes will be identified by rescue of mutant embryonic stem cells after transfection with either individual cDNA or multiple cDNA clones.

描述（改编自研究者的摘要）：这是修订版 应用程序研究 MSD 基因座在小鼠发育中的功能。 MSD 突变体表型包括无法形成中胚层和 位点是由白化基因内一系列重叠的缺失定义的 复杂的。 拟议研究的总体目标是了解 中胚层分化的遗传调控。 该计划的具体目标 建议 1) 确定 MSD 突变是否破坏生长因子 发信号。 2) 评估msd在开发过程中是否还有其他作用 除了参与中胚层形成之外，3) 克隆 msd 基因。 对于特定目标1，标记的野生型ES细胞将被注射到msd中 突变囊胚以确定 MSD 内胚层是否能够产生 中胚层诱导信号。 此外，Nodal 的表达模式， 将在突变型 msd 胚胎中检查 BMP-4 和 Bmpr，以确定 msd 是否 通过阻断已知中胚层诱导物质的产生或接收来发挥作用 信号。 在一系列相关的实验中，调节异位表达 中胚层信号通路的已知下游成分，例如 Mad1， 将检查 T 和 Map 激酶绕过中胚层的能力 阻止 MSD 突变体。 第 2 节中 MSD 的附加特征 表型将包括解决细胞周期长度的实验 早期原肠胚形成，原位杂交研究以确定是否早期 前后轴形成的标记在 MSD 突变体中表达 胚胎，含有突变 MSD 细胞的嵌合胚胎的生产 野生型背景，以及在 MSD 中新 ENU 诱导突变的分离 基因。 在资助的第三部分中，克隆 MSD 区域的方法 被描述。 它们包括跨越 MSD 的重叠群的分离 通过 cDNA 识别重叠群内的转录单位 选择。 MSD基因将通过拯救突变体来鉴定 用单个 cDNA 或转染后的胚胎干细胞 多个 cDNA 克隆。