GENETIC ANALYSIS OF MESODERM INDUCTION IN MICE

小鼠中胚层诱导的遗传分析

• 批准号: 2910217
• 负责人: BERNADETTE C HOLDENER
• 金额: $ 24.21万
• 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 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910217
• 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（根据调查员的摘要改编）：这是一个修订版 用于研究MSD基因座在小鼠发育中的功能的应用。 MSD突变表型包括未能形成中胚层和 基因座是由白化病中的一系列重叠缺失定义的 复杂的。 拟议研究的广泛目标是了解 中胚层分化的遗传调节。 特定目标 建议是1）确定MSD突变是否破坏生长因子 信号。 2）评估MSD在开发过程中是否具有其他角色 除了它参与中胚层形成，以及3）克隆MSD基因。 对于特定的AIM 1，标记的野生型ES细胞将被注入MSD 突变的胚泡以确定MSD内胚层是否能够产生 中胚层诱导信号。 另外，节点的表达模式， BMP-4和BMPR将在突变的MSD胚胎中检查，以确定MSD是否 通过阻断已知中胚层诱导的生产或接收来实现功能 信号。 在相关的一系列实验中，调控异位表达 中胚层信号通路（例如MAD1）的已知下游成分， T和MAP激酶将检查其绕过中胚层的能力 MSD突变体中的块。 在第2节中，MSD的其他表征 表型将包括解决细胞周期长度的实验 早期胃胃，原位杂交研究，以确定早期是否早期 前后轴形成的标记在MSD突变体中表达 胚胎，在A中含有突变MSD细胞的嵌合胚胎的产生 野生型背景，并在MSD中隔离新ENU诱导的突变 基因。 在赠款的第三部分中，克隆MSD区域的方法 描述。 它们包括跨MSD的重叠群的隔离 区域和cDNA重叠群中转录单元的识别 选择。 MSD基因或基因将通过拯救突变体鉴定 用单个cDNA或 多个cDNA克隆。