GENETIC ANALYSIS OF MESODERM INDUCTION IN MICE

描述（改编自研究者摘要）：这是一份修订版 应用于研究msd基因座在小鼠发育中的功能。 msd突变体表型由不能形成中胚层和 基因座由白化病内的一系列重叠缺失定义 复杂. 拟议研究的广泛目标是了解 中胚层分化的遗传调控。 的具体目标 建议是1）确定msd突变是否破坏生长因子 发信号。 2)评估msd在开发过程中是否有其他角色 除了它参与中胚层形成之外，和3）克隆msd基因。 对于具体目标1，将标记的野生型ES细胞注射到msd中， 突变胚泡，以确定MSD内胚层是否能够产生 中胚层诱导信号。 此外，Nodal， 将在突变msd胚胎中检查BMP-4和Bmpr，以确定msd 通过阻断已知的中胚层诱导因子的产生或接收来发挥功能 信号. 在一系列相关的实验中， 中胚层信号通路的已知下游组分如Mad 1， 将检查T和Map激酶绕过中胚层的能力 在msd突变体中阻断。 在第2节中，msd的其他表征 表型将包括实验，以解决细胞周期的长度， 早期原肠胚形成，原位杂交研究，以确定是否早期 前后轴形成的标记物在msd突变体中表达 胚胎，在a中生产含有突变msd细胞的嵌合胚胎 野生型背景，并在msd中分离新的ENU诱导的突变 基因 在本研究的第三部分中， 描述了 它们包括跨越msd的重叠群的分离 区域和通过cDNA鉴定重叠群内的转录单位 选择. msd基因或基因将通过突变体的拯救来鉴定 胚胎干细胞在用单独的cDNA或 多个cDNA克隆。

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.