TISSUE SPECIFIC CONTROL OF CELL PROLIFERATION

描述(改编自调查员摘要)：一个基本组成部分 组织形态发生、功能和动态平衡的区别在于 控制不同细胞类型的细胞增殖，这些细胞类型包括 组织。拟议研究的长期目标是了解 这种细胞类型特异性调控的分子机制 扩散。作为鉴定基因的起点， 参与特定细胞类型的细胞增殖调控， Gossler选择专注于影响增殖的小鼠突变 特定细胞类型或组织的。在这项提议中，老鼠的突变 正在研究的是针尾(Pt)。小尾巴是从一只 用致癌物质甲基胆三烯治疗的小鼠。它是一个半显式 4号染色体上的突变，它特异性地影响细胞的增殖 脊索细胞呈剂量依赖关系。对铂的描述性研究， 近40年前发表的研究表明，有丝分裂细胞在 脊索缩小到正常值的50%和30%左右， 分别为杂合子和纯合子。因此，脊索是 明显变小或完全不在后部 胚胎；这反过来会产生骨骼异常。 拟议的实验分为四个具体目标。特定目标 1进一步探讨了铂的表型。首先，分析了BrdU 将整合到突变型和野生型胚胎的脊索细胞中 用于定义铂细胞周期的G1、S、G2和M参数 突变细胞。此外，还将对以下内容进行表情分析 细胞周期各阶段的标志物。第二，嵌合体将是 将PtES细胞注射到野生型囊胚中构建 评估铂突变的细胞自主性。具体目标2建议 至少使用以下方法建立针尾突变的精细遗传图谱 2000只回交动物由两个不同的亚种间产生 回传。利用遗传图谱和染色体行走技术，一种 将为PT关键序列构建物理地图和BAC重叠群 时间间隔。铂基因将在特定目标3中使用 候选基因方法、cDNA选择和/或a&lt；50kb的测序 临界区。铂基因的真实性将通过 表达分析、测序和适当的转基因或敲除 实验。在具体目标4中，将采用转基因方法来 功能分析针尾基因及其在细胞类型特异性中的作用 控制细胞增殖。

DESCRIPTION (Adapted from investigator's abstract): An essential component of tissue morphogenesis, function, and homeostasis is the differential control of cell proliferation in the different cell types that comprise a tissue. The long term goal of the proposed studies is to understand the molecular mechanisms underlying such cell type-specific controls of cell proliferation. As a starting point toward the identification of genes that participate in the cell type-specific regulation of cell proliferation, Gossler has chosen to focus on mouse mutations that affect the proliferation of particular cell types or tissues. In this proposal, the mouse mutation under study is Pintail (Pt). Pintail was isolated from the offspring of mice treated with the carcinogen methylcholantrene. It is a semidominant mutation on chromosome 4 and it specifically affects the proliferation of notochord cells in a dose dependent manner. A descriptive study of Pt, published nearly 40 years ago, showed that the frequency of mitotic cells in the notochord was reduced to approximately 50% and 30%, of normal values in, respectively, heterozygotes and homozygotes. As a result, the notochord is significantly smaller or completely absent in the posterior portion of the embryo; this in turns generates skeletal abnormalities. The proposed experiments are divided into four specific aims. Specific Aim 1 further explores the Pt phenotype. First, an analysis of BrdU incorporation into notochord cells of mutant and wild type embryos will be performed to define the G1, S, G2, and M parameters of the cell cycle in Pt mutant cells. In addition, an expression analysis will be conducted on markers for defined phases of the cell cycle. Second, chimeras will be constructed by the injection of Pt ES cells into wild type blastocysts to assess the cell autonomy of the Pt mutation. Specific Aim 2 proposes to establish a fine scale genetic map of the Pintail mutation using at least 2000 backcross animals generated from two different intersubspecific backcrosses. Using the genetic map and chromosomal walking techniques, a physical map and a BAC contig will be constructed for the Pt critical interval. The Pt gene will be identified in Specific Aim 3 using the candidate gene approach, cDNA selection, and/or sequencing of a <50 kb critical region. The authenticity of the Pt gene will be verified by expression analyses, sequencing, and appropriate transgenic or knockout experiments. In Specific Aim 4, transgenic approaches will be undertaken to functionally analyze the pintail gene and its role in cell type specific control of cell proliferation.