CLONING PALLID A MUTANT AFFECTING INNER EAR AND BLEEDING

克隆 PALLID A 突变体影响内耳和出血

• 批准号: 3330185
• 负责人: ROSEMARY W ELLIOTT
• 金额: $ 16.11万
• 依托单位: ROSWELL PARK CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3330185
• 关键词: artificial chromosomes; ataxia; blood coagulation; cell line; chromosome walking; computer assisted sequence analysis; disease /disorder model; embryo /fetus tissue /cell culture; fungal genetics; gene complementation; gene mutation; genetic library; genetic markers; hemostasis; laboratory mouse; linkage mapping; melanocyte; melanosomes; molecular cloning; mutant; otocyst /otolith; pigmentation disorders; platelet disorder; platelets; polymerase chain reaction; pulsed field gel electrophoresis; recombinant DNA; restriction mapping; southern blotting; transfection

The goal of this project is to clone a gene that complements the mouse pallid mutant. This mutant is one of a group of eleven genetically distinct mutants affecting coat color and bleeding time. These effects are mediated through alterations in melanosomes and platelet dense granules, both of which are present, but defective in the mutant mice. Some of the mutants, including pallid, also have some effects on the function of a third organelle, the lysosome. Pallid is one of three mutants that affect the inner ear, and the mutants display ataxia because the otoliths, required for balance, are missing. Cloning the normal gene will allow analysis of the normal function of the gene in all parts of its complex phenotype, both at the molecular and cellular level, and analysis of the mutant phenotype. The proposed approach to cloning this gene involves identifying the molecular marker closest to the pallid mutation on mouse chromosome 2. This will be done using an interspecific backcross (C57BL/6J-pa a1/pa a1 X M. spretus-+ A/+ A)F1 X C57BL/6J-pa a1/pa a1, that has already produced 123 progeny. Preliminary data suggest that beta2-microglobulin is a very close marker. A YAC clone containing the selected marker will be obtained and used to start the walk to pallid. The ends of the YAC will be cloned and sequenced to identify overlapping YACs. Backcross animals containing recombination breakpoints will be identified and used first to determine the direction of the walk and later to monitor progress between the starting point and the target. YAC clones overlapping the interval between the recombination breakpoints flanking the pallid mutation will be candidates for the desired clones. To determine whether any of the genes on the YAC are expressed in megakaryocytes (platelet precursors) or in skin, cDNA libraries from skin and bone marrow cells will be made in a 'fragmenting vector' which contains a selectable yeast marker not present in the YAC and a yeast telomere. The libraries will be transformed into yeast cells containing the candidate YAC and recombinants between individual cDNAs and genes on the YAC will be selected. This will allow identification of one or more candidate genes, as regions between the candidate gene and the telomere will be deleted in the recombinant YACs, being replaced by vector sequences. Experiments to detect the function of the locus involve transformation of DNA from the candidate YAC or its cosmid subclones into bone marrow cells and reintroduction of these cells into mice to determine whether the fraction of platelets with filled dense granules is increased. To determine whether the pigment dilution can be corrected, transgenic mice will be made by injecting the same constructs into fertilized eggs homozygous for the mutant.

这个项目的目标是克隆一种与小鼠互补的基因 苍白的变种人。这个突变体是由11个基因组成的群体中的一个 影响毛色和出血时间的不同突变体。这些影响是 通过黑素小体和血小板致密颗粒的改变， 这两种基因都存在，但在突变小鼠中是有缺陷的。其中一些 突变体，包括苍白的，也有一些影响的功能 第三细胞器，溶酶体。苍白是影响人类健康的三个突变体之一 内耳和突变体表现出共济失调，因为耳石， 平衡所必需的，都缺失了。克隆正常基因将使 基因在其复合体各部分的正常功能分析 在分子和细胞水平上的表型，以及对 突变表型。 建议的克隆该基因的方法包括识别 与小鼠2号染色体上苍白突变最接近的分子标记。 这将使用种间回交(C57BL/6J-pa A1/pa A1 X)来完成 Spretus-+A/+A)F1×C57BL/6J-pa A1/pa A1，已产生123个 后代。初步数据表明，β2-微球蛋白是一种非常接近 记号笔。将获得包含所选标记的YAC克隆 用来开始走向苍白。YAC的末端将被克隆并 测序以识别重叠的YAC。回交动物包含 重组断点将首先被识别和使用，以确定 行走的方向，并在稍后监视 起点和目标。YAC克隆重叠的间隔 苍白突变两侧的重组断裂点将是 所需克隆的候选者。 为了确定YAC上的任何基因是否在 巨核细胞(血小板前体)或皮肤中，来自皮肤的cDNA文库 骨髓细胞将在一种含有以下物质的“碎片化载体”中制造 YAC中不存在的可选择酵母标记和酵母端粒。这个 文库将转化为含有候选YAC的酵母细胞 而YAC上单个cDNA和基因之间的重组体将是 被选中了。这将允许识别一个或多个候选基因， 因为候选基因和端粒之间的区域将在 重组的YAC被载体序列取代。实验以实现 检测与DNA转化有关的基因座的功能 候选YAC或其粘粒亚克隆进入骨髓细胞并 将这些细胞重新引入小鼠体内，以确定该组分是否 填充致密颗粒的血小板数量增加。以确定是否 色素稀释可以纠正，转基因小鼠将通过 将相同的结构注射到纯合子受精卵中 变种人。