ALBINO-DELETION COMPLEX AND EARLY MOUSE DEVELOPMENT

白化病缺失复合体和早期小鼠发育

• 批准号: 2199186
• 负责人: TERRY MAGNUSON
• 金额: $ 19.94万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2199186
• 关键词: albinism; chromosome aberrations; chromosome complement; chromosome deletion; chromosome walking; complementary DNA; developmental genetics; early embryonic stage; endonuclease; gel electrophoresis; gene expression; gene mutation; genetic mapping; genetic markers; genetic regulation; genetic transcription; genetically modified animals; genome; homeobox genes; in situ hybridization; laboratory mouse; mammalian embryology; molecular cloning; molecular genetics; northern blottings; nucleic acid sequence; polymerase chain reaction; radionuclides; restriction fragment length polymorphism

The long-term goal of this proposal is to understand the molecular basis for the genetic control of mammalian development. The albino-deletion complex represents one of few regions of the mouse genome where a set of specific developmental defects are associated with a series of overlapping chromosomal deletions. A total of 37 deletions exist, all of which remove the region of mouse chromosome 7 that surrounds and includes the albino coat color locus. The complementation patterns among these deletion chromosomes indicate hat at least 9 distinct genetic units exist in this region. Four of these units are needed for normal embryonic development during the preimplantation or early postimplantation stages. The work proposed here will concentrate on a molecular genetic analysis of two of these units, each of which is known to contain a gene(s) whose expression is required during the time that the basic body plan is being established in the early postimplantation mouse embryo. The homozygous (null) lethal phenotype associated with deletions that remove this areal has been determined and molecular markers for this region have been generated by chromosome microdissection and microcloning. Chromosome walking and pulsed-field gel electrophoresis will be used to isolate and physically map additional markers. Transcription units will be identified by screening cDNA libraries made from early postimplantation embryos and also by NOrthern blot analysis using RNA from embryo-derived stem cells. Once a transcription unit has been identified, its tissue- and stage-specific pattern of expression will be determined either by Northern blot analysis or by in situ hybridization to tissue sections. A molecular characterization of cDNAs and corresponding genomic sequences will be done with the purpose of identifying putative protein coding regions, direction of transcription, 5' and 3' ends, and intron/exon boundaries. Antibodies will be made to synthetic peptides with the purpose of locating the protein product in the embryo. Transgenic mice will be produced to determine if any of the identified transcription units can complement the homozygous defect. Analysis of the lethal phenotype associated with the e=homozygous state of the five deletions of interest will be extended by examining specific gene expression in mutant embryos. Homeobox gene expression in wild-type mouse embryos has been extensively studied, and it is known that some of these genes begin to be expressed at a time which is coincident with the appearance of abnormalities in the homozygous individuals. Therefore, by determining the pattern of homeobox gene expression in the mutant embryos, a more accurate assessment of their developmental potential can be made.

该提议的长期目标是了解分子基础 用于哺乳动物发育的遗传控制。 白化病 复合物代表了一组一组小鼠基因组的少数区域之一 特定的发育缺陷与一系列重叠有关 染色体缺失。 总共存在37个删除，所有删除 围绕并包括白化病的小鼠染色体区域 外套颜色基因座。 这些删除之间的互补模式 染色体表明帽子至少有9个不同的遗传单位 地区。 正常胚胎开发需要这些单元中的四个 在植入前或植入后阶段。 工作 此处提出的将集中于两个分子遗传分析 这些单元，每个单元都包含一个表达的基因 在建立基本身体计划的过程中需要 在早期植入后小鼠胚胎中。 纯合（无效）致命 与删除该区域的缺失相关的表型已经 该区域的确定和分子标记已经由 染色体显微解剖和微量成分。 染色体步行和 脉冲场凝胶电泳将用于隔离和物理映射 附加标记。 转录单元将通过筛选确定 由早期植入后胚胎制成的cDNA库，也由 使用来自胚胎衍生的干细胞的RNA进行北印迹分析。 一次 已确定了转录单元，其组织和阶段特异性 表达方式将通过北印迹分析确定 或通过原位杂交与组织切片。 分子 将完成CDNA和相应基因组序列的表征 为了识别推定的蛋白质编码区域，方向 转录，5'和3'末端以及内含子/外显子边界。 抗体 将制定为合成肽，目的是定位蛋白质 胚胎中的产品。 将产生转基因小鼠以确定是否 任何已确定的转录单元都可以补充纯合子 缺点。 与E =纯合子相关的致命表型的分析 五个感兴趣删除的状态将通过检查来扩展 突变胚胎中的特定基因表达。 同型基因表达 野生型小鼠胚胎已经进行了广泛的研究，众所周知 这些基因中的一些开始表达 纯合个体出现异常。 因此，通过确定同源基因表达的模式 突变胚胎，对其发育潜力的更准确评估 可以做。