GENE EXPRESSION DURING MAMMALIAM YOLK SAC HEMATOPOIESIS

哺乳动物卵黄囊造血过程中的基因表达

• 批准号: 2222263
• 负责人: James Palis
• 金额: $ 11.57万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-05 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222263
• 关键词: DNA binding protein; complementary DNA; developmental genetics; early embryonic stage; embryonic stem cell; gene expression; genetic library; genetic manipulation; hematopoiesis; histochemistry /cytochemistry; histogenesis; immunocytochemistry; in situ hybridization; laboratory mouse; mammalian embryology; mesoderm; messenger RNA; molecular cloning; neoplastic cell; northern blottings; nucleic acid sequence; polymerase chain reaction; southern blotting; tissue /cell culture

A major goal of developmental research has been the understanding of the mechanisms by which cells become committed to particular fates and the sequence of events through which they then differentiate into morphologically and functionally distinct cell types. In the mammalian embryo, the visceral yolk sac (VYS) is the first morphologic and functional development of two interrelated tissues arising from newly proliferating mesoderm: blood cells and endothelial cells. Blood cells are functionally evident in the mouse embryo by day 6-7, and morphologically evident by day 8. Yet, nothing is known about the signals which trigger these events. The overall aim of this study is to begin to elucidate the molecular events underlying the initiation of hematopoiesis in the mammalian embryo. We hypothesize that genes transiently expressed during early tissue development may be involved with the process of lineage specification, rather than being the products of the differentiated phenotype. We will, therefore, isolate genes expressed during the initial stages of embryonic hematopoiesis by subtractive screening of a PCR amplified cDNA library. The spatial and temporal pattern of expression of these genes will be investigated by in situ hybridization and Northern blots. To identify the function of selected genes, full length transcripts will be sequenced and analyzed for functional motifs. Their expression will be investigated in both murine erythroleukemia cells (MELC) and the murine genetic mutant W, with altered hematopoiesis. Antisense oligomers of selected genes will be added to MELC and to embryonic stem cell cultures. If induced blood cell formation is blocked or altered, then a direct role for these genes in hematopoietic development can be postulated. The size of the protein products of these genes as well as their temporal and spatial patterns of expression will be determined by Western blots and immunostaining using antibodies raised to fusion proteins. This information will provide clues to their regulation as well as their function, eg. nuclear vs secreted proteins. We expect that these clones will be the earliest lineage-specific hematopoietic genes available. Even if they are not directly involved with lineage specification, they will be invaluable markers for the study of lineage development in the mouse, since no early markers for mesoderm or hematopoietic tissue as yet exist. The study of these genes may also provide a better understanding of clonal disorders such as CML and erythroleukemia.

发展研究的主要目标是理解 细胞致力于特定命运的机制和 事件序列，然后它们分化为 形态学和功能上不同的细胞类型。 在哺乳动物中 胚胎，内脏蛋黄囊（VYS）是第一个形态学和功能 由新增殖引起的两个相互关联的组织的发展 中胚层：血细胞和内皮细胞。 血细胞在功能上是 在第6-7天，在小鼠胚胎中明显，并且在形态上明显明显 8。然而，触发这些事件的信号一无所知。 这项研究的总体目的是开始阐明分子事件 在哺乳动物胚胎中造血的启动。 我们 假设在早期组织中瞬时表达的基因 发展可能与谱系规范过程有关， 而不是作为分化表型的产物。 因此，我们将在初始阶段表达的分离基因 通过减去PCR扩增cDNA的胚胎造血症 图书馆。 这些基因表达的空间和时间模式 将通过原位杂交和北印迹进行调查。 为了识别所选基因的功能，全长成绩单将是 测序并分析了功能基序。 他们的表达将是 在鼠红血球细胞（MELC）和鼠中都进行了研究 遗传突变体W，造血改变。 选定基因的反义寡聚物将添加到MELC，并将其添加到 胚胎干细胞培养物。 如果诱导的血细胞形成被阻塞 或改变，然后这些基因在造血发育中的直接作用 可以假设。 这些基因及其时间的蛋白质产物的大小 和表达的空间模式将由蛋白质印迹和 使用融合蛋白提出的抗体进行免疫染色。 这 信息将为他们的法规及其法规提供线索 功能，例如。核与分泌的蛋白质。 我们希望这些克隆将是最早的谱系特定的 可提供造血基因。 即使他们不直接参与 血统规范，它们将是研究的宝贵标记 小鼠的谱系发展，因为没有中胚层的早期标记或 造血组织还存在。 这些基因的研究也可能 对CML等克隆疾病有更好的了解 红血球血症。