Developing a competitive hematopoietic repopulating assay in zebrafish

开发斑马鱼竞争性造血再生试验

• 批准号: 8772416
• 负责人: Jill L de Jong
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8772416
• 关键词: Address; Adult; Animal Model; Biological Assay; Biology; Blood Cells; Bone Marrow; Bone Marrow Transplantation; Cell Transplants; Cell physiology; Cells; Characteristics; Chimerism; Complex; Complex Mixtures; Confounding Factors (Epidemiology); Data; Decision Making; Defect; Embryo; Engraftment; Equilibrium; Fishes; Flow Cytometry; Fluorochrome; Funding; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Programming; Genome; Gold; HDAC1 gene; Hematological Disease; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Homeostasis; Housing; Human; Immune; Individual; Injury; Insertional Mutagenesis; Laboratories; MYB gene; Malignant Neoplasms; Marrow; Methods; Modeling; Monitor; Mus; Mutagenesis; Organism; PLC gamma1; Pancytopenia; Partner in relationship; Phenocopy; Play; Process; Proto-Oncogene Proteins c-myb; Radiation Injuries; Recovery; Regulator Genes; Relative (related person); Role; Siblings; Site; Speed; Stem Cell Development; Testing; Time; Transgenic Organisms; Transplantation; Ubiquitin; Work; Zebrafish; abstracting; base; cell type; cost effective; human HDAC1 protein; human disease; in vivo; in vivo Model; insight; leukemia; leukemogenesis; mutant; novel; precursor cell; programs; public health relevance; research study; screening; self-renewal; stem cell differentiation; tool; vector

DESCRIPTION (provided by applicant): Developing a competitive hematopoietic repopulating assay in zebrafish Project Summary/Abstract Hematopoietic stem cells (HSCs) precisely regulate the balance between self-renewal and differentiation to generate appropriate numbers of mature blood cells required by an organism. When genes that direct these decisions are dysregulated, unchecked self-renewal can result in hematopoietic malignancy. Characterizing the decision-making processes regulating HSC function is critically important to understand leukemogenesis and also to enhance HSC transplantation strategies used to treat many human cancers and blood diseases. My laboratory is capitalizing on the zebrafish animal model to uncover new genetic regulators of HSCs. I have developed an immune- matched transgenic zebrafish model to phenocopy murine models of competitive repopulation using clonal CG2 zebrafish. To compare the relative engraftment potential of mutant zebrafish HSCs with wild type HSCs, transgenic CG2:ubiquitin-GFP and CG2:ubiquitin-DsRedE2 donor fish with fluorochrome positive marrow cells are transplanted into fluorochrome negative clonal recipients, resulting in long term, multi-lineage engraftment. A preliminary pilot screen of zebrafish mutants with abnormal embryonic HSC development identified two haploinsufficient mutants with impaired recovery of hematopoietic precursor cells in the marrow after sublethal radiation injury. Both phospholipase C gamma1 (plc?1) and histone deacetylase 1 (hdac1) have previously been identified as playing a role in self-renewal, and our data suggest they may be important for regulating HSC homeostasis. I will characterize and refine our competitive hematopoietic repopulating assay by testing the hypothesis that PLC?1 and HDAC1 haploinsufficiency will result in impaired hematopoietic engraftment. In addition, using random insertional mutagenesis of clonal zebrafish donors, I will perform an unbiased forward mutagenesis screen to identify genes that alter HSC engraftment. Marrow from heterozygous and/or homozygous mutant adults will be used in my competitive hematopoietic repopulating assay to identify those mutants with skewed marrow repopulation favoring one of the competitive donors. I am confident this assay will identify known and unknown genes important for multiple aspects of hematopoietic engraftment. This work will generate mutant zebrafish to use as tools to dissect the complex regulatory network that determines HSC function. Characterizing these genes will accelerate our understanding of normal HSC function in vivo and after transplantation, as well as providing new insights into the mechanisms that cause leukemia.

描述（由申请人提供）：在斑马鱼中开发竞争性造血再生试验项目摘要/摘要造血干细胞（HSC）精确调节自我更新和分化之间的平衡，以产生生物体所需的适当数量的成熟血细胞。当指导这些决定的基因失调时，不受控制的自我更新可能导致造血系统恶性肿瘤。表征调节 HSC 功能的决策过程对于了解白血病发生以及增强用于治疗许多人类癌症和血液疾病的 HSC 移植策略至关重要。我的实验室正在利用斑马鱼动物模型来发现新的造血干细胞遗传调节因子。我开发了一种免疫匹配的转基因斑马鱼模型，使用克隆 CG2 斑马鱼对竞争性繁殖的小鼠模型进行表型复制。为了比较突变型斑马鱼 HSC 与野生型 HSC 的相对植入潜力，将具有荧光染料阳性骨髓细胞的转基因 CG2：泛素-GFP 和 CG2：泛素-DsRedE2 供体鱼移植到荧光染料阴性克隆受体中，从而产生长期的多谱系植入。对胚胎 HSC 发育异常的斑马鱼突变体进行初步筛选，发现两种单倍体不足的突变体在亚致死辐射损伤后骨髓中造血前体细胞的恢复受损。磷脂酶 C gamma1 (plc?1) 和组蛋白脱乙酰酶 1 (hdac1) 先前已被确定在自我更新中发挥作用，我们的数据表明它们可能对调节 HSC 稳态很重要。我将通过测试 PLC?1 和 HDAC1 单倍体不足将导致造血移植受损的假设来表征和完善我们的竞争性造血再生测定。此外，利用克隆斑马鱼供体的随机插入诱变，我将进行无偏正向诱变筛选，以识别改变 HSC 植入的基因。来自杂合和/或纯合突变体成人的骨髓将用于我的竞争性造血再增殖测定，以鉴定那些骨髓再增殖偏向竞争性供体之一的突变体。我相信该测定将识别对造血移植多个方面重要的已知和未知基因。这项工作将产生突变斑马鱼作为工具来剖析决定 HSC 功能的复杂调控网络。表征这些基因将加速我们对体内和移植后正常 HSC 功能的理解，并为导致白血病的机制提供新的见解。