Mapping the functional major histocompatibility complex genes in zebrafish

绘制斑马鱼功能主要组织相容性复合体基因图谱

• 批准号: 8094702
• 负责人: Jill L de Jong
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-19 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8094702
• 关键词: Address; Alleles; Antigen Presentation; Antigens; Applied Genetics; Autologous; Biological Assay; Biology; Candidate Disease Gene; Cell Transplants; Cell surface; Cells; Cessation of life; Chimerism; Chromosome Mapping; Chromosomes; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 19; Chromosomes, Human, Pair 8; Data; Disease; Engraftment; Evaluation; Fishes; Generations; Genes; Genetic Models; Genetic Screening; Graft Rejection; Haplotypes; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Human; Hybrids; Immune; Immunologics; Immunology; Inherited; Injection of therapeutic agent; Knowledge; Link; Lymphocyte Activation; MYC gene; Major Histocompatibility Complex; Major Histocompatibility Complex Gene; Malignant Neoplasms; Mammals; Maps; Methods; Modeling; Mus; Natural Killer Cells; Neoplasm Transplantation; Parents; Partner in relationship; Peptides; Play; Research Personnel; Role; Screening procedure; Sequence Homology; Series; Siblings; Signal Transduction Pathway; T-Cell Leukemia; Tissue Donors; Tissues; Transplant Recipients; Transplantation; Tumor Biology; Vertebrate Biology; Work; Zebrafish; base; body system; c-myc Genes; chemical genetics; expression vector; human disease; in vivo; infectious disease model; leukemia; overexpression; positional cloning; research study; tumor; vector

DESCRIPTION (provided by applicant): The zebrafish (Danio rerio) is a powerful genetic model to study many features of vertebrate biology in vivo. However, transplantation methods have been lagging in the zebrafish due to lack of knowledge about the zebrafish Major Histocompatibility Complex (MHC) genes. In order to apply the genetic and screening advantages of the zebrafish model to questions involving all aspects of transplantation biology, the functional MHC genes in the zebrafish must be identified. Unlike mice and humans where both Class I and Class II MHC genes are linked to a single chromosomal locus inherited as a haplotype from each parent, there appear to be at least three chromosomal loci in the zebrafish with putative MHC genes by sequence homology. Despite substantial work mapping these MHC genes, there are almost no data characterizing their function. It is unclear which genes are actually expressed on the cell surface, playing a functional role presenting peptide antigens to recognize foreign tissues. We propose to map the functional MHC genes in the zebrafish that are important for immune matching in transplantation. This will be addressed in Specific Aim 1 using a candidate gene approach. Putative zebrafish MHC genes will be individually cloned into an overexpression vector. Zebrafish T cell leukemias will be generated by co-injecting the MHC expression vector and the murine c-Myc oncogene into single-cell clonal CG1 fish. These leukemias will be transplanted into CG1 recipient fish. If the putative MHC gene is functional, the tumor will be rejected. If the MHC gene is not functional, the leukemia will engraft, causing the recipient's death. Specific Aim 2 will use an unbiased approach to map the genes required for rejection of a transplanted tumor. In this case tumors will be generated by injection of the murine c-Myc oncogene into single-cell progeny from serial crosses of clonal CG1 fish and AB/CG1 hybrid fish. Each subsequent generation will have fewer AB MHC alleles, and eventually some progeny will carry only CG1 MHC alleles. These MHC-matched tumors will engraft into CG1 recipients while mismatched tumors are rejected. Standard positional cloning methods will be used to map the relevant gene in tumors that do not engraft compared with tumors from sibling fish that do engraft. Together these aims will identify the functional zebrafish MHC genes, opening the door to numerous transplantation experiments that have previously not been possible. Such experiments would harness the advantages of the zebrafish model, and hence address questions that are difficult or impossible to ask in mammals. Because thousands of sibling progeny can be generated in a few weeks from a single mating pair of zebrafish, large scale chemical and genetic screens related to hematopoietic and tumor transplantation could be performed. In addition, immunology experiments studying antigen presentation, activation of lymphocytes and natural killer cells, and infectious disease models in the zebrafish would become possible. Discovery of the functional MHC genes would open the door for zebrafish researchers studying many aspects of transplantation and vertebrate biology. PUBLIC HEALTH RELEVANCE: Signal transduction pathways, tumor biology, hematopoiesis and many organ systems are highly conserved in the zebrafish in comparison to mammals. Understanding the zebrafish MHC genes important for immune matching will enable experiments which would otherwise be possible to model many human diseases, including hematopoietic diseases and cancer.

描述（由申请人提供）：斑马鱼（Danio rerio）是一种研究体内脊椎动物生物学的许多特征的有力遗传模型。然而，由于缺乏有关斑马鱼主要组织相容性复合物（MHC）基因的知识，斑马鱼的移植方法一直在斑马鱼中滞后。为了将斑马鱼模型的遗传和筛查优势应用于涉及移植生物学各个方面的问题，必须确定斑马鱼中的功能性MHC基因。与小鼠和人类不同的是，I类和II类MHC基因都与每个父母的单倍型相关的单个染色体基因座，似乎在斑马鱼中至少有三个染色体基因座，并由序列MHC基因通过序列同源性。尽管绘制了这些MHC基因的大量工作，但几乎没有表征其功能的数据。目前尚不清楚哪些基因实际上在细胞表面表达，发挥了功能作用，表现出肽抗原以识别异物组织。我们建议绘制斑马鱼中的功能性MHC基因，对于移植中的免疫匹配很重要。这将在特定的目标1中使用候选基因方法解决。假定的斑马鱼MHC基因将分别克隆到过表达载体中。斑马鱼T细胞白血病将通过将MHC表达载体和鼠C-Myc癌基因共同注入单细胞克隆CG1鱼而产生。这些白血病将被移植到CG1接受者鱼中。如果推定的MHC基因功能性，则将拒绝肿瘤。如果MHC基因不起作用，则白血病会植入，从而导致接受者的死亡。特定的目标2将使用公正的方法来绘制拒绝移植肿瘤所需的基因。在这种情况下，将通过将鼠C-myc癌基因从克隆CG1鱼和AB/CG1杂交鱼的连续十字中注入鼠C-myc癌基因产生。随后的每一代将具有较少的AB MHC等位基因，最终某个后代只会携带CG1 MHC等位基因。这些MHC匹配的肿瘤会植入CG1受体中，而错配肿瘤被拒绝。标准位置克隆方法将用于绘制与植入的同胞鱼的肿瘤相比，肿瘤中相关基因的绘制。这些目标共同确定斑马鱼MHC基因，为许多以前无法实现的移植实验打开了大门。这样的实验将利用斑马鱼模型的优势，因此解决了在哺乳动物中难以或不可能提出的问题。由于可以在几周内通过一对斑马鱼，大规模的化学和遗传筛选与造血和肿瘤移植有关的一对兄弟后代。此外，研究抗原表现，淋巴细胞激活和天然杀伤细胞的免疫学实验以及斑马鱼中的传染病模型将成为可能。功能性MHC基因的发现将为研究移植和脊椎动物生物学的许多方面的斑马鱼研究人员打开大门。 公共卫生相关性：与哺乳动物相比，在斑马鱼中，信号转导途径，肿瘤生物学，造血系统和许多器官系统在斑马鱼中是高度保守的。了解斑马鱼MHC基因对免疫匹配很重要的基因将实现实验，否则可以对许多人类疾病进行建模，包括造血疾病和癌症。