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）是一种强大的遗传模型，可用于研究体内脊椎动物生物学的许多特征。然而，由于缺乏对斑马鱼主要组织相容性复合体（MHC）基因的了解，斑马鱼的移植方法一直滞后。为了将斑马鱼模型的遗传和筛选优势应用于涉及移植生物学各个方面的问题，必须鉴定斑马鱼中的功能性 MHC 基因。小鼠和人类的 I 类和 II 类 MHC 基因均与作为单倍型遗传自父母双方的单个染色体位点相连，而斑马鱼与此不同，斑马鱼中似乎至少有 3 个染色体位点具有序列同源性推定的 MHC 基因。尽管对这些 MHC 基因进行了大量工作，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，发挥呈递肽抗原以识别外来组织的功能作用。我们建议绘制斑马鱼功能性 MHC 基因图谱，这些基因对于移植中的免疫匹配很重要。这将在具体目标 1 中使用候选基因方法来解决。假定的斑马鱼 MHC 基因将被单独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 癌基因共同注射到单细胞克隆 CG1 鱼中而产生。这些白血病将被移植到CG1受体鱼中。如果推定的 MHC 基因具有功能，肿瘤就会被排斥。如果 MHC 基因不起作用，白血病就会植入，导致受体死亡。具体目标 2 将使用公正的方法来绘制排斥移植肿瘤所需的基因。在这种情况下，将小鼠 c-Myc 癌基因注射到克隆 CG1 鱼和 AB/CG1 杂交鱼连续杂交的单细胞后代中，从而产生肿瘤。随后的每一代都会有更少的 AB MHC 等位基因，最终一些后代将只携带 CG1 MHC 等位基因。这些 MHC 匹配的肿瘤将被移植到 CG1 受体中，而不匹配的肿瘤将被排斥。将使用标准的定位克隆方法来定位未移植的肿瘤与移植的同胞鱼的肿瘤中的相关基因。这些目标共同将鉴定出功能性斑马鱼 MHC 基因，为许多以前不可能的移植实验打开大门。此类实验将利用斑马鱼模型的优势，从而解决在哺乳动物中很难或不可能提出的问题。由于一对斑马鱼可以在几周内产生数千个兄弟后代，因此可以进行与造血和肿瘤移植相关的大规模化学和遗传筛选。此外，研究斑马鱼抗原呈递、淋巴细胞和自然杀伤细胞激活以及传染病模型的免疫学实验也将成为可能。功能性 MHC 基因的发现将为斑马鱼研究人员研究移植和脊椎动物生物学的许多方面打开大门。 公共卫生相关性：与哺乳动物相比，斑马鱼的信号转导途径、肿瘤生物学、造血和许多器官系统高度保守。了解对免疫匹配很重要的斑马鱼 MHC 基因将使实验成为可能，从而模拟许多人类疾病，包括造血系统疾病和癌症。