Mapping the functional major histocompatibility complex genes in zebrafish

描述(申请人提供)：斑马鱼(Danio Rerio)是一种强大的遗传模型，可以在体内研究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处于滞后状态。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，必须确定斑马鱼中具有功能的MHC基因。与小鼠和人类不同的是，第一类和第二类MHC基因都连接到从各自亲本那里以单倍型遗传的单个染色体位点，根据序列同源性，斑马鱼中似乎至少有三个染色体位点与推测的MHC基因有关。尽管对这些MHC基因进行了大量的绘制工作，但几乎没有数据来描述它们的功能。目前尚不清楚哪些基因实际上在细胞表面表达，发挥功能作用，呈递识别异物组织的多肽抗原。我们建议对斑马鱼的MHC功能基因进行定位，这些基因对于移植中的免疫匹配非常重要。这将在具体目标1中使用候选基因方法加以解决。可能的斑马鱼MHC基因将被单独克隆到一个超表达载体中。将MHC表达载体和小鼠c-Myc癌基因共注射到单细胞克隆性CG1 FISH中，将产生斑马鱼T细胞白血病。这些白血病将被移植到CG1受体鱼体内。如果假定的MHC基因具有功能，肿瘤将被排斥。如果MHC基因不起作用，白血病就会植入，导致接受者死亡。特殊目标2将使用一种无偏见的方法来绘制移植肿瘤排斥所需的基因图谱。在这种情况下，通过将小鼠c-Myc癌基因注射到克隆CG1 FISH和AB/CG1杂交FISH的单细胞后代中，将产生肿瘤。每一代都会有较少的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相合的肿瘤将被移植到CG1受体体内，而不相合的肿瘤被排斥。标准的位置克隆方法将被用来定位没有移植的肿瘤中的相关基因，而不是移植的近亲鱼类的肿瘤。这些目标将共同识别斑马鱼的功能性MHC基因，为许多以前不可能进行的移植实验打开大门。这样的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。由于一对交配的斑马鱼可以在几周内产生数千个兄弟姐妹后代，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，研究斑马鱼的抗原呈递、淋巴细胞和自然杀伤细胞的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在移植和脊椎动物生物学的许多方面打开大门。

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.