利用基因编辑技术构建tet基因家族斑马鱼突变体

发布时间：2018-02-09 21:28

本文关键词： TALEN CRISPR-Cas9 斑马鱼 tet 去甲基化　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：在脊椎动物中,造血系统的发育是一个有序且复杂的动态过程,其中包括各种血液细胞的发育,成熟等方面,并且同时受许多转录因子的调控。在血液里面,造血干细胞(hematopoietic stem cells,HSCs)是各谱系血细胞的共同祖先。在造血发育过程中,造血干细胞的早期发育过程是最为关键。造血干细胞(HSC)的正常产生、维持、增殖以及顺利迁移、定位,对机体胚胎期的器官发生以及成体中组织稳态的维持以及损伤修复不可或缺。在造血过程中,造血干细胞首先分化成各系血祖细胞和造血前体细胞并最终分化为各谱系的成熟细胞,这一过程受到多种机制的精密调控。当这些造血细胞发育出现缺陷时,就可能诱发各种细胞类型的白血病,如髓系白血病和淋系白血病。在人类肿瘤中最常见的变化之一就是DNA的异常甲基化,它在白血病发生和发展中对基因的表达、调控、基因的结构稳定等方面发挥着关键作用。DNA的甲基化指的是在CpG二核苷酸中胞嘧啶的5号位碳原子上加上一个甲基,生成5-甲基胞嘧啶(5mc)。DNA甲基化影响基因组的稳定性与基因表达调控,跟肿瘤发生密切相关。近年来的研究表明,DNA异常甲基化在人类白血病中普遍存在[3]。TET(ten-eleven translocation)蛋白是生物体内普遍存在的一种α-酮戊二酸(α-KG)和Fe2+依赖的双加氧酶。TET蛋白可以催化5-甲基胞嘧啶(5-mC)转化为5-羟甲基胞嘧啶(5-hmC),是DNA在进行去甲基化过程中不可或缺的一种酶。人类TET基因的突变可以引起多种肿瘤,特别是血液系统方面的肿瘤。人的TET蛋白家族有三个成员,分别为TET1、TET2和TET3,7ET1是在研究一例存在t(10;11)(q22;q23)异位的白血病患者时鉴定成功的,因此得名。TET1最早被鉴定有催化5-mC羟基化的酶活性,进一步研究发现,三种TET蛋白均具有将5-mC转化为5-hmC的能力。深入研究TET家族作用的分子机制有助于进一步了解和阐明白血病致病机理和发展过程。方法:利用TALEN和CRISPR-Cas9基因靶向敲除技术敲除野生型斑马鱼AB品系的tet家族中的tet1、tet2和tet3基因,筛选出突变体,进行血液系统表型和功能研究,以探究tet家族基因造成的甲基化异常与造血发育之间的关系。结果:我们获得了 TALEN和CRISPR-Cas9靶向敲除的tet2、tet3突变体,并在发育早期分析了造血相关的表型,与野生型斑马鱼相比无明显差异。结论:我们成功构建出tet2、tet3斑马鱼突变体,为进一步研究tet基因家族对血液系统疾病发生发展的作用提供了良好的动物模型。
[Abstract]:In vertebrates, the development of the hematopoietic system is an orderly and complex dynamic process, including the development and maturation of various blood cells, and is also regulated by many transcription factors. Hematopoietic stem cells are the common ancestor of hematopoietic stem cells. The early development process of hematopoietic stem cells is the most important in hematopoietic development. The normal production, maintenance, proliferation, smooth migration and localization of hematopoietic stem cells. Essential for organogenesis of the body at the embryonic stage, as well as the maintenance of the steady-state tissue in the adult body, as well as the repair of damage. In the process of hematopoiesis, Hematopoietic stem cells first differentiate into progenitor cells and hematopoietic progenitor cells, and eventually differentiate into mature cells of various lineages, a process that is carefully regulated by multiple mechanisms. When these hematopoietic cells develop defective, One of the most common changes in human tumors is abnormal methylation of DNA, which regulates gene expression in the genesis and progression of leukemia. DNA methylation refers to the addition of a methyl group to the 5 carbon atom of cytosine in CpG dinucleotides. The formation of 5-methylcytosine 5mc.DNA methylation affects the stability of the genome and the regulation of gene expression. Recent studies have shown that aberrant methylation of Fe2 is a common protein in human leukemia, which is a kind of 伪 -ketoglutaric acid (伪 -KGG) and Fe2 dependent dioxygenase. TET protein can be found in human leukaemia. [3] .TETten-eleven translocation protein is a kind of 伪 -ketoglutaric acid (伪 -KG) and Fe2 dependent dioxygenase. The conversion of 5-methylcytosine 5-mCin to 5-hydroxymethylcytosine 5-hmChe is an indispensable enzyme of DNA in the process of demethylation. The mutation of human TET gene can cause many kinds of tumors. In particular, tumors in the blood system. The human TET protein family has three members, TET1, TET2 and TET3O7ET1, which were successfully identified in a study of an ectopic leukemic patient with tnlO10, 11, Q22, Q23). Therefore, the title. TET1 was first identified as an enzyme that catalyzes the hydroxylation of 5-mC, and further studies show that, All three TET proteins have the ability to convert 5-mC to 5-HMC. Further study on the molecular mechanism of TET family is helpful for further understanding and elucidating the pathogenesis and development of leukemia. Methods: targeting knockout of TALEN and CRISPR-Cas9 genes is used to further understand and elucidate the pathogenesis of leukemia. The tet1 tet2 and tet3 genes in the tet family of wild type zebrafish AB strain were knocked out. The mutant was screened for phenotypic and functional studies of the blood system to explore the relationship between methylation abnormalities caused by tet family genes and hematopoietic development. Results: we obtained tet2tet3 mutants targeted for TALEN and CRISPR-Cas9 knockout. The phenotypes associated with hematopoiesis were analyzed at the early stage of development and there was no significant difference compared with wild type zebrafish. Conclusion: the mutant tet2tet3 was successfully constructed. It provides a good animal model for the further study of the role of tet gene family in the development of hematological diseases.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;R733.7

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