本文關(guān)鍵詞： TALEN CRISPR-Cas9 斑馬魚 tet 去甲基化　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：在脊椎動(dòng)物中,造血系統(tǒng)的發(fā)育是一個(gè)有序且復(fù)雜的動(dòng)態(tài)過程,其中包括各種血液細(xì)胞的發(fā)育,成熟等方面,并且同時(shí)受許多轉(zhuǎn)錄因子的調(diào)控。在血液里面,造血干細(xì)胞(hematopoietic stem cells,HSCs)是各譜系血細(xì)胞的共同祖先。在造血發(fā)育過程中,造血干細(xì)胞的早期發(fā)育過程是最為關(guān)鍵。造血干細(xì)胞(HSC)的正常產(chǎn)生、維持、增殖以及順利遷移、定位,對(duì)機(jī)體胚胎期的器官發(fā)生以及成體中組織穩(wěn)態(tài)的維持以及損傷修復(fù)不可或缺。在造血過程中,造血干細(xì)胞首先分化成各系血祖細(xì)胞和造血前體細(xì)胞并最終分化為各譜系的成熟細(xì)胞,這一過程受到多種機(jī)制的精密調(diào)控。當(dāng)這些造血細(xì)胞發(fā)育出現(xiàn)缺陷時(shí),就可能誘發(fā)各種細(xì)胞類型的白血病,如髓系白血病和淋系白血病。在人類腫瘤中最常見的變化之一就是DNA的異常甲基化,它在白血病發(fā)生和發(fā)展中對(duì)基因的表達(dá)、調(diào)控、基因的結(jié)構(gòu)穩(wěn)定等方面發(fā)揮著關(guān)鍵作用。DNA的甲基化指的是在CpG二核苷酸中胞嘧啶的5號(hào)位碳原子上加上一個(gè)甲基,生成5-甲基胞嘧啶(5mc)。DNA甲基化影響基因組的穩(wěn)定性與基因表達(dá)調(diào)控,跟腫瘤發(fā)生密切相關(guān)。近年來的研究表明,DNA異常甲基化在人類白血病中普遍存在[3]。TET(ten-eleven translocation)蛋白是生物體內(nèi)普遍存在的一種α-酮戊二酸(α-KG)和Fe2+依賴的雙加氧酶。TET蛋白可以催化5-甲基胞嘧啶(5-mC)轉(zhuǎn)化為5-羥甲基胞嘧啶(5-hmC),是DNA在進(jìn)行去甲基化過程中不可或缺的一種酶。人類TET基因的突變可以引起多種腫瘤,特別是血液系統(tǒng)方面的腫瘤。人的TET蛋白家族有三個(gè)成員,分別為TET1、TET2和TET3,7ET1是在研究一例存在t(10;11)(q22;q23)異位的白血病患者時(shí)鑒定成功的,因此得名。TET1最早被鑒定有催化5-mC羥基化的酶活性,進(jìn)一步研究發(fā)現(xiàn),三種TET蛋白均具有將5-mC轉(zhuǎn)化為5-hmC的能力。深入研究TET家族作用的分子機(jī)制有助于進(jìn)一步了解和闡明白血病致病機(jī)理和發(fā)展過程。方法:利用TALEN和CRISPR-Cas9基因靶向敲除技術(shù)敲除野生型斑馬魚AB品系的tet家族中的tet1、tet2和tet3基因,篩選出突變體,進(jìn)行血液系統(tǒng)表型和功能研究,以探究tet家族基因造成的甲基化異常與造血發(fā)育之間的關(guān)系。結(jié)果:我們獲得了 TALEN和CRISPR-Cas9靶向敲除的tet2、tet3突變體,并在發(fā)育早期分析了造血相關(guān)的表型,與野生型斑馬魚相比無明顯差異。結(jié)論:我們成功構(gòu)建出tet2、tet3斑馬魚突變體,為進(jìn)一步研究tet基因家族對(duì)血液系統(tǒng)疾病發(fā)生發(fā)展的作用提供了良好的動(dòng)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 偽 -ketoglutaric acid (偽 -KGG) and Fe2 dependent dioxygenase. TET protein can be found in human leukaemia. [3] .TETten-eleven translocation protein is a kind of 偽 -ketoglutaric acid (偽 -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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;R733.7

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相關(guān)期刊論文 前2條

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本文編號(hào)：1498899