發(fā)布時(shí)間：2018-05-15 06:03

  本文選題：CRISPR-Cas9 + TALEN　； 參考：《南方醫(yī)科大學(xué)》2016年碩士論文

【摘要】：研究背景血液系統(tǒng)疾病(如地中海貧血、免疫缺陷和白血病等)一直是影響我國人民健康的嚴(yán)重疾病,其發(fā)病率居高不下給整個(gè)社會(huì)的醫(yī)療保障體系帶來沉重負(fù)擔(dān)。究其深層次的原因,血液疾病發(fā)病率居高不下反映了人類對于造血調(diào)控和相關(guān)疾病發(fā)生的分子機(jī)制和病理機(jī)制認(rèn)識(shí)不足,因?yàn)檠合到y(tǒng)正確作用與維持穩(wěn)態(tài)依賴于各種血細(xì)胞的正常發(fā)育,而造血過程缺陷會(huì)導(dǎo)致多種血細(xì)胞或血管發(fā)育障礙。因此,在分子和細(xì)胞水平更好地了解造血細(xì)胞的發(fā)育與造血過程,將有助于逐步明晰血液系統(tǒng)疾病發(fā)病機(jī)理,從而拓展臨床治療血液系統(tǒng)疾病的應(yīng)用潛能。在脊椎動(dòng)物中,造血系統(tǒng)發(fā)育過程是各種血細(xì)胞發(fā)育、成熟,同時(shí)受到多個(gè)因子調(diào)控的復(fù)雜而又有序的動(dòng)態(tài)過程。盡管各種血細(xì)胞組分的生理功能不同,但是它們有著同一祖先——造血干細(xì)胞(hematopoietic stem cells,HSCs)。造血系統(tǒng)的發(fā)育中,造血干細(xì)胞早期發(fā)育是最核心的過程。HSC的正確產(chǎn)生、維持、增殖以及順利遷移及定位,對于動(dòng)物體的胚胎期的器官發(fā)生以及成體中組織穩(wěn)態(tài)的維持以及損傷修復(fù)均必不可缺[1,2]。從HSC向成熟血細(xì)胞分化的過程中,經(jīng)歷了造血祖細(xì)胞階段和造血前體細(xì)胞階段,最終分化生成所有譜系成熟血細(xì)胞。造血干細(xì)胞向各譜系發(fā)育分化的過程中需要多種調(diào)節(jié)分子的參與,轉(zhuǎn)錄因子在此過程中起了重要的調(diào)控作用,并且轉(zhuǎn)錄因子之間發(fā)生相互調(diào)控,從而形成復(fù)雜的調(diào)控網(wǎng)絡(luò)。轉(zhuǎn)錄因子在造血細(xì)胞發(fā)育分化過程中起著重要的作用,各轉(zhuǎn)錄因子在細(xì)胞發(fā)育的不同時(shí)間及空間表達(dá)增強(qiáng)或減弱,使得造血干細(xì)胞或祖細(xì)胞向著某一譜系分化。Gfi1作為一種參與造血調(diào)控的轉(zhuǎn)錄抑制因子,該基因缺失或突變的病人表現(xiàn)為粒細(xì)胞成熟缺陷,阻滯于早幼粒階段。雖然在Gfi1基因敲除小鼠實(shí)驗(yàn)中未發(fā)生白血病,但是由于GFI1功能的缺失,有助于白血病的發(fā)生,特別是髓系細(xì)胞白血病的發(fā)生,如AML。經(jīng)典的模式生物如線蟲、果蠅等與人類同源性相差甚遠(yuǎn),而傳統(tǒng)模式動(dòng)物小鼠等哺乳動(dòng)物繁殖速度較慢,且體積相對較大,難以實(shí)施高通量造血缺陷突變體的篩選。而且哺乳動(dòng)物為子宮內(nèi)發(fā)育,不利于觀察胚胎期造血的表型。斑馬魚作為脊椎動(dòng)物,在器官發(fā)育、造血調(diào)控、疾病發(fā)生等方面與人類高度相似。特別地,斑馬魚的血液和心血管系統(tǒng)早期發(fā)育與人類極為相似,不論是血液成分的組成,還是發(fā)育過程都非常相似,而且該系統(tǒng)的發(fā)育缺陷突變體仍然可以存活數(shù)天,為人們研究胚胎早期造血調(diào)控提供了極為有利的條件[3]。本論文前一部分分別應(yīng)用TALEN靶向敲除技術(shù)、CRISPR-Cas9敲除技術(shù),成功對斑馬魚AB品系gfi1aa基因、gfi1b基因進(jìn)行敲除,獲得多種不同基因型的gfi1aako突變體、gfi1bko突變體。并詳細(xì)研究突變體在造血過程(原始造血和永久造血)中各個(gè)譜系標(biāo)記物的時(shí)空表達(dá)情況以及各個(gè)譜系細(xì)胞的發(fā)育情況,旨在探討gfi1aa基因以及gfi1b基因調(diào)控紅系造血發(fā)育的機(jī)理。論文后一部分主要探索a-catenin、β-catenin對胚胎期造血干細(xì)胞遷移的作用,以期進(jìn)一步明確造血干細(xì)胞遷移過程的相關(guān)細(xì)胞和分子調(diào)控機(jī)制。論文內(nèi)容共分為兩個(gè)部分:第一部分參加全國"斑馬魚1號染色體全基因敲除"項(xiàng)目及gfi1aa調(diào)控斑馬魚造血發(fā)育的機(jī)理研究;第二部分a-catenin、β-catenin對胚胎期造血干細(xì)胞遷移的作用研究。第一部分參加全國"斑馬魚1號染色體全基因敲除"項(xiàng)目及gfi1基因調(diào)控斑馬魚造血發(fā)育的機(jī)制研究1.目的本部分首先介紹CRISPR-Cas9及TALEN基因編輯技術(shù),然后以斑馬魚為模式生物,利用這兩種技術(shù)完成了以下兩項(xiàng)工作:1)利用CRISPR-Cas9技術(shù),參與全國"斑馬魚1號染色體全基因敲除"項(xiàng)目,對1號染色體上的ZKO編號為863～873的11個(gè)基因(vps37c、tmem132a、dnaja1、smu1b、rps6、btr01、c1h20orf27、muc13a、si:ch211-239f4.6、mucms1、si:ch211-239f4.4)進(jìn)行基因敲除,篩選穩(wěn)定遺傳的F2代突變體。2)通過TALEN靶向基因敲除技術(shù),利用斑馬魚模式生物對其造血系統(tǒng)發(fā)育基因gfi1aa進(jìn)行敲除,獲得造血發(fā)育缺陷的gfi1aa突變體。同時(shí)利用CRISPR-Cas9敲除技術(shù),對gfi1家族另一個(gè)gfi1b基因進(jìn)行敲除并獲得gfi1bko突變體。最后,利用這兩個(gè)突變體進(jìn)行表型與功能研究,探索gfi1基因調(diào)控造血發(fā)育的細(xì)胞以及分子機(jī)理。2.方法1)主要采用CRISPR-Cas9基因組編輯系統(tǒng),利用gRNA的靶向識(shí)別及Cas9的核酸切割作用。通過NCBI、Ensemb1和Smart網(wǎng)站查找相關(guān)基因的基本信息,對ZKO編號為863～873的11個(gè)基因進(jìn)行靶點(diǎn)設(shè)計(jì),合成相應(yīng)的引物。體外轉(zhuǎn)錄得到相應(yīng)的gRNA和Cas9 mRNA,混合共同顯微注射到one-cell的野生型AB斑馬魚胚胎中,得到F0代嵌合體的斑馬魚,將養(yǎng)大的FO(Founder)與野生型AB成魚外交得到F1 embryos,通過對F1代胚胎進(jìn)行PCR、酶切、測序的方法篩選出生殖細(xì)胞中帶有靶位點(diǎn)突變的F0成魚。將這樣的F0外交得到的F1養(yǎng)大,再通過剪尾逐條檢測F1的靶位點(diǎn)序列篩選可穩(wěn)定遺傳的斑馬魚個(gè)體。2)分別應(yīng)用TALEN、CRISPR-Cas9基因敲除技術(shù),對斑馬魚AB品系造血發(fā)育相關(guān)基因gfi1aa基因、gfi1b基因進(jìn)行敲除,以期獲得gfi1aako突變體、gfi1bko突變體。然后針對突變體在造血過程(原始造血和永久造血)中各個(gè)譜系標(biāo)記物的時(shí)空表達(dá)情況以及各個(gè)譜系細(xì)胞的發(fā)育情況,探討gfi1aa基因以及gfi1b基因調(diào)控造血發(fā)育的機(jī)理。3.結(jié)果1)利用Cas9基因敲除技術(shù),得到單種基因型突變的F1代斑馬魚,然后再與AB外交得到F2代,通過PCR、酶切、測序的方法得到F2代雜合突變體斑馬魚,并將部分上交到國家斑馬魚資源中心。然后對剩下的F2代突變體斑馬魚進(jìn)行intercross,通過原位雜交、蘇丹黑B染色、中性紅染色等方法進(jìn)行表型篩選。2)利用TALEN靶向基因敲除技術(shù)對斑馬魚gfi1aa進(jìn)行敲除,得到缺失23個(gè)堿基(5'-TCTTCTCAACAGTCCCCGCTGAG-3')的gfi1aako 突變體。表型分析結(jié)果顯示:在斑馬魚造血發(fā)育早期,gfi1aa影響紅細(xì)胞和中性粒細(xì)胞發(fā)育,而不影響巨噬細(xì)胞發(fā)育。而gfi1aa影響早期紅系發(fā)育的細(xì)胞學(xué)機(jī)理主要是通過抑制紅細(xì)胞的增殖,而并不影響紅細(xì)胞前體細(xì)胞和紅細(xì)胞的細(xì)胞形態(tài)。在50 hpf時(shí)期,gfi1aako純合突變體血紅蛋白表達(dá)量恢復(fù)到與野生型相當(dāng)?shù)乃�。gfi1b是gfi1aa的同源基因,也高表達(dá)于紅系及巨核系細(xì)胞,為了驗(yàn)證在紅細(xì)胞發(fā)育的定向造血階段是受gfi1b的調(diào)控,我們利用CRISPR-Cas9基因敲除技術(shù)獲得gfi1bko的突變體斑馬魚,通過雙突變體的實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)在定向造血階段,gfi1的家族基因gfi1b會(huì)補(bǔ)償gfi1aa基因的功能。第二部分a-cateninn、β-catenin對胚胎期造血干細(xì)胞遷移的作用研究1.目的造血干細(xì)胞(HSC)發(fā)育缺陷會(huì)引起各種血細(xì)胞或血管發(fā)育障礙,并與很多血液系統(tǒng)疾病密切相關(guān)。而胚胎期造血干細(xì)胞的遷移是HSC正確發(fā)育的必經(jīng)過程,然而人們對于胚胎發(fā)育期HSC遷移的機(jī)制知之甚少。目前與胚胎期造血干細(xì)胞遷移調(diào)控相關(guān)的分子研究較少,他們是黏附分子、細(xì)胞因子和趨化因子[4]。而β-catenin作為細(xì)胞內(nèi)骨架的一種功能性蛋白,能和a-catenin及γ-catenin結(jié)合到黏附分子VE-cadherin上,作為蛋白復(fù)合體參與細(xì)胞間的黏附[5]。Takahashi等研究發(fā)現(xiàn),惡性黑色素瘤B16細(xì)胞沉默β-catenin基因后促進(jìn)腫瘤細(xì)胞遷移[6]。而前期我們對cMyb在胚胎期HSC遷移調(diào)控的報(bào)道[7],是cmyb基因功能研究中前所未有的發(fā)現(xiàn)。本部分我們以cmybhk=3斑馬魚缺陷突變體為模型,深入研究斑馬魚模型的a-catenin、β-catenin對于胚胎期HSC遷移及定位的機(jī)理,逐步完善胚胎期HSC發(fā)育調(diào)控的分子信號調(diào)控網(wǎng)絡(luò)。2.方法首先,為了檢測斑馬魚中a-catenin、β-catenin在HSC中的表達(dá)是否受到cmyb突變的影響,我們利用Tg(cd4Ⅰ:eGFP)轉(zhuǎn)基因胚胎中GFP+來標(biāo)記HSC,通過QRT-PCR比較了 Cd41-GFP信號分選出的cmybh-3突變體與其同胞的HSC中a-catenin(ctnnaⅠ)、β-catenin(ctnnb1)的表達(dá)情況,發(fā)現(xiàn) ctnna1、ctnnb1在cmybhk=3突變體中的表達(dá)比在其同胞中表達(dá)高;其次,運(yùn)用morpholino基因敲低拯救實(shí)驗(yàn),分別顯微注射ctnna1-MO、ctnnb1-MO 到 Tg(cd41:eGFP)/cmybhk=3intercross的后代中,通過confocal實(shí)時(shí)觀察比較cmybhk=3突變體及其同胞VDA區(qū)域的HSC數(shù)目以及單位時(shí)間內(nèi)(4h)從VDA區(qū)遷出的HSC數(shù)目。最后,利用β-catenin的抑制劑氯喹(CQ)處理斑馬魚,觀察cmybhk=3突變體VDA區(qū)的HSC數(shù)目。3.結(jié)果通過QRT-PCR實(shí)驗(yàn),發(fā)現(xiàn)ctnna1、ctnnb1在cmybhk=-3突變體中的表達(dá)比在其同胞中表達(dá)高,說明a-catenin、β-catenin可能是cmyb基因的下游調(diào)控基因,參與cMyb調(diào)控HSC的遷移。顯微注射ctnna1-MO和ctnmb1-MO,cmybhk=3純合突變體VDA區(qū)HSC的數(shù)目減少到與其同胞相當(dāng)水平,而且單位時(shí)間內(nèi)遷出的HSC數(shù)目也增加。CQ處理后,能促進(jìn)cmybh=3突變體VDA中滯留的HSC發(fā)生遷移,使VDA區(qū)域的HSC減少。全文結(jié)論第一部分1)CRISPR-Cas9技術(shù)介導(dǎo)的斑馬魚基因組定點(diǎn)突變,對1號染色體上的ZKO編號為863～873的11個(gè)基因進(jìn)行基因敲除,獲得穩(wěn)定遺傳的F2代突變體。2)在斑馬魚早期造血發(fā)育過程中,主要是gfi1aa基因而不是gfi1b基因調(diào)控紅系的發(fā)育過程;3)gfi1aa基因主要通過調(diào)控紅細(xì)胞增殖來影響早期紅系造血過程,而gfi1aa基因缺失前體細(xì)胞發(fā)育正常、紅細(xì)胞的細(xì)胞形態(tài)也正常。4)在定向造血階段,gfi1的家族基因gfi1b會(huì)補(bǔ)償gfi1aa基因的功能。第二部分5)α-catenin、β-catenin是cmyb基因的下游調(diào)控基因,共同參與cMyb調(diào)控HSC的遷移。6)α-catenin、β-catenin蛋白可以通過改變與細(xì)胞骨架的結(jié)合,影響胚胎期HSC的遷移。
[Abstract]:Background blood system diseases (such as thalassemia, immunodeficiency and leukemia) have been a serious disease affecting the health of the people in our country. The high incidence of the disease has brought a heavy burden to the health care system of the whole society. The deep reason is that the high incidence of blood diseases reflects the human regulation of hematopoiesis. The molecular and pathological mechanisms of related diseases are poorly understood, because the correct and steady state of the blood system depends on the normal development of various blood cells, and the deficiency of the hematopoietic process leads to a variety of blood cells or vascular dysplasia. Therefore, the development and hematopoiesis process of hematopoietic cells is better understood at the molecular and cellular levels. It will help to gradually clarify the pathogenesis of blood system diseases, thus expanding the application potential of clinical treatment of blood system diseases. In vertebrates, the development process of hematopoietic system is the complex and orderly dynamic process of various blood cell development, mature, and regulated by multiple factors. In spite of the physiological functions of various blood cell components Different, but they have the same ancestor -- hematopoietic stem cells (HSCs). In the development of the hematopoietic system, the early development of hematopoietic stem cells is the core process of the correct production of.HSC, maintenance, proliferation, and smooth migration and localization, the organogenesis of the embryonic stage of the animal body and the stability of the tissue in the adult body. Holding and injury repair are essential for [1,2]. to differentiate from HSC to mature blood cells, experiencing the stage of hematopoietic progenitor cells and the stage of hematopoietic progenitor cells, and eventually differentiating into all lineage mature blood cells. The transcriptional factors play an important role in the development and differentiation of hematopoietic cells. The transcription factors play an important role in the development and differentiation of hematopoietic cells. The transcription factors are enhanced or weakened at different time and space of the cell development, making the hematopoietic stem cells or progenitor cells to a certain extent. Genealogical differentiation.Gfi1 is a transcriptional inhibitor that participates in the regulation of hematopoiesis. The deletion or mutation of the gene is characterized by granulocyte maturation defects and retarding in the early promyelocytic stage. Although leukaemia is not occurring in the Gfi1 knockout mice experiment, the absence of GFI1 function contributes to the occurrence of leukaemia, especially myeloid cells. The occurrence of leukemia, such as AML. classic model organisms such as nematodes and Drosophila, is quite different from human homology, while the traditional model animal mice are very slow and relatively large, and it is difficult to carry out the screening of high throughput hemopoiesis defect mutants. Moreover, mammalian animals are intrauterine, which is not conducive to the observation of embryo hematopoiesis. Zebrafish, as a vertebrate, is highly similar to human beings in organ development, hematopoiesis, and disease. In particular, the early development of the blood and cardiovascular system of zebrafish is very similar to that of human beings, whether the composition of the blood components and the development process are very similar, and the developmental defect mutants of the system still remain. Although it can survive several days, it provides a very favorable condition for people to study the regulation of early embryo hematopoiesis. [3]. in the first part of this paper, TALEN target knockout technique, CRISPR-Cas9 knockout technique, the gfi1aa gene of zebrafish AB strain and GFI1B gene are knocked out successfully, and a variety of different genotypes of gfi1aako mutants are obtained, gfi1bko process The temporal and spatial expression of various genealogical markers in the hematopoiesis process (primary hematopoiesis and permanent hematopoiesis) and the development of various genealogical cells are studied in detail. The purpose of this study is to explore the mechanism of gfi1aa gene and GFI1B gene regulation of erythropoiesis development. The latter part of the paper mainly explores a-catenin, beta -catenin to the embryo period The role of hematopoietic stem cell migration, in order to further clarify the related cell and molecular regulation mechanism of hematopoietic stem cell migration, is divided into two parts: the first part participates in the national "total gene knockout of zebrafish chromosome 1" and the mechanism of gfi1aa regulation of zebrafish blood development; the second part of a-catenin, beta -cat Study on the effect of enin on the migration of hematopoietic stem cells at embryonic stage. Part 1 participates in the national "total gene knockout of zebrafish chromosome 1" and the study on the mechanism of Gfi1 gene regulation of zebrafish hematopoiesis. The purpose of this part is to introduce the CRISPR-Cas9 and TALEN gene editing techniques first, and then use these two techniques with zebrafish as model organisms. The following two tasks were completed: 1) using CRISPR-Cas9 technology to participate in the national "total gene knockout" of zebrafish chromosome 1, and to knock on the 1 chromosome with 11 genes (vps37c, tmem132a, DnaJA1, smu1b, RPS6, btr01, c1h20orf27, muc13a, si:ch211-239f4.6, mucms1). The hereditary F2 generation mutant.2) using the TALEN targeting gene knockout technique, using zebrafish model organisms to knock out the hematopoietic phylogenetic gene gfi1aa, and obtain the gfi1aa mutant of the hematopoietic development defect. At the same time, the CRISPR-Cas9 knockout technique is used to knock out another GFI1B gene of the Gfi1 family and obtain the gfi1bko mutant. Finally, the CRISPR-Cas9 knockout technique is used. Using these two mutants to study the phenotype and function, explore the Gfi1 gene regulating the hematopoietic development cells and the molecular mechanism.2. method 1) mainly using the CRISPR-Cas9 genome editing system, using gRNA target identification and Cas9 nucleic acid cutting. NCBI, Ensemb1 and Smart sites search for the basic information of the related genes, to ZKO compiling. The 11 genes of 863~873 were designed to target the target, and the corresponding primers were synthesized. The corresponding gRNA and Cas9 mRNA were transcribed in vitro, and the mixed microinjection of the wild type AB zebrafish embryos with one-cell was obtained. The zebrafish of the F0 chimera was obtained, and the big FO (Founder) and the wild type AB adult fish were obtained F1 embryos, through the F1 generation embryos. PCR, enzyme digestion and sequencing were used to screen out F0 adult fish with mutation of target loci in germ cells. The F1 of such F0 diplomacy was raised, and the stable genetic zebrafish individual.2 was screened by the sequence of F1 target loci to be screened by scissors, and the TALEN and CRISPR-Cas9 gene knockout techniques were applied to the hematopoiesis development of zebrafish AB strains. The related gene gfi1aa gene and GFI1B gene are knocked out in order to obtain the gfi1aako mutants and gfi1bko mutants. Then the gfi1aa gene and the GFI1B gene regulate the development of the hematopoiesis according to the temporal and spatial expression of various genealogical markers in the hematopoietic and permanent hematopoiesis and the development of each lineage cell. Mechanism.3. result 1) Cas9 gene knockout technique was used to get F1 generation zebrafish with single genotypic mutation, and then F2 generation with AB diplomacy. The zebrafish of F2 generation heterozygous mutant was obtained by PCR, enzyme digestion and sequencing, and a part of the zebrafish of the national zebrafish was given in part, and intercros of the remaining F2 generation mutant zebrafish was then carried out. S, through in situ hybridization, Sultan black B staining, neutral red staining and other methods for phenotypic screening.2), TALEN targeting gene knockout technique was used to knock out zebrafish gfi1aa, and a gfi1aako mutant with 23 bases (5'-TCTTCTCAACAGTCCCCGCTGAG-3') missing was obtained. Phenotypic analysis showed that gfi1aa affected the red fine in the early development of zebrafish hemopoiesis. The development of cell and neutrophils does not affect the development of macrophages. The cytological mechanism that gfi1aa affects the early red lineage is mainly by inhibiting the proliferation of red blood cells, but does not affect the cell morphology of the erythrocyte precursor cells and red cells. In the 50 HPF period, the expression of gfi1aako homozygous hemoglobin was restored to the wild type phase. When.Gfi1b is a homologous gene of gfi1aa, it is also highly expressed in red and megakaryocyte cells. In order to verify that the hematopoietic stage of erythrocyte development is regulated by GFI1B, we use CRISPR-Cas9 knockout technique to obtain the mutant zebra fish of gfi1bko. The results of the double mutant body were found to be in the directional hematopoiesis stage, Gfi1. The family gene GFI1B compensates the function of the gfi1aa gene. Second part a-cateninn, the effect of beta -catenin on the migration of hematopoietic stem cells in embryo stage; 1. the development defects of hematopoietic stem cells (HSC) may cause various blood cell or vascular dysplasia and are closely related to many blood system diseases. The migration of embryonic stem cells is HS C is a necessary process for correct development. However, little is known about the mechanism of HSC migration during embryonic development. There are few molecular studies related to the regulation of embryonic hematopoietic stem cell migration. They are adhesion molecules, cytokines and chemokines [4]., while beta -catenin is a functional protein of the cytoskeleton, and can be used with a-catenin and gamma. Catenin combined with adhesion molecule VE-cadherin, as protein complex involved in intercellular adhesion [5].Takahashi, and other studies found that malignant melanoma B16 cells silenced the beta -catenin gene to promote tumor cells to migrate [6]. and we reported [7] on the regulation of HSC migration in the embryonic stage of cMyb in the early stage, which is unprecedented in the study of cmyb gene function. In this part, we use the cmybhk=3 zebrafish defect mutants as a model to study the mechanism of a-catenin, beta -catenin for the migration and localization of HSC in the embryonic period, and gradually improve the molecular signaling network.2. method for the regulation of HSC development at the embryonic stage, and the detection of a-catenin in zebrafish, beta -catenin in HSC. Whether the expression is affected by cmyb mutation, we use GFP+ to label HSC in Tg (CD4 I: eGFP) transgenic embryos, and compare the expression of cmybh-3 mutant of Cd41-GFP signal and a-catenin (ctnna I) and beta of HSC in Cd41-GFP signal by QRT-PCR. Second, using the morpholino gene knockout experiment, microinjection of ctnna1-MO, ctnnb1-MO to Tg (cd41:eGFP) /cmybhk=3intercross, the number of cmybhk=3 mutants and the number of HSC in the VDA region of the cmybhk=3 and the unit time (4h) from the VDA region were observed in real time by confocal. The -catenin inhibitor chloroquine (CQ) was used to treat zebrafish, and the number of HSC in the cmybhk=3 mutant VDA region was observed by QRT-PCR experiment. The expression of ctnna1 and CTNNB1 in cmybhk=-3 mutants showed higher expression than in their siblings, indicating that a-catenin, beta -catenin may be the downstream regulation gene of the gene. Microinjection of ctnna1-MO and ctnmb1-MO, the number of HSC in the VDA region of the cmybhk=3 homozygous mutant decreased to a considerable level with its compatriots, and the number of HSC in the unit time increased by.CQ processing, which could promote the migration of HSC in the cmybh=3 mutant VDA, and reduce the HSC in VDA region. The first part of the full text conclusion 1) Directed mutagenesis of zebrafish genome, gene knockout of 11 genes with ZKO number of 863~873 on chromosome 1, and a stable genetic F2 generation mutant.2). During the early hematopoiesis of zebrafish, the major gfi1aa gene was not the GFI1B gene regulating the development of the red system; 3) the gfi1aa gene was mainly controlled by the regulation of red fine. The cell proliferation affects the early erythropoiesis process, while the gfi1aa gene deletion precursor cells develop normally, the cell morphology of the red cells is also normal.4). The family gene GFI1B of Gfi1 will compensate for the function of the gfi1aa gene. Second part 5) alpha -catenin, the beta -catenin is the downstream regulation gene of the cmyb gene, and participates in cMyb regulation HSC. The migration of.6) alpha -catenin and beta -catenin can affect the migration of HSC in embryonic stage by changing the binding with cytoskeleton.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R331

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