本文選題：CRISPR-Cas9技術(shù)　切入點：Cre-loxP重組系統(tǒng)　出處：《上海師范大學》2017年碩士論文

【摘要】：白血病或腫瘤的發(fā)病過程通常伴隨著融合基因的產(chǎn)生,融合基因可促進腫瘤的產(chǎn)生和發(fā)展,并可作為腫瘤的分子診斷和治療靶標。MLL-AF9基因融合在急性髓系白血病(AML)病人中較為常見,構(gòu)建MLL-AF9的細胞模型對研究AML的發(fā)病機理與藥物發(fā)現(xiàn)有很重要的意義。CRISPR-Cas9是近年來發(fā)展起來的一種新型基因編輯技術(shù),其構(gòu)建和使用非常方便并且成本較低,可同時對多個基因進行編輯,已用于構(gòu)建多種基因融合模型,但是打靶效率較低,小于10%,亟需提高其打靶效率。本文提出了一種利用CRISPR-Cas9技術(shù)在體外高效構(gòu)建MLL-AF9融合基因的新策略。(1)在MLL基因的第10個內(nèi)含子與AF9基因的第5個內(nèi)含子中設(shè)計并篩選出高效作用的sgRNA。(2)設(shè)計一種全新的打靶載體p EASY-Blunt-MLL-GFP-AF9(2Loxp),以p EASY-Blunt作為載體,分別插入MLL和AF9基因的同源臂序列來介導同源重組,并且該載體中含有可敲除的GFP篩選基因。(3)將高效的MLL sgRNA,AF9 sgRNA和打靶載體同時轉(zhuǎn)染人HEK293T細胞,兩條sgRNA分別在MLL和AF9基因的相應(yīng)位點造成切口,在打靶載體的作用下,通過同源重組使MLL基因前10個外顯子和AF9基因的第6-7個外顯子融合在一起形成新的融合基因,最后通過Cre-Loxp系統(tǒng)將GFP基因敲除。(4)通過293T細胞基因組DNA的PCR及測序來鑒定發(fā)生MLL-AF9重組的293T細胞,從目前結(jié)果來看重組效率可高達30%,比以前的方法有極大的提高。(5)通過熒光原位雜交技術(shù)觀察在293T細胞中MLL和AF9基因的狀態(tài),但是并沒有檢測到融合基因的存在,可是由于293T細胞為多倍體,無法準確的檢測到所有的染色體,可通過換一個兩倍體的細胞系來進一步觀察。本文創(chuàng)立了一種利用CRISPR-Cas9技術(shù)高效構(gòu)建基因融合的方法。從PCR實驗結(jié)果來看,設(shè)計的新的打靶載體可通過介導同源重組來有效地提高融合效率,我們后續(xù)會通過更換其他細胞系來進一步優(yōu)化實驗條件體系。該策略的成功建立,將顯著提高融合基因的打靶效率,為構(gòu)建染色體基因融合細胞模型提供新的技術(shù)手段,并為研究腫瘤等相關(guān)疾病的作用機制奠定基礎(chǔ)。
[Abstract]:The pathogenesis of leukemia or tumor is usually accompanied by the production of fusion genes, which can promote the production and development of tumors. MLL-AF9 gene fusion is common in patients with acute myeloid leukemia (AMLL). The construction of MLL-AF9 cell model is of great significance to study the pathogenesis of AML and drug discovery. CRISPR-Cas9 is a new gene editing technology developed in recent years, its construction and use is very convenient and low cost. Multiple genes can be edited at the same time, which has been used to construct multiple gene fusion models, but the efficiency of targeting is low. It is urgent to improve the efficiency of target shooting. This paper proposes a new strategy of efficiently constructing MLL-AF9 fusion gene in vitro by using CRISPR-Cas9 technology. We design and screen the 10th intron of MLL gene and the fifth intron of AF9 gene. A novel targeting vector, p EASY-Blunt-MLL-GFP-AF9 / 2Loxphe, was designed, and p EASY-Blunt was used as the carrier. The homologous arm sequences of MLL and AF9 genes were inserted to mediate homologous recombination, and the vector contained knockout GFP screening gene. The vector transfected high efficient MLL sgRNA-AF9 sgRNA and targeting vector into human HEK293T cells at the same time. Two sgRNA fragments were cut at the corresponding sites of MLL and AF9 genes. By homologous recombination, the first 10 exons of MLL gene and the 6-7 exons of AF9 gene were fused together to form a new fusion gene. Finally, GFP gene was knockout by Cre-Loxp system. 293T cells were identified by PCR and sequencing of genomic DNA of 293T cells. According to the present results, the efficiency of recombination can be as high as 30%, which is much higher than the previous method.) the status of MLL and AF9 genes in 293T cells was observed by fluorescence in situ hybridization (Fish), but the presence of fusion genes was not detected. But because 293T cells are polyploid, it is impossible to detect all chromosomes accurately. We have established a method for efficient construction of gene fusion by using CRISPR-Cas9 technology. From the results of PCR experiments, we can observe further by changing a diploidy cell line. The designed targeting vector can effectively improve the fusion efficiency by mediating homologous recombination. We will further optimize the experimental condition system by replacing other cell lines. It will significantly improve the targeting efficiency of the fusion gene, provide a new technique for the construction of chromosome gene fusion cell model, and lay a foundation for the study of the mechanism of tumor and other related diseases.
【學位授予單位】：上海師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.71

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相關(guān)期刊論文 前1條

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本文編號：1656693