应用CRISPR-Cas9技术高效构建MLL-AF9基因融合模型

发布时间：2018-03-24 04:21

本文选题：CRISPR-Cas9技术　切入点：Cre-loxP重组系统　出处：《上海师范大学》2017年硕士论文

【摘要】：白血病或肿瘤的发病过程通常伴随着融合基因的产生,融合基因可促进肿瘤的产生和发展,并可作为肿瘤的分子诊断和治疗靶标。MLL-AF9基因融合在急性髓系白血病(AML)病人中较为常见,构建MLL-AF9的细胞模型对研究AML的发病机理与药物发现有很重要的意义。CRISPR-Cas9是近年来发展起来的一种新型基因编辑技术,其构建和使用非常方便并且成本较低,可同时对多个基因进行编辑,已用于构建多种基因融合模型,但是打靶效率较低,小于10%,亟需提高其打靶效率。本文提出了一种利用CRISPR-Cas9技术在体外高效构建MLL-AF9融合基因的新策略。(1)在MLL基因的第10个内含子与AF9基因的第5个内含子中设计并筛选出高效作用的sgRNA。(2)设计一种全新的打靶载体p EASY-Blunt-MLL-GFP-AF9(2Loxp),以p EASY-Blunt作为载体,分别插入MLL和AF9基因的同源臂序列来介导同源重组,并且该载体中含有可敲除的GFP筛选基因。(3)将高效的MLL sgRNA,AF9 sgRNA和打靶载体同时转染人HEK293T细胞,两条sgRNA分别在MLL和AF9基因的相应位点造成切口,在打靶载体的作用下,通过同源重组使MLL基因前10个外显子和AF9基因的第6-7个外显子融合在一起形成新的融合基因,最后通过Cre-Loxp系统将GFP基因敲除。(4)通过293T细胞基因组DNA的PCR及测序来鉴定发生MLL-AF9重组的293T细胞,从目前结果来看重组效率可高达30%,比以前的方法有极大的提高。(5)通过荧光原位杂交技术观察在293T细胞中MLL和AF9基因的状态,但是并没有检测到融合基因的存在,可是由于293T细胞为多倍体,无法准确的检测到所有的染色体,可通过换一个两倍体的细胞系来进一步观察。本文创立了一种利用CRISPR-Cas9技术高效构建基因融合的方法。从PCR实验结果来看,设计的新的打靶载体可通过介导同源重组来有效地提高融合效率,我们后续会通过更换其他细胞系来进一步优化实验条件体系。该策略的成功建立,将显著提高融合基因的打靶效率,为构建染色体基因融合细胞模型提供新的技术手段,并为研究肿瘤等相关疾病的作用机制奠定基础。
[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.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

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