兰州大学机构库 >生命科学学院
CRISPR/Cas9基因编辑实验系统建立及CBX2调控性别发育基因的功能初探
Alternative TitleA pilot study on regulatory mechanisms of CBX2 in controlling sexual development genes using CRISPR/Cas9 based gene editing system
白米雪
Thesis Advisor程博
2017-05-20
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordCBX2 性别决定 性别分化 CRISPR/Cas9
Abstract

CBX蛋白作为哺乳动物多梳抑制复合体PRC1的核心组分在干细胞干性维持和胚胎发育过程中发挥着重要的作用。哺乳动物CBX同源蛋白Cbx2敲除型小鼠出现雄性-雌性的性别逆转,类似的发育异常现象在人类Cbx2点突变的病例中也有报道。目前有一定实验证据表明CBX2可能通过调控性别决定基因Sry及Nr5a1基因的表达调控性别发育,但由于其效应显示为基因激活作用,与已知的PRC1抑制基因表达的效应不吻合,具体的调控机制目前还不明确。

为了深入探究CBX2调控性别发育的机制,对Cbx2进行敲除和过表达等基因操作必不可少。而此时一种新型基因编辑系统CRISPR/Cas9应运而生,操作简便快速高效,因此本论文尝试应用了多种CRISPR/Cas9体系去进行Cbx2基因的敲除。通过建立并不断完善本实验室CRISPR/Cas9系统,得到了高效率的瞬时转染实验系统和慢病毒载体感染系统,并且构建了可诱导表达Cas9的多种细胞系,能够较为成熟简便地使用CRISPR/Cas9对人胚肾细胞HEK293T、小鼠胚胎干细胞E14TG2a和小鼠睾丸畸胎瘤细胞F9等不同细胞系进行基因编辑。目前已使用瞬时转染系统得到了纯合型和杂合型的Cbx2 KO HEK293T单克隆细胞系,并且确立了在基因测序和蛋白水平上进行了敲除效率验证的实验体系。另外,我们对收集到的一例性别发育障碍(DSD)患者病例的外周血样品中Cbx2外显子进行了扩增后测序,发现了一个点突变(L4M)。同时我们也对建立检测CBX2靶向募集特异性的报告基因实验系统进行了初步的前期摸索。

本论文的工作不仅在方法学上成功建立了多种CRISPR/Cas9实验体系进行基因敲除,更重要的是所建立的相关Cbx2敲除细胞系为进一步揭示CBX2在胚胎性别决定与分化过程中的调控机制以及是否依赖PRC1发挥调控作用奠定了良好基础。

Other Abstract

CBX proteins play important roles in stem cell self-renewal and embryonic development as core components of mammalian Polycomb Repressive Complex PRC1. Knockout of mammalian CBX protein Cbx2 in mouse exhibit male-female sex reversal, similar to the reports of developmental abnormalities in human Cbx2 mutation cases. There is some experimental evidence suggesting that CBX2 regulates sex development by up-regulating the expression of Sry and Nr5a1 genes, which is inconsistent with the known repressive functions of PRC1, and the precise regulation mechanism is yet unclear.

In order to further explore the roles of CBX2 in sex development, Cbx2 knockout, overexpression or other gene manipulations are necessary. As a new gene editing system, CRISPR/Cas9 has become a popular choice for mammalian gene manipulation. The benificial features such as simple, quick and efficient, attracted us to use the CRISPR/Cas9 system to carry out Cbx2 gene knockout. Through the establishment and continuous improvement of the CRISPR/Cas9 system, a high efficiency transient transfection experiment system and a lentiviral transfection system were developed, and a variety of cell lines capable of inducing expression of Cas9 were constructed, e.g. in human embryonic kidney cell line HEK293T, mouse embryonic stem cell line E14TG2a, mouse testicular teratoma cell line F9 etc. At present, homozygotic and heterozygotic Cbx2 KO HEK293T cell lines have been established using our transient transfection system, and an experimental system has been established to verify the occurrence of knockout at either genomic sequence level or at protein level. In addition, we identified a new mutation (L4M) by sequencing the Cbx2 exons in the peripheral blood sample collected from a patient with sex developmental disorder (DSD). We also carried out some preliminary studies on a reporter system for the examination of CBX2 targeting specificity.

This paper details the establishment of a variety of CRISPR/Cas9 experimental systems for gene knockout, and more importantly, the establishment of Cbx2 knockout cell lines may further reveal the roles of CBX2 in embryonic sex determination and differentiation, and whether its function relies on the canonical repressive function of PRC1.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222028
Collection生命科学学院
Recommended Citation
GB/T 7714
白米雪. CRISPR/Cas9基因编辑实验系统建立及CBX2调控性别发育基因的功能初探[D]. 兰州. 兰州大学,2017.
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