【作者】 王佳贤；

【导师】 朱建伟；

【作者基本信息】 上海交通大学 ， 药学， 2016， 硕士

【摘要】 背景和目的:Crispr(Clustered Regularly Interspaced Short Palindrome Repeats)称为规律成簇的间隔短回文重复序列,最初发现于多种古生物与细菌基因组中,被认为与其免疫系统相关。随着研究的深入,人们发现Crispr可用于基因工程,并将其开发成为一种强大的基因编辑技术。与早些年兴起的基因编辑技术锌指核酸酶(ZFNs)和转录激活样效应因子核酸酶(TALENs)相比,Crispr/Cas9系统操作更简单,所需时间少,编辑效率高且成本更低,因此近年来逐渐成为主流的基因编辑技术。在本文中,我们采用Crispr/Cas9系统在工程细胞株中华仓鼠卵巢细胞(CHO)和人胚胎肾细胞(HEK293T)中进行靶向的基因编辑,并取得了成功。目前市场上有一半以上的治疗用重组蛋白药物都是通过哺乳动物细胞表达系统进行生产,但哺乳动物细胞株存在不稳定性,随着培养时间的延长,有的细胞株产量会显著降低。研究表明这种不稳定性有可能和细胞的表观遗传学修饰有关,特别是DNA的甲基化。目前在哺乳动物细胞中,已发现有三种控制DNA甲基化的酶,我们选择了其中一种,即DNA甲基转移酶3a(DNMT3a)进行研究,通过Crispr/Cas9技术将控制此酶表达的基因敲除,并初步研究了该基因被敲除后细胞株的生长特性及外源蛋白的表达情况。另一方面,目前构建重组细胞株的方法主要是随机整合的方式,而这种随机整合的细胞株由于无法控制目的基因在基因组中插入的位置,会造成称之为“位置效应”的问题,为克服此种情况的发生,学者纷纷转向在基因组中定点整合目的基因。由于Crispr/Cas9技术的独特优势,有很多研究开始尝试利用其进行细胞基因组中目的基因的定点插入。在本文中,我们就利用Crispr技术在CHO细胞的岩藻糖转移酶(FUT8)基因座中定点插入了细胞毒T淋巴细胞相关抗原4(CTLA4Ig)基因。方法:在CHO细胞和HEK293T细胞中敲除DNMT3a基因:针对CHO细胞和HEK293T细胞DNMT3a蛋白的关键作用位点设计两对短向导RNA(sgRNA),构建成功后将两对sgRNA共转染到相应的细胞中,然后用有限稀释法在96孔板中铺单克隆并筛选到基因敲除的单克隆细胞株。用绿色荧光蛋白(GFP)作为模式蛋白来验证在DNMT3a基因敲除的细胞株中,外源蛋白的转染及表达与野生型细胞是否具有差异。在CHO细胞FUT8基因座中定点整合CTLA4Ig基因:在FUT8基因的起始密码子ATG附近设计五对sgRNA,然后挑选效率最高的两对sgRNA,将其连同构建的供体质粒共转染到CHO细胞,经过加药筛选后,用有限稀释法铺单克隆培养板,挑取单克隆并进行检测,成功得到CTLA4Ig基因定点整合的细胞株。结果:在CHO细胞和HEK293T细胞中敲除DNMT3a基因:最终通过有限稀释法筛选得到了一株DNMT3a基因完全敲除的CHO细胞株(后面简称DNMT3a-CHO)和一株DNMT3a基因完全敲除的HEK293T细胞株(后面简称DNMT3a-HEK293T)。应用超高效液相色谱-四级杆飞行时间质谱仪(UMS)对细胞全基因组的胞嘧啶甲基化情况进行分析,结果表明相比于野生型CHO细胞(后面简称WT-CHO),DNMT3a-CHO细胞基因组甲基化胞嘧啶的含量降低了27%,而DNMT3a-HEK293T细胞基因组中甲基化的胞嘧啶含量要比野生型的HEK293T细胞(后面简称WT-HEK293T)降低了22%。在生长性质方面,DNMT3a-CHO和WT-CHO生长特性无显著差异,而DNMT3a-HEK293T较WT-HEK293T倍增时间延长。应用GFP作为模式蛋白验证在DNMT3a基因敲除的细胞株中,外源蛋白的产量是否得到了提高,结果表明在CHO细胞中,敲除DNMT3a基因对GFP的表达量没有显著影响,而在HEK293T细胞中,敲除DNMT3a基因则会显著减少GFP的单位表达量。在CHO细胞FUT8基因座中定点整合CTLA4Ig基因:所设计的5条sgRNA编辑效率从17.7%到31.7%不等,在ATG的上下游分别选择了一条效率最高的sgRNA进行下一步的实验。通过对转染了sgRNA和供体质粒的CHO细胞进行加药筛选,最终我们获得了56株单克隆细胞株,通过聚合酶链式反应(PCR)和蛋白印迹(Western Blot)检测,我们从56株单克隆细胞株中挑选出了19株成功定点整合的细胞,阳性率达到了33.9%。更多还原

【Abstract】 Background and Purpose:Crispr,short for Clustered Regularly Interspaced Short Palindrome Repeats,was originally found in the genome of a number of archaea and bacterials,is considered to be relevant to immune responses of these prokaryotes.With the research about it going deeper,Crispr/Cas9 has been adapted to a popular gene editing technology gradually.Compared with previously developed genome engineering technologies such as zinc finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs),Crispr/Cas9 system is easier to use,less time-consuming,cost-effective,as well as with a higher editing efficiency.In our research,Crispr/Cas9 system was successfully employed to targeted engineer the genome of two most widely used cell lines for biopharmaceuticals: Chinese Hamster Ovary(CHO)cells and Human Embryo Kidney(HEK293T)cells.Currently more than a half of the therapeutic recombinant proteins approved on market are produced in mammalian cells,however,there is a major drawback with the mammalian cell expression system.It is the instability of recombinant cell lines.With the culture time extending,the productivity of the cell line will drop significantly.Some evidence shows that this instability may be related with epigenetic modifications,especially DNA methylation.So far only three classes of enzymes responsible for DNA methylation are found in mammalian cells.And one of the three enzymes,DNA methyltransferases 3a(DNMT3a)was chose by us for further study.The gene encoding this enzyme was first disrupted with Crispr/Cas9 system and then alterations were observed before and after the gene was knockout.And we mainly investigated whether the productivity of exogenous proteins was elevated when the gene was disrupted.At the meantime,recombinant cell lines were constructed mainly by random integration at present.But random integration will give rise to unwanted problems due to lack of control of the gene insertion-termed the “position effect”.In order to circumvent such a situation,scholars are focusing on targeted integration.Since Crispr/Cas9 system has unique advantages in gene editing,a variety of researchers are using this technology to insert the gene of interest(GOI)into the specific locus in the genome.In our research,we utilized the Crispr technology to targeted integrate the cytotoxic T Lymphocyte Antigen 4(CTLA4Ig)gene into the fucosyltransferases 8(FUT8)locus in CHO cells.Methods:Disruption of DNMT3 a gene in both CHO cells and HEK293 T cells: Two pairs of sgRNA targeting the key functional regions of DNMT3 a protein were designed and constructed.Then the two pairs of sgRNA were co-transfected into CHO cells,following limiting dilution to plate transfected cells into 96 well-plates to obtain single cell clones.Green fluorescence protein(GFP)was used as the model protein to assess whether the productivity of transgene was improved or not when the DNMT3 a gene was disrupted.Targeted integration of CTLA4 Ig gene into the FUT8 locus of CHO cells: A total of five pairs of sgRNA targeting the sites around the start codon ATG were designed and their indel efficiency was evaluated.The two most efficient sgRNA pairs were chose for further use.The two picked sgRNA pairs together with the donor plasmid were co-transfected into CHO cells.Transfected cells were subjected to limiting dilution to obtain single cell clones followed by a drug-selection process.Single cell clones were picked up and detected to obtain correctly targeted cell lines.Results:Disruption of DNMT3 a gene in both CHO cells and HEK293 T cells: Finally one CHO cell line with DNMT3 a gene completely disrupted(referred to as DNMT3a-CHO)as well as one HEK293 T cell line(referred to as DNMT3a-HEK293T)with DNMT3 a gene completely disrupted was obtained through limiting dilution.UMS was applied to analyze the methylated cytosine content present in the whole genome.And results showed that the methylated cytosine in DNMT3a-CHO genome decreased by about 27% compared with that of wild type CHO(referred to as WT-CHO).While in DNMT3a-HEK293 T genome,methylated cytosine decreased by approximate 22% compared to wild type HEK293T(referred to as WT-HEK293T).As for growth profile,there is no difference between WT-CHO and DNMT3a-CHO cell line,however,DNMT3a-HEK293 T cell line grows much slower than WT-HEK293 T cell line.When GFP was applied as the model protein to evaluate whether transgene in DNMT3 a disrupted cell line is improved,results demonstrated that the productivity was not affected for CHO cell line while in HEK293 T cell line,loss of DNMT3 a gene caused great loss of GFP.Targeted integration of CTLA4 Ig gene into the FUT8 locus of CHO cells: The editing efficiency of the designed five pairs of sgRNA ranged from 17.7% to 31.7%.The most efficient sgRNA upstream or downstream of the start codon ATG were picked up for transfection.56 single cell clones were finally obtained after drug was applied into the cells transfected with sgRNA and donor plasmid.19 cell lines of the 56 single cell clones were identified as correctly targeted events after detection with polymerase chain reaction(PCR)and Western Blot.The targeting efficiency reached up to 33.9%.更多还原