检索结果-南通市图书馆

The collagen type Ⅰ alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

Journal of Integrative Agriculture 2023年第1期22卷 202-213页

作者： XIANG Guang-ming ZHANG Xiu-ling XU Chang-jiang FAN Zi-yao XU Kui WANG Nan WANG Yue CHE Jing-jing XU Song-song MU Yu-lian LI Kui LIU Zhi-guo State Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China Institute of Animal SciencesChinese Academy of Agricultural SciencesBeijing 100193P.R.China School of Life Science and Engineering Foshan UniversityFoshan 528231P.R.China Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs Agricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518120P.R.China

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.Safe harbor loci in the animal genome enable consistent overexpression of foreign genes,without side effects.However,relatively few safe harbor loci are available in pigs,a fact which has impeded the development of multi-transgenic pig research.We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain(COL1A1)gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.After the knock-in of a 2A peptide-green fluorescence protein(2A-GFP)transgene in the last codon of COL1A1 in multiple porcine cells,including porcine kidney epithelial(PK15),porcine embryonic fibroblast(PEF)and porcine intestinal epithelial(IPI-2I)cells,quantitative PCR(qPCR),Western blotting,RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.The qPCR results showed that the GFP knock-in had no effect(P=0.29,P=0.66 and P=0.20 for PK15,PEF and IPI-2I cells,respectively)on the mRNA expression of COL1A1 gene.Similarly,no significant differences(P=0.64,P=0.48 and P=0.80 for PK15,PEF and IPI-2I cells,respectively)were found between the GFP knock-in and wild type cells by Western blotting.RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation(Pknock-in did not alter the global expression of endogenous genes.Furthermore,the CCK8 assay showed that the GFP knock-in events had no adverse effects(P_(24)h=0.31,P_(48)h=0.96,P_(72)h=0.24,P_(96)h=0.17,and P_(120)h=0.38)on cell proliferation of PK15 cells.These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.

关键词： COL1A1 gene safe harbor knock-in CRISPR/Cas9 pig

来源：

维普期刊数据库

同方期刊数据库评论

在线全文

学校读者我要写书评

暂无评论

利用Easi-CRISPR技术建立Gtl2转录终止的knock-in小鼠模型

利用Easi-CRISPR技术建立Gtl2转录终止的Knock-in小鼠模型

引用

作者：刘炬杭哈尔滨工业大学

学位级别：硕士

长非编码RNA（lncRNA）Gtl2是位于Dlk1-Dio3印记基因簇上的印记基因,具有母本表达而父本印记的印记模式,对于小鼠胚胎发育具有重要的作用。目前对于lncRNA Gtl2在小鼠胚胎发育中的调节作用没有明确的结论,两项针对Gtl2基因的敲除研究给... 详细信息

长非编码RNA（lncRNA）Gtl2是位于Dlk1-Dio3印记基因簇上的印记基因,具有母本表达而父本印记的印记模式,对于小鼠胚胎发育具有重要的作用。目前对于lncRNA Gtl2在小鼠胚胎发育中的调节作用没有明确的结论,两项针对Gtl2基因的敲除研究给出了互相矛盾结果。虽然在两篇文章中均出现了胚胎致死表型,证明了Gtl2是对小鼠胚胎发育及其重要的基因,但报道的致死时间与致死机制彼此不同,大规模的基因敲除会删除Gtl2-DMR与mi RNA等重要原件是造成结果矛盾的一条重要原因。此外,有大量的研究表明lncRNA的基因序列具有调控作用,基因敲除会混淆基因序列与lncRNA本身的功能,带来不可靠结果,不适用于lncRNA功能的研究。本研究采用了poly A转录终止法结合Easi-CRISPR技术,构建Gtl2转录终止的小鼠模型,在不删除任何DNA序列的情况下,达到基因敲除等同的效果。深度分析Gtl2的基因序列与转录本信息,将sg RNA的选择范围固定在前三个外显子和前两个内含子序列当中,选择较低活性无功能的DNA区域作为sg RNA靶向位点,避开对调控元件的破坏。根据Easi-CRISPR技术所需要的donor设计原则装配ss DNA donor,通过体内与体外编辑的方式验证sg RNA指导Cas9蛋白编辑基因组的能力,同时检测ss DNA donor中3×poly A序列的转录终止能力。采用原核注射的方式对B6D2F1品系小鼠的受精卵及进行编辑,手术法移植囊胚至代孕小鼠的子宫中,成功获得5只带有阳性插入片段的F0代小鼠,无脱靶现象,成功构建出GTR-poly A小鼠模型。遗传学杂交显示,没有纯和小鼠出生,而且出生幼鼠的基因型比例严重偏离孟德尔遗传定律。此外,本研究优化了基因插入体系,探究了显微注射方式与注射时机对基因敲入效率的影响。设计了5种可能提高基因插入效率的注射方式:受精卵胞质注射（1CC）、受精卵原核注射（1CN）、2-细胞期双胞质注射（2CDC）、2-细胞期单核注射（2CMN）和2-细胞期双核注射（2CDN）。通过每组3次的重复实验总结出,采用原核注射的方式可以得到最高为67%的基因插入效率,而且2-细胞对受精卵进行显微注射的效果会优于受精卵胞质注射。综上,本课题制作了GTR-poly A小鼠模型,遗传杂交实验结果严重偏离了理论比例,提示了lncRNA Gtl2的重要作用,为证实lncRNA Gtl2对小鼠发育的功能奠定了基础。

关键词： lncRNA Gtl2 Easi-CRISPR knock-in 原核注射 2-细胞注射

来源：

同方学位论文库评论

在线全文

同方学位论文库

学校读者我要写书评

暂无评论

Genetic approach to track neural cell fate decisions using human embryonic stem cells

引用

Protein & Cell 2014年第1期5卷 69-79页

作者： Xuemei Fu Zhili Rong Shengyun Zhu Xiaocheng Wang Yang Xu Blue B. Lake Shenzhen Children's Hospital Shenzhen 518026 China Section of Molecular Biology Division of Biological Sciences University of California San Diego 9500 Gilman Drive LaJolla CA 92093-0322 USA

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells （hESCs） hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells （NPCs）, we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro （embryoid bodies） and in vivo （ter- atomas）. This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry＊ NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.

关键词： Nestin knock-in human embryonic stemcells neural progenitor cells

来源：

维普期刊数据库评论

在线全文

维普期刊数据库

学校读者我要写书评

暂无评论

Use of gene-editing technology to introduce targeted modifications in pigs

引用

Journal of Animal Science and Biotechnology 2018年第2期9卷 268-277页

作者： Junghyun Ryu Randall S.Prather Kiho Lee Department of Animal and Poultry Sciences Virginia Tech175 W.Campus DriveBlacksburgVA 24061USA Division of Animal Science University of Missouri-Columbia920 East Campus DriveColumbiaMO 65211USA National Swine Resource and Research Center University of Missouri-ColumbiaColumbiaMO 65211USA

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress relate

关键词： CRISPR/Cas9 Gene-editing Genetic engineering knock-in knockout Pig TALEN ZFN

来源：

维普期刊数据库

同方期刊数据库评论

在线全文

学校读者我要写书评

暂无评论

TMEM43-S358L mutation enhances NF-κBTGFp signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy

引用

Protein & Cell 2019年第2期10卷 104-119页

作者： Guoxing Zheng Changying Jiang Yulin Li Dandan Yang Youcai Ma Bing Zhang Xuan Li Pei Zhang Xiaoyu Hu Xueqiang Zhao Jie Du Xin Lin Tsinghua University-Peking University Joint Center for Life Sciences Beijing 100084China Institute for Immunology Department of Basic Medical SciencesSchool of MedicineTsinghua UniversityBeijing 100084China The 7th Affiliated Hospital of Sun Yat-Sen University ShenzhenGuangdong 510275China Department of Molecular and Cellular Oncology The University of TexasMD Anderson Cancer CenterHoustonTX 77030USA Beijing Anzhen Hospital Capital Medical UniversityThe Key Laboratory of Remodeling-Related Cardiovascular DiseasesMinistry of EducationBeijing Collaborative Innovation Center for Cardiovascular DisordersBeijing Institute of HeartLung&Blood Vessel DiseaseBeijing 100029China

Arrhythmogenic right ventricular dysplasia/cardiomyopathy(ARVD/C)is a genetic cardiac muscle disease that accounts for approximately 30%sudden cardiac death in young adults.The Ser358Leu mutation of transmembrane protein 43(TMEM43)was commonly identified in the patients of highly lethal and fully penetrant ARVD subtype,ARVD5.Here,we generated TMEM43 S358L mouse to explore the underlying mechanism.This mouse strain showed the classic patholo.gies of ARVD patients,including structural abnormalities and cardiac fibrofatty.TMEM43 S358L mutation led to hyper-activated nuclear factor kB(NFkB)activation in heart tissues and primary cardiomy.ocyte cells.Importantly,this hyper activation of NF-κB directly drove the expression of pro-fibrotic gene,transforming growth factor beta(TGFβ),and enhanced downstream signal,indicating that TMEM43 S358L mutation up-regulates NF-κB-TGFβ signal cascade during ARVD cardiac fibrosis.Our study partially reveals the regulatory mechanism of ARVD development.

关键词： TMEM43 ARVD NF-κB TGFβ fibrosis knock-in mouse

来源：

维普期刊数据库评论

在线全文

维普期刊数据库

学校读者我要写书评

暂无评论

TALEN-Mediated FLAG-Tagging of Endogenous Histone Methyltransferase DOT1L

引用

Advances in Bioscience and Biotechnology 2017年第9期8卷 311-323页

作者： Cheng An Guangjing Zhu Suzanne N. Martos Xue Feng Haimou Zhang Yankai Jia Zhibin Wang Guang’an Men Hospital China Academy of Chinese Medical Sciences Beijing China Laboratory of Human Environmental Epigenomes Department of Environmental Health Sciences Bloomberg School of Public Health Johns Hopkins University Baltimore MD USA School of Life Sciences Hubei University Wuhan China GENEWIZ Suzhou Suzhou China Fenxian Central Hospital Shanghai China Department of Oncology and Sidney Kimmel Comprehensive Cancer Center School of Medicine Johns Hopkins University Balti-more MD USA

Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, colorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.

关键词： DOT1L Flag TALEN knock-in

来源：

维普期刊数据库评论

在线全文

维普期刊数据库

学校读者我要写书评

暂无评论

The Transactivating Function 2 (AF-2) of Estrogen Receptor (ER) <i>α</i>Is Indispensable for ER<i>α</i>-Mediated Physiological Responses and AF-1 Activity

引用

Open Journal of Endocrine and Metabolic Diseases 2013年第4期3卷 12-19页

作者： Yukitomo Arao Katherine J. Hamilton Kenneth S. Korach Receptor Biology Section Laboratory of Reproductive and Developmental Toxicology National Institute of Environmental Health Sciences/NIH Research Triangle Park North Carolina USA

Estrogen has various physiological functions and the estrogen receptor (ER) is a key regulator of those functions. ERα is a ligand-dependent transcription factor and that activity is mediated by the transactivating function-1 (AF-1) in the N-terminal domain and transactivating function-2 (AF-2) in the C-terminal ligand-binding domain. The functions of ERα AF-1 and AF-2 have been characterized by various in vitro experiments, however, there is still less information about the in vivo physiological functions of ERα AF-1 and AF-2. Recently, we established a genetically mutated ERα AF-2 knock-in mouse (AF2ERKI) that possessed L543A, L544A mutated-ERα. This AF-2 core mutation disrupted AF-2 function and resulted in ERα null phenotypes. This mouse model revealed that proper AF-2 core structure and function were indispensable for ERα-mediated physiological responses and AF-1 functionality. AF2ER mutation reverses the ERα antagonists to agonists and that activity is mediated by AF-1 solely. The pure antagonist, ICI182780/fulvestrant, activated several estrogen-mediated physiological responses in the AF2ERKI mouse. The AF2ERKI mouse model will be useful to discern estrogen physiological functions which involve AF-1.

关键词： knock-in Mouse SERMs Antagonist Reversal

来源：

维普期刊数据库评论

在线全文

维普期刊数据库

学校读者我要写书评

暂无评论

基于CRISPR/Cas9技术的荧光标记IFNGR2敲入细胞系构建

基于CRISPR/Cas9技术的荧光标记IFNGR2敲入细胞系构建

引用

作者：樊浩军事科学院

学位级别：硕士

干扰素γ(IFNγ)信号通路在免疫反应、炎性反应、抗肿瘤免疫等过程中发挥至关重要的作用,其功能状态是决定肿瘤免疫治疗成败的关键。IFN-γ发挥作用主要是通过结合膜表面受体IFN-γR并激活其下游的信号分子,调节相关基因的转录和表达。I... 详细信息

干扰素γ(IFNγ)信号通路在免疫反应、炎性反应、抗肿瘤免疫等过程中发挥至关重要的作用,其功能状态是决定肿瘤免疫治疗成败的关键。IFN-γ发挥作用主要是通过结合膜表面受体IFN-γR并激活其下游的信号分子,调节相关基因的转录和表达。IFN-γR由两种亚单位组成:IFNγR1(IFNGR1)和IFNγR2(IFNGR2)。IFNGR1在细胞中表达相对过剩,IFNGR2的表达受到机体严格的调控。IFNGR2的表达水平直接决定了IFNγ信号通路反应性,因此调控IFNGR2的表达是提高免疫治疗效果的潜在突破口,阐明IFNGR2的调控机制有助于深入理解IFNγ信号通路有关的免疫反应和疾病发生过程,具有潜在的临床意义。为了发现调控IFNGR2表达和IFNγ信号通路的基因、小分子化合物等,寻找研究其调控方式及其分子机制的新思路和新策略,本文采用CRISPR/Cas9基因编辑技术,在IFNGR2基因末端原位敲入融合表达的绿色荧光蛋白(EGFP),建立IFNGR2报告细胞系,通过绿色荧光强度表征活细胞中内源IFNGR2的表达情况,实现对IFNGR2受调控程度的快速、通量化监测。我们采取了两种策略来实现EGFP的敲入,分别为CRISPR介导的微同源末端连接技术(microhomology-mediated end-joining,MMEJ)和CRISPR介导的同源重组技术(homologous recombination,HR)。两种敲入策略均通过CRISPR-Cas9系统造成IFNGR2基因最后一个外显子末端的DNA双链断裂,同时引入供体载体中含有EGFP编码序列以及与DNA断裂位置的同源序列,运用细胞内的多种DNA修复机制实现基因敲入。两种方法的主要区别在于,MMEJ利用细胞内的微同源重组修复机制来完成基因编辑,所需同源序列长度为20bp左右;HR利用细胞内同源重组修复机制,需要的同源序列较长,为200bp左右。微同源末端连接不受细胞周期的限制,敲入效率更高;而同源重组因为有更长的同源序列,可以更为灵活地选择CRISPR-Cas9靶位,两种策略各具优势,具有一定互补性。在构建knock-in细胞系时,我们分别选取了小鼠黑色素瘤细胞系B16和小鼠细胞前列腺癌细胞系My C-Ca P,以及人源结肠癌细胞系HCT116。我们将Cas9载体(携带靶向IFNGR2基因sg RNA)和供体载体(携带EGFP编码序列及敲入位点同源序列)同时转入细胞系,利用抗生素抵抗基因筛选获得候选细胞克隆。通过提取基因组DNA和PCR鉴定,明确EGFP编码序列正确插入;对候选克隆进行EGFP表达筛选,选取EGFP表达水平相对较高的克隆;最后先验证这些克隆具有正常的IFNγ反应性,确定稳定细胞系构建成功;在此基础上,进一步检测IFNGR2调控因子对细胞系IFNGR2和EGFP表达水平的调控作用。我们发现肿瘤坏死因子(tumor necrosis factor alpha,TNF-α)和奥沙利铂(Oxaliplatin)可以明显促进敲入细胞系中Ifngr2和EGFP表达水平。TNF-α是一类十分重要的细胞因子,调控各种免疫过程并可诱导肿瘤细胞凋亡。有研究提示TNF-α可以通过NF-κB信号通路调控IFNGR2表达。Oxaliplatin作为一种抗肿瘤药物,广泛地使用在多种癌症的治疗方案中,最新研究表明其具有明显促进抗肿瘤免疫治疗的作用。我们的结果提示Oxaliplatin具有促抗肿瘤免疫的作用,是通过调控肿瘤细胞IFNGR2表达进而提高肿瘤细胞对IFNγ的敏感性实现的。本研究基于CRISPR/Cas9基因编辑技术,在My C-Ca P前列腺癌细胞系和小鼠B16黑色素瘤细胞系和人结肠癌细胞系HCT116中Ifngr2末端敲入EGFP基因。基因组水平确认成功将EGFP基因敲入B16细胞系和My C-Ca P细胞系Ifngr2基因末端,其中B16细胞中可检测到绿色荧光的表达。进一步的,我们在构建的B16 Ifngr2-EGFP细胞系中对细胞因子和抗肿瘤药物调控IFNGR2表达进行了初步研究。结果表明,B16 Ifngr2-EGFP细胞系中,TNFα和Oxaliplatin可以上调IFNGR2的表达,绿色荧光蛋白的表达变化能够反映IFNGR2的表达情况。本文建立的细胞系,为深入研究IFNGR2表达、IFNγ信号通路调控和免疫调控活性药物筛选等研究提供了有力的技术支撑。

关键词：干扰素γ信号通路 IFNGR2 knock-in 表达调控

来源：

同方学位论文库评论

在线全文

同方学位论文库

学校读者我要写书评

暂无评论

基于CRISPR/Cas9靶向基因组编辑技术的HEK293-MMP12-T2A-luciferase-KI细胞系建立及实验研究

基于CRISPR/Cas9靶向基因组编辑技术的HEK293-MMP12-T2A-lucifera...

引用

作者：杜春花陕西师范大学

学位级别：硕士

基质金属蛋白酶12（Matrix metalloproteinase 12,MMP12）是基质金属蛋白酶家族（Matrix metalloproteinases,MMPs）的成员之一,在正常生理条件下,可以维持细胞外基质（Extracellularmatrix,ECM）的正常结构和功能,在细胞外基质的降解和重构等方面发挥着重要的作用。近年来多项研究结果表明,MMP12参与炎症、心血管疾病和肿瘤发生等病理过程。因此,研究MMP12表达调控机制将为其功能研究及相关疾病的治疗提供新的思路和有力的科学依据。CRISPR/Cas9基因编辑技术为靶向基因组编辑提供了一种简便、高效的方法,可以在真核细胞中实现高度灵活和特异的基因组编辑,目前已经广泛用于基因功能的研究。为了研究MMP12基因的转录调控及其功能,本课题在HEK293细胞系中,利用CRISPR/Cas9技术将荧光素酶（luciferase）报告基因定点整合到MMP12基因下游,经过正负药筛以及有限稀释法克隆化后,对细胞系进行基因型检测及测序鉴定,证实了 HEK293细胞系中外源荧光素酶报告基因的定点插入,成功构建了受内源性MMP12基因启动子调控荧光素酶活性的HEK293-MMP12-T2A-luciferase-KI 细胞系。为了验证HEK293-MMP12-T2A-luciferase-KI细胞系的可靠性,我们首先选择了可能对MMP12有激活作用的三个转录因子STAT3、AP-1、SP-1分别转染该细胞系,对细胞系中luciferase的表达活性进行检测,并将三个转录因子转染HEK293细胞,通过检测MMP12基因的mRNA表达加以验证,结果显示,HEK293-MMP12-T2A-luciferase-KI细胞系中luciferase的表达与野生型HEK293细胞系中mRNA的表达趋势一致,表明HEK293-MMP12-T2A-luciferase-KI细胞系中荧光素酶的表达能准确反映内源性MMP12基因启动子活性,证明了实验方法的可行性和所构建细胞系的可靠性;接下来,我们利用构建好的细胞系进行了与炎症相关的小分子药物筛选,通过luciferase活性检测,发现一种小分子药物Tanshinone I可以抑制MMP12基因的表达,与Tanshinone I处理后的HEK293细胞系中MMP12的mRNA表达趋势一致,这进一步证明了 HEK293-MMP12-T2A-luciferase-KI细胞系的可靠性,与此同时,筛选出的小分子药物为研究以MMP12为治疗靶点的潜在药物开发提供了新的思路。因此,这一新型细胞系的构建将有助于研究MMP12基因的转录调控及其生物功能,在筛选作用于MMP12基因的转录因子和小分子药物方面具有较大的潜力。本课题的具体实验流程和研究内容分为以下几个部分:1.打靶载体的构建:为了构建打靶载体,我们在sgRNA在线设计网站（http://crispr-era.***.edu/index.jsp）设计并选择了四个针对 MMP12 基因终止子下游区域的sgRNA,每个sgRNA被克隆到pU6/sgRNA1.0载体,并利用T7E1对四个sgRNA1-4分别进行生物活性检测,选择生物活性最高的sgRNA3,连入载体 pUC19/CMV-Cas9,获得打靶载体 pUC19/CMV-Cas9-U6-MMP12 sgRNA3。2.靶向MMP12基因的供体载体的构建:在NCBI查找获知MMP12基因序列,将PCR获得的针对MMP12打靶区域的上游、下游同源臂,分步连入实验室现有的载体,成功构建了靶向MMP12基因的供体载体。其中上游同源臂后连着 T2A-luciferase 元件和正筛元件 CMV-eGFP-T2A-Neomycin-SV40pA,然后是下游同源臂以及紧随其后的负筛元件PGK-TK-T2A-mCherry-SV40pA。由于供体载体含有报告基因和正、负筛元件,因此可以通过直接观察eGFP及mCherry的表达区分随机整合细胞和同源重组细胞,通过TK自杀基因能剔除随机整合的细胞,通过Neomycin筛选基因能富集同源重组成功的细胞,从而有效提升同源重组细胞的占比。3.HEK293-MMP12-T2A-luciferase-KI细胞系的构建及鉴定:首先,将打靶载体pUC 19/CMV-Cas9-U6-MMP 12 sgRNA3 和供体载体用脂质体法共转染HEK293细胞,通过药物G418和GCV分别进行正、负筛选后,将生长稳定的细胞进行有限稀释法克隆化。然后,对获得的多个单克隆细胞系进行luciferase活性检测,选择luciferase表达活性较高的几个单克隆细胞系提基因组,通过PCR进行外源基因定点插入检测。最后,选择PC.R鉴定阳性的单克隆细胞进行进一步测序检测,成功获得外源基因定点整合的HEK293-MMP12-T2A-lucifer

关键词： MMP12 CRISPR/Cas9技术 knock-in 转录调控药物筛选

来源：

同方学位论文库评论

在线全文

同方学位论文库

学校读者我要写书评

暂无评论

CRISPR-Cas9-mediated genome editing in widespread pathogen Eimeria tenella

CRISPR-Cas9-mediated genome editing in widespread pathogen E...

引用

第四届全国禽病分子生物技术青年工作者会议

作者： Xinming Tang Xianyong Liu Jingxia Suo Xiuling Tian Xun Suo National Animal Protozoa Laboratory & College of Veterinary Medicine China Agricultural University

The prokaryotic CRISPR(clustered regularly interspaced short palindromic repeat)-Cas9,an RNA-guided endonuclease,is a new powerful technique for genome editing and has been widely employed to study gene function in a wide variety of organisms.Eimeria tenella,causes coccidiosis in chickens,is a model organism for studying the protozoan parasites and both transient and stable transfection system were development successfully.In the present study,the CRISPR-Cas9 system has been adapted to E.tenella.We introduced the Cas9 nucleaseEYFP into E.tenella and generated guide RNA(gRNA)expression vector by using the E.tenella U6 promoter.E.tenella histone 4 promoter and a 90-bp nuclear localization sequence(NLS)were introduced into the vector to regulate Cas9-EYFP expression.We demonstrated that Cas9 is able to introduce site-specific DNA doublestrand breaks in the E.tenella genome that can be repaired through homologous recombination by red fluorescent protein(RFP)reporter gene knock-in the sporozoites surface antigen 13(SAG 13)open reading frame(ORF).We selected two specific sites in SAG 13 ORF as the efficiency of reporter gene knock-in the target site is relatively low by CRISPR-Cas9 system.We also introduced TgDHFR gene into the donor plasmid to improve the selection efficiency as TgDHFR was promoted by endogenous SAG 13 promoter which exhibits resistance to pyrimethamine.We cotransfected the E.tenella sporozoites with the Cas9 plasmid(HCEA-U6-sgRNA)and the donor fragment with 700bp homologous repair templates from PCR products using nuclear transfection.We inoculated two-day-old AA broilers with a dosage of 2 x 10 transfected sporozoites via cloacal route.We got a co-expressing EYFP and RFP E.tenella population from fecal samples demonstrated that CRISPR-Cas9 efficient in reporter gene knock-in E.tenella genome.The efficacy of CRISPR-Cas9 and target gene deletion using CRISPR-Cas9 in E.tenella parasite should be further investigated.Our results establish methodologies for introducing desired modifications in the E.tene

关键词： CRISPR-Cas9 Eimeria tenella knock-in genome editing

来源： cnki会议评论

在线全文

cnki会议

学校读者我要写书评

暂无评论

欢迎您,

建议与咨询留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

时间限定

文献类型

馆藏选择

核心期刊

语言

文献类型

帮助

文字说明：

检索规则说明：

检索范例：

分类表

所选分类

限定检索结果

文献类型

馆藏范围

日期分布

学科分类号

主题

机构

作者

语言

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

请选择保存的检索档案：

请选择收藏分类：

通借通还

欢迎您,

建议与咨询 留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

时间限定

文献类型

馆藏选择

核心期刊

语言

文献类型

帮助

文字说明：

检索规则说明：

检索范例：

分类表

所选分类

限定检索结果

文献类型

馆藏范围

日期分布

学科分类号

主题

机构

作者

语言

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

在线全文

请选择保存的检索档案： 新增检索档案 确定 取消

请选择收藏分类： 新增自定义分类 确定 取消

通借通还

建议与咨询留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

请选择保存的检索档案：

请选择收藏分类：