Live imaging of X chromosome inactivation and reactivation dynamics

The epigenetic phenomenon called X chromosome inactivation plays critical roles in female development in eutherian mammals, and has attracted attention in the fields of developmental biology and regenerative biology in efforts to understand the pluripotency of stem cells. X chromosome inactivation is routinely studied after cell fixation, but live imaging is increasingly being required to improve our understanding of the dynamics and kinetics of X chromosome inactivation and reactivation processes. Here, we describe our live imaging method to monitor the epigenetic status of X chromosomes using a gene knock‐in mouse strain named “Momiji” and give an overview of the application of this strain as a resource for biological and stem cell research.     

Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene

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CRISPR/dCas9系统在活细胞成像领域的研究进展

研究不同基因、染色体以及基因与染色体之间的时空关系在遗传学、发育生物学和生物医学等领域具有重要意义。CRISPR/Cas9基因编辑技术具有优异的靶向性,已经成为应用最广泛的基因编辑工具。近年来,研究人员基于Cas9的核酸酶失活突变体dCas9发展了一系列先进的活细胞成像技术,为染色质、基因组特定位点的高分辨成像提供了快速、方便的研究工具。文中从细胞递送方式、荧光信号优化以及正交多色成像3个方面对CRISPR/dCas9系统在活细胞成像中的研究进展进行了综述,并对该领域的发展趋势进行了展望。     

组蛋白去乙酰化酶抑制剂调控干细胞分化与体细胞重编程的研究进展

表观遗传调控,如组蛋白乙酰化修饰,是决定干细胞分化方向的重要机制。组蛋白去乙酰化酶抑制剂(HDACi)通过影响不同亚类的组蛋白去乙酰化酶(HDAC)活性,提高组蛋白乙酰化水平,调控基因表达,从而影响胚胎干细胞自我更新,以及沿神经元、心肌和造血等细胞谱系的定向分化。HDACi类小分子化合物在体细胞重编程中也有广泛的应用,可替代致癌因子c-Myc和Klf4,促进体细胞克隆。研究显示,HDACi的效应与药物剂量、细胞类型和细胞分化状态密切相关。本文主要阐述了HDACi在干细胞分化和体细胞重编程中的应用进展,并对所涉及的分子通路进行讨论,有助于揭示干细胞定向分化的关键分子机制,优化干细胞定向分化诱导策略,对干细胞诱导分化具有重要的理论和实用价值。     

X chromosome reactivation and regulation in cloned embryos

Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.     

Generation of genome-modified Drosophila cell lines using SwAP

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.     

SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre‐implantation embryo development

ObjectivesGene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species.Materials and methodsTo analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR.ResultsOur results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.     

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18 Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.     

Deciphering the heterogeneity in DNA methylation patterns during stem cell differentiation and reprogramming

Background

Human induced pluripotent stem cells (iPSCs) have a wide range of applications throughout the fields of basic research, disease modeling and drug screening. Epigenetic instable iPSCs with aberrant DNA methylation may divide and differentiate into cancer cells. Unfortunately, little effort has been taken to compare the epigenetic variation in iPSCs with that in differentiated cells. Here, we developed an analytical procedure to decipher the DNA methylation heterogeneity of mixed cells and further exploited it to quantitatively assess the DNA methylation variation in the methylomes of adipose-derived stem cells (ADS), mature adipocytes differentiated from ADS cells (ADS-adipose) and iPSCs reprogrammed from ADS cells (ADS-iPSCs).

Results

We observed that the degree of DNA methylation variation varies across distinct genomic regions with promoter and 5’UTR regions exhibiting low methylation variation and Satellite showing high methylation variation. Compared with differentiated cells, ADS-iPSCs possess globally decreased methylation variation, in particular in repetitive elements. Interestingly, DNA methylation variation decreases in promoter regions during differentiation but increases during reprogramming. Methylation variation in promoter regions is negatively correlated with gene expression. In addition, genes showing a bipolar methylation pattern, with both completely methylated and completely unmethylated reads, are related to the carbohydrate metabolic process, cellular development, cellular growth, proliferation, etc.

Conclusions

This study delivers a way to detect cell-subset specific methylation genes in a mixed cell population and provides a better understanding of methylation dynamics during stem cell differentiation and reprogramming.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-978) contains supplementary material, which is available to authorized users.     

利用CRISPR/Cas9系统构建稳定敲除TrxR1基因的HCT-116细胞株北大核心CSCD

为更好地研究靶向硫氧还蛋白还原酶1的小分子化合物的细胞内靶点选择性,利用CRISPR/Cas9系统构建稳定敲除TrxR1基因(编码硫氧还蛋白还原酶1)的HCT-116细胞株。首先根据TrxR1基因序列和CRISPR/Cas9靶点设计原则,设计并选择合适的敲除位点,再根据敲除位点序列设计敲除TrxR1基因的sgRNA干扰序列,以pCasCMV-Puro-U6空质粒载体为骨架构建能表达该sgRNA干扰序列的重组质粒。质粒共转染至HCT-116细胞后,利用嘌呤霉素筛选TrxR1敲除的HCT-116细胞,通过DNA测序、免疫蛋白印迹、TRFS-green荧光探针和细胞内TrxR1酶活力检测等方法鉴定和验证HCT-116细胞的TrxR1基因敲除效果。进一步通过CCK-8实验初步研究靶向TrxR1小分子化合物对细胞内TrxR1酶活力和细胞增殖力抑制的相关性。结果显示,表达sgRNA干扰序列的重组质粒可以敲除HCT-116细胞中TrxR1基因,筛选获得的稳定敲除细胞HCT116-TrxR1-KO中无TrxR1蛋白表达,而靶向TrxR1小分子抑制剂对该细胞无TrxR1酶活力和细胞增殖力抑制效果。本研究利用CRISPR/Cas9系统成功构建了HCT-116的TrxR1基因敲除的稳定细胞株,为进一步研究TrxR1在相关疾病的发生机制和治疗中的作用奠定了基础。     

Live cell imaging of the intracellular compartmentalization of the contaminate benzo[a]pyrene

This study investigates the cellular response of murine hepatoma cells to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) using two‐photon and confocal laser scanning microscopy. The intracellular distribution of B[a]P and the B[a]P/AhR complex was visualized time‐ and concentration‐dependent for up to 48 h of exposure. B[a]P was predominantly found in lipid droplets, endoplasmic reticulum and lysosomes, where B[a]P is collected and forms large aggregates. Changes in mitochondrial membrane potential and bleb formation due to high B[a]P concentrations were observed. The imaging data presented in this study provide new insights into the systemic cellular regulation following B[a]P exposure. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

CRISPR/Cas9介导的RPSA基因敲除细胞系的建立及其应用

[目的]利用CRISPR/Cas9技术建立RPSA基因缺失的乳仓鼠肾细胞(baby hamster kidney cells,BHK21)细胞系,为开展RPSA调控病毒复制机制研究提供工具;同时,初步探究RPSA对塞内卡病毒复制的影响.[方法]根据GenBank中仓鼠的RPSA基因序列找到产生不同转录本的共同外显子段,...     

Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as β-lactamase.     

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.     

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.     

利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

LMNA基因编码A型和C型核纤层蛋白,参与细胞核核膜的组织,影响基因组稳定性并对细胞分化产生影响。人类肿瘤中LMNA表达异常普遍存在,其突变造成多种核纤层蛋白病,如Emery-Dreifuss肌营养不良症(Emery-Dreifussmusculardystrophy,EDMD)、扩张型心肌病(dilatedcardiomyopathy,DCM)和儿童早老症(Hutchinson-Glifordprogeriasyndrome,HGPS)等。为进一步研究LMNA在细胞内的功能,本研究利用CRISPR/Cas9技术对体外培养的293T与HepG2细胞株的LMNA基因进行编辑,获得两株LMNA基因敲除(LMNA KO)的稳定细胞系。与野生型相比,LMNAKO细胞系增殖能力相对减弱,凋亡增加。同时,细胞形态上也发生显著改变,核膜凹凸不平。本研究首次报道了LMNA KO永生细胞系构建和形态研究结果,为后续LMNA基因功能研究和致病突变体研究奠定基础。     

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT‐PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild‐type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto‐ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G₂/M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G₂/M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.