微细胞介导人14号染色体自A9细胞转移至DT40细胞

为了获得含人14号染色体的DT40细胞,用于人抗体基因的表达研究.本研究利用微细胞介导的染色体转移技术,将A9细胞中的人14号染色体转移至DT40细胞中.首先,摸索秋水仙胺诱导A9细胞微核形成最佳浓度与最佳时间,以终浓度为10 mg/mL的细胞松驰素B破坏细胞骨架,离心分离微细胞,获得的微细胞依次经8μm、5μm、3μm滤膜过滤后与受体细胞DT40融合,细胞铺板后加入G418筛选.然后,对长出的抗性克隆进行基因组DNA检测及FISH杂交,分析人14号染色体在DT40杂合细胞克隆中的存在情况.结果显示,成功获得含人14号染色体的DT40(#14)细胞,三轮试验共获得抗性克隆30个,人14号染色体有效转移率为1×10-6.实验结果表明,人14号染色体完整的自A9细胞转移至DT40细胞,获得的DT40(#14)细胞可用于制备含人抗体基因的人类人工染色体,用于人抗体基因的表达研究.     

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.     

鼻咽癌细胞系与永生化的鼻咽上皮细胞系蛋白质表达差异分析

鼻咽癌对我国南部居民的健康造成严重的威胁.为了研究鼻咽癌的发病机理,本研究采用了蛋白质组学技术分析和比较了鼻咽癌细胞系（HNE1和CNE1）与永生化的鼻咽上皮细胞系的蛋白质表达谱.采用双向凝胶电泳分离提取的全细胞蛋白质,通过PDQuest软件分析找出在肿瘤中表达变化的蛋白质点,用基质辅助激光解析电离飞行时间串联质谱(MALDI- TOF/TOF-MS)进行鉴定.共得到了15个在肿瘤细胞系中表达上调和18个在肿瘤细胞系中表达下调的蛋白质,并对其中一些蛋白质的表达进行免疫印迹的验证.这些表达差异的蛋白质与细胞的增殖和调亡、癌症的转移,细胞骨架,信号传导等有关.本研究鉴定了一批可能作为鼻咽癌治疗的药物靶标的蛋白质,并对研究鼻咽癌发病机理提供了相关的线索.     

The Symplechosome: a Unique Cell Organelle of some Basidiomycetes*

An unusual cell organelle of some basidiomycetes, the symplechosome, is described and illustrated in detail using Saccoblastia farinacea as an example. Symplechosomes are structurally similar, but not identical to “classical” dictyosomes of green plants and animals. As is typical for dictyosomes, each symplechosome consists of a stack of platelike cisternae. The central portions of the symplechosome-cisternae are flattened, and adjacent cisternae are separated in the mid-region by an intercisternal space of constant width. In contrast to dictyosomes, the intracisternal spaces are completely obliterated in the central area, and hexagonally arranged bars extend between adjacent cisternae. Identical bars often connect the symplechosomes with mitochondria. Symplechosomes are highly complex-structured organelles which differ significantly from the simple individual Golgi cisternae or “Golgi bodies” observed in asco- and basidiomycetes.     

Biotechnology and the chicken B cell line DT40

Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection.     

The avian chB6 alloantigen induces apoptosis in DT40 B cells

In avian species, B-lymphocytes develop in the bursa of Fabricius. Cells developing in the bursa are subject to signals regulating their survival, with the majority of cells dying by apoptosis within the bursa. However, the molecules delivering the signals influencing this life and death decision remain enigmatic. We have previously shown that antibodies against the chB6 alloantigen present on avian B-lymphocytes can induce a rapid form of cell death. Here we extend this finding by showing that anti-chB6 antibodies induce true apoptosis in DT40 cells without visible membrane damage. This apoptosis results in DNA degradation and morphologic changes characteristic of apoptosis. Furthermore, this apoptosis is coincident with a loss of mitochondrial membrane potential and is inhibited by either overexpression of bcl-x(L) or the presence of inhibitors of caspase 8, 9, or 3 activity. Collectively these data argue that chB6 may function as a novel death receptor on avian B-lymphocytes and support the use of DT40 as an amenable model to study the signaling involved in chB6-induced apoptosis.     

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies

Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce Ig M-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line(DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.     

The chicken HPRT gene: a counter selectable marker for the DT40 cell line

We describe the cloning, characterisation and chromosomal mapping of the chicken hprt gene together with the construction of two counter selectable hprt-/- DT40 derived cell lines. One of these cell lines contains a stably integrated gene encoding a conditionally active cre recombinase and thus allows efficient manipulation of targeted loci by site-specific recombination. These cell lines will enhance the utility of the hyper-recombinogenic DT40 cell line as a system for the genetic analysis of cell autonomous functions in vertebrates and as a tool for mammalian chromosome engineering.     

Acetyl-L-carnitine suppresses apoptosis of thioredoxin 2-deficient DT40 cells

To elucidate the mechanism by which l-carnitine and related metabolites inhibited mitochondria-dependent apoptosis, we used conditional TRX2-knockout DT40 cells (TRX2^−/−) and compared the properties of signaling pathways leading to apoptosis in the wild and TRX2^−/− cells. Caspase-3 and 9, but not caspase-8, were strongly activated in TRX2^−/− cells but not in wild cells. TRX2^−/− cells generated large amounts of reactive oxygen species that markedly decreased cellular glutathione levels both in cytosol and mitochondria. We found that the critical thiol groups of adenine nucleotide translocator (ANT) were oxidized more easily in TRX2^−/− cells than in wild cells and that the reduced form, but not oxidized form, of ANT selectively bound to TRX2. Cytochrome c and SOD1 were released from mitochondria more easily in TRX2^−/− cells than in wild cells. All these phenomena observed with TRX2^−/− cells were effectively inhibited by acetyl-l-carntine but not l-carnitine. Thus, acetyl-l-carnitine effectively suppressed the oxidative stress in and around mitochondria thereby preventing mitochondrial signaling pathway leading to apoptosis.     

The DT40 web site: sampling and connecting the genes of a B cell line

Thousands of new vertebrate genes have been discovered and genetic systems are needed to address their functions at the cellular level. The chicken B cell line DT40 allows efficient gene disruptions due to its high homologous recombination activity. However, cloning the gene of interest is often cumbersome, since relatively few chicken cDNA sequences are present in the public databases. In addition, the accumulation of multiple mutations within the same cell clone is limited by the consumption of one drug-resistance marker for each transfection. Here, we present the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html), which includes a comprehensive database of chicken bursal ESTs to identify disruption candidate genes and recyclable marker cassettes based on the loxP system. These freely available resources greatly facilitate the analysis of genes and genetic networks.     

Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing

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Highlights

• •Quantitative proteome of neonatal, young, and aged OPCs.
• •50% of the proteome is differentially expressed between neonatal and adult OPCs.
• •Myelin proteins are increased, and cholesterol synthesis proteins decreased with age.
• •Proteins associated with other neurodegenerative diseases are increased in aged OPCs.

     

Development of a Targeted Flip-in System in Avian DT40 Cells

Gene-targeting to create null mutants or designed-point mutants is a powerful tool for the molecular dissection of complex phenotypes involving DNA repair, signal transduction, and metabolism. Because gene-targeting is critically impaired in mutants exhibiting attenuated homologous recombination (HR), it is believed that gene-targeting is mediated via homologous recombination, though the precise mechanism remains unknown. We explored gene-targeting in yeast and avian DT40 cells. In animal cells, gene-targeting is activated by DNA double strand breaks introduced into the genomic region where gene-targeting occurs. This is evidenced by the fact that introducing double strand breaks at targeted genome sequences via artificial endonucleases such as TALEN and CRISPR facilitates gene-targeting. We found that in fission yeast, Schizosaccharomyces pombe, gene-targeting was initiated from double strand breaks on both edges of the homologous arms in the targeting construct. Strikingly, we also found efficient gene-targeting initiated on the edges of homologous arms in avian DT40 cells, a unique animal cell line in which efficient gene-targeting has been demonstrated. It may be that yeast and DT40 cells share some mechanism in which unknown factors detect and recombine broken DNA ends at homologous arms accompanied by crossover. We found efficient targeted integration of gapped plasmids accompanied by crossover in the DT40 cells. To take advantage of this finding, we developed a targeted flip-in system for avian DT40 cells. This flip-in system enables the rapid generation of cells expressing tag-fused proteins and the stable expression of transgenes from OVA loci.     

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP₃R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP₃R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP₃R-dependent Ca²⁺ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca²⁺ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP₃Rs may play a role in the delayed apoptotic response observed in these cells.     

Protein evolution by hypermutation and selection in the B cell line DT40

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible.     

Study on Organelle DNA Within the Generative Cell and Sperm Cells in Pharbitis

The organelle DNA in generative cell and its behavior during spermatogenesis in Pharbitis limbata and P. purpurea were observed by epifluorescence microscopy stained with 4',-6-diamidino-2-phenylindole (DAPI). In these two species, the generative cell is long and thin in which a great amount of cytoplasmic DNA is present. Most pairs of sperm cells are isomorphic, in which one end is obtuse and the other is elongate, but in a few pairs dimorphi sperms are present. The nucleus is located at one end of the cell. A lot of cytoplasmic DNA are distributed randomly throughout the cytoplasm. The size of organelle nucleoids and their fluorescence intensity are different in a sperm cell. The features of generative cell and sperm cell, and behavior of cytoplasmic DNA are similar in P. limbata and P. purpurea. The obvious differences between them are that the size and fluorescence intensity of organelle nucleoids in P. purpurea are respectively smaller and weaker than in P. limbata. The results showed that morning glory has potential of biparental or paternal cytoplasmic in heritance. Isomorphism and dimorphism of sperms, and the relationship between the ratio of nucleus and cytoplasm in sperm cell and the plastid biparental inheritance are discussed.     

The Legionella pneumophila Effector VipA Is an Actin Nucleator That Alters Host Cell Organelle Trafficking

Legionella pneumophila, the causative agent of Legionnaires'' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.     

Changes in Organelle Movement in the Nuclear Region during the Cell Cycle of Adiantum Protonema

Movements of organelles in the nuclear region as the cell cycleprogresses in single-celled protonemata of Adiantum capillus-veneriswere examined by digital image processing techniques and microscopyof particle movement. Organelles in the nuclear region werenot very crowded and moving directionally along the longitudinalaxis of the filamentous cell in the G₁ and S phases. They beganto gather and accumulate in the nuclear region in early G₂ phase,after which directional movement changed to undirectional Brownianmotion-like movement in late G₂ phase. Movement of organelleslocated on the lateral surface of the nucleus slowed after premitoticpositioning of nucleus and lasted until the nucleolus disappeared.Movement of organelles in the cytoplasm surrounding the nucleoplasmresumed just after the nucleolus disappeared, whereas organelleslocated in the outer regions of the apical and basal surfacesof the nucleus moved rapidly during prophase but did not moveduring metaphase, movement being resumed after chromosome separation.Thus, organelle movement in the nuclear region showed temporaland spatial change during the cell cycle. (Received August 24, 1983; Accepted December 28, 1983)