这项工作得到了CIHR和加拿大NSERC资助。 VIT是一个新的CIHR调查和Concordia大学基因组学研究主席，细胞生物学和老化。

材料和方法酵母菌株和生长条件。 丰富YEPD培养基中生长的野生型菌株BY4742（MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0）（1％酵母提取物，2％蛋白胨，2％葡萄糖） 。细胞培养在30 ° C旋转摇晃锥形瓶在200 RPM“瓶体积/中等音量”5:1的比例。 原油线粒体分数隔离<ol><li>长为48小时1升的野生型菌株BY4742文化</li><li>倒入500毫升NALGENE离心管文化。平衡离心负载和收获细胞在室温下在3000 XG 5分钟。 </li><li>倒出上清液在250毫升的dH2O颗粒和悬浮细胞。在3000 XG在室温下5分钟，离心沉淀细胞。 </li><li>倒出上清液在250毫升的dH2O颗粒和悬浮细胞。在3000 XG在室温下5分钟，离心沉淀细胞。 </li><li>确定细胞沉淀湿重。 </li><li>重悬浮颗粒细胞，在数码地面电视的缓冲缓冲/克（湿重）细胞[2毫升]。 </li><li>将细胞悬液50毫升猎鹰塑料管。 </li><li> 70在摇床转速在30 ° C 20分钟的旋转管</li><li>在3000 XG在室温下5分钟，离心收集细胞。 </li><li> Zymolyase缓冲区中的颗粒细胞重悬于无Zymolyase毫升缓冲/克（湿重）细胞[7 ]。 </li><li>在3000 XG在室温下5分钟，离心沉淀细胞。 </li><li> Zymolyase缓冲区中的颗粒细胞重悬于无Zymolyase毫升缓冲/克（湿重）细胞[7 ]。 </li><li>细胞悬液转移到一个玻璃瓶中。 </li><li>添加粉末Zymolyase - 100T [1毫克Zymolyase - 100T /克（湿重）的细胞，细胞悬液。 </li><li> 70在摇床转速旋转烧瓶与细胞悬液，在30 ° C 30分钟</li><li>传输原生质球形成由于Zymolyase - 100T至50毫升塑料离心管细胞壁的消化。 </li><li>佩莱原生质球离心8分钟在2200 XG在4 ° C </li></ol> **所有后续步骤，应在4℃或冰面上进行。原生质球悬浮应轻轻削减提示使用移液器，以避免破坏细胞器处理** <ol start="18"><li>在同质化冰冷的缓冲缓冲/克（湿重）细胞[6.5毫升]重悬沉淀的原生质球。 </li><li>佩莱原生质球离心8分钟在2200 XG在4 ° C </li><li>在同质化冰冷的缓冲缓冲/克（湿重）细胞[6.5毫升]重悬沉淀的原生质球。 </li><li>转移到玻璃匀浆冰预冷的细胞。 </li><li>使用一紧杵，均质15招杵细胞。 </li><li>加入1体积冰冷的同质化缓冲区。 </li><li>同质化原生质球转移至50毫升塑料离心管。 </li><li>颗粒完整细胞，细胞核，并在1500 XG 5分钟离心4大碎片° C。 </li><li>离心机3000 XG 5分钟产生的上清液在4 ° C。 </li><li> 15分钟产生的上清液离心12000 XG在4 ° C。 </li><li>倒出上清液，沉淀重悬在冰冷的同质化缓冲区[6.5毫升缓冲/克（湿重）细胞]。 </li><li> 3000 XG 5分钟离心4 ° C。 </li><li> 15分钟产生的上清液离心12000 XG在4 ° C。 </li><li>倒出上清液，重悬沉淀在冰冷的扫描电镜缓冲区3毫升。 </li></ol> **虽然这种悬挂在线粒体丰富，还含有其它细胞器，如内质网（微粒），高尔基体和空泡。为了得到纯净的线粒体，这种原油的线粒体分数可以受到进一步的分馏，如下所述** 线粒体的没有污染的其他细胞器的分离纯化<ol><li>广场1.5毫升冰冷的60％（W / V）蔗糖的EM缓冲区成贝克曼超清晰的离心管。 </li><li>覆盖60％（W / V）4毫升32％的蔗糖，1.5毫升23％，1.5毫升15％的蔗糖（所有WT / V在EM缓冲区）。 </li><li>将3毫升15％（W / V）蔗糖顶部原油在SEM中的缓冲区的线粒体的一小部分。 </li><li>在一个贝克曼SW41钛摆动斗1小时134000 XG（33000 RPM）在4 ° C转子离心机</li><li>完整的线粒体形式在60％/ 32％的蔗糖界面的棕色带。 </li><li>小心取出，直到达到线粒体乐队的蔗糖。 </li><li>删除一个切尖线粒体的乐队，用吸管和一个MLS - 5转子放入一个贝克曼离心管。 </li><li>用SEM缓冲区的填充管。 </li><li> MLS - 5 10000 XG（31000 RPM）为30分钟30分钟的转子离心沉淀的纯线粒体在4 ° C。 </li><li>倒出上清液采用吸管。 </li><li>使用纯线粒体线粒体功能，线粒体蛋白质组和lipidome和线粒体DNA的分析。 </li></ol>试剂<ol><li>数码地面电视的缓冲区[Tris/H2SO4（pH 9.4）的100毫米，10毫米二硫苏糖醇（15毫升） <ul style="list-style-type:none;"><li> 1.5毫升1个M Tris/H2SO4缓冲液（pH值9.4） </li><li> 150μL的1 M数码地面电视</li><li>卷15毫升</li></ul></li><li> Zymolyase缓冲液[20毫米磷酸钾（pH值7.4），1.2米山梨醇]（100毫升） <ul style="list-style-type:none;"><li> 2毫升1 M磷酸钾缓冲液（pH 7.4） </li><li> 60毫升的2米山梨醇</li><li>容至100 ml </li></ul></li><li>同质化缓冲液[10毫米的Tris /盐酸（pH值7.4），0.6米山梨糖醇，1 mM的EDTA，0.2％（W ​​/ V）牛血清白蛋白（250毫升] <ul style="list-style-type:none;"><li> 2.5毫升的1 M Tris / HCl缓冲液（pH值7.4） </li><li> 75毫升的2米山梨醇</li><li> 500μL500 mM的EDTA </li><li> 0.5克的BSA </li><li>容积250毫升</li></ul></li><li>扫描电镜缓冲区[10毫米公开资讯观测站/氢氧化钾（pH值7.2），蔗糖250毫米，1毫米EDTA]（250毫升） <ul style="list-style-type:none;"><li> 2.5毫升1个M MOPS /氢氧化钾缓冲液（pH值7.2） </li><li> 31.25毫升2米蔗糖</li><li> 500μL500 mM的EDTA </li><li>容积250毫升</li></ul></li><li>电磁缓冲液[10毫米公开资讯观测站/氢氧化钾（pH值7.2），1 mM的EDTA]（250毫升） <ul style="list-style-type:none;"><li> 2.5毫升1个M MOPS /氢氧化钾缓冲液（pH值7.2） </li><li> 500μL500 mM的EDTA </li><li>容积250毫升</li></ul></li></ol>

<ol>
	<li>Voet, D., Voet, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry.">Biochemistry.</a> , (2004).</li><li>Suen, D. -. F., Norris, K. L., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+and+apoptosis.">Mitochondrial dynamics and apoptosis.</a> Genes Dev. 22, 1577-1590 (2008).</li><li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F. &. a. m. p. ;. a. m. p., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Detmer, S. A., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+dysfunctions+of+mitochondrial+dynamics.">Functions and dysfunctions of mitochondrial dynamics.</a> Nat. Rev. Mol. Cell Biol. 8, 870-879 (2007).</li><li>McBride, H. M., Neuspiel, M., Wasiak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+more+than+just+a+powerhouse.">Mitochondria: more than just a powerhouse.</a> Curr. Biol. 16, R551-R560 (2006).</li><li>Balaban, R. S., Nemoto, S., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria,+oxidants,+and+aging.">Mitochondria, oxidants, and aging.</a> Cell. 120, 483-495 (2005).</li></ol>

The authors have nothing to disclose.

这种方法使高产的隔离从酵母细胞中的纯线粒体。通过这种方法纯化的线粒体其他细胞器的污染基本上是免费的，其净化后保留其结构和功能的完整性。所述的方法产生的线粒体适用于无细胞重组基本线粒体过程。这些高度纯净的线粒体也可以用于线粒体结构和功能，线粒体蛋白质组和lipidome，和线粒体DNA的分析。

purification of mitochondria from yeast cells

线粒体是ATP生产的主要场所，在真核细胞非光合细胞的有氧代谢<sup&gt; 1</sup&gt;。这些复杂的细胞器在细胞凋亡中发挥重要作用<sup&gt; 2</sup&gt;，细胞的生存<sup&gt; 3</sup&gt;，哺乳动物的发展<sup&gt; 4</sup&gt;，神经细胞的发育和功能<sup&gt; 4</sup&gt;，细胞内的信号<sup&gt; 5</sup&gt;和长寿调控<sup&gt; 6</sup&gt;。我们的线粒体控制这些复杂的生物过程的理解，依靠强大的方法，以评估它们的形态，其蛋白质和脂质成分，它们的DNA的完整性，他们的许多重要职能。芽殖酵母<em&gt;酿酒酵母</em&gt;，一种基因和生物化学来操作带注释的基因组和蛋白质组定义良好的，单细胞真核生物是一个有价值的模型研究线粒体的重要生物功能的分子和细胞的内在机制，。对于这些类型的研究，这是至关重要的高纯度线粒体。在这里，我们提出了一个迅速和有效的方法纯化的酵母线粒体的详细描述。这种方法使其他细胞器的污染基本上是免费的，保留其净化后的结构和功能完整性的高度纯净的线粒体的隔离。通过这种方法纯化线粒体是适合无细胞重组基本线粒体过程，可用于线粒体结构和功能，线粒体的蛋白质和lipidome，和线粒体DNA的分析。

Watch this Scientific Journal Video about Purification of Mitochondria from Yeast Cells at JoVE.com

Purification of Mitochondria from Yeast Cells

我们描述了一个快速和有效的方法纯化的酵母线粒体<em&gt;酿酒酵母</em&gt;。这种方法使高产纯线粒体隔离，其他细胞器的污染基本上是免费的，并保留其净化后的结构和功能完整性。

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

purification-of-mitochondria-from-yeast-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

25.0K 次观看.  Concordia University. 我们描述了一个快速和有效的方法纯化的酵母线粒体酿酒酵母。这种方法使高产纯线粒体隔离，其他细胞器的污染基本上是免费的，并保留其净化后的结构和功能完整性。

视频: Purification of Mitochondria from Yeast Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

从酵母细胞线粒体的纯化

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