Name: reconstitution of cell-cycle oscillations in microemulsions of cell-free xenopus egg extracts
Uploaded: 2018-09-27T14:30:00
Description: Watch this Scientific Journal Video about Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts at JoVE.com

我们感谢玛德琳路建设 securin-mCherry 质粒, 圈人李, 肯尼斯和艾伦 P 刘讨论关于水滴生成, 杰里米 b. 张和詹姆斯 e. 费雷尔 Jr 提供 GFP-NLS 结构。这项工作得到了国家科学基金会 (早期职业补助金 #1553031)、国立卫生研究院 (米拉 #GM119688) 和斯隆研究金的支持。

这里描述的所有方法都得到了密歇根大学机构动物护理和使用委员会 (IACUC) 的批准。
1. 细胞周期重建和检测材料的制备
<ol><li>
securin-mCherry 质粒 DNA 构建及 mRNA 纯化的吉布森组装克隆
	<ol>
<li>通过聚合酶链反应 (PCR) 和凝胶纯化21,22, 制备三个 DNA 片段, 包括 pMTB2 向量主干、securin 和 mCherry。</li>
		<li>使用 fluorospectrometer 测?...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cyclin+synthesis+and+degradation+in+the+control+of+maturation+promoting+factor+activity.">The role of cyclin synthesis and degradation in the control of maturation promoting factor activity.</a> Nature. 339, 280-286 (1989).</li><li>Yang, Q., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cdk1-APC/C+cell+cycle+oscillator+circuit+functions+as+a+time-delayed,+ultrasensitive+switch.">The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch.</a> Nature Cell Biology. 15, 519-525 (2013).</li><li>Chang, J. B., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+trigger+waves+and+the+spatial+coordination+of+the+Xenopus+cell+cycle.">Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle.</a> Nature. 500, 603-607 (2013).</li><li>Trunnell, N. B., Poon, A. C., Kim, S. Y., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitivity+in+the+Regulation+of+Cdc25C+by+Cdk1.">Ultrasensitivity in the Regulation of Cdc25C by Cdk1.</a> Molecular Cell. 41, 263-274 (2011).</li><li>Kim, S. Y., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+competition+as+a+source+of+ultrasensitivity+in+the+inactivation+of+Wee1.">Substrate competition as a source of ultrasensitivity in the inactivation of Wee1.</a> Cell. 128, 1133-1145 (2007).</li><li>Pomerening, J. R., Kim, S. Y., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems-level+dissection+of+the+cell-cycle+oscillator:+bypassing+positive+feedback+produces+damped+oscillations.">Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations.</a> Cell. 122, 565-578 (2005).</li><li>Pomerening, J. R., Sontag, E. D., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+cell+cycle+oscillator:+hysteresis+and+bistability+in+the+activation+of+Cdc2.">Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2.</a> Nature Cell Biology. 5, 346-351 (2003).</li><li>Telley, I. A., Gaspar, I., Ephrussi, A., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aster+migration+determines+the+length+scale+of+nuclear+separation+in+the+Drosophila+syncytial+embryo.">Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo.</a> The Journal of Cell Biology. 197, 887-895 (2012).</li><li>Telley, I. A., Gaspar, I., Ephrussi, A., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+Drosophila+embryo+extract+for+the+study+of+mitosis+ex+vivo.">A single Drosophila embryo extract for the study of mitosis ex vivo.</a> Nature Protocols. 8, 310-324 (2013).</li><li>Tsai, T. Y. C., Theriot, J. A., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+Oscillatory+Dynamics+in+the+Cell+Cycle+of+Early+Xenopus+laevis+Embryos.">Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos.</a> PLoS Biology. 12, e1001788 (2014).</li><li>Chang, J. B., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+trigger+waves+and+the+spatial+coordination+of+the+Xenopus+cell+cycle.">Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle.</a> Nature. 500, 603-607 (2013).</li><li>Yang, Q., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cdk1-APC/C+cell+cycle+oscillator+circuit+functions+as+a+time-delayed,+ultrasensitive+switch.">The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch.</a> Nature Cell Biology. 15, 519-525 (2013).</li><li>Kumagai, A., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claspin,+a+novel+protein+required+for+the+activation+of+Chk1+during+a+DNA+replication+checkpoint+response+in+Xenopus+egg+extracts.">Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.</a> Molecular Cell. 6, 839-849 (2000).</li><li>Chen, R. H., Murray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+spindle+assembly+checkpoint+in+Xenopus+egg+extracts.">Characterization of spindle assembly checkpoint in Xenopus egg extracts.</a> Methods in Enzymology. 283, 572-584 (1997).</li><li>Chen, R. H., Waters, J. C., Salmon, E. D., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+spindle+assembly+checkpoint+component+XMAD2+with+unattached+kinetochores.">Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores.</a> Science. 274, 242-246 (1996).</li><li>Minshull, J., Sun, H., Tonks, N. K., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+MAP+kinase-dependent+spindle+assembly+checkpoint+in+Xenopus+egg+extracts.">A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts.</a> Cell. 79, 475-486 (1994).</li><li>Good, M. C., Vahey, M. D., Skandarajah, A., Fletcher, D. A., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+Volume+Modulates+Spindle+Size+During+Embryogenesis.">Cytoplasmic Volume Modulates Spindle Size During Embryogenesis.</a> Science. 342, 856-860 (2013).</li><li>Hazel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+cytoplasmic+volume+are+sufficient+to+drive+spindle+scaling.">Changes in cytoplasmic volume are sufficient to drive spindle scaling.</a> Science. 342, 853-856 (2013).</li><li>Garibyan, L., Avashia, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+Techniques+Made+Simple:+Polymerase+Chain+Reaction+(PCR).">Research Techniques Made Simple: Polymerase Chain Reaction (PCR).</a> The Journal of Investigative Dermatology. 133, e6 (2013).</li><li>Hecker, K. H., Roux, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+and+low+annealing+temperatures+increase+both+specificity+and+yield+in+touchdown+and+stepdown+PCR.">High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR.</a> BioTechniques. 20, 478-485 (1996).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6, 343-345 (2009).</li><li>Froger, A., Hall, J. 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我们提出了一种新的方法来开发一个高通量人工细胞系统, 使体外重建和长期跟踪自我持续的细胞周期振荡的单细胞水平。有几个关键步骤使此方法成功。首先, 与下蛋相比, 新鲜挤压的爪蛋具有良好的品质, 往往会产生具有更持久振荡活性的萃取物。其次, 在表面活性剂稳定的微环境中, 萃取物的封装对于保持振荡活性至关重要。第三, 将油滴孵化成一根薄薄的管子, 将水滴限制在一层...

由爪蟾卵制备的细胞质提取物是细胞周期生化研究中最主要的模型之一, 考虑到卵母细胞的体积大、细胞周期的快速发展以及重组的能力。体外有丝分裂事件1,2。该系统允许初步发现和机械特性的关键细胞周期调节器, 如成熟促进因子 (MPF) 以及下游有丝分裂过程, 包括主轴组装和染色体分离1 ,2,3,4,5,6,7,8,9,10, 11。爪蟾卵提取物也被用来详细解剖细胞周期时钟8,12,13,14的调节网络和 DNA 损伤的研究/复制检查点15和有丝分裂主轴组件检查点 16、17、18。
这些研究的细胞周期使用爪蟾卵提取物主要是基于散装测量。然而, 常规的散装反应化验可能不会模仿真实的细胞行为, 因为它们的维度和亚细胞空间划分的反应分子的主要差异。此外, 在快速阻尼8之前, 对有丝分裂活动的大量测量往往会给出有限的周期数。大量反应的缺点使萃取系统无法进一步了解复杂时钟的动态特性和功能。最近的研究已经封装了无细胞 cytostatic 因子 (CSF)爪蟾提取物 19,20到大小定义的细胞样车厢, 这有助于阐明如何调整主轴尺寸细胞质容积。然而, 这种体外系统是在减数分裂的中期被逮捕的 cytostatic 因子1的作用, 一个能够长期持续振荡的单细胞水平, 以进一步调查细胞周期振荡 器。
为了研究细胞周期振荡的单细胞分辨率, 我们开发了一个细胞规模, 高通量系统的重建和同时测量多个自持续有丝分裂振荡过程中的个别微乳液液 滴。在详细的视频协议中, 我们演示了在10到300µm 的微乳液中封装循环爪蟾卵胞浆的人工有丝分裂振荡系统的建立。在该系统中, 有丝分裂振荡包括染色体凝结和脱凝、核包络击穿和改造, 以及后期基质的降解和合成 (如本协议中的 securin mCherry) 是成功重组。

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			<td height="43" style="height:43px;width:215px;">Xenopus laevis frogs</td>
			<td style="width:93px;">Xenopus-I Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">QIAprep spin miniprep kit</td>
			<td style="width:93px;">QIAGEN</td>
			<td style="width:119px;">27104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">QIAquick PCR Purification Kit (250)</td>
			<td style="width:93px;">QIAGEN</td>
			<td style="width:119px;">28106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">BL21 (DE3)-T-1 competent cell</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">B2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Calcium ionophore</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">A23187</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Hoechst 33342</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">B2261</td>
			<td style="width:167px;">Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Trichloro</td>
			<td rowspan="2" style="width:93px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">448931</td>
			<td rowspan="2" style="width:167px;">Toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">(1H,1H,2H,2H-perfluorooctyl) silane</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">PFPE-PEG surfactant</td>
			<td style="width:93px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant-2wtH-50G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">GE Healthcare Glutathione Sepharose 4B beads</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">GE17-0756-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">PD-10 column</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">GE17-0851-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">VitroCom miniature hollow glass tubing</td>
			<td style="width:93px;">VitroCom</td>
			<td style="width:119px;">5012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Olympus SZ61 Stereo Microscope</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Olympus IX83 microscope</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Olympus FV1200 confocal microscope</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">NanoDrop spectrophotometer</td>
			<td style="width:93px;">Thermofisher</td>
			<td style="width:119px;">ND-2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">0.4 mL Snap-Cap Microtubes</td>
			<td style="width:93px;">E&#38;K Scientific</td>
			<td style="width:119px;">485050-B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="34">
			<td height="54" rowspan="2" style="height:55px;width:215px;">&#160;PureLink RNA Mini Kit</td>
			<td rowspan="2" style="width:93px;">ThermoFisher&#65288;Ambion&#65289;</td>
			<td rowspan="2" style="width:119px;">12183018A</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fisherbrand Analog Vortex Mixer</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">2215365</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Imaris</td>
			<td style="width:93px;">Bitplane</td>
			<td style="width:119px;">Version 7.3</td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

reconstitution of cell-cycle oscillations in microemulsions of cell-free xenopus egg extracts

实时测量单细胞水平的振荡, 对于揭示生物钟的机理具有重要意义。虽然从爪蟾卵制备的大块提取物在细胞周期发展的基础上解剖生物化学网络中具有强大的功能, 但它们的整体平均测量通常会导致阻尼振荡, 尽管每个单个振荡器被持续。这是由于噪声生物系统中单个振荡器之间的完美同步的困难。为了检索振荡器的单细胞动力学, 我们开发了一个以液滴为基础的人工细胞系统, 它能在细胞状腔内重组爪蟾卵的循环细胞质提取物的有丝分裂周期。这些简单的胞浆细胞表现出30多个周期的持续振荡。为了构建更复杂的细胞核细胞, 我们添加了 demembranated 精子染色质, 以触发系统中的核自组装。我们观察到染色体凝结/decondensation 和细胞核包络破裂/改革的周期性进展, 就像在真实细胞中。这表明有丝分裂振荡器的功能忠实地推动多个下游有丝分裂事件。利用多通道时程荧光显微镜, 我们同时跟踪了单个液滴的有丝分裂振荡器和下游过程的动力学。人工细胞周期系统提供了一个高通量的框架, 用于定量操作和分析单细胞分辨率的有丝分裂振荡, 这可能提供了重要的洞察力的调节机制和功能时钟。

在图 2中, 我们表明该协议在简单的无核细胞和复杂的细胞核中产生有丝分裂振荡, 振荡器驱动原子核形成和变形的循环交替。无核的水滴产生的有丝分裂振荡高达30无阻尼吸循环超过92小时的时间跨度, 这表明由周期性合成和退化的荧光记者 securin-mCherry (图 2A)。Securin 是后期促进复合 APC/C 的基底。securin-mCherry 在后期的降解从而?...

Watch this Scientific Journal Video about Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts at JoVE.com

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

本文提出了在油中微乳液中对爪蟾卵提取物进行单细胞水平的体外自持续有丝分裂振荡的生成方法。

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

该方法有助于回答系统生物学领域中的关键问题，如线粒体网络背后的调节机制以及细胞周期时钟的动态特性和功能。该技术的主要优点是，它将生成大量具有多个细胞周期的液滴，为我们所有人提供定量操作和分析的高通量框架。首先，丢弃动物和植物杆之间界限不清的鸡蛋批次，或在动物杆顶上出现不规则的白点。接下来，从一只雌性青蛙中选择一批合适的卵子，将卵子转移到600毫升的烧杯中。倒出多余的0.2xMMR缓冲液，并轻轻地将半胱氨酸加入1x提取物缓冲液中。之后，用手大力摇动烧杯，取出鸡蛋的果冻涂层。重复三次，用总共800毫升的半胱氨酸缓冲液，或直到鸡蛋聚集并沉淀在烧杯的底部。去除果冻涂层后，在一升 0.2 倍 MMR 溶液中清洗鸡蛋四次。使用玻璃移液器拾取并丢弃变白的鸡蛋。将MMR缓冲液从烧杯中倒出来，将200毫升的钙离子磷溶液加入鸡蛋中。使用玻璃移液器轻轻搅拌鸡蛋，直到它们被激活，并去除钙离子磷溶液。将鸡蛋留在钙离子磷溶液中太久会引发凋亡。我们搅拌鸡蛋1.5分钟，并检查激活效率。如果未激活，请继续搅拌，并更频繁地检查，直到实现 90% 激活。鸡蛋在烧杯底部沉淀后，检查激活效率。活性良好的鸡蛋有大约25%的动物杆和75%的植物杆。接下来，用总体积为50毫升的提取物缓冲液洗两次鸡蛋，并辅以蛋白酶抑制剂。然后，小心地将鸡蛋转移到0.4毫升的扣盖微管中。在200克时将微管离心60秒，然后在600克离心30秒，以包装鸡蛋。然后，使用玻璃巴氏移液器去除鸡蛋顶部的多余的缓冲液后，每一步。之后，在15，000克的鸡蛋中，在4摄氏度下粉碎10分钟。使用剃刀刀片切割管子，分离三层碎鸡蛋，并保持粗细胞质在中间层。然后，将粗细胞质转移到清洁超离心管。补充细胞质蛋白酶抑制剂和细胞链酶B，每毫升10微克。将粗细胞质在15，000克和4摄氏度下离心5分钟。然后，使用剃刀刀片切割管子，并保持细胞质在中间层。将新鲜准备好的提取物放在冰上。首先，在准备好的提取物中加入安全素-mCherry mRNA。接下来，在提取物mRNA混合物中加入200微升2%PFPE-PEG氟化物。使用涡流混合器生成液滴，根据需要调整涡流速度和持续时间。然后，目视检查液滴，确保液滴的大小均匀。使用 200 微升移液器，将漂浮在顶部的液滴和等量的表面活性剂转移到 PCR 管中。将玻璃管浸入液滴层，等待液滴填充约 75% 的管子。然后，将管子推入表面活性层，并完全填充管子。接下来，用矿物油填充玻璃底盘。然后，使用细钳子将充满液滴的管子浸入油中。使用玻璃移液器清除任何可见的碎屑或泄漏的液滴。为避免液滴的移动和泄漏，应小心地将装满液滴的玻璃管浸入矿物油下面。我们可以平衡地将管子推到两端，或者先在管子的两端同时添加几滴矿物油。在此之后，使用配备电动 xy 舞台的荧光显微镜对液滴进行成像。每六到九分钟录制一次亮场和多个荧光通道中的延时视频，直到液滴失去活动。利用这种协议，在简单、无核的细胞和具有核的复杂细胞中产生了线粒体振荡。无核液滴在 92 小时内产生长达 30 个未采样的滴位振荡。还研究了线粒体振荡器驱动下游线粒体事件的能力。自我维持的线粒体振荡器通过不同细胞周期阶段的自主变化，推动着核形态的变化。在尝试此过程时，重要的是要记住制作提取物时的时间，因为它对时间敏感，并且始终将鸡蛋和提取物放在冰上。该技术开发后，为系统生物学领域的研究人员探索复杂时钟特性的原理铺平了道路，如Xenopus laevis的可调性和随机性。

Research

JoVE Journal

Biochemistry

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

科研

生物化学

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...