Name: stable isotopic profiling of intermediary metabolic flux in developing and adult stage caenorhabditis elegans
Uploaded: 2011-02-27T14:00:00
Duration: 12 min 10 s
Description: Watch this Scientific Journal Video about Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans at JoVE.com

这项工作是由美国国立卫生研究院（K08 - DK073545和赞助NICHD的智力和发育残疾研究中心新研究者奖），费城基金会，宾夕法尼亚大学麦凯布奖（MJF），以及特里斯坦部分经费马伦基金（MJF和MY）。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

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	<li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans: A practical approach. , (1999).</li><li>Dingley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+dysfunction+variably+increases+oxidant+stress+in+Caenorhabditis+elegans.">Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans.</a> Mitochondrion. 10, 125-136 (2010).</li><li>Jones, B. N., Gilligan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=o-Phthaldialdehyde+precolumn+derivatization+and+reversed-phase+high-performance+liquid+chromatography+of+polypeptide+hydrolysates+and+physiological+fluids.">o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids.</a> Journal of chromatography. 266, 471-482 (1983).</li><li>Falk, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+pathway+profiling+of+mitochondrial+respiratory+chain+mutants+in+C.+elegans.">Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans.</a> Molecular genetics and metabolism. 93, 388-397 (2008).</li><li>Yudkoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+in+vivo+ureagenesis+with+stable+isotopes.">Measuring in vivo ureagenesis with stable isotopes.</a> Molecular genetics and metabolism. 100, 37-41 (2010).</li><li>Tsang, W. Y., Sayles, L. C., Grad, L. I., Pilgrim, D. B., Lemire, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+deficiency+in+Caenorhabditis+elegans+results+in+developmental+arrest+and+increased+life+span.">Mitochondrial respiratory chain deficiency in Caenorhabditis elegans results in developmental arrest and increased life span.</a> J Biol Chem. 276, 32240-32246 (2001).</li></ol>

应用质谱测量同位素丰度的中间代谢产物，提供了一次关键的生化改变5奇妙的详细图片。在这里，我们提供详细的协议，利用这个高度敏感和特异的方法，以评估中介代谢通量基因通用的模型动物，C. 线虫 。事实上，饲养动物活体与稳定同位素标记的代谢前体（ 如 13 C -葡萄糖）允许将中介代谢途径流量新颖的见解，测量时在下游代谢产物的同位素富集。我们已经开发出可靠的方法，获得通过糖酵解，丙酮酸代谢，三羧酸循环强大的分析通量。要做到这一点无论是在美联储稳定同位素幼体早期发育阶段的动物，或在有丝分裂后的成年期开始。可以结合全虫游离氨基酸分析研究代谢产物在不同物种的同位素富集高效液相色谱法，如前面所述4，确定在不同物种免费蠕虫丙氨酸，天门冬氨酸，谷氨酸，谷氨酰胺绝对同位素富集。事实上，一贯的同位素富集，是获得在1,000至2,000个年轻的成年同步蠕虫等分测量时，氨基酸和有机酸分析物。此种方法能灵敏地歧视中介通量异常核基因为基础的线粒体突变株与野生型蠕虫。 这项技术拥有价值调查相关的具体生化途径的通量变化常见的代谢与生俱来的错误。与稳定同位素标记的葡萄糖的利用率，特别是通过线粒体疾病相关的中央的代谢途径通知碳通量。事实上，我们的数据表明，应用此种方法能灵敏地歧视中介通量异常核基因为基础的线粒体复合三亚基突变株（ISP - 1（qm150））相对于野生型蠕虫。 重要的是要认识到，由于同位素曝光超过数天完成了一个周期，量化同位素富集代表超过规定的时间内可能是在细胞模型确定的“稳定状态”浓缩的价值，而不是量化通量暴露的同位素几分钟到几小时内。另一个潜在的局限性，对线虫的发展过程中询问途径通量与幼体发育的不同长度，在发生特定的突变株。例如，许多严重的线粒体基因突变是已知的导致第三（L3）的幼虫期 6逮捕。因此，只在动物体内存活到成年的同位素掺入的分析可能无法代表整个突变人口。然而，这种方法可以适用于评估中介代谢产物在一个特定的幼虫阶段（如二级），而不是等待的动物，以达到成年阶段的同位素掺入。同样，进入另一种幼虫阶段可能作为的dauer已知的动物有明显改变（可能降低）代谢通量相对动物进行直接通过四个幼虫。未来的研究也可以进行直接评估在不同遗传背景的dauer阶段动物的同位素掺入。动物暴露到一个固定时间段开始，一旦动物达到年轻的成年阶段（在这里，24至48小时）的稳定同位素（详见协议乙）删除变量发育时期的影响。虽然只询问13的C -葡萄糖糖酵解，丙酮酸代谢和三羧酸循环的通量，其他同位素示踪剂可能被评定为通过其他途径线虫利益的生化途径通量询问。例如，15的N -甘氨酸可能被用来评估线虫氨基酸周转。 这种方法的另一个潜在的局限性代谢物检测的灵敏度水平。我们的经验表明，至少500名年轻的成年线虫需要，这里的HPLC和GC / MS方法，成功地量化同位素掺入。使用仪器具有更大的敏感性，如超高效液相色谱（UPLC），，可能会允许研究的动物数量进一步减少，尽管我们预期这是不可能的，允许可靠的定量的代谢物和同位素掺入的代谢产物种类从单一的蠕虫的。我们研究了蠕虫人口1,000元实验动物，我们发现，可靠地检测每一株低丰度代谢物。然而，如GABA的一些代谢物，只能是可靠的量化线虫更大的人口，秩序1 × 10 6动物4。 总之，稳定同位素分析提供了一种非侵入性和安全（非放射性）的方法，早已被用来研究先天性代谢缺陷。这种方法应用到 C 线虫提供了一种新的在体内，实时，发生在整体动物水平的代谢改变，这可能会导致从个人的遗传性疾病和/或药物剂风险能力进行调查。

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		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>S. basal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1 - page 59</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Cholesterol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-8503</td>
<td>Protocol B</td>
</tr>
<tr>
<td>OP50 E. coli</td>
<td>&nbsp;</td>
<td>Caenorhabditis Genetics Center</td>
<td>&nbsp;</td>
<td>Protocols A and C</td>
</tr>
<tr>
<td>K12 E. coli</td>
<td>&nbsp;</td>
<td>Caenorhabditis Genetics Center</td>
<td>&nbsp;</td>
<td>Protocol B</td>
</tr>
<tr>
<td>NGM agar</td>
<td>&nbsp;</td>
<td>Research Products INT</td>
<td>N81800-1000.0</td>
<td>Protocols A, B, and C</td>
</tr>
<tr>
<td>13C glucose (universally labeled)</td>
<td>&nbsp;</td>
<td>Cambridge Isotope labs</td>
<td>CLM-1396</td>
<td>Protocol A and C</td>
</tr>
<tr>
<td>13C glucose (1,6 labeled)</td>
<td>&nbsp;</td>
<td>Cambridge Isotope labs</td>
<td>CLM-2717</td>
<td>Protocol B</td>
</tr>
<tr>
<td>60% Perchloric acid (PCA)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>MK2766500</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>&epsilon;-aminocaproic acid (Internal Standard)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-2504</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>MTBSTFA</td>
<td>&nbsp;</td>
<td>Regis</td>
<td>270243</td>
<td>Protocol E</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Regis</td>
<td>270010</td>
<td>Protocol E</td>
</tr>
<tr>
<td>AG 1-X8, 100-200, Chloride form resin</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>140-1441</td>
<td>Protocols A and B (organic acid extraction)</td>
</tr>
<tr>
<td>AG 50W-X8, 100-200, Hydrogen form resin</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>142-1441</td>
<td>Protocols A and B (amino acid extraction)</td>
</tr>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-8875</td>
<td>Protocol C</td>
</tr>
<tr>
<td>NaOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8045</td>
<td>Protocol C</td>
</tr>
<tr>
<td>3N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol D</td>
</tr>
<tr>
<td>1N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol A</td>
</tr>
<tr>
<td>0.1 N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol D</td>
</tr>
<tr>
<td>4N NH4OH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>30501</td>
<td>Protocol D</td>
</tr>
<tr>
<td>4N KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>484016</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Deionized water</td>
<td>&nbsp;</td>
<td>Milli Q Biocel</td>
<td>ZMQA60F01</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Helium</td>
<td>&nbsp;</td>
<td>Airgas</td>
<td>24001364</td>
<td>Protocol C2</td>
</tr>
<tr>
<td>SMZ-800 Zoom Stereo-Microscope</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>NI MNA41000</td>
<td>Protocols A, B, and C</td>
</tr>
<tr>
<td>pH meter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S68167</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Table top centrifuge - 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>05-400-93</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Incubated platform shaker</td>
<td>&nbsp;</td>
<td>New Brunswick</td>
<td>M1324-0004</td>
<td>Protocol B</td>
</tr>
<tr>
<td>Mini LabRoller Rotator</td>
<td>&nbsp;</td>
<td>Labnet</td>
<td>H-5500</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Pestles</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>K749520-0090</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Drill</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>K749540-0000</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>1 L glass beaker</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-539P</td>
<td>&nbsp;</td>
<tr>
<td>1 L Erlenmeyer Flasks</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-368</td>
<td>&nbsp;</td>
<tr>
<td>25 mL Erlenmeyer Flasks</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-356</td>
<td>Protocol B</td>
</tr>
<tr>
<td>7ml round-bottom glass tubes + rubber stopper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VT6431</td>
<td>Protocols A, B, and C1,C2</td>
</tr>
<tr>
<td>10 ml round-bottom glass tubes + rubber stopper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VT6430</td>
<td>Protocol C2</td>
</tr>
<tr>
<td>Blue top tubes</td>
<td>&nbsp;</td>
<td>Labco</td>
<td>438B</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical plastic tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>14-959-49A</td>
<td>All protocols</td>
</tr>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-681-320</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Syringes (1 mL, 10 mL, 20 mL)</td>
<td>&nbsp;</td>
<td>BD Medical</td>
<td>301025, 301029, 301031</td>
<td>Protocols C1 and C2</td>
</tr>
<tr>
<td>25 gauge needles</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-253-131</td>
<td>Protocols C1 and C2</td>
</tr>
<tr>
<td>Poly-Prep Chromatography Columns</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>731-1550</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14673-043</td>
<td>Protocol D</td>
</tr>
<tr>
<td>60mm Petri dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25373-085</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Glass chamber (cut bottom of 1 L flask) fitted with grease-sealed 3-way stopcock</td>
<td>&nbsp;</td>
<td>Custom Made in Glass Shop</td>
<td>&nbsp;</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Optically transparent glass plate</td>
<td>&nbsp;</td>
<td>Custom Made in Glass Shop</td>
<td>&nbsp;</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Reacti-Vap III Evaporator</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>18826</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Cotton</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14224-516</td>
<td>Protocol D</td>
</tr>
<tr>
<td>High Vacuum Grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>Protocol C1</td>
</tr>
<tr>
<td>Aluminum Foil</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>01-213-18</td>
<td>Protocol B</td>
</tr>
<tr>
<td>Timer</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>T-2504-5</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Gas-Ratio Mass Spectrometer</td>
<td>&nbsp;</td>
<td>Thermophinigan</td>
<td>&nbsp;</td>
<td>Protocol D</td>
</tr>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>HP</td>
<td>5980/5971</td>
<td>Protocol D</td>
</tr>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6980N/5973N</td>
<td>Protocol D</td>
</tr>
<tr>
<td>HPLC</td>
<td>&nbsp;</td>
<td>Varian</td>
<td>9010</td>
<td>Protocol D</td>
</tr>		</tbody>
		</table>
		</div>

stable isotopic profiling of intermediary metabolic flux in developing and adult stage caenorhabditis elegans

稳定同位素分析早已允许在细胞和哺乳动物模型中的基因突变和/或药物疗法的代谢后果的敏感调查。在这里，我们描述了详细的方法来执行的线虫 ，线虫的中间代谢及代谢通量的稳定同位素分析。全虫，氨基酸，标记的二氧化碳，有机酸标记，标记的氨基酸分析中的稳定同位素线虫生长培养基琼脂板或青壮年开始从早期的发展，而暴露在各种药物治疗的动物的描述方法在液体培养。游离氨基酸是由高性能液相色谱法（HPLC）在整个蠕虫分装在4％高氯酸提取量化。通用标记13的C -葡萄糖或1,6 - 13彗星2 -葡萄糖利用稳定同位素前体，其标示碳质谱（大气和溶解二氧化碳）以及在代谢物通量的指示通过糖酵解追溯到丙酮酸代谢，三羧酸循环。代表结果证明野生型线虫同位素照射时间的影响，各种细菌的结算协议，并替代的蠕虫病毒破坏方法，以及同位素掺入在线粒体复合物III突变蠕虫的相对程度（ISP - 1 （qm150） ）相对于野生型蠕虫。生活线虫的稳定同位素分析中的应用提供了一种新颖的能力，以调查在整体动物水平的实时代谢是由个人的遗传性疾病和/或药物治疗引起的改建。

Watch this Scientific Journal Video about Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans at JoVE.com

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

稳定同位素分析气相色谱质谱中介代谢通量的质谱分析中所述的线虫，<em&gt;线虫</em&gt;。详细的评估中的二氧化碳，有机酸，氨基酸和同位素暴露在琼脂平板上的发展，或在液体培养成年的同位素富集的方法。

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Research

JoVE Journal

Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...