Name: dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans
Uploaded: 2013-11-29T16:00:00
Description: Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

我们感谢Chris Webster对于执行图代表结果的3和Jason Watts和克里斯·韦伯斯特所示的初步实验对稿件有用的意见。资助这项研究是通过提供赠款来自美国国立卫生研究院（美国）（R01DK074114）到仲量行。在这项工作中所使用的一些线虫菌株是由线虫遗传学中心，这是由研究基础设施计划的美国国立卫生研究院办公室（P40 OD010440）提供资助。

多不饱和脂肪酸是对热，光和氧敏感。因此，必须制备脂肪酸补充剂板时，应考虑这样的脂肪酸是不暴露在过量的热和光。含有0.1％的Tergitol（NP-40）NGM介质被高压灭菌和部分冷却，之后，脂肪酸钠盐中加入并不断搅拌。所述板被允许在黑暗中进行干燥。脂肪酸由C摄取线虫培养于这些板然后可以通过气相色谱法来监测。 1。脂肪酸补充培养基的制备<ol><li>测量出...

<ol>
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这里我们描述的补充C的方法线虫与食物中的不饱和脂肪酸。如上所述，必须注意在多不饱和脂肪酸补充板的准备，因为在多不饱和脂肪酸的双键的反应自然会导致这些不饱和脂肪酸，通过热和光11是氧化敏感。为了避免氧化，该多不饱和脂肪酸加入到液体琼脂培养基后，媒体已经冷却至55℃，并存储板在黑暗环境中是很重要的。 他人已经介绍的游离脂肪?...

脂肪酸是膜的必要的结构部件，以及有效的能量储存分子。此外，脂肪酸可以从细胞膜裂解通过脂肪酶和酶促修饰以产生信号的效应1。天然存在的多不饱和脂肪酸（PUFA），含有两个或多个顺式双键。 ω-3脂肪酸和ω-6脂肪酸是彼此区分的基础上的双键相对于脂肪酸的甲基端的位置。健康饮食需要两个ω-6和ω-3脂肪酸。然而，西方的饮食是特别丰富的omega-6脂肪酸，差ω-3脂肪酸。高ω-6与ω-3脂肪酸的比例是与心血管和炎性疾病的风险增加，但是，具体的脂肪酸的精确有益和有害功能都不能很好地理解2相关联。蛔虫秀丽隐杆线虫elega因为它综合了所有必要的酶来生产一系列的ω-6和ω-3脂肪酸，包括ω-3去饱和酶的活动，是在缺席的大多数动物3,4纳秒是研究脂肪酸的功能是有用的。突变体缺乏脂肪酸去饱和酶的酶不能产生特定的PUFA，从而导致一系列的发育和神经缺陷4-6。 探讨膳食脂肪酸的生理效应，我们已经开发出一种生化检测与遗传分析兼容同时使用突变和RNAi敲除技术在C线虫 。补充特定的多不饱和脂肪酸是通过将脂肪酸的钠盐溶液的琼脂培养基之前浇注来实现。这将导致在多不饱和脂肪酸的摄取由大肠杆菌大肠杆菌的食物来源，它积聚在细菌膜。C.线虫摄取的含PUFA的细菌，这饮食补充足够的抢救有缺陷的多不饱和脂肪酸缺乏的突变体TS。大多数脂肪酸补充有对野生型动物没有有害的影响，但是，具体的ω-6脂肪酸，尤其是二高-γ-亚麻酸（DGLA，20点03的n-6）引起℃的永久性破坏线虫生殖细胞7,8。 气相色谱法是用于监视补充脂肪酸的细菌食物源（OP50或HT115）以及线虫的吸收。在加入洗涤剂的Tergitol（NP-40）中的媒体的允许通过整个板和的脂肪酸由大肠杆菌更高效摄取均匀分布的脂肪酸大肠杆菌和线虫。我们已经发现，不饱和脂肪酸可以容易地采取了由细菌和C。线虫 ，但是饱和脂肪酸的摄取是非常低效率的。本文将描述一步一步如何与脂肪酸补充琼脂培养基，以及如何监测在日脂肪酸的摄取Ë线虫用气相色谱法。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

脂肪酸是必需的许多细胞功能。他们作为高效的储能分子，补膜的疏水核心，并参与各种信号通路。 秀丽隐杆线虫综合了所有必要的酶来生产一系列的ω-6和ω-3脂肪酸。此，结合了简单的解剖学和可遗传工具的范围，使之成为有吸引力的模型来研究脂肪酸功能。为了研究介导的膳食脂肪酸的生理作用的遗传途径，我们已开发出一种方法，以补充C。线虫的饮食与不饱和脂肪酸。补充剂是改变蠕虫的脂肪酸组合物，一种有效的手段，也可用于抢救中脂肪酸的缺失突变体的缺陷。我们的方法用线虫生长培养基琼脂（NGM），补充有脂肪acidsodium盐。在补充板的脂肪酸成为incorpora特德进入细菌的食物来源，然后采取了由C的膜线虫饲料的补充细菌。我们还描述了一种气相色谱协议来监控发生在补充蠕虫中脂肪酸组成的变化。这是为了补充C的大和小群体的饮食的有效方法线虫 ，允许的范围内对本方法的应用。

在C的补充线虫饮食是由细菌食物源的摄取和结合脂肪酸到细菌膜的能力的限制。要确定大肠杆菌的能力大肠杆菌 OP50吸收各种脂肪酸进入其的膜，OP50被镀到无补充剂，0.1毫米和硬脂酸（18:0），油酸钠（18:1 N-9），和钠DGLA（20 0.3mM的浓度媒体：3N-6）。平板在室温下干燥在黑暗中3天，并保温在20℃下进行3天。细菌草坪被轻轻地刮草坪成水与火焰消毒压舌板收集。细菌通过?...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

的&lt;em&gt;线虫实验&lt;/ em&gt;的是一个有用的模型，以探讨发展和生理学的多不饱和脂肪酸的功能。这个协议描述补充的&lt;em&gt; C的一种有效的方法线虫&lt;/ em&gt;的饮食与多不饱和脂肪酸。

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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 ...

科研

生物学

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 ...

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...