Name: quantification of ethanol levels in zebrafish embryos using head space gas chromatography
Uploaded: 2020-02-11T14:00:00
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本文中介绍的研究得到了美国国家卫生研究院/国家牙科和颅面研究所（NIH/NIDCR）R01DE020884对J.K.E的先前资助。 以及国家卫生研究院/国家酒精滥用和酒精中毒研究所（NIH/NIAAA）F32A021320至C.B.L.以及国家卫生研究院/国家酒精滥用研究所（NIH/NIAAA）R00A023560向C.B.L.提供的赠款。我们感谢鲁本·冈萨雷斯提供和协助进行气致度表分析。我们感谢蒂娜·奥尼特罗斯和吉娜·诺布尔斯博士的写作协助。

在这个程序中使用的所有斑马鱼胚胎是在既定的IACUC协议27下培育和繁殖的。这些协议得到了德克萨斯大学奥斯汀分校和路易斯维尔大学的批准。
注：本研究28使用斑马鱼线Tg（fli1：EGFP）y1。 本程序中使用的所有水都是无菌反渗透水。所有统计分析均使用图形板棱镜 v8.2.1 执行。
1. 制造胚胎介质
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斑马鱼作为一种开发模型系统，非常适合研究环境因素对发展的影响。他们产生大量的外部受精胚胎，这允许在乙醇研究中精确计时和剂量范式。这与活成像能力以及人类的遗传和发育保护相结合，使斑马鱼成为teratology研究的强大模型系统。本文使用头空间气相色谱法测量斑马鱼胚胎的胚胎乙醇浓度。
确定胚胎乙醇浓度对于了解乙醇对发育中的胚胎的影响至关重要。在啮齿?...

胎儿酒精谱系障碍（FASD）描述了与胚胎乙醇暴露相关的多种神经损伤和颅面畸形。多种因素，包括乙醇暴露的时间和剂量以及遗传背景，都促成了FASD2，3的变化。在人类中，这些变量的复杂关系使得研究和理解FASD的病因变得具有挑战性。动物模型已经证明对于我们理解乙醇致畸性的机械基础至关重要。各种各样的动物模型系统已经被用来研究FASD的多个方面，结果已经非常一致，在人类接触发现4。啮齿动物模型系统用于检查FASD的许多方面，其中小鼠是最常见的5，6，7。这项工作的大部分重点是发育缺陷，以早期乙醇暴露8，虽然后来接触乙醇已被证明会导致发育异常以及9。此外，小鼠的遗传能力大大有助于我们探索FASD10、11的遗传基础的能力。这些小鼠研究强烈表明，有基因-乙醇相互作用与声波刺猪通路， 视黄酸信号，超氧化物歧化酶， 一氧化二氮合成酶 I， Aldh2和Fancd2 8，10，11，12，13，14，15，16，17，1819，20，21.这些研究表明，动物模型对于促进我们对FASD及其基本机制的理解至关重要。
斑马鱼已成为一个强大的模型系统，以检查乙醇畸变的许多方面22，23。由于其外受精、高肥力、遗传可遗传性和活成像能力，斑马鱼非常适合研究乙醇畸变的时序、剂量和遗传学等因素。乙醇可以施用精确分期胚胎，然后可以成像研究乙醇在发育过程中的直接影响。这项工作可以直接与人类有关，因为斑马鱼和人类之间的遗传开发程序高度保守，因此可以帮助指导FASD人类研究24。虽然斑马鱼已经被用来检查乙醇畸形的产生，但缺乏共识报告胚胎乙醇浓度，使得与人类相比困难25。在哺乳动物系统中，血液-酒精水平与组织乙醇水平直接相关26。许多斑马鱼研究在胚胎的循环系统完全形成之前治疗。由于没有母体样本进行检测，需要一个评估乙醇浓度的过程来量化胚胎内的乙醇水平。在这里，我们描述了一个过程，以量化乙醇浓度在正在发展的斑马鱼胚胎使用头空间气相色谱。

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	<tbody>
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			<td>Air</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>VWR</td>
			<td>10204-962</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoSampler, CP-8400</td>
			<td>Varian</td>
			<td></td>
			<td>Gas Chromatograph Autosampler</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatograph vial with polytetrafluoroethylene/silicone septum and plastic cap 2 mL</td>
			<td>Agilent</td>
			<td>8010-0198</td>
			<td>Can reuse the vials after cleaning, but not the caps/septa</td>
		</tr>
		<tr>
			<td>Gas Chromatograph, CP-3800</td>
			<td>Varian</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Helium</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
		</tr>
		<tr>
			<td>HP Innowax capillary column</td>
			<td>Agilent</td>
			<td>19095N-123I</td>
			<td>30 m x 0.53 mm x 1.0 &#956;m film thick</td>
		</tr>
		<tr>
			<td>Hyrdogen</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (Heptahydrate)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>2682002</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 10 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 1000 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611127</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 200 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611112</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 100 mm</td>
			<td>Fisher Scientific</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p1000L Micropipette</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p200L Micropipette</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p2L Micropipette</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene/silicone septum and plastic cap</td>
			<td>Agilent</td>
			<td>5190-7021</td>
			<td>Replacement caps/septa for gas chromatograph vials</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Dibasic)</td>
			<td>VWR</td>
			<td>BDH9266-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>VWR</td>
			<td>97062-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Beads .5 mm</td>
			<td>Biospec Products</td>
			<td>11079105z</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Beads 1.0 mm</td>
			<td>Biospec Products</td>
			<td>11079110z</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>VWR</td>
			<td>BDH9280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid-phase microextraction fiber assembly Carboxen/Polydimethylsiloxane</td>
			<td>Millipore Sigma</td>
			<td>57343-U</td>
			<td>Replacement fibers</td>
		</tr>
		<tr>
			<td>Star Chromatography Workstation</td>
			<td>Varian</td>
			<td></td>
			<td>Chromatography software</td>
		</tr>
		<tr>
			<td>Thermogreen Low Bleed (LB-2) Septa</td>
			<td>Millipore Sigma</td>
			<td>23154</td>
			<td>Replacement inlet septa</td>
		</tr>
	</tbody>
</table>

quantification of ethanol levels in zebrafish embryos using head space gas chromatography

胎儿酒精谱系障碍 （FASD） 描述了乙醇引起的发育缺陷的高度可变连续体，包括面部畸形和神经损伤。与复杂的病理学，胎儿外病影响大约每100个孩子在美国出生每年。由于FASD的高度可变性，动物模型在我们目前对乙醇引起的发育缺陷的机械理解中已被证明是至关重要的。越来越多的实验室专注于使用斑马鱼来检查乙醇引起的发育缺陷。斑马鱼产生大量外部受精、遗传可移植的半透明胚胎。这使得研究人员能够精确控制多种遗传环境中乙醇暴露的时间和剂量，并通过活成像技术量化胚胎乙醇暴露的影响。这结合人类对遗传学和发育的高度保护，已经证明斑马鱼是研究乙醇致畸性力学基础的有力模型。然而，不同斑马鱼研究之间的乙醇接触方案各不相同，这混淆了这些研究中对斑马鱼数据的解释。下面是使用头空间气相色谱对斑马鱼胚胎中的乙醇浓度进行量化的协议。

血液乙醇水平不能确定在早期胚胎斑马鱼，因为它们缺乏一个完全形成的循环系统。为了确定斑马鱼胚胎中的乙醇浓度水平，乙醇水平直接从均质胚胎组织测量。为了正确测量胚胎乙醇浓度，必须考虑胚胎体积。胚胎（附着的蛋黄）位于被胚胎外流体包围的胆囊（蛋壳）内（图1）。胚胎的任何体积测量都必须在胚胎暴露时进行，因为胚胎体积会随着胚胎大小的增大而改变。...

Watch this Scientific Journal Video about Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography at JoVE.com

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

这项工作描述了一种使用头部空间气相色谱从适当的暴露方法到胚胎加工和乙醇分析来量化斑马鱼胚胎中乙醇水平的协议。

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

该协议意义重大，因为它有助于了解乙醇在开发斑马鱼胚胎时的药代动力学，这将有助于在斑马鱼研究中标准化乙醇暴露制度。我们技术的主要优点是，它允许研究人员在胚胎发育期间快速、可重复地测量乙醇水平，特别是在没有血液供应的胚胎中。该方法可以方便地应用于其他模型系统以及其他环境因素，并采用适当的技术和设备设置。我预计首次执行此协议的个人将难以在胚胎加工过程中以必要的速度防止乙醇蒸发。首先将10个胚胎与额外的胚胎液体转移到1.5毫升的微离心管中，其标记的线为250微升。向填充线添加水。为脱光样品准备额外的微离心管。为了去除胆小菜，在100毫米培养皿中孵育胚胎，每毫升蛋白酶鸡尾酒在胚胎培养剂中孵育10分钟。每隔几分钟轻轻旋转一次盘子，以打破菜。一旦所有的胚胎都摆脱胆小球，从蛋白酶鸡尾酒中去除它们，用新鲜介质洗两次。然后将胚胎移植到100毫米的新鲜培养皿中。当所有样品都准备好进行体积测量时，使用具有最小尖端的 P200 微移子，小心地从胚胎周围取出所有液体。以小于 0.1 毫克的精度称水，然后通过减去 250 微升去除的水的重量来确定 10 个胚胎的体积。要确定单个胚胎的体积，请将差值除以 10。从交配罐中收集胚胎，并把它们放在一个标准的100毫米培养皿中，然后在28.5摄氏度下孵育。受精后6小时，将多达100个胚胎移植到具有胚胎培养和1%乙醇或培养新培养皿中。盖上，但不要密封培养皿，并把它们放在28.5摄氏度的培养箱18小时或直到它们达到24小时后受精。使用两个 P200 微管设置为 50 微升，将蛋白酶鸡尾酒溶液吸进一个，然后放入第二个溶液中。然后用玻璃移液器将10个胚胎快速放在1.5毫升的微离心管中，并关闭盖子。对所有样品进行测试、控制和乙醇处理胚胎重复此过程。一次使用一个样本，快速打开瓶盖，用 P200 微移子设置为 200 微升来吸气所有残留胚胎介质。快速但轻轻地添加和去除50微升的水，然后添加50微升蛋白酶鸡尾酒，并关闭管盖。让样品在室温下坐10分钟，然后快速加入450微升的5摩尔氯化钠，并关闭管盖。通过涡旋10分钟使样品均质，将两个微升上流液转移到气相色谱小瓶中。用聚四氟乙烯盖密封小瓶。完成启动方法后，加载 436 电流 SPME 乙醇两到五分钟 RG 运行。从分析软件中对样本列表中每个样本的方法菜单中的冰毒。从空气、水和五个摩尔氯化钠蛋白酶鸡尾酒空白开始填写样品列表。然后按0.3125到40毫摩尔的顺序输入乙醇标准。遵循标准与第二轮空白。输入所有均质胚胎上能样品，从最低到最高预测乙醇浓度进行测试，然后进入第三轮空白。将气相色谱小瓶按样品输入的顺序添加到自动取样器中，让它们加热 10 到 15 分钟。然后使用软件开始示例运行。完成所有运行后，通过在示例列表中添加最终示例并运行备用文件来激活关闭方法。备份在示例运行期间获取的所有数据。关闭方法完成后，单击打开的色谱图并打开包含结果的文件夹。样品会自动集成到此程序中。在结果中，确保已集成正确的峰值。确认所有样品后，打印或导出结果。在图形上绘制乙醇标准的峰值高度，然后计算斜率、y-intercept 和 r 平方值。通过从标准的 y-intercept 中减去样品的峰值高度，并除以标准的斜率，获取每个样品的乙醇值。要计算胚胎中的乙醇浓度，计算每个样品的稀释因子，并乘以该稀释因子的样品乙醇值。最后，通过将介质乙醇值乘以稀释因子10来计算介质参考样本。为了正确计算胚胎乙醇的浓度，测量了胚胎在胚胎中和从卵子中去除的体积。在这项分析中，胚胎占胆子内总体积的56%。根据先前发表的作品，73.6%作为所有提出的分析的胚胎含水量。这导致水包括胚胎体积1.1微升的0.82微升。在总的体积中，49%包含在胚胎中，51%包含在外质流体中。在气相色谱分析中，对两组15个样品及其处理介质进行了每组测量5次。第一组的介质乙醇水平为143.6毫摩尔，第二组为133.6毫摩尔。第一组和第二组胚胎的乙醇浓度平均为63.5毫摩尔和53.1毫摩尔。计算了每个样品的胚胎乙醇浓度与乙醇介质水平的比率。第一组有44%的介质乙醇水平，而组有40%的媒体乙醇水平。未经处理的控制胚胎以零毫摩尔介质乙醇浓度测量。尝试此过程时，要记住的最重要的事情是，正确处理胚胎进行GC分析至关重要，因为乙醇是挥发性且会迅速蒸发的。描述执行此步骤的最佳方法，但需要练习以发展适当的肌肉记忆。这一程序将有助于规范斑马鱼的乙醇处理制度，并允许今后的工作解剖乙烷致畸的遗传和细胞机制。

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...