这项工作得到了来自美国国立糖尿病和消化和肾脏疾病研究所的奖项编号 P30DK056336 的支持, 该研究中心是在伯明翰的阿拉巴马大学营养和肥胖研究所提供的试验补助金。

满足要求由果蝇活动监控单元 (用于源信息, 请参阅材料表) 安装在由马达单元控制的旋转臂上 (图1)。活动监视器确定在给定时间范围内, 在检测管中间的激光束阵列被打乱的频率。有关详细的绘图和深入的描述, 请参见我们以前的出版物9。虽然我们的系统使用 LAM25H 单元, 满足要求可以修改, 以适应其他果蝇活动监测单位以及。
1. 测试满足要求的设置
<ol><li>负载测试飞入玻璃标本管通过麻醉动物与 CO2或其他选择的方法来处理, 移动到空管, 并盖管。</li>
	<li>将封闭的玻璃试样管插入活动监控单元的插槽中。使用橡胶 O 形环 (17-18 规) 和/或橡胶带在单位两侧确保管子在旋转过程中不移动:<ol>
<li>在试样管上滑动一 o 形环, 并将 o 形环定位在管的中点附近。将试样管插入到活动监视器的插槽中, 并将其居中。</li>
		<li>根据需要移动 o 形环, 并确保一旦试样管居中, O 形环就会尽可能靠近活动监视器单元滑动。通过在玻璃管背面滑动第二 o 形环来固定试样管, 再次将 o 形环移动到活动监视器单元的附近。</li>
	</ol>
</li>
	<li>通过翻转设备前面的电源开关来启动满足要求的旋转。在本示例中, 将设备前面的刻度盘调整为所需的速度 (4 rpm (每分钟旋转)。 注意: 此示例中的旋转速度被选择为与以前发布的研究7相匹配。由于不同的转速将导致不同的动物活动水平, 当启动一项新的研究, 优化的转速为特定的实验设置 (基因型, 运动回合的长度,等.) 可能是必要的。 注意: 重要的是要确保管道正确定位, 使它们不会沿满足要求单位底部拖动旋转。</li>
	<li>通过打开 DAMSystem308 软件并确保连接指示灯保持绿色以进行多个旋转, 测试与果蝇活动监视单元提供的数据收集系统的连接。软件将开始记录数据到文本文件 "monitorX" (X 是单位号, 如果多台显示器正在使用) 时立即启动。记录频率可在 "首选项" 选项卡中进行调整;我们通常以5分钟的间隔记录。</li>
	<li>调查由 DAMSystem308 软件生成的文本文件, 以确保数据连接正常工作。连接的问题导致了切口, 时间点为满足要求中的所有位置记录了0活动 (请参见代表结果和表2)。如果出现切口, 请调整导致旋转电话插孔的数据连接, 如在旋转期间, 此连接可能会松动或扭曲。我们发现, 稳定与磁带的连接有助于防止此问题。</li>
</ol>2. 动物的制备
注意: 所有动物在标准条件下被饲养和测试在一个孵化器 (25 °c, 60-70% 湿气, 12 h 光/黑暗周期) 在糖蜜或玉米粉媒介10。
<ol><li>两周前的计划实验, 建立了小瓶与控制动物密度收集实验苍蝇从;我们通常会建立一个小瓶与7男性和10女性。一个健康的股票, 从一个单一的瓶子, 大约15处女男性和15女性可以收集在4天的时间内。根据化验所需的动物数量, 调整所建立的小瓶数量;一个典型的化验在我们的实验室包括10套10处女和雌性每种基因型 (100 男性和100女性)。 注: 非处女蝇可根据具体实验问题使用。如果对非处女动物进行长期试验, 爬行幼虫可能干扰准确的活动监测。</li>
	<li>在设置瓶子后一周内移除母蝇。</li>
	<li>从10天开始, 在设置了瓶子后, 从瓶子里收集处女苍蝇, 然后把它们按性别分开存放。</li>
	<li>收集足够的动物为实验和年龄他们根据需要, 3 天在这个例子。 注意: 我们通常用 CO2麻醉动物进行处理。CO2已知会在治疗后的较长时间内影响动物活动水平 (例如, 请参阅et . 201511)。如果 CO2效果干扰计划的下游化验或分析, 请使用不同的麻醉方法, 如冰麻醉。</li>
</ol>3. 与满足要求的数据收集
<ol><li>麻醉使用 CO2或另一种麻醉方法对动物进行定量研究。根据需要将它们分成组, 并将这些动物群装入活动监视器的空玻璃管中;在这个例子中, 同一年龄的10只动物被装入每个玻璃管, 10 个复制为每种动物类型 (性/基因型)。一定要注意每个管子里装的是哪种动物类型。 注: 对于较长的实验, 食品可以包括在标本管中。在这种情况下, 重要的是食物是安全的, 并不会在旋转过程中脱落。</li>
	<li>将玻璃化验管装入满足要求单元, 并用橡胶 O 形环将其固定。如果用一个以上的性别/基因型, 使用随机块设计或随机化的动物在满足要求的位置将消除任何潜在的位置影响。随机化可以通过给每个小瓶分配一个随机数来实现, 例如从基于 web 的随机数生成器 (例如, https://www.randomizer.org/) 或电子表格中, 然后根据随机数订购瓶子。</li>
	<li>将满足要求放入孵化器, 确保恒温、湿度和光照条件。确保数据和电源线都已正确连接。</li>
	<li>允许动物从麻醉中恢复, 适应新的环境1小时。</li>
	<li>开始实验, 启动满足要求的旋转, 以期望的速度。 注意: 另一种选择是先从动物开始旋转前收集基线活动数据, 然后记录活动水平以响应旋转刺激。</li>
	<li>通过打开 DAMsystem308 软件 (只需打开软件启动数据收集) 启动数据收集。数据被写入一个文本文件在设定的间隔 (这里, 5 分钟);如果需要, 请更改 "首选项" 选项卡中的间隔设置 (请参见步骤 1.4)。</li>
	<li>要确保数据成功传输到计算机, 请在经过一个或两个时间间隔后打开由 DAMsystem308 软件生成的文本文件, 并确认数据正在写入该文件。关闭并重新打开此文件以查看任何其他时间点的数据。收集所需时间量的数据;在本例中, 2 h。 注意: 在化验期间不要打开孵化器, 因为动物对任何干扰都非常敏感, 并且可能会随着活动的增加而反应。</li>
	<li>在练习的最后, 通过关闭 DAMsystem308 软件终止数据收集, 然后关闭满足要求。将动物从玻璃化验管中取出, 然后清洗管子。如果需要不同年龄的重复措施, 这些动物可以被移回食物瓶中。 注: 如果死亡发生在演习的制度, 它应该被记录, 因为存在的死苍蝇可以影响活动计数。在我们的经验与 DGRP2 飞线12,13, 演习在 4 rpm 2 小时没有导致任何死亡, 但较弱的菌株可能反应不同。</li>
</ol>4. 数据分析
<ol><li>打开 DAMSystem308 软件生成的标记为 "Monitor1" 的 .txt 文件。</li>
	<li>检查数据文件中是否存在数据捕获过程中可能发生的任何问题 (遗漏的时间点、等;表 1)。如有必要, 请在录制开始和结束时删除数据点, 对文件进行审查。</li>
	<li>使用您选择的统计软件 (例如, R), 分析所收集的数据。生成描述性统计数据并进行方差分析, 以调查性别和基因型等影响, 如果这些资料是正常分布的。如果数据通常不分布, 请使用非参数方法 (如克鲁斯卡尔-沃利斯测试) 来比较组。所需的具体分析将取决于具体的科学问题和实验设计。</li>
</ol>

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作者没有什么可透露的。

如有代表性的结果表明, 满足要求能够准确测量果蝇的运动活动。满足要求是灵活的, 并允许研究人员解决与运动生物学或运动干预有关的各种研究问题。要突出显示的协议中有两个关键步骤。首先, 必须测试满足要求的设置, 以确保从满足要求到 DAMSystem308 的数据传输正常工作。如果没有正确设置, 数据线可能会在旋转过程中发生纠缠, 有时满足要求和 DAMSystem308 之间的连接由于磨损而在旋转连接处中断 (尽管几乎每天都使用满足要求, 但我们不得不两年内更换连接器一次)。保持手头的零件更换是谨慎的。其次, 环境参数的一致性是实验成功的必要条件。果蝇对噪声或振动非常敏感, 因此在数据采集过程中不干扰实验是很重要的。因此, 理想情况下, 实验是在一个专门的孵化器中运行的, 在实验运行过程中是无法访问的。注意这两个关键步骤将确保从满足要求收集高质量的数据。
当我们在这里演示如何满足要求可以用来测量动物活动期间2小时连续运动治疗从小组的动物, 满足要求是灵活的, 因为运动的时间和强度可以调整为各种运动治疗。此外, 根据所使用的活动监测单元, 可以对其进行修改, 以测量单苍蝇或非常大的动物种群的活动。此外, 满足要求可用于测试运动体制的设计和优化。它也可以用来测量额外的变量, 如一天的时间, 动物年龄, 饮食, 人口大小, 或药物治疗对诱发活动和运动反应的影响。根据确切的实验设置,即,运动机制的长度和时间, 满足要求也可以用来干扰果蝇的自然睡眠模式。这些例子说明了满足要求的多功能性和果蝇研究中的一些潜在用途。其他小动物研究团体也可能有兴趣调整满足要求的目的, 从而扩大这一新工具的效用。
目前, 满足要求的一个限制是, 在给定时间可以处理的样本数量有限, 这是由所使用的活动监视器所决定的, 它最多可检测32个样本。虽然使用多个满足要求单位是可能的, 以允许满足要求用于大规模的基因屏幕或类似的应用, 开发一个更高的吞吐量版本的满足要求是理想的。
由于它能够测量与 Treadwheel 相同的方式诱导的活动水平, 满足要求可以与 Treadwheel 结合使用, 这允许更高的吞吐量 (一次可以处理48个样本)。可以使用满足要求优化运动协议, 然后在 Treadwheel 上进行进一步的研究。因此, Treadwheel 和满足要求可以作为具体研究设计所需要的互补。
满足要求是果蝇运动研究中的重要一步, 因为它允许对诱发活动进行量化。有一台能同时诱发运动和测量这项运动的机器是果蝇运动领域的一个明显的需要, 因为德国科学家组独立开发了一种非常相似的装置, 称为 "摇摆艇", 它还利用活动在旋转14引起的运动过程中测量活动的监视器。"摇摆船" 不使用完全旋转, 而是在旋转轴周围来回摆动活动监视器单元大约30度。因此, "荡舟", 就像满足要求一样, 使用旋转来不断诱发消极的 geotaxis 反应和增加动物活动。满足要求和 "摇摆船" 补充了现有的视频跟踪方法, 用于在刺激后检测果蝇, 如果蝇唤醒跟踪系统 (DART) 15.满足要求和 "摇摆船" 在像飞镖这样的系统上都有改进, 只有在刺激停止后才会跟踪活动。因此, 满足要求和 "摇摆船" 是果蝇运动领域研究人员的重要新工具, 可与现有的 Treadwheel 和动力塔装置结合使用。

最近, 研究人员已经开始使用果蝇果蝇来研究运动生物学。"黑腹"是100年来1、2的遗传模型系统。然而, 果蝇的研究不仅为遗传学做出了贡献, 也为其他学科包括神经生物学、行为生物学和生理学提供了帮助3。2009年, 第一台用于果蝇的运动机器被描述为4。电力塔利用动物的消极 geotaxis 反应。当扰时 , 果蝇倾向于移动到其外壳的顶端。这种反应是建立良好的, 是流行的 "环" (快速迭代阴性 Geotaxis5) 的基础, 用于估计果蝇的攀爬能力和/或体能。该电源塔使用与电机单元相连的机械臂, 在其外壳内反复举起一组动物, 并将它们放回地面以诱导负 geotaxis 响应 (Tinkerhess et 20126提供一个视频说明使用的电源塔)。在电力塔上进行长时间的治疗, 从而增加了动物的体力活动量 (跑步或飞行), 与未经处理的控制动物相比, 随着时间的推移, 提高了身体健康的环检测的性能4。因此, 这项工作证明了利用果蝇作为运动生物学模型的可行性。
为了扩大果蝇运动研究的可用工具, 在 2016年, 门德斯和同事们描述了第二个果蝇运动机器, Treadwheel7。与动力塔类似, Treadwheel 利用果蝇的负 geotaxis 反应。然而, 这种反应是由动物外壳的持续旋转引起的, 而不是像在动力塔中那样把它们举起来。这种感应方法是温和的, 并允许一个更耐用的运动机制, 以避免任何物理创伤可能发生在电力塔运动 (见 Katzenberger, r.j. et 等20138的影响反复物理果蝇健康的创伤)。与动力塔4类似, 在 Treadwheel 上对动物的运动治疗会导致多种生理反应, 包括身体健康、甘油三酯水平和体重7的变化。因此, 果蝇生物学家研究运动有两种互补的方法。
动力塔和 Treadwheel 的一个限制是无法测量运动治疗引起的活动量。从 Treadwheel 的录像分析显示, 不同果蝇菌株对运动治疗的反应有显著性差异7。具体而言, 研究的菌株在受刺激的7时所执行的额外活动有多大不同。这一观察促使我们开发了第三个运动系统, 即旋转运动定量系统 (满足要求), 它允许我们在旋转诱导运动中测量动物活动水平 9.满足要求利用一个商业上可用的活动监测单元, 安装在旋转臂上, 通过旋转来刺激运动, 如在 Treadwheel 中。与满足要求的初步工作证实, 基因上不同的果蝇菌株-和性别-对旋转刺激有明显不同的反应, 因此, 不同基因型的运动所引起的运动量不完全相同 9.因此, 满足要求现在使果蝇生物学家能够测量治疗引起的运动量, 在运动领域开辟了各种新的研究途径。
这里我们详细描述了如何使用满足要求来量化旋转运动。满足要求诱导旋转运动, 同时测量被治疗动物的活动水平。满足要求可以容纳各种各样的运动项目, 从简单的2小时连续锻炼机制这里展示到更加复杂的间隔训练方法如门德斯和同事7所描述, 并且刺激可以通过调整通过转速 (约1-13 旋转每分钟)。根据用于生产满足要求的活动监测单元, 该方法适用于对单飞蝇或大型动物种群的分析。由于这种多功能性, 满足要求为果蝇研究人员提供了一系列的机会来研究, 例如, 不同的锻炼制度, 饮食干预, 或人口密度的影响。

<table border="0" cellpadding="0" cellspacing="0" width="769">
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			<td height="21" style="height:21px;width:216px;">Drosophila Activity Monitor&#160;</td>
			<td style="width:147px;">Trikinetics</td>
			<td style="width:119px;">LAM25H</td>
			<td style="width:287px;">REQS component</td>
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			<td height="21" style="height:21px;width:216px;">Telephone Cord Detangler</td>
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			<td>uv20170719</td>
			<td>REQS component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vial closures (flugs)</td>
			<td style="width:147px;">Genesee Scientific</td>
			<td style="width:119px;">49-102</td>
			<td style="width:287px;">Drosophila culture supplies</td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Vials&#160;</td>
			<td style="width:147px;">Genesee Scientific</td>
			<td>32-120</td>
			<td style="width:287px;">Drosophila culture supplies</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Drosophila culture netting</td>
			<td style="width:147px;">Carolina Biological Supply</td>
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			<td style="width:287px;">Drosophila culture supplies</td>
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			<td height="21" style="height:21px;width:216px;">Cornmeal</td>
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			<td>43375</td>
			<td>Drosophila media</td>
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			<td height="21" style="height:21px;width:216px;">Molasses</td>
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			<td>BLA-GAL</td>
			<td>Drosophila media</td>
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			<td height="21" style="height:21px;width:216px;">Agar</td>
			<td style="width:147px;">Apex Bioresearch</td>
			<td style="width:119px;">66-103</td>
			<td>Drosophila media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inactive Dry Yeast</td>
			<td style="width:147px;">Genesee Scientific</td>
			<td style="width:119px;">62-106</td>
			<td>Drosophila media</td>
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			<td height="21" style="height:21px;width:216px;">Tegosept</td>
			<td style="width:147px;">Apex Bioresearch</td>
			<td style="width:119px;">20-258</td>
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			<td height="21" style="height:21px;width:216px;">Propionic acid</td>
			<td style="width:147px;">Genesee Scientific</td>
			<td style="width:119px;">20-271</td>
			<td>Drosophila media</td>
		</tr>
	</tbody>
</table>

measuring exercise levels in drosophila melanogaster using the rotating exercise quantification system (reqs)

果蝇是运动生物学研究的一个新的模型有机体。迄今为止, 已经介绍了两个主要的运动系统、电力塔和 Treadwheel。然而, 还缺乏一种方法来测量通过运动治疗引起的额外动物活动量。旋转运动量化系统 (满足要求) 填补了这种需求, 为经历旋转运动的动物提供了动物活动的量度。本协议详细介绍了如何使用满足要求来评估旋转运动过程中的动物活动, 并说明了可以生成的数据类型。在这里, 我们展示了如何使用满足要求来衡量在运动诱发活动中的性别和应变特定的差异。满足要求还可以用来评估不同的实验参数, 如年龄, 饮食, 或人口大小对运动诱发活动的影响。此外, 它还可用于比较不同运动训练方案的功效。重要的是, 它提供了一个机会, 以规范的运动治疗之间的菌株, 使研究员能够达到同等数量的活动之间的群体, 如果需要的话。因此, 满足要求是一个值得注意的新资源为运动生物学家与果蝇模型系统和补充现有的运动系统。

使用满足要求的单个运行的输出是 DAMSystem308 软件生成的数据表, 默认情况下将标记为 "Monitor1.txt" (例如, 请参见补充文件1)。此类表的摘录显示在表 1中。每一列都包含来自单个检测管的数据, 而行包含从实验开始 (顶端) 到末尾 (底部) 的每个时间间隔中测量的活动。如果有数据切口 (如表 2中所示), 则应监视前三个数据点, 在这种情况下, 必须调整电缆。
图 2显示了比较四种不同基因的果蝇线 (DGRP2 线371、703、810和 89712、13) 的实验结果。对于四行中的每条线, 按照《议定书》一节中所述, 收集了十份来自两性的十只处女蝇组的复制量测量。从 DAMSystem308 软件的输出文件中, "每10只苍蝇平均活动5分钟" 的计算方法是在整个2小时的时间跨度内平均进行活动测量。因此, 每一个柱的平均值为每个化验室产生一个度量值。表 3中提供了图 2所基于的摘要表。
对表 3中提供的数据进行了方差分析、基因型、性别和性别与基因型交互作用的测试。由于只有基因型的影响是显著的 (p < 2 x 10-16), 所以两性在图 2中显示的图表中被组合在一起。方差分析发现了基因型的强烈效应, 这在图中是明显的。各四基因型平均运动活动水平差异显著 (p < 0.05;Tukey 的 HSD), 因此,图 2说明了如何使用满足要求来检测基因型对运动活动水平的影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57751/57751fig1.jpg"> 图 1: 满足要求。这张照片中显示的是这个过程中使用的满足要求。活动监视器单元 (A) 围绕其由旋转臂 (B) 驱动的水平轴旋转。旋转速度可通过拨号 (C) 进行调整, 并且计算机的操作由开/关开关 (D) 控制。插入 (E) 显示活动监视器单元和旋转电话插孔之间的数据连接的特写。<a href="/files/ftp_upload/57751/57751fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57751/57751fig2.jpg"> 图 2: 在四种不同 DGRP 菌株中满足要求测量的运动活动的比较。X 轴: 动物基因型。Y 轴: 动物活动测量为每10只动物每5分钟的横梁横穿。A: DGRP2 897 号线;B: DGRP2 810 号线;C: DGRP2 703 号线;D: DGRP2 371 号线。这里显示的图表结合了男性和女性的数据, 因为对这些特定的线条没有显著的性别效应 (方差分析;p = 0.557)。然而, 有一个强烈的基因型效应 (方差分析;p < 2 x 10-16), 并且在四基因型之间的所有个体比较是高度重要的 (Tukey 的 HSD;p < 0.05)。黑金刚石: 基因型平均值;黑线: +/-一 SD (标准差)。<a href="/files/ftp_upload/57751/57751fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>时间</td>
			<td>活动 (1)</td>
			<td>活动 (2)</td>
			<td>活动 (3)</td>
			<td>活动 (4)</td>
			<td>活动 (5)</td>
		</tr>
<tr>
<td>12:25:00</td>
			<td>21</td>
			<td>86</td>
			<td>48</td>
			<td>32</td>
			<td>76</td>
		</tr>
<tr>
<td>12:30:00</td>
			<td>31</td>
			<td>55</td>
			<td>58</td>
			<td>74</td>
			<td>119</td>
		</tr>
<tr>
<td>12:35:00</td>
			<td>27</td>
			<td>45</td>
			<td>47</td>
			<td>80</td>
			<td>125</td>
		</tr>
<tr>
<td>12:40:00</td>
			<td>28</td>
			<td>55</td>
			<td>34</td>
			<td>83</td>
			<td>91</td>
		</tr>
<tr>
<td>12:45:00</td>
			<td>36</td>
			<td>56</td>
			<td>45</td>
			<td>67</td>
			<td>103</td>
		</tr>
</tbody></table>表 1: DAMSystem308 软件中准确数据的输出示例.每个 "活动 (#)" 列代表一小瓶10只苍蝇, 活动记录在5分钟的间隔。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>时间</td>
			<td>活动 (1)</td>
			<td>活动 (2)</td>
			<td>活动 (3)</td>
			<td>活动 (4)</td>
			<td>活动 (5)</td>
			<td>故障？</td>
		</tr>
<tr>
<td>12:00:00</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>是的</td>
		</tr>
<tr>
<td>12:05:00</td>
			<td>98</td>
			<td>1</td>
			<td>36</td>
			<td>0</td>
			<td>8</td>
			<td>不</td>
		</tr>
<tr>
<td>12:10:00</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>是的</td>
		</tr>
<tr>
<td>12:15:00</td>
			<td>88</td>
			<td>24</td>
			<td>44</td>
			<td>1</td>
			<td>9</td>
			<td>不</td>
		</tr>
<tr>
<td>12:20:00</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>是的</td>
		</tr>
<tr>
<td>12:25:00</td>
			<td>106</td>
			<td>51</td>
			<td>41</td>
			<td>0</td>
			<td>15</td>
			<td>不</td>
		</tr>
</tbody></table>表 2:DamSystem308 软件中带有数据包丢失的错误数据的输出示例.每列代表一小瓶10只苍蝇, 活动记录在5分钟的间隔。行12:00:00、12:10:00 和12:20:00 在所有列中显示 "0" 活动录制, 表明数据连接有问题 (故障？"是" 在最后一列中)。
表 3: 用于生成的示例数据图 2。<a href="/files/ftp_upload/57751/Table_3.xlsx">请单击此处下载此文件.</a>
补充文件 1:DAMSystem308 软件生成的未处理的输出文件。列1记录时间点编号。列2记录实验的日期, 3 列记录每次记录时间点的时间。不使用列 4-10, 列11-42 表示活动监视器32插槽中的录制。<a href="/files/ftp_upload/57751/Supplemental_File.txt">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS at JoVE.com

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

旋转运动定量系统 (满足要求) 可以通过旋转来诱导果蝇运动, 同时测量动物的活动量. 在这里, 我们提出一个点对点协议, 详细说明如何测量动物的活动水平, 使用满足要求的旋转运动治疗。

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

measuring-exercise-levels-drosophila-melanogaster-using-rotating

Nanyang Technological University

Research

JoVE Journal

Genetics

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

7.4K 次观看.  University of Alabama at Birmingham. 旋转运动定量系统 (满足要求) 可以通过旋转来诱导果蝇运动, 同时测量动物的活动量. 在这里, 我们提出一个点对点协议, 详细说明如何测量动物的活动水平, 使用满足要求的旋转运动治疗。

视频: Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

量化腹部色素沉着<em&gt;黑腹果蝇</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

科研

遗传学

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

在酵母S.酵母缺氧反应期间, 测量一段时间内的 mRNA 水平

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

TreadWheel:果蝇轻度诱导运动的间歇训练协议

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.