Name: quantification of information encoded by gene expression levels during lifespan modulation under broad-range dietary restriction in c. elegans
Uploaded: 2017-08-16T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans at JoVE.com

我们感谢巴格曼和霍维茨的实验室试剂。毛细管气相色谱法，这由美国国立卫生研究院办公室的研究基础设施项目 (P40 OD010440) 提供了一些毒株。我们还感谢 M.Lipovsek 对手稿 》 的评论。这项研究受维康信托基金会 (项目赠款 087146 向王室法律顾问)、 BBSRC （BB/H020500/1 和 BB/M00757X/1 对王室法律顾问）、 欧洲研究理事会 (NeuroAge 242666 向王室法律顾问)、 美国国立卫生研究院 （R01AG035317 和 R01GM088333 到 H.L.） 和美国国家自然科学基金 (0954578 到 H.L.，分别被称为 M.Z 到 0946809 GRFP)。

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作者没有透露。

在这里，我们提出新方法封装广泛得多的食物浓度比以前发布协议的饮食限制。此方法链接两个以前单独现象见于线虫博士文学、 细菌剥夺和古典的饮食限制，允许两个饮食的影响，要研究下一个协议。我们使用新的宽范围博士范式，目前检查单个细胞基因表达在特定的环境提示响应和确定此单元格如何对信息进行编码的一般框架。我们的框架包括说明如何执行寿命和定量成像，分别的两个实验协议下宽范围, 从这些实验协议博士数据然后可以检查与中提供的计算分析这一框架进行量化编码的基因的表达水平或寿命的变化跨不同的食物条件的信息。
使用宽范围博士范式的寿命实验涉及六个独特的食物水平 (表 1)。这就需要比检查较少的食物水平，如膳食剥夺10，11下长寿或使用吃 2遗传背景35更劳动密集型的解决办法。然而，审查在单一条件下的寿命可以限制博士的基因作用的解释。例如，我们最近表明， daf 7突变体食物浓度相比野生型动物12 （图 1A） 响应双向衰减。在没有食物的情况下， daf 7突变体显示野生型动物相比其寿命的缩短。如果我们只考虑膳食剥夺，我们将解释， daf 7基因作为必要只寿命延长，时事实上daf 7作用是更复杂。因此，关键的结果议定书 》 的这一部分是寿命的去确定是否感兴趣的基因参与调制在食物丰富整体响应的变化。
本议定书与其他方法相比的一大优点是，它使用一种新的方法来消除后代生产在经历寿命分析的动物。大多数研究使用药物 FuDR 成人呈现他们不育系的增殖抑制作用。然而，最近的研究表明 FuDR 治疗可以有条件和基因特异性影响寿命17，，1819，20，21，调用到质疑其普遍适用性。在本议定书，消除后代的生产通过 24 小时实现治疗的动物与 rna 干扰靶向的鸡蛋 5基因，抑制几丁质蛋壳的形成受精线虫卵母细胞导致他们死亡22，23。此方法的优点是寿命的它是寿命的非常晚代理，所以不会干扰的胚，是寿命的一个主要调节器，线虫。
一个潜在的警告，宽范围博士议定书 》 是对使用的抗生素来控制细菌的增殖，从而确保严格控制空气细菌浓度的依赖。蠕虫在肠道内的细菌扩散是已知事件中线虫16的主要死因。因此，抑菌抗生素，如羧苄青霉素 NGM 琼脂中的使用可以防止细菌扩散，增加寿命的蠕虫相比，非抗生素控制16。某些类型的抗生素，如利福平36和四环素家庭37，38，成员已被证明延长寿命中的线虫独立及其对细菌的影响扩散。然而，尚无证据在文献中羧苄青霉素或链霉素可增加长寿独立及其对细菌增殖的影响。
使用寿命可以看作一个复杂的计算的输出地方环境信息，通过基因表达中的神经网络，路由转交生理学。我们的协议提供了一种方法来理解特定基因如何影响这流动的环境信息。为了解决这个问题，我们需要可靠的图像处理，以确定在单个细胞水平基因表达响应的分布。能够估计不仅对基因表达的平均响应更改在食物丰富，也从大量人口的全面统计分布表示适用方法的一项重要要求。食物丰富基因表达回应此准确描述允许应用程序信息理论的量化编码的特定神经元的信息，以及神经电路所采用的编码战略。
在本议定书中概述的方法的成像和计算方面都适用于生物背景一大套。我们的工作，我们重点放在小的神经网络所涉及的食物传感，然而，信息处理功能的分析并不限于特定的单元格类型或具体环境线索。在将来，这些方法可能给予的输入变量，影响任何生理的输出更大的品种。这些方法将有助于更好地理解基因调控网络是如何编码的过程和传输信息。

所有的有机体需要能够感知和响应变化的环境，以确保他们的生存。在动物中，中枢神经系统原发性探测器和换能器的有关环境的信息和协调对任何可能会影响生物的生存1的变化的生理反应。食物丰富度是环境的提示，在多个上下文中具有很好的研究，不仅调节食品相关的行为，如觅食2，而且也会影响长寿的动物。寿命的食物丰富度变化的调制是一种现象称为饮食限制 (DR) 和具有广泛的进化环境保育3。
秀丽隐杆线虫线虫是一个强大的模型系统为解决基本的生物学问题。大量的技术已经发展到了允许蠕虫病毒基因组，如编辑技巧的 RNAi 和体内基因操纵。小的物理尺寸的蠕虫和光学透明度也把自己借给体内成像的两个转录和翻译荧光记者和实用的高通量技术如微流体4。在一起，可以利用这些工具来检查如何神经电路直接动物行为。
线虫是 bacterivore 和几个已发表的方法，允许食物丰富度的精确控制，操纵8，细菌浓度5，，67.线虫研究社群，博士研究了在两个不同的上下文中。第一次可称之为 '古典博士'，它反映了在降低食品中其他生物的响应中看到的变化。在这方面，减少食物丰富度从随意水平导致了越来越多的使用寿命，直到达到最佳之后这点长寿随进一步减少食品6，7， 9。第二个背景下的线虫研究了博士是膳食剥夺蠕虫的寿命增加任何细菌的食物源10，11完全去除。在 Entchev 等人 （2015 年）12，我们表明博士因这两种不同范式的复杂性可以被审查同时语境下我们称之为 '宽范围博士'。通过使用协议概述如下，我们确定了一类新的基因参与博士那双向调节对食物丰富的寿命响应并参与感觉食物12 (图 1) 的神经回路。
动物对环境变化的响应集成链接到复杂的监管交互环境信息传达给生理感官系统的生物学过程的序列。虽然这种"信息流"的机械细节往往是未知的遗传的工具可以用于获得深入了解这个复杂的计算如何组织之间不同的生物成分。在我们最近的工作中，我们展示了daf 7和tph 1参与有关通过调节线虫12寿命食物传感神经电路的食物丰富的环境信息的传输,13.通过应用信息理论14的数学框架，我们就能够量化的环境信息，在位，由在daf 7和基因表达的变化表示 tph 1在跨不同食品级别的特定神经元。从这一点，我们当时无法揭示编码策略受雇于该神经回路和基因控制的方式 (图 2)。
在以下协议中，我们概述了解什么是基因的兴趣在特定神经元中表达的影响以及如何他们参与食品信息流向从环境到生命周期所需的步骤。广义地说，我们的框架分为两个实验和计算的工作流。实验方面，它是关键有突变的基因的兴趣，可以在宽范围下审查博士忠实转录记者也是必要的不同食品级基因的表达水平进行量化。能够进行了计算分析，讨论在我们的方法，数据集需要足够的尺寸，以便提供有意义的表达分布的估计。尽管我们为分析提供模板源代码，用户需要熟悉信息理论，广泛应用在我们计算框架的语言。源代码是 R 和 c + + 写的。因此，有一定的编程能力也是需要在有意义的方式加以应用的。

<table><tbody><tr><td>Carbenicillin di-Sodium salt</td><td>Sigma-Aldrich</td><td>C1389-5G</td><td>Antibiotic</td></tr><tr><td>Streptomycin Sulphate salt</td><td>Sigma-Aldrich</td><td>S6501-50G</td><td>Antibiotic</td></tr><tr><td>&amp;nbsp;Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I6758-10G</td><td>Inducer for RNAi plates</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>71380-1KG-M</td><td>Used in S basal, and NGM agar</td></tr><tr><td>di-Potassium Hydrogen Phosphate(K&lt;span class=&quot;font9&quot;&gt;&lt;sub&gt;2&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;HPO&lt;/span&gt;&lt;span class=&quot;font9&quot;&gt;&lt;sub&gt;4&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;)&lt;/span&gt;</td><td>Sigma-Aldrich</td><td>1.05104.1000</td><td>Used in S basal, and NGM agar</td></tr><tr><td>Potassium di-Hydrogen Phosphate (KH&lt;span class=&quot;font10&quot;&gt;&lt;sub&gt;2&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;PO&lt;/span&gt;&lt;span class=&quot;font10&quot;&gt;&lt;sub&gt;4&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font11&quot;&gt;)&lt;/span&gt;</td><td>Sigma-Aldrich</td><td>P9791-1KG</td><td>Used in S basal, and NGM agar</td></tr><tr><td>Magnesium Sulphate (MgSO&lt;span class=&quot;font9&quot;&gt;&lt;sub&gt;4&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;)&lt;/span&gt;</td><td>Sigma-Aldrich</td><td>M2643-1KG</td><td>Used in NGM agar</td></tr><tr><td>Calcium Chloride (CaCl&lt;span class=&quot;font9&quot;&gt;&lt;sub&gt;2&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;)&lt;/span&gt;</td><td>Sigma-Aldrich</td><td>C5670-500G</td><td>Used in NGM agar</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>71687-500G</td><td>Used for bleaching</td></tr><tr><td>Pluronic-F127</td><td>Sigma-Aldrich</td><td>P2443-1KG</td><td>Used in imaging</td></tr><tr><td>Sodium Hypochlorite (NaClO)</td><td>Sigma-Aldrich</td><td>1.05614.2500</td><td>Used for bleaching</td></tr><tr><td>LB Broth</td><td>Invitrogen</td><td>12780052</td><td>Used to grow bacteria</td></tr><tr><td>Adavanced TC 6 cm Tissue Culture plates</td><td>Greiner Bio-One</td><td>628960</td><td>Plates for lifespan</td></tr><tr><td>CellStar 10cm Tissue Culture plates</td><td>Greiner Bio-One</td><td>664160</td><td>Plates for imaging</td></tr><tr><td>Low Retention P200 tips</td><td>Brandt</td><td>732832</td><td>Tips for handling worms in liquid</td></tr><tr><td>Agar</td><td>BD</td><td>214510</td><td>Agar for NGM, RNAi and NSC plates</td></tr><tr><td>Bacto-peptone</td><td>BD</td><td>211820</td><td>Peptone for NGM, RNAi and NSC plates</td></tr></tbody></table>

quantification of information encoded by gene expression levels during lifespan modulation under broad-range dietary restriction in c. elegans

感官系统允许动物进行检测，处理，及对他们的环境作出反应。食物丰富度是环境的提示，有深远的影响，对动物生理和行为。最近，我们表明，长寿的秀丽隐杆线虫线虫的食物丰富度调制比以前认为的更复杂。寿命到食物水平变化的响应能力是由作用是通过控制信息处理内神经电路的特定基因决定的。我们的框架结合遗传分析、 高通量定量成像和信息理论。在这里，我们描述如何使用这些技术来描述任何具有宽范围限制饮食的生理相关性的基因。具体来说，此工作流被为了揭示如何感兴趣的基因调节寿命下宽范围限制饮食;然后建立基因的表达是如何随食物水平;和最后，提供的信息量客观的量化传达的关于食物丰富度环境中的基因表达。当几个基因同时神经电路的语境下，检查此工作流可以发现电路所采用的编码战略。

通过进行突变体基因的兴趣与野生型 N2 应变寿命实验，一个可以建立是否这些基因在宽范围博士食品响应中发挥作用。野生型反应应该类似于图 1所示。这种反应的突变体，反映了非均匀效应的整个食品的条件，任何调制指示这些基因是如何影响该蠕虫能够正确响应更改，在食物丰富度，在哪点进一步调查对宽范围博士的这些基因表达响应被必要。如果，然而，突变体的长寿反应并不明显不同于野生型基因有没有转导的宽范围博士影响至少一级的平均使用寿命的作用。如果基因突变导致的整个寿命响应统一转变基因有长寿食品独立影响。这并不排除出感兴趣的基因的表达是食物敏感，在这种情况下，由这些基因的信息不会传递给使用寿命的可能性。
议定书 》 的下一个阶段是兴趣的确定如何表达水平改变下宽范围博士为基因。在图 1B，我们说明这通过daf-7，在食物中 ASI 感觉神经元水平显示响应更改转录记者的表达水平。在一个daf-7(-)突变体，改变了转录记者表达响应。如果感兴趣的基因是真正食物快速响应的寿命一级然后就可以预期他们表达也会改变与食物。相应地，在突变体为背景的转录记者应有表达改变配置文件宽范围博士回应。但是，它也可能是基因的野生型背景感兴趣转录记者中不显示任何食物反应的变化，在表达式中。在这种情况，这可能表明本议定书范围之内的转录调节作用。
在戴安娜等人 (2017)13，我们提取表达式值为daf 7在 ASI 和 ADF 和 NSM tph 1 。在图 2A，我们说明了 ASI 和 ADF 为给定的食物水平的表达分布的估计。有多个读数从单一蠕虫图像使我们能够分析不仅独立编码的每个神经元，但整个神经电路 (图 2B-2 C) 的组合信息的信息量。结合这两项的信息理论措施允许我们来表征系统的神经元的编码策略来传达有关食物的信息。在电路中的冗余量可以通过采取互信息的每个神经元数目和联合互信息 （信道容量） 通过考虑电路的组合读数中减去。这种差异为正值表示冗余字符的编码策略因为零部件之间的累积信息大于编码由整个电路的实际信息。相反一个负的值反映了协同的战略因为真实信息编码大于其组件 (图 2B) 的总和。信息和冗余可以跨不同的基因型，以探讨可能高顺序作用的基因调控，例如在戴安娜等人进行比较(2017)13的daf 7突变影响切换到协同 (图 2C-2D) 从冗余编码策略。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56292/56292fig1.jpg"> 
图 1: 响应的寿命和基因表达下宽范围博士(A) 平均寿命的野生型 N2 应变 （黑线） 显示宽范围博士的复杂反应。此反应的规模被衰减在空的突变体的daf 7基因 （红线）。误差线代表的意思，从 Entchev等人汇集数据的标准误差（2015 年）12.(B) 平均表达水平的野生型背景 （黑线） daf 7基因转录的记者也显示宽范围博士非单调的复杂反应。Daf-7(-)遗传背景中的转录记者表达高度减毒和在食品水平显示小响应的变化。误差线代表的意思，数据从 Entchev等人单独审判的标准误差（2015 年）12.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56292/56292fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56292/56292fig2.jpg"> 
图 2:计算方法.(A) 插图的二维密度估计的tph-1表达的 ADF 和daf 7表达的 ASI 所得 'ks' R 包使用网格尺寸 30 × 30。(B) 可视化的信息编码的tph 1和daf 7 （整体） 的联合表达和单独 （部分的总和） ADF、 ASI 和 NSM 神经元。冗余和协同字符编码的表示由堆积条形图右边的高度和信息编码的完整电路之间的区别。(C) 比较食品信息编码的野生型动物和daf-7(-)突变体。(D) 减少互信息在突变体中观察到的被伴随着对协同编码开关。小组 B-D 都改编自戴安娜等.(2017)13.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56292/56292fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>细菌浓度 （细胞/毫升）</td>
			<td>光密度 （600nm 处）</td>
			<td>稀释因子 （从以前的）</td>
		</tr>
		<tr>
			<td>1.12E + 10</td>
			<td>56.000</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>2.00E + 09</td>
			<td>10.000</td>
			<td>5.60</td>
		</tr>
		<tr>
			<td>6.32E + 08</td>
			<td>3.160</td>
			<td>3.16</td>
		</tr>
		<tr>
			<td>6.32E + 07</td>
			<td>0.316</td>
			<td>10.00</td>
		</tr>
		<tr>
			<td>2.00E + 07</td>
			<td>0.100</td>
			<td>3.16</td>
		</tr>
		<tr>
			<td>0 (S 基)</td>
			<td>0.000</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>
表 1: 食物水平及稀释因素在宽范围博士用细菌l 浓度 （细胞/毫升） 使用在宽范围博士协议，以及他们各自的 OD600测量和实现每个浓度从上一个所需的稀释倍数。
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="7">实验温度为寿命</td>
		</tr>
		<tr>
			<td>一天</td>
			<td>12.5 ° C</td>
			<td>15 ° C</td>
			<td>17.5 ° C</td>
			<td>20 ° C</td>
			<td>22.5 ° C</td>
			<td>25 ° C</td>
			<td>27.5 ° C</td>
		</tr>
		<tr>
			<td>-12</td>
			<td colspan="7">大块新鲜 NGM 板所有菌株和保持在 20 ° C</td>
		</tr>
		<tr>
			<td>-11</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-10</td>
			<td colspan="7">设置 P0 daf-7(-) 菌株的生成和维护在 20 ° C （每板 1 L4、 5 版）</td>
		</tr>
		<tr>
			<td>-9</td>
			<td colspan="7">设置 P0 一代的野生型菌株和保持在 20 ° C （每板 1 L4、 5 版）</td>
		</tr>
		<tr>
			<td>-8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-5</td>
			<td colspan="7">设置 daf-7(-) 菌株的 F1 代和保持在 20 ° C （每板 1 L4、 90 板）</td>
		</tr>
		<tr>
			<td>-4</td>
			<td colspan="7">建立了野生型菌株的 F1 代和保持在 20 ° C （每板 1 L4、 30 个板块）</td>
		</tr>
		<tr>
			<td>-3</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0</td>
			<td colspan="7">拿到 F2 L4 幼虫 &#62; 蛋 5 RNAi 板和保持在 20 ° C (15 L4 每板，24 片)</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="7">1 日龄成虫移动到 NSC 板与 2.0 e + 9 细胞/毫升食品级种子，并保持在 20 ° C</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="7">移动 2 日龄成虫，到实验温度和 NSC 板实验食品条件下种子</td>
		</tr>
		<tr>
			<td>3</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
		</tr>
		<tr>
			<td>4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
		</tr>
		<tr>
			<td>6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
		</tr>
		<tr>
			<td>8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>9</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
		</tr>
		<tr>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>11</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td></td>
		</tr>
		<tr>
			<td>12</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>13</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td>转让</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>17</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18</td>
			<td>转让</td>
			<td>转让</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>19</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>21</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>22</td>
			<td>转让</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
表 2: 在不同温度下的寿命的时间表进行。步骤来设置宽范围博士寿命在不同温度下，使用daf-7(-)和野生型菌株作为例子的实验所需的纲要。数量转移到新鲜板的每个实验食品水平随温度的升高。这是占动物在较高温度下老化的速度快的事实，所以更倾向于物理伤害，每份转让。
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>天</td>
			<td>Ima</td></tr></tbody></table>ging 管道
		
		<tr>
			<td>-14</td>
			<td>大块记者菌株 daf(-) 背景中。保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-13</td>
			<td>大块记者株野生型背景中。保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-12</td>
			<td>设置 P0 一代的 daf-7(-) 记者株。使用 3 L4 幼虫每 10 厘米 NGM 板。使用 2 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-11</td>
			<td></td>
		</tr>
		<tr>
			<td>-10</td>
			<td>设置 P0 代的记者的野生型菌株。使用 3 L4 幼虫每 10 厘米 NGM 板。使用 2 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-9</td>
			<td></td>
		</tr>
		<tr>
			<td>-8</td>
			<td>建立了 F1 代的 daf-7(-) 记者株。使用 3 L4 幼虫每 10 厘米 NGM 板。使用 12 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-7</td>
			<td></td>
		</tr>
		<tr>
			<td>-6</td>
			<td>建立了 F1 代的记者的野生型菌株。使用 3 L4 幼虫每 10 厘米 NGM 板。使用 4 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-5</td>
			<td></td>
		</tr>
		<tr>
			<td>-4</td>
			<td></td>
		</tr>
		<tr>
			<td>-3</td>
			<td>在下午漂白 daf-7(-) 记者株 (~ 5 pm) 和在 3 10 厘米 NGM 板上产卵并保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-2</td>
			<td>在早上 （上午 ~ 10） 和存款的漂白剂记者野生型菌株鸡蛋上 3 10 厘米 NGM 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>-1</td>
			<td></td>
		</tr>
		<tr>
			<td>0</td>
			<td>到 10 厘米鸡蛋 5 RNAi 板洗 L4。使用每株 3 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>1</td>
			<td>洗到 NSC 印版与 2.0 e + 9 细胞种子 1 天成人/毫升使用每株 3 板和保持在 20 ° c。</td>
		</tr>
		<tr>
			<td>2</td>
			<td>NSC 板洗 2 天成人种子与实验粮食水平。使用实验温度 3 板每应变和转变。</td>
		</tr>
		<tr>
			<td>3</td>
			<td>转移到新鲜 NSC 板。使用每株 3 板和维护在实验温度。</td>
		</tr>
		<tr>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td>转移到新鲜 NSC 板。使用每株 3 板和维护在实验温度。</td>
		</tr>
		<tr>
			<td>6</td>
			<td>拿掉板动物和准备成像。</td>
		</tr>
	

表 3: 成像管道时间表。步骤来设置成像实验使用荧光转录记者株在不同温度下的daf-7(-)和野生型背景作为例子的宽范围博士所需的纲要。

Watch this Scientific Journal Video about Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans at JoVE.com

线虫大范围饮食限制下寿命调节过程中基因表达水平编码信息的量化遗传学 |视频

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

在这里，我们提出一个框架，以宽范围饮食限制涉及基因表达和使用寿命。我们描述协议为宽范围限制饮食和定量成像的这个范式下的基因表达。我们进一步列出了计算分析，以揭示潜在的信息处理功能的基因电路参与食品遥感。

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

quantification-information-encoded-gene-expression-levels-during

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Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Gene Expression

SCIENTISTS IN ACTION

8.0K 次观看.  King's College London. 在这里，我们提出一个框架，以宽范围饮食限制涉及基因表达和使用寿命。我们描述协议为宽范围限制饮食和定量成像的这个范式下的基因表达。我们进一步列出了计算分析，以揭示潜在的信息处理功能的基因电路参与食品遥感。

视频: Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

的转录分析<em&gt;ç</em&gt;。<em&gt;线虫</em&gt; RNA测序数据通过炫套房的银河项目

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

科研

遗传学

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

测量 秀丽隐杆 线虫食物摄入量和寿命的高通量测定

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

作者聚焦：探索脂毒性与 HFpEF 之间的关系 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

行为学

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

生物工程

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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 range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

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 ...

生物学

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

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 ...

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

摘要

探索更多视频

此视频中的章节

的转录分析

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

秀丽线虫纵向成像的微流控平台

实心板为基础的饮食限制在线虫

线虫的核苷酸和蛋白质提取联合

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

摘要

探索更多视频

此视频中的章节

的转录分析

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

秀丽线虫纵向成像的微流控平台

实心板为基础的饮食限制在线虫

线虫的核苷酸和蛋白质提取联合

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量