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)。

<ol>
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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 style="border-collapse:collapse;width:465pt" width="620">
	<colgroup>
		<col style="width:177pt" width="236" />
		<col style="width:67pt" width="89" />
		<col style="width:68pt" width="91" />
		<col style="width:153pt" width="204" />
	</colgroup>
	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:177pt;height:30.0pt" width="236">Carbenicillin di-Sodium salt</td>
			<td class="xl18" style="width:67pt" width="89">Sigma-Aldrich</td>
			<td class="xl18" style="width:68pt" width="91">C1389-5G</td>
			<td class="xl18" style="width:153pt" width="204">Antibiotic</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Streptomycin Sulphate salt</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">S6501-50G</td>
			<td class="xl18">Antibiotic</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">&#160;Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">I6758-10G</td>
			<td class="xl18">Inducer for RNAi plates</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Sodium Chloride (NaCl)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">71380-1KG-M</td>
			<td class="xl18">Used in S basal, and NGM agar</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">di-Potassium Hydrogen Phosphate(K2HPO4)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">1.05104.1000</td>
			<td class="xl18">Used in S basal, and NGM agar</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Potassium di-Hydrogen Phosphate (KH2PO4)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">P9791-1KG</td>
			<td class="xl18">Used in S basal, and NGM agar</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl19" height="40" style="width:177pt;height:30.0pt" width="236">Magnesium Sulphate (MgSO4)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">M2643-1KG</td>
			<td class="xl18">Used in NGM agar</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl19" height="40" style="width:177pt;height:30.0pt" width="236">Calcium Chloride (CaCl2)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">C5670-500G</td>
			<td class="xl18">Used in NGM agar</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl19" height="40" style="width:177pt;height:30.0pt" width="236">Sodium Hydroxide (NaOH)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">71687-500G</td>
			<td class="xl18">Used for bleaching</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl19" height="40" style="width:177pt;height:30.0pt" width="236">Pluronic-F127</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">P2443-1KG</td>
			<td class="xl18">Used in imaging</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl19" height="40" style="width:177pt;height:30.0pt" width="236">Sodium Hypochlorite (NaClO)</td>
			<td class="xl18">Sigma-Aldrich</td>
			<td class="xl18">1.05614.2500</td>
			<td class="xl18">Used for bleaching</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">LB Broth</td>
			<td class="xl18">Invitrogen</td>
			<td class="xl20">12780052</td>
			<td class="xl18">Used to grow bacteria</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Adavanced TC 6 cm Tissue Culture plates</td>
			<td class="xl18">Greiner Bio-One</td>
			<td class="xl21" style="width:68pt" width="91">628960</td>
			<td class="xl18">Plates for lifespan</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">CellStar 10cm Tissue Culture plates</td>
			<td class="xl18">Greiner Bio-One</td>
			<td class="xl20">664160</td>
			<td class="xl18">Plates for imaging</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Low Retention P200 tips</td>
			<td class="xl18">Brandt</td>
			<td class="xl20">732832</td>
			<td class="xl18">Tips for handling worms in liquid</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Agar</td>
			<td class="xl18">BD</td>
			<td class="xl20">214510</td>
			<td class="xl18">Agar for NGM, RNAi and NSC plates</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="height:30.0pt">Bacto-peptone</td>
			<td class="xl18">BD</td>
			<td class="xl20">211820</td>
			<td class="xl18">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/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/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 ...

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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

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

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 ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

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

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

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

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

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 ...

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 ...

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

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

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