Name: rapid analysis of circadian phenotypes in arabidopsis protoplasts transfected with a luminescent clock reporter
Uploaded: 2016-09-17T15:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter at JoVE.com

We would like to thank Prof. David Somers and Dr. Jeongsik Kim for the luciferase reporter plasmid and initial method development. Prof. Karen Halliday kindly provided all the circadian mutant lines used in this study. This research was supported by Royal Society research grants RS120372 and RS140275. GvO is a Royal Society University Research Fellow (UF110173).

注意:在这个协议中，所描述的试剂和卷每被测试拟南芥系中表达，并且将导致该基因型六个复制孔中。与行数乘以材料分析多于一行时被分析。在视频中，野生型拟南芥会相比，生物钟突变系ZTL（尽管从附加线路代表性的结果将随后提供）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 酶液 </td><td></td></tr><tr><td>纤维素酶</td><td> 0.5％（重量/体积） </td></tr><tr><td>果胶酶R10 </td><td> 0.25％（重量/体积） </td></tr><tr><td> D-甘露糖醇</td><td> 400毫米</td></tr><tr><td> 氯化钙 </td><td> 10毫</td></tr><tr><td>氯化钾</td><td> 20毫米</td></tr><tr><td>牛血清白蛋白</td><td> 0.1％（重量/体积） </td></tr><tr><td> MES，pH值5.7 </td><td> 20毫米</td></tr><tr><td></td><td></td></tr><tr><td> W5的解决方案 </td><td></td></tr><tr><td>氯化钠</td><td> 150毫米</td></tr><tr><td> 氯化钙 </td><td> 125毫米</td></tr><tr><td>氯化钾</td><td> 5毫米</td></tr><tr><td> MES，pH值5.7 </td><td> 2毫米</td></tr><tr><td>葡萄糖</td><td> 5毫米</td></tr><tr><td></td><td></td></tr><tr><td> MMG解决方案 </td><td></td></tr><tr><td> MES，pH值5.7 </td><td> 4毫米</td></tr><tr><td> D-甘露糖醇</td><td> 400毫米</td></tr><tr><td> 氯化镁 </td><td> 15毫米</td></tr><tr><td></td><td></td></tr><tr><td> PEG解决方案 </td><td></td></tr><tr><td> PEG4000 </td><td> 40％（重量/体积） </td></tr><tr><td> D-甘露糖醇</td><td> 200毫米</td></tr><tr><td> 氯化钙 </td&gt; <td> 100毫米</td></tr><tr><td></td><td></td></tr><tr><td> 成像解决方案 </td><td></td></tr><tr><td> W5的解决方案</td><td>至最终体积</td></tr><tr><td>胎牛血清</td><td> 5％（V / V） </td></tr><tr><td>荧光素</td><td> 1.2毫米</td></tr><tr><td>氨苄青霉素</td><td> 50毫克/毫升</td></tr></tbody></table> 表1: 溶液的列表。 1.材料和缓冲液的制备<ol><li>植物材料<ol><li>保持拟南芥山口-0在水的种子在1.5ml管中，在4℃在黑暗中四天。移液器种子的土壤上在一个锅里，并在21°C转移到漫长的一天条件（16小时光照8小时黑暗）7天。留在锅里盖子的前三天，以确保高湿度。移植6苗到一个新的8厘米×13厘米×5厘米的花盆并继续anoth在相同条件下生长的植物呃21天。确保土壤湿润各地。 </li></ol></li><li>记者质粒<ol><li>净化CCA1pro:从细菌培养LUC报告质粒19预先，使用根据制造商的说明的DNA分离试剂盒。 注:20微克报告质粒是必需的（10微升在卫生署2 O 2微克/微升）的转染。 </li></ol></li><li>解决方案 注意:对于这个协议都需要5的解决方案:酶溶液，清洗5（W5）溶液，甘露醇镁（MMG）溶液，聚乙二醇（PEG）溶液，和成像溶液（ 表1）。在继续原生质体分离步骤之前准备这些。 <ol><li>制备的10ml酶溶液按表1中 ，添加酶最后，并过滤消毒通过0.22μm注射器过滤该溶液。 </li><li>在下面的卷准备其他的解决方案:15毫升W5的解决方案，将10毫升MMG小号olution，将1ml PEG溶液和1.5毫升成像溶液按表1中 。 注:该PEG溶液制备不下转染前一小时，以确保PEG已完全溶解是很重要的。过滤消毒通过0.22微米注射器过滤器的成像溶液。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54586/54586fig1.jpg" /> 图1:原生质体分离成人拟南芥（A）的叶子被固定在与下表皮层朝上（B）釜磁带。 （C）的魔术胶带轻轻压到用15毫升锥形管的尖端的下表皮层。 （D）的魔术胶带除去以暴露叶肉细胞（E）。 （F）与假釜胶带条附s的浮在含有纤维素酶和果胶酶的酶溶液，释放原生质体在溶液中。 （G）骨架留在釜磁带酶消化后叶被丢弃，留下酶溶液（H）的原生质体。 （I）最终导致原生质体（比例尺= 50微米）。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54586/54586fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.原生质体分离<ol><li>标注培养皿加入10 mL过滤灭菌酶溶液。 </li><li>固定高压釜胶带的四个条带的粘朝上放在干净的实验室表面，并标记的条带培养皿的大小。 </li><li>切4周龄植物（ 图1a）的叶子，然后按上表皮到磁带釜（ 即下表皮面朝上（ <stroNG&gt;图1b））。 </li><li>轻压使用一个15毫升的锥形管魔术胶带条到下表皮表面。小心不要粉碎叶组织（ 图1c）。小心地将魔术贴关闭剥去表皮下层（ 图1d）和暴露的叶肉细胞（ 图1E）。 </li><li>切断高压釜胶带以适合培养皿，使用镊子将磁带移动到培养皿并漂浮在酶溶液（ 图1F），叶朝下。旋转在一个平台摇床进行60分钟，在此期间，将原生质体将被释放到溶液中以60rpm。 </li><li>使用镊子除去并丢弃高压釜胶带（ 图1G）的条和吸管将含有原生质体（ 图1小时 ）的溶液到50毫升锥形管中。使用大孔枪头（如P5000笔尖或与端P1000的前端切断）。 </li><li>离心原生质体以100×克在4℃下3分钟，并用移液管去除上清。请注意，由于颗粒是非常宽松的。 </li><li>通过轻轻摇动管加入10 mL W5解决原生质体和悬浮。 </li><li>搁置在冰的原生质体30-60分钟。在此步骤中，评估通过用血细胞计数器（ 图1i）中计数原生质体的产率。 </li><li>收集离心原生质体在100×g下进行3分钟，4℃，并用移液管去除上清。请注意，由于颗粒是非常宽松的。 </li><li>悬浮原生质体，以在MMG溶液5×10 5原生质体/ ml的浓度。 </li></ol> 3.原生质体转染<ol><li>加10微升报道质粒（2微克/微升）到15毫升锥形管中。 </li><li>添加400μl的原生质体的管。 </li><li>添加一个卷（410微升）PEG溶液中颠倒试管12次转染原生质体混合。 </li><li>后8-15分钟INCubation在室温下，稀有四个卷（1640微升）W5溶液的原生质体的DNA-PEG混合物，并通过反相轻轻六倍混合。 </li><li>收集通过离心转染的原生质体，在100×g离心在室温下2分钟，并用移液管去除上清。再次，照顾，因为颗粒是非常宽松的。 </li><li>重悬1250微升成像解决方案的原生质体。 </li><li>等份加入200μl成六个复制一个96孔板的孔中，填充W5溶液中的任何空的孔，并密封与适于发光成像的粘合剂清楚盖板。 </li></ol> 4.成像<ol><li>板块转移到适合植物成像任何发光成像设置。变化对板块10-15分钟粘接盖转让后，以避免井之间的凝聚和压力差，然后开始成像。读板在室温下5天每〜45分钟（每孔3秒）。 注意:板的频率读出并每孔的读出时间可能取决于成像设置。 </li></ol> 5.数据分析<ol><li>分析使用任何分析软件（例如，生物节律分析软件系统（黄铜）22）的发光的结果。比较突变品系与野生型对照以显示昼夜缺陷。 </li></ol>

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The authors have nothing to disclose.

在这里，我们提出了一个快速检测筛选拟南芥线昼夜周期缺陷，包括原生质体分离，转染，并luminecent成像。在野生型的昼夜周期拟南芥和时钟突变CCA1 / LHY，ZTL 和 CCA1 -OX被从一个CCA1pro发光的测量计算:LUC报告并发现与使用更多时间从整个植物产生的公布的数据一致耗时转基因方法（ 表2）。使用该测定以筛选拟南芥属突变体避免了初始要求产生转基因发光线，这是耗时，并且可能仅揭示的特定突变显示出野生型的节奏。该协议的一个明显优势是任何拟南芥行可以在很短的时间进行筛选，改变昼夜节律的转录，这将有助于影响植物报时更多基因的鉴定。 26,27。随后的消化需要至少3小时温育在黑暗中的原生质体释放到酶溶液中。当不采用真空浸渗，孵化时间长达18小时建议。在这里介绍的协议，因为难以渗透到酶表皮细胞层通过胶带除去真空渗入是不必要的。当通过切割植物材料产生的原生质体，有必要从消化后的细胞壁碎片分离原生质体;这通常是通过过滤所述溶液或纯化它的糖梯度27,26完成。这里，任何未消化的组织将保持高压釜后胶​​带上消化，所以可以省略原生质体的过滤和糖梯度纯化。还有的是，从长期的原生质体程序产生的应力影响细胞活力和生物钟的机会。这里可视化的基于磁带的原生质体法20产生了大量的原生质体;和给定细胞的存活率超过昼夜时间序列和原生质体和整个幼苗（ 表2）的匹配期间的长度，在这里所描述的方法优于其他方法来产生原生质体。 该协议的转染步骤是关键步骤，关于原生质体的生存力的影响。所述PEG溶液使在该溶液中被传递到细胞中的DNA，并且两个孵育时间（步骤3.4），并应根据经验确定的PEG的百分比。标准浓度为20％，用较低的浓度降低了转化效率和高浓度降低了生存能力28。孵育时间短五分钟可产生原生质体27的有效转染。 原生质体可以在任何发光成像平台进行成像。在此视频，我们已经使用配备有外部光的发光板读数器（红色和蓝色发光二极管，630和470处，分别在〜20μE的组合强度），其允许高通量筛选和频繁的测量。其他成像设备，其检测发光，例如基于一个电荷耦合器件（CCD）照相机替代板读取器或设置，可以应用同样只要照明可用于光合作用。使用安装在一可控制柜CCD照相机的优点在于，光线和温度可被编程。一个缺点是较长的捕获时间，引入黑暗的长时间可导致应力到原生质体中除扰乱ING内源性报时。无论选择哪个实验平台，设置调整可能相比，苗或成熟植物设置是必要的。根据我们的经验，在成像缓冲器转染和/或萤光素过程中增加的报告质粒的浓度可能会解决可能发生在最初的实验中的任何不稳定或快速衰减的节奏。 在以后的实验中，这里描述的方法可以用于研究任何突变拟南芥线的昼夜表型。稍作修改，EG的各种药物对计时的影响可能在此设置进行研究。此外，转染可被修改以包括用于基因沉默19人工微RNA，进一步扩大该协议的通用性。协议生成大米原生质体成熟29和这里介绍的成像协议可能同样适用于我们进一步ü在经济上重要的单子叶作物植物时钟nderstanding。

大多数生物体具有内源性计时员，以帮助每日变动对环境的有效协商。此计时，生物钟，调节生物体的代谢和生理学的许多方面以预料的外部环境可预见的变化。在例如植物，在时间上可预见的病原体或草食动物攻击的预期强烈降低了整体的易感性1 - 4。淀粉代谢紧紧生物钟调节到最后保证淀粉的储备，直到天亮在或长或短日照条件下5是否增长。事实上，与之搭配的24小时环境显示出增加的增长率生物钟植物固碳率和存活率相比，运行时间不匹配6的时钟植物。昼夜节律在所有这些过程中广泛的影响产生在很大程度上从多达三分之一有节奏的调控在拟南芥7的总转录。这些节律的基因产物参与广泛的细胞过程，包括代谢途径，激素信号，和应激反应7。最重要的是，蛋白质功能的直接昼夜调节是通过磷酸化8昼夜调控最有可能其他翻译后后修饰成立。 在驱动这每天转录重新编程生物钟的中心位于转录网络/翻译反馈回路（TTFLs），包括早晨表达基因生物钟ASSOCIATED 1（CCA1）和LATE芒胚轴（LHY），该抑制的表达CAB的1（TOC1），GIGANTEA（GI）和晚上复杂（EC）的成员日晚基因时序 ; LUX ARRHYTHMO（LUX），早开花3（ELF3）和ELF4 9 - 11。一起机智小时假响应调节 -genes（PRR3，5，7和9），TOC1压制CCA1 / LHY表达而EC充当阳性调节器12 - 14。所述TTFL网络组件的附加反馈机制，转录后和翻译后调节起着调谐昼夜节律的重要作用。到目前为止，上确定生物钟蛋白质最丰富的PTM是磷酸化15。 CCA1由酪蛋白激酶2（CK2）的磷酸化会影响其结合靶子16和为生物钟17的温度补偿重要。该PRR蛋白磷酸化差异在昼夜周期，TOC1的磷酸化影响其负调节因子的相互作用，晚上分阶段时钟组件ZEITLUPE（ZTL）18。这些实施例说明如何翻译后修饰和蛋白质 - 蛋白质相互作用调TTFL网络到24小时的转录振荡器。对于更详细的介绍了转录时钟网络及其调控，近期出色的评语是可用的（ 如参考15）。 然而，仍不太清楚的是，以有节奏调节过程和细胞代谢的其它方面反馈到计时机构本身的程度。时钟缺陷拟南芥突变体的筛选粗放可能获得进一步的了解它的机制或信号通路可能在24小时节律的产生涉及从TTFL网络。为此，金和索先前发表的时钟功能的任何拟南芥线19中的高效率和快速分析的突破方法。基于原生质体采用瞬时表达，这种方法超越了稳定的转基因株系的需要或超过到发光的时钟标记线。在这里，我们可视化高产原生质体隔离和与优化的协议合并正方法20筛选通过的瞬时表达的时钟标记发光成像昼夜缺陷在96孔板阅读器。 我们将野生型协作0拟南芥比较良好的特点时钟突变体，确认这里描述成功检测改变昼夜节律的方法。从这些行衍生的原生质体转染表达与昼夜CCA1子萤火虫荧光素酶报道构建; CCA1pro:LUC 19。萤光素酶催化的多步反应，其中萤光素转化成电子激发氧化萤光素发射光21，其被成像在一段时间，以评估在这些原生质体的期间，振幅和转录节律的相位。 该协议包括三个主要部分组成;分离的原生质体，用报道质粒，和发光即时转染的原生质体老化。这三个部分应该总是在一天用新鲜制备的缓冲液和试剂进行。植物生长，并有足够量的报道质粒的纯化应提前进行，并且在该协议中不可见。

<table><tbody><tr><td>CELLULYSIN Cellulase, &lt;em&gt;Trichoderma viride&amp;nbsp;&lt;/em&gt;</td><td>Merck Millipore</td><td>219466</td><td></td></tr><tr><td>Pectinase R10</td><td>Sigma Aldrich</td><td>P2401</td><td></td></tr><tr><td>D-mannitol</td><td>Sigma Aldrich</td><td>M4125&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C4901</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A4503</td><td></td></tr><tr><td>MES</td><td>Acros Organics</td><td>327765000</td><td></td></tr><tr><td>NaCl</td><td>Acros Organics</td><td>327300010</td><td></td></tr><tr><td>Glucose</td><td>Fischer Scientific</td><td>G/0500/60</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M2670</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 4000</td><td>Acros Organics</td><td>434630010</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Sigma Aldrich</td><td>F4135</td><td></td></tr><tr><td>D-Luciferin, Firefly</td><td>Biosynth AG</td><td>L-8200</td><td>Dissolve luciferin powder in 0.1 M trisphosphate buffer pH 8.0 to a concentration of 50 mM</td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>A9618</td><td></td></tr><tr><td>Comply Steam Indicator Tape</td><td>3M</td><td>1222</td><td></td></tr><tr><td>Magic Tape</td><td>3M</td><td>819-1460</td><td></td></tr><tr><td>Petri Dishes</td><td>Scientific Laboratory Supplies</td><td>SLS2002</td><td></td></tr><tr><td>Microplate, 96 well, flat bottom, white</td><td>Greiner Bio-One</td><td>655075</td><td></td></tr><tr><td>TopSeal-A, adhesive lids for 96-well plates</td><td>Perkin Elmer</td><td>6050195</td><td></td></tr><tr><td>Syringe, 10 ml, w/o needle</td><td>BD Plastipak</td><td>302188</td><td></td></tr><tr><td>Syringe filter 0.22 &amp;micro;m</td><td>Merck Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Biological Rhythms Analysis Software System (BRASS)</td><td></td><td></td><td>Free download at amillar.org</td></tr><tr><td>QIAfilter Maxiprep Kit</td><td>Qiagen</td><td>12262</td><td></td></tr><tr><td>Modified Fuch-Rosenthal counting chamber, double cell</td><td>Hawksley</td><td>AC6000</td><td></td></tr><tr><td>Bright field microscope</td><td>Optika Microscopes</td><td>B-150POL-B</td><td></td></tr><tr><td>LB942 TriStar&lt;sup&gt;2&lt;/sup&gt; multimode reader, with luminescence module</td><td>Berthold Technologies Ltd</td><td>57947/57948</td><td></td></tr></tbody></table>

rapid analysis of circadian phenotypes in arabidopsis protoplasts transfected with a luminescent clock reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

野生型拟南芥的原生质体相比，已知的时钟突变CCA1 / LHY（短周期突变体2），ZTL（长周期突变体23），和过表达线CCA1 -OX（心律失常24）。所有瞬时的CCA1pro转:LUC报道，并在恒光超过5天的成像板读卡器。 在这里，我们表明，在CCA1 / LHY突变体，昼夜节律控制基因表达的自由运行周期被缩短（ 图2a），符合由发光时钟的稳定的转基因表达标志物2,9-分析发布期间。昼夜振荡在ZTL突变体的原生质体的期间是比野生型更长（ 图2b），根据从幼苗先前发表的结果，细胞悬液文化前进资源和原生质体19,23,25。 CCA1的表达锁定在早上相时钟和全球转录成为心律失常24。一致，我们观察到在CCA1pro没有振荡:在CCA1 -OX 原生质体 （ 图2c）LUC表达。 <img alt="图2" src="/files/ftp_upload/54586/54586fig2.jpg" /> 图2:0协作，CCA1 / LHY，ZTL和CCA1的原生质体昼夜节律原生质体-OX 瞬时转化与CCA1pro:LUC建设和发光昼夜节律成像5天以上（A - C）。 （ 四 ）根据发光痕迹上校-0，CCA1 / LHY和ZTL昼夜周期估算（A - B）。我一个±SEM，N = 3-5，t检验p值所示。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54586/54586fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 基因型 </td><td> 周期（原生质体） </td><td> 周期（转基因） </td><td> 报道: </td><td> 参考 </td></tr><tr><td>同事0 </td><td> 23.0±0.21 </td><td> 24.4±0.09 </td><td> pCCA1:LUC在山口-0 </td><td> （2） </td></tr><tr><td> CCA1 / LHY </td><td> 20.6±0.13 </td><td> 19.9±0.11 </td><td> pCCA1:LUC在山口-0 </td><td> （2） </td></tr><tr><td></td><td></td><td> 〜18 </td><td> RNA期L 呃背景</td><td> （9） </td></tr><tr><td&gt; ZTL </td><td> 25.1±0.12 </td><td> 27.4±0.5 </td><td> CAB2:LUC在C24生态型</td><td> （22） </td></tr><tr><td> CCA1 -OX </td><td>心律失常</td><td>心律失常</td><td> pCCA1:LUC（LD数据只） </td><td> （2） </td></tr><tr><td></td><td></td><td>心律失常</td><td> RNA和蛋白胶原-0背景</td><td> （23） </td></tr></tbody></table> 表2:原生质体昼夜节律相比转基因幼苗 pCCA1的昼夜周期:以首次报道的突变期的长度和（如果可用）相比，原生质体LUC的表达，与pCCA1:在山口-0 LUC表达。

Watch this Scientific Journal Video about Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter at JoVE.com

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

生物钟的调节有关拟南芥转录组的三分之一，但基因的反馈到报时的比例尚不清楚。在这里，我们可视化的方法来使用一昼夜记者在原生质体瞬时表达的荧光成像拟南芥突变体的任何行快速评估昼夜的表型。

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

rapid-analysis-circadian-phenotypes-arabidopsis-protoplasts

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.7K 次观看.  University of Edinburgh. 生物钟的调节有关拟南芥转录组的三分之一，但基因的反馈到报时的比例尚不清楚。在这里，我们可视化的方法来使用一昼夜记者在原生质体瞬时表达的荧光成像拟南芥突变体的任何行快速评估昼夜的表型。 

视频: Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

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

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

生物学

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

神经科学

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant environmental parameters that synchronize animal circadian clocks. In Drosophila circadian photo-responses are mediated by a solely blue light photoreceptor named CRYPTOCHROME (CRY). Upon photoreception, CRY changes its conformation and initiates the proteosomal dependent degradation of TIMELESS (TIM). TIM is an important pacemaker protein, thus degradation of TIM will reset the circadian clock. Under constant light conditions (LL), wild type flies quickly become arrhythmic because of the constant degradation of TIM, while flies bearing defects with circadian photo-responses will still be rhythmic. Thus LL triggered arrhythmicity has been used for screening of components in circadian light input pathways. A brief short light pulse in the night can also dramatically shift phases of circadian rhythms. As expected, this phase shift response is reduced in flied with defects in circadian photoresponse. Thus analyzing locomotion behavior rhythmicity under LL or phase changes after short light pulse in constant darkness (DD) are two major methods to study circadian photoresponse. Here we describe how to design and analyze LL and phase response experiments. LL arrhythmicity is suitable for screening light input pathways mutants, whereas phase response validates the results and provide further information for light sensitivity.

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

优化人肠道类肠生物用于昼夜节律研究

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

作者聚焦：多细胞系统中昼夜节律的体外研究

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

使用类器官中的生物发光报告基因监测昼夜节律振荡

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

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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

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

使用类器官中的生物发光报告基因监测昼夜节律振荡

使用类器官中的生物发光报告基因监测昼夜节律振荡