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://www.jove.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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELLULYSIN Cellulase, Trichoderma viride&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</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>MgCl2</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&#160;</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 0.1M trisphosphate buffer pH 8.0 to a concentration of 50mM</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 &#181;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 TriStar2 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://www.jove.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

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

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

科研

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

口面表型的定量<em&gt;爪蟾</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.