Name: in vivo monitoring of circadian clock gene expression in the mouse suprachiasmatic nucleus using fluorescence reporters
Uploaded: 2018-07-04T12:30:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters at JoVE.com

我们感谢张实验室的各位成员提供了激动人心的讨论, 以及站内实验室的成员提供技术援助。这项研究由中国国家自然科学基金、2012CB837700 (E.E.Z. 和霍山) 资助的 31500860 (到霍山) 的973个项目中的 m.o.s. T, 并由北京市政府提供资金支持。E.E.Z. 得到中国 "全球青年专家招募计划" 的支持。

本议定书的所有程序都是根据中国政府条例在北京国家生物科学研究所的机构动物保育和使用委员会 (IACUC) 的批准下进行的。
1. Cry1荧光记者的构建
注: 先前的昼夜节律研究使用生物发光系统2,12,13融合一个昼夜启动器与不稳定的荧光素酶 (dLuc), 以诱导有节奏的dLuc表达。为了开发荧光记者, 不稳定的荧光素酶取代了不稳定的萤光蛋白 (dVenus), 以诱导韵律dVenus表达。P (Cry1) d金星记者的世代被描述如下, 它的结构当前被递交到 Addgene 并且将很快是可利用的为学术用途。
<ol><li>放大功能小鼠Cry1启动子 (+ 328 到-1208 Cry1基因的 bp 区域, 转录起始点被指定为 "-1")。使用 pcr 酶 (点歌加新), 引物 F1 和 R1 (见表 2), 小鼠基因组作为模板和制造商的 PCR 协议, 以放大Cry1启动子 DNA 片段。</li>
	<li>放大Cry1基因的内含子 336, 这对于Cry114的昼夜节律功能是重要的。使用 pcr 酶, 引物 F2 和 R2, 小鼠基因组作为模板和制造商的 PCR 协议, 以放大内含物 336 DNA 片段。</li>
	<li>放大金星。以 pcr 酶、引物 F3 和 R3、pVENUS-N1 质粒为模板, 并利用制造商的 pcr 协议放大金星DNA 片段。</li>
	<li>放大 NLS-D2 DNA 片段。以 pcr 酶、引物 F4 和 R4、pcDNA3.3-d2eGFP 质粒为模板, 并利用制造商的 pcr 协议对 NLS-D2 DNA 片段进行放大。</li>
	<li>合成 DNA 片段1作为外显子1。</li>
	<li>将 dna 片段2合成为内含子336与金星dna 片段之间的链接器。</li>
	<li>用 mCherry 和 WPRE 酶切 pAAV-EF1a-double floxed-hChR2 (H134R)-HGHpA-MluI-EcoRI 载体质粒。使用凝胶萃取试剂盒净化较大的 DNA 片段。使用制造商的协议。</li>
	<li>使用吉布森装配组合及其标准协议, 组装所有的 DNA 片段一起生产 pAAV (Cry1)-intron336-Venus-NLS-D2-WPRE-HGHpA 记者。使用制造商的协议。</li>
</ol>2. 腺相关病毒的生产15
<ol><li>制备以下质粒 DNA 储存: 31.25 µg pRV1、pH21 31.25 µg、µg 125 pFdelta6 和µg-P (pAAV) Cry1 intron336-Venus-NLS 的 62.5 D2-WPRE-HGHpA。</li>
	<li>准备五15厘米293T 细胞的盘子, 等到细胞变成90% 汇合。</li>
	<li>染的质粒进入297T 细胞后转染试剂协议。对于每道菜, 使用6.25 µg 的 pRV1, 6.25 µg pH21, 25 µg pFdelta6, 12.5 µg 的 pAAV P (Cry1) intron336-Venus-NLS-D2-WPRE-HGHpA, 120 µL 的转染试剂。</li>
	<li>72 h 在转染后, 用吸管枪将每个盘子中的细胞分离出来;将细胞分成两个50毫升的试管。</li>
	<li>将细胞以 800 x g 为10分钟, 然后丢弃上清。将20毫升磷酸盐缓冲的生理盐水 (PBS) 放入每管中冲洗细胞;将细胞以 800 x g 为10分钟, 并丢弃上清。</li>
	<li>并用重悬颗粒在150毫米氯化钠和20毫米三溶液, pH 8.0;使用25毫升的溶液/管。在每管中加入1.25 毫升新鲜制备的10% 脱氧钠 (dH2O)。添加 benzonase 核酸酶到最终浓度为每毫升50单位, 并彻底混合。孵育37°c 为1小时。</li>
	<li>用 3000 x g 离心15分钟, 去除细胞碎片。通过0.45 微米注射器过滤器, 进一步确保所有碎片被去除;这一步骤将有助于防止肝素柱堵塞。</li>
	<li>用蠕动泵建立肝素柱;将流量设置为1毫升/分钟, 确保不会将气泡引入列中。</li>
	<li>平衡柱与10毫升的150毫米氯化钠, 20 毫米三溶液, pH 值8.0。将包含病毒的解决方案添加到每一列。用20毫升的100毫米氯化钠, 20 毫米三溶液, pH 8.0 来洗涤柱。然后, 使用5毫升注射器, 用1毫升的200毫米氯化钠和20毫米三溶液 pH 值8.0 来冲洗柱, 接着是1毫升氯化钠和300毫米三溶液, pH 20。</li>
	<li>使用5毫升注射器洗脱的病毒与3毫升的400毫米氯化钠, 20 毫米三溶液, pH 8.0, 其次是4毫升的一个450毫米氯化钠, 20 毫米三溶液, pH 8.0, 最后由 3 ml 的500毫米氯化钠, 20 毫米三溶液, pH 值8.0。收集提取的材料。</li>
	<li>对于重组腺相关病毒 (rAAV) 浓度, 加载提取的材料, 每次4毫升, 进入离心过滤器单位的10万分子量截止。在室温下离心每4毫升样品 2000 x 克, 以5分钟的温度, 丢弃流经。</li>
	<li>当所有提取的材料都经过离心过滤单元, 加入4毫升的 PBS 和离心机的混合物约 250 ul。去除 rAAVs (病毒滴度应该是大约 2-8 x 1012病毒微粒每毫升) 从离心过滤器单位, 整除并且存放在-80 °c 直到使用。在这个阶段, 病毒可以储存几个月。</li>
</ol>3. rAAV 注射和光纤插入成年小鼠 SCN
<ol><li>rAAVs 的昼夜荧光报告携带者注射入 SCN<ol>
<li>负载5µL 的 rAAV (病毒滴度是大约 2-8 x 1012病毒粒子每毫升) 成一个微型注射器。</li>
		<li>麻醉一只成年老鼠 (2-5 月大, C57BL/6 背景) 与戊巴比妥 (80 毫克/千克) 通过腹腔注射。检查是否有足够的麻醉深度, 因为没有脚趾捏的反应。</li>
		<li>使用剪刀从鼠标头上取出皮毛, 然后将鼠标装入立体定向设备。通过循环热水毯或另一种加热装置, 为麻醉动物提供热源。 注: 或者, 使用脱毛奶油或电剪剪去去除毛皮。</li>
		<li>用适当的消毒剂清洁手术部位。例如, 这可能包括无菌水或酒精和碘, betadine, 或稀释洗必泰在交替磨砂。重复至少3次。把手术部位悬垂起来。</li>
		<li>用剪刀在头皮上做个切口, 露出头骨。</li>
		<li>调整立体定向装置, 使 bregma 和 lambda 在同一水平平面上;将两个对称点 bregma 在同一水平平面上。</li>
		<li>用不育的棉签浸泡在4% 小时2o2/小时2o 腐蚀脑骨膜。然后使用干净, 不育的棉签清除和干燥头骨表面。</li>
		<li>使用微钻, 使一个孔约1毫米直径0.46 毫米后, 0.25 毫米侧向 bregma。</li>
		<li>
fi使用干净的棉花消毒拭子与生理盐水溶液清除骨碎片从暴露的表面的大脑。</li>
		<li>将微型注射器插入大脑, 确保微注射器的尖端是0.46 毫米后部和0.25 毫米侧向 bregma, 5.7 毫米深的头骨表面。</li>
		<li>注入 500 rAAV (50 nl 分钟-1) 进入 SCN, 留下微注射器到位10分钟后注射, 以确保完全扩散的 rAAV。</li>
		<li>慢慢地取出微注射器。</li>
	</ol>
</li>
	<li>将光纤插入 SCN 区域。<ol>
<li>使用光纤刀, 在长度上切割17毫米光纤, 确保光纤的每一侧都是平滑的。</li>
		<li>将光纤插入陶瓷插针中, 并将光纤固定在带有甲基乙基酮过氧化物 (AB 胶) 的陶瓷箍上, 确保光纤和陶瓷插针的表面是冲洗的。</li>
		<li>用冷消毒剂溶液或环氧乙烷清洗纤维。</li>
		<li>rAAV 注射 (步骤 3.1.11) 后, 将陶瓷插箍光纤插入 SCN。</li>
		<li>用牙科树脂将陶瓷插针和光纤固定在头骨上, 使其干燥。</li>
		<li>用后指甲油涂抹牙科树脂的表面。重复3.1.2 中概述的步骤 3.2.5, 省略实际注入 rAAV 控制。</li>
	</ol>
</li>
	<li>术后护理<ol>
<li>为小鼠提供术后镇痛药, 如丁丙诺啡2毫克/千克体重, 一天两次。</li>
		<li>把老鼠放在康复笼里, 当他们从麻醉中出来的时候。</li>
		<li>小鼠单独在12小时的光/暗条件下 (光强度约100莱克丝在笼子底部) 与食物/水可用的广告随意。在进一步试验前, 应分配八天时间, 以便能有足够的恢复期, 并确保最佳荧光报告的表达。</li>
	</ol>
</li>
</ol>4.体内荧光信号监测和数据采集
<ol><li>手术后八天, 用荧光信号监测器将鼠标头上的纤维连接起来(例如, 基因观察者)。检查控制鼠的 SCN 中的荧光信号, 建立背景信号。利用荧光信号监测仪对该信号进行观测和测量。</li>
	<li>用控制鼠标的相同操作对实验小鼠的荧光信号进行评价。排除已接收病毒载体但只显示分析背景信号的老鼠, 因为这可能表明光纤的尖端已经错过了 SCN。在这些初步措施之后, 把老鼠送回自己的笼子3周, 让荧光信号稳定下来。</li>
	<li>3周后, 用荧光信号监测器将小鼠连接起来。每10分钟测量荧光量为十五年代, 频率为100赫兹。在这些测量过程中, 老鼠可以自由地在笼子里移动, 食物/水可用的广告随意。使用100莱克丝的笼子底部的光强度, 并在光纤尖端的激光功率在 15-20 µW 之间。</li>
	<li>所有录音完成后, 灌注小鼠与4% 多聚甲醛, 并删除和切片的大脑检查放置的光纤。排除小鼠的分析, 如果光纤尖端没有正确植入的 SCN。</li>
</ol>5. 数据分析和介绍
<ol><li>测量荧光信号每10分钟为十五年代, 频率为100赫兹, 产生1500个数据点。这1500点的平均值在任何给定的时间点转换为荧光信号。随着时间的推移, 绘制多个平均值会产生一个信号-时间曲线, 它对应于自由移动小鼠的Cry1下的控制光条件下的转录节律。</li>
</ol>

<ol>
	<li>Welsh, D. K., Takahashi, J. S., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suprachiasmatic+nucleus:+cell+autonomy+and+network+properties.">Suprachiasmatic nucleus: cell autonomy and network properties.</a> Annual Review of Physiology. 72, 551-577 (2010).</li><li>Zhang, E. E., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clocks+not+winding+down:+unravelling+circadian+networks.">Clocks not winding down: unravelling circadian networks.</a> Nature Reviews Molecular Cell Biology. 11, 764-776 (2010).</li><li>Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+architecture+of+the+mammalian+circadian+clock.">Transcriptional architecture of the mammalian circadian clock.</a> Nature Reviews Genetics. 18, 164-179 (2017).</li><li>Yoo, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proceedings of the National Academy of Sciences U S A. 101, 5339-5346 (2004).</li><li>Davidson, A. J., Castanon-Cervantes, O., Leise, T. L., Molyneux, P. C., Harrington, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+jet+lag+in+the+mouse+suprachiasmatic+nucleus+and+peripheral+circadian+timing+system.">Visualizing jet lag in the mouse suprachiasmatic nucleus and peripheral circadian timing system.</a> European Journal of Neuroscience. 29, 171-180 (2009).</li><li>Savelyev, S. A., Larsson, K. C., Johansson, A. S., Lundkvist, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+preparation,+organotypic+tissue+culturing+and+luciferase+recording+of+clock+gene+activity+in+the+suprachiasmatic+nucleus.">Slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Yamaguchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression:+View+of+a+mouse+clock+gene+ticking.">Gene expression: View of a mouse clock gene ticking.</a> Nature. 409, 684-684 (2001).</li><li>Ono, D., Honma, K. I., Honma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+and+ultradian+rhythms+of+clock+gene+expression+in+the+suprachiasmatic+nucleus+of+freely+moving+mice.">Circadian and ultradian rhythms of clock gene expression in the suprachiasmatic nucleus of freely moving mice.</a> Science Reports. 5, 12310 (2015).</li><li>Ono, D., Honma, S., Honma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+PER2::LUC+rhythms+in+the+olfactory+bulb+of+freely+moving+mice+depend+on+the+suprachiasmatic+nucleus+but+not+on+behaviour+rhythms.">Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms.</a> European Journal of Neuroscience. 42, 3128-3137 (2015).</li><li>Ono, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+Per1+and+Bmal1+circadian+rhythms+in+the+suprachiasmatic+nucleus+in+parallel+with+behavioral+outputs.">Dissociation of Per1 and Bmal1 circadian rhythms in the suprachiasmatic nucleus in parallel with behavioral outputs.</a> Proceedings of the National Academy of Sciences U S A. 114, E3699-E3708 (2017).</li><li>Reppert, S. M., Weaver, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+circadian+timing+in+mammals.">Coordination of circadian timing in mammals.</a> Nature. 418, 935-941 (2002).</li><li>Liu, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundant+function+of+REV-ERBalpha+and+beta+and+non-essential+role+for+Bmal1+cycling+in+transcriptional+regulation+of+intracellular+circadian+rhythms.">Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.</a> PLoS Genetics. 4, e1000023 (2008).</li><li>Maywood, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+core+circadian+feedback+loop+in+suprachiasmatic+nucleus+of+mCry1-luc+transgenic+reporter+mouse.">Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse.</a> Proceedings of the National Academy of Sciences U S A. 110, 9547-9552 (2013).</li><li>Ukai-Tadenuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+in+feedback+repression+by+cryptochrome+1+is+required+for+circadian+clock+function.">Delay in feedback repression by cryptochrome 1 is required for circadian clock function.</a> Cell. 144, 268-281 (2011).</li><li>McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+titering+of+recombinant+adeno-associated+viral+vectors.">Production and titering of recombinant adeno-associated viral vectors.</a> Journal of Visualized Experiments. 57, e3348 (2011).</li><li>Yamaguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+genetically+deficient+in+vasopressin+V1a+and+V1b+receptors+are+resistant+to+jet+lag.">Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag.</a> Science. 342, 85-90 (2013).</li><li>Nagano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abrupt+shift+in+the+day/night+cycle+causes+desynchrony+in+the+mammalian+circadian+center.">An abrupt shift in the day/night cycle causes desynchrony in the mammalian circadian center.</a> Journal of Neuroscience. 23, 6141-6151 (2003).</li><li>Golombek, D. A., Rosenstein, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+Circadian+Entrainment.">Physiology of Circadian Entrainment.</a> Physiological Reviews. 90, 1063-1102 (2010).</li><li>Mei, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+recording+of+circadian+rhythms+in+brains+of+freely+moving+mice.">Long-term in vivo recording of circadian rhythms in brains of freely moving mice.</a> Proceedings of the National Academy of Sciences. 115, 4276-4281 (2018).</li></ol>

作者没有披露的利益冲突。

与前体方法相比, 如切片培养4,5, rt-pcr16,原位杂交17, 这要求动物被杀死,体内记录方法允许研究者们研究活着的动物的昼夜节律基因表达。因此, 这种技术提供了评估不同物理扰动 (如睡眠剥夺、压力、食物摄入等) 对神经昼夜节律的影响的能力。与体内生物发光7</su...

在哺乳动物中, 视交叉上核 (SCN) 支配着整个身体的昼夜节律, 以协调个体对外部环境变化的反应 (如光照、温度、压力等)1。核心时钟组件由Per1-3、 Cry1-2、时钟和Bmal1组成, 在调节每个细胞的昼夜节律时起着中枢作用。每个细胞在 SCN 包含转录激活剂, CLOCK/BMAL1, 作为一个 heterodimer, 以诱导表达的每和哭。每/哭复合, 然后抑制 CLOCK/BMAL1 的功能, 形成一个转录转换反馈回路, 约24小时完成2,3。
以往对 scn 的研究主要采用了前体4、5、6的体外营养切片培养方法, 虽然这种方法提供了有价值的信息, 但它的局限性抑制了我们获取有关其他脑核对 SCN 的影响的数据, 以及外源性刺激 (如光照) 对居住在这个关键区域的细胞的影响。在 2001年, 仁冈村的组是第一个使用生物发光系统在体内监测昼夜节律基因表达的 SCN 在自由移动小鼠7。肯-本间的小组在过去几年里进一步开发了生物荧光在体内记录系统在 SCN8,9,10。这些研究共同为研究人员提供了在恒定的黑暗或光脉冲后监测生物钟的能力。然而, 由于生物发光太暗, 无法在光/暗循环期间进行连续监测, 加上光是11昼夜节律性时钟所需的主要信号, 因此有不断增加的需求, 开发的实验方法, 克服了与生物发光记录相关的限制。本报告描述了一种基于荧光的系统, 用于监测自由移动小鼠体内的 SCN 生物钟。这种易于使用的方法允许在光/暗循环期间进行连续监测, 并允许在实时和高时间分辨率下长期观察 SCN 中生物钟基因的转录。

<table><tbody><tr><td>KOD Plus Neo</td><td>TOYOBO</td><td>KOD-401</td><td>Reagent</td></tr><tr><td>pVENUS-N1</td><td>addgene</td><td>#61854</td><td>Plasmid</td></tr><tr><td>pcDNA3.3_d2eGFP</td><td>addgene</td><td>#26821</td><td>Plasmid</td></tr><tr><td>pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA</td><td>addgene</td><td>#20297</td><td>Plasmid</td></tr><tr><td>MluI</td><td>Thermo Scientific</td><td>FD0564</td><td>Reagent</td></tr><tr><td>EcoRI</td><td>Thermo Scientific</td><td>FD0274</td><td>Reagent</td></tr><tr><td>Gibson Assembly Mix</td><td>NEB</td><td>E2611s</td><td>Reagent</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Scientific</td><td>12566014</td><td>Reagent</td></tr><tr><td>Syringe Filter</td><td>EMD Millipore</td><td>SLHV033RS</td><td>0.45&amp;nbsp;&amp;micro;m&amp;nbsp;</td></tr><tr><td>HiTrap heparin columns</td><td>gelifesciences</td><td>17-0406-01</td><td>1 mL&amp;nbsp;</td></tr><tr><td>Amicon ultra-4 centrifugal filter</td><td>EMD Millipore</td><td>&amp;nbsp;UFC810024</td><td>100,000 MWCO</td></tr><tr><td>Benzonase nuclease</td><td>Sigma-Aldrich</td><td>E1014</td><td>Reagent</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D5670</td><td>Make fresh solution for each batch</td></tr><tr><td>mouse stereotaxic apparatus</td><td>B&amp;amp;E TEKSYSTEMS LTD</td><td>#SR-5M/6M</td><td>Equipment</td></tr><tr><td>pentobarbital</td><td>SigmaAldrich</td><td>#1507002</td><td>Reagent</td></tr><tr><td>mouse stereotaxic apparatus</td><td>B&amp;amp;E TEKSYSTEMS LTD</td><td>#SR-5M/6M</td><td>Equipment</td></tr><tr><td>Hydrogen peroxide solution</td><td>SigmaAldrich</td><td>#216763</td><td>Reagent</td></tr><tr><td>Optical Fiber</td><td>Thorlabs</td><td>FT200EMT</td><td>0.39 NA, &amp;Oslash;200 &amp;micro;m</td></tr><tr><td>microsyringe pump</td><td>Nanoliter 2000 Injector, WPI</td><td></td><td>Equipment</td></tr><tr><td>ceramic ferrule</td><td>Shanghai Fiblaser</td><td></td><td>230&amp;thinsp;&amp;mu;m I.D., 2.5 mm O.D.</td></tr><tr><td>Gene Observer</td><td>BiolinkOptics</td><td></td><td>Equipment</td></tr></tbody></table>

in vivo monitoring of circadian clock gene expression in the mouse suprachiasmatic nucleus using fluorescence reporters

该技术将光纤介导荧光记录与重组腺相关病毒基因记者的精确交付结合在一起。这种新的和易于使用的体内荧光监测系统, 以记录的转录节律的时钟基因, Cry1, 在视交叉上核 (SCN) 的自由移动小鼠。为此, Cry1转录荧光报告器被设计和包装成腺相关病毒。纯化, 浓缩病毒注射到小鼠的 SCN, 然后插入一根光纤, 然后固定在大脑的表面上。这些动物被送回自己的笼子, 并允许1月的手术后恢复期, 以确保足够的记者表达。荧光然后记录在自由移动的小鼠通过体内监测系统, 在我们的机构建设。在体内记录系统中, 488 nm 激光与 1 x 4 光束分配器耦合, 将光分成四个同等功率的激光励磁输出。这个设置使我们能够同时记录四动物。每个发射的荧光信号都是通过光电倍增管和数据采集卡收集的。与先前的活体生物钟记录技术的生物发光相比, 这种荧光体内记录系统允许在光周期中记录生物钟基因的表达。

图 1A显示了Cry1的荧光记者设计。使用上述方法, 500 rAAV (Cry1) intron336-Venus-NLS-D2 成功地注入成年鼠的 SCN, 并表现出强健的金星表达 (图 1B, 1C)。在12小时/12 小时的光/暗 (LD) 和暗/暗 (DD) 条件下记录的荧光信号 (图 2) 在两种情况下产生了强健的昼夜节律。最后, 将荧光信号记录在长周期...

Watch this Scientific Journal Video about In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters at JoVE.com

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

这种新开发的荧光技术能够长期监测自由运动小鼠视交叉上核 (SCN) 中昼夜节律基因的转录。

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 ...

in-vivo-monitoring-circadian-clock-gene-expression-mouse

Research

JoVE Journal

Neuroscience

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

8.2K 次观看.  Peking University. 这种新开发的荧光技术能够长期监测自由运动小鼠视交叉上核 (SCN) 中昼夜节律基因的转录。

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

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 ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

神经科学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

发育生物学

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 ...

生物学

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.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

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 ...

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 ...