Name: an optogenetic method to control and analyze gene expression patterns in cell-to-cell interactions
Uploaded: 2018-03-22T14:00:00
Description: Watch this Scientific Journal Video about An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions at JoVE.com

这项工作得到了 JST (人工智能) 的支持, 进化科学和技术的核心研究 (JPMJCR12W2 (警服)), 为创新领域的科学研究提供资助 (教育部、文化部、体育、科学和技术部 (下个), 日本26119708 (人工智能) 和 16H06480 (警服)), 科学研究 (a) (日本促进科学学会 (jsp) 24240049 (警服)) 和年轻科学家 (A) (jsp 15H05326 (人工智能)) 和一个资助为创新领域的科学研究 "荧光"现场成像" 的下个, 日本和平台的动态方法的生活系统从下个, 日本。

1. Tol2 系统生成稳定的细胞系
<ol><li>
将 Tol2-based optogenetic 模块的染质粒向量 (图 1A) 与 transposase (Tol2) 表达式向量 (pCAGGS-mT2TP) 结合到 C2C12 单元格中。在所有步骤中, 培养细胞与 DMEM 培养基补充10% 胎牛血清 (血清) 和青霉素-链霉素在37°c (表 1), 在存在 5% CO2, 否则表示。
	<ol>
<li>将 trypsinized 单元格计数为单元格计数器, 并将 5 x 104 C2C12 单元格中的每?...

<ol>
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L., Malapert, P., McGrew, M., Pourqui&#233;, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+inhibition+by+Lunatic+Fringe+underlies+the+chick+Segmentation+Clock.">Periodic inhibition by Lunatic Fringe underlies the chick Segmentation Clock.</a> Nature. 421, 275-278 (2003).</li><li>Okubo, Y., Sugawara, T., Abe-Koduka, N., Kanno, J., Kimura, A., Saga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lfng+regulates+the+synchronized+oscillation+of+the+mouse+segmentation+clock+via+trans-repression+of+Notch+signalling.">Lfng regulates the synchronized oscillation of the mouse segmentation clock via trans-repression of Notch signalling.</a> Nat. Commun. 3, 1141 (2012).</li><li>Shimojo, H., Isomura, A., Ohtsuka, T., Kori, H., Miyachi, H., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+control+of+Delta-like1+in+cell+interactions+regulates+dynamic+gene+expression+and+tissue+morphogenesis.">Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis.</a> Genes Dev. 30, 102-116 (2016).</li><li>Isomura, A., Ogushi, F., Kori, H., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+perturbation+and+bioluminescence+imaging+to+analyze+cell-to-cell+transfer+of+oscillatory+information.">Optogenetic perturbation and bioluminescence imaging to analyze cell-to-cell transfer of oscillatory information.</a> Genes Dev. 31, 524-535 (2017).</li><li>Wang, X., Chen, X., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+gene+expression+by+a+light-switchable+transgene+system.">Spatiotemporal control of gene expression by a light-switchable transgene system.</a> Nat. 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我们展示了一种控制基因表达动力学的方法, 周期为2到3小时。这个时间尺度比其他传统系统中的周期要短得多, 包括 LightOn 系统和原始的系统。达到超时间尺度的关键参数是光诱导的分子产物、基因和蛋白质的半衰期。这些动力学参数可能取决于细胞类型和种类。为调整动力学, 替换 Hes1 3 ' UTR 序列与其他是一个直接向前的方式, 因为它不改变目标蛋白质的序列和作用。为了寻求其他 3 ' UTRs 获得理...

细胞对细胞的通信在胚胎模式的发展过程中起着至关重要的作用。在脊椎动物胚胎中, 称为胚胎背部的 metameric 结构沿前后体轴形成, 在时间保持时钟的控制下精确的时间精度, 称为分段时钟 1.在此过程中, 一组 presomitic 胚层 (PSM) 单元格以同步方式定期转换为胚胎背部。这一过程涉及同步振荡基因表达和 PSM 细胞, 振荡的阶段形成相同的胚胎背部。在小鼠中振荡基因表达的周期约为2至3小时, 斑马鱼中约30分钟。当离解时, PSM 单元格会丢失同步2、3, 但当重新聚合时, 它们可以自组织并恢复人口同步4, 这表明单元格耦合是同步振荡.
广泛的努力表明, 信号分子在三角洲凹槽通路是紧密相连的同步振荡的分割时钟基因。无论是药理抑制剂或凹槽信号的基因突变 desynchronize 的振荡器的数量。在斑马鱼中, 凹槽信号元件 (如 DeltaC、DeltaD 和 Notch1a) 的突变体显示异步振荡5,6。在小鸡或小鼠胚胎中, 不仅有凹槽配体 Delta-like1 (Dll1), 而且还需要有凹槽调制器 (Lfng), 同步振荡7,8,9。然而, 由于基因调控动力学的常规扰动的时间分辨率不足以研究这种分子在细胞到细胞间的动态信息传递的功能能力, 因此很难检测2–3 h (超节律) 的尺度过程。
我们最近开发了一种综合的方法来控制和监测哺乳动物细胞中的基因表达模式10。该技术使超时间尺度上周期性光照对基因表达脉冲的诱导。该协议代表了在细胞间通信的环境下, 通过活细胞发光监测来建立光敏细胞线和观察细胞动态反应的方法。该方法适用于其它许多信号通路的分析。

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">FACS</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:180px;">FACSAriaII SORP</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Camera</td>
			<td>Andor</td>
			<td>iKon M-934</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microscope</td>
			<td>Olympus</td>
			<td>IX-81 ZDC</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">PMT device</td>
			<td>Churitsu eletric corp.</td>
			<td>CL24B-LIC/B</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Blue LED illuminator</td>
			<td>OptoCode</td>
			<td>LEDB-SBOXH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM</td>
			<td>Nacalai</td>
			<td>08459-35&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Penicillin-streptomycin</td>
			<td>Nacalai</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal bovine serum</td>
			<td>Sigma</td>
			<td>172012</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KRYSTAL24 (black 24 well plate )</td>
			<td>Hi-tech</td>
			<td>303012</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">D-Luciferin Potassium Salt</td>
			<td>Nacalai</td>
			<td>20028-24&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Light meter</td>
			<td>LI-COR Biosciences</td>
			<td>LI-250A</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">anti-HA-Peroxidase antibody</td>
			<td>Roche</td>
			<td>clone 3F10</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">anti-Actin-Peroxidase antibody</td>
			<td>Wako</td>
			<td>clone 2F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optogenetic method to control and analyze gene expression patterns in cell-to-cell interactions

细胞应适当地对世俗变化的环境作出反应, 而这种情况受周围细胞各种因素的影响。凹槽信号通路是细胞间通信必不可少的分子机制之一, 在胚胎的正常发育中起着关键作用。这一途径涉及细胞对细胞传递的振荡信息与超的节奏, 但尽管分子生物学技术的进展, 它一直是挑战, 以阐明多细胞相互作用对振荡基因的影响动力学.在这里, 我们提出一个协议, 允许 optogenetic 控制和实时监测的基因表达模式, 在一个精确的时间方式。该方法成功地揭示了凹槽信号的胞内细胞和细胞间周期输入 entrain 在单细胞分辨率下的频率调谐和相移的固有振荡。该方法适用于分析各种信号通路的动态特性, 为多细胞系统动态基因表达程序的功能意义测试提供了独特的平台。

我们适应了 LightOn 系统11,12, 这使得在哺乳动物细胞中的照片诱导的基因表达, 研究的基因振荡器与 2-到 3-小时周期性。该系统包括两部分: 光诱导转录激活剂 hGAVPO 和一个无人机启动盒, 以推动转录的任意基因的兴趣。为了加速光诱导基因表达的脉动动力学, 波利亚 Hes1 3 ' UTR 取代了无人参与启动盒中的序列, 缩短了小鼠成纤维细?...

Watch this Scientific Journal Video about An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions at JoVE.com

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

本文提出了一种通过 optogenetic 控制和实时监测基因表达来分析振荡信息的细胞间传递的协议。这种方法为测试多细胞系统动态基因表达程序的功能意义提供了一个独特的平台。

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

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

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