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 单元格中的每个井在 12-井板中, 在转染前一天。</li>
		<li>用 pAI218 试剂共染0.375 微克的 Tol2-based optogenetic 载体 (pAI177 或 pAI170), 0.125 微克的药物选择载体 (pCAGGS-mT2TP) 和0.5 微克的 lipofection 载体。</li>
		<li>胰蛋白酶处理转染的细胞, 并将其板成100毫米的培养皿后的一天。</li>
		<li>交换培养基为转染的细胞到培养基补充100毫克/毫升潮霉素。</li>
		<li>培养细胞3天, 以消除未转染的细胞。请注意, 中等变化是不必要的。</li>
	</ol>
</li>
	<li>胰蛋白酶处理转染细胞, 并纯化细胞的数量, 表达荧光蛋白的选择。对于携带 pAI177 的接收细胞, 将分选门设置为绿色 PE 通道, 以净化 mCherry 阳性细胞种群。对于携带 pAI218 的光敏发件人细胞, 将分拣门设置为 APC 通道以净化 iRFP713-positive 细胞种群。</li>
	<li>选)采用标准方法建立克隆细胞种群, 如有限稀释或单细胞分选。</li>
</ol>2. 在人口层面评价工程细胞的光敏感度
注: 本部分描述了一种通过生化检测 (如 qPCR 和西方印迹) 检查 Dll1 配体蛋白在蓝光照射下动态诱导的协议。
<ol><li>设置两种类型的孵化器, 有或没有光源的光诱发条件或暗条件, 分别。设置光强度的蓝色 LED 反式照明灯, 如图 1B所示, 使用测光表。程序的定时和光照持续时间通过将控制脚本加载到单板微控制器, 允许可编程的照明计划 (图 1C)。</li>
	<li>使用单元格计数器计数 trypsinized 单元格, 1.0 x 105感光发件人细胞携带 pAI218 和 pAI170 在35毫米直径的塑料培养皿上 (超过12个盘子为光情况和1盘为黑暗的情况) 和设置菜在单独的孵化器为黑暗/光的条件。设置餐具后, 保持孵化器的门关闭, 直到收集细胞裂解物。</li>
	<li>电镀后一天半, 开始照明 (细胞预计将超过80% 汇合)。不要把细胞暴露在光线下, 以避免不良的照片刺激。 注意: 照明的时间表取决于实验的目的。请注意, 持续光照条件 (e. g. 1 分钟持续时间和10分钟间隔, 40 µmol/米2/s 光强度) 足以进行简单的照片感应检查, 而且强光照射对细胞有害。因此, 请确保选择的光照的无害参数。</li>
	<li>电镀后约2天, 用30分钟间隔制备细胞裂解液。将样品碟从孵化器移到冰上, 并开始准备细胞裂解物进行进一步分析。</li>
</ol>3. 人口水平动态发件人-接收器分析: PMT 对光刺激时细胞反应的实时监测
<ol><li>用标准方法胰蛋白酶处理和计数发送方和接收单元的数字。准备1毫升的混合悬浮液的总体积 2.5 x 104接收机和 1.25 x 105发件人单元 (1:5 比率)。 注意: 2:1、1:1 或1:2 发件人-接收器比率还可以使用 1.5 x 105的总单元格数。10
</li>
	<li>24井黑板各井中的板块混合单元, 培养基中含有1毫米荧光素的培养基。</li>
	<li>对平板阅读器中的细胞进行荧光素酶检测, 并确认输出信号对记录系统不太高。 注: 如果输出信号高于 1 x 106光子计数/秒, 则它们可能已饱和。在这种情况下, 将荧光素的浓度降低到最佳值, 以便输出信号低于 1 x 10 6 光子计数/秒.</li>
	<li>在录音系统上设置车牌, 然后启动录制程序 (图 1D)。例如, 设置录音后, 在18小时开始照明。 注意: 时间滤波, 如 detrending 信号的运动平均和 Savitzky-Golay 滤波, 对于波形和峰值检测的可视化是有用的。</li>
</ol>4. 单细胞水平动态发件人-接收器检测: Optogenetic 摄动控制下单细胞反应的实时成像
<ol><li>板 0.5 x 105接收机和 2.5 x 105发件人单元格到27毫米直径-玻璃基35毫米直径菜肴。确保正确调整混合比率和总细胞数 (细胞密度)。</li>
	<li>电镀后的一天, 交换介质与2毫升的记录介质 (苯酚-红色自由 DMEM 培养基补充5% 的血清, 青霉素-链霉素和1毫米荧光素), 并设置玻璃基盘在显微镜上。如有必要, 用 PBS (-) 冲洗死细胞的碎片。 注意: 最好在黑暗条件下执行此步骤。</li>
	<li>在37摄氏度和 5% CO2上安装一个环境室的倒置显微镜。</li>
	<li>设置一个冷却的 CCD 相机。确保 CCD 传感器的温度降低到目标值 (通常为-90 摄氏度)。</li>
	<li>
在自动采集软件中设置 "多维 Timelapse" 窗口的参数, 以5分钟间隔和288倍以上 (一夜录音) 捕捉图像。
	<ol>
<li>打开 "多维 Timelapse" 窗口, 从下拉菜单中选择一个发光通道和一个慢速 50 kHz 读出模式, 并设置 4 x 4 binning, 其曝光率为4分钟。确保读出模式设置为慢速50赫模式, 这对于减少读出的噪声以检测弱发光的生物发光光至关重要。</li>
		<li>打开 "多维 Timelapse" 窗口, 从下拉菜单中选择 "荧光通道" 和 "快速 1 MHz 读出" 模式, 并设置 2 x 2 binning 和 400 ms 曝光。确保读出模式设置为快速 1 MHz 模式, 以减少荧光成像所需的时间。</li>
		<li>通过单击 "获取" 按钮开始定时记录。</li>
	</ol>
</li>
	<li>用图像分析软件从时间推移电影中提取出发光通道的单细胞痕迹, 如下所示。首先, 将图像数据导入图像分析软件, 使发光和荧光叠加图像。对于发光图像, 通过比较在世俗相邻图像中像素的强度, 将像素值替换为上一个和下一个帧的时间平均值 (实现为 "SpikeNoise 过滤器"), 删除来自宇宙射线的热像素,将这些已处理的图像应用于空间平滑滤镜, 并从每个帧中减去外场视图的背景像素值。然后, 在每个时间点确定荧光图像上每个单元格的 "感兴趣区域" (roi), 将 roi 应用于发光图像, 并测量每个 ROI 的发光强度。</li>
</ol>

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作者没有什么可透露的。

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 获得理想的脉动模式, 在特定的细胞, 光诱导的活细胞监测 dLuc 是一个良好的出发点, 并用于筛选和验证。
最常问的问题之一是对光敏感细胞的日常处理。在动态发件人-接收器检测的缺口信号, 我们能够做每日细胞通道下的房间灯, 因为我们的系统 (照片诱导 dLuc 或 Dll1 细胞和接收细胞) 不改变细胞增殖和迅速恢复休息状态后6小时内的细胞被移动到一个黑暗的条件。如果感兴趣的基因是命运决定因素, 漏表达可以诱导细胞分化或防止细胞的扩张, 因此, 建立一个红光环境, 以避免不良的 optogenetic 摄动是必要的, 在细胞处理。
在本协议中, 我们提出了用光诱导基因表达盒生成稳定细胞系的程序。这是在动态发件人-接收器检测中获得可靠结果的关键步骤。人们可以考虑将 optogenetic 模块引入到细胞中的瞬变转染, 但是它在我们的手里并不能很好地测量超脉冲的数量。我们发现, 携带无人能启动盒的质粒的瞬态转染, 即使在黑暗条件下, 也会导致相对较高水平的漏表达。这种漏表达导致了高背景, 没有任何光刺激, 阻碍了可靠的时间路线测量。建立稳定单元的另一种方法是慢病毒北疆系统, 它对于不适用于转染12 的单元格类型很有用。克隆细胞系比异质细胞系呈现更均匀的反应, 但即使在克隆种群中, 细胞反应也不总是一致的: 有些细胞不响应光刺激, 因此呈现出一种分布的光诱导水平。蛋白表达。这可能是由于 hGAVPO 蛋白的可变表达水平和/或可变内源丰度的黄素, 生色所需的光传感 hGAVPO。
本文提出的协议为基因表达动力学分析提供了有力的工具, 但也存在一些局限性。一个缺点是, 一些荧光通道的活细胞显微镜是不兼容蓝光刺激。蓝光照明不仅激活了蓝光敏感蛋白, 而且还活化了荧光蛋白, 包括绿色和黄色荧光蛋白 (GFPs 和 YFPs), 它们容易被光漂白。相反, GFP 的可视化需要蓝光激发, 这可能会在捕捉荧光图像的过程中触发感光细胞的不良活化。光场 (相对比) 成像的环境光源也不兼容蓝光 optogenetic 实验, 但你可以绕过这个问题, 使用绿色或更长的波长光源的成像。
动态发件人-接收器检测将有助于调查其他信号通路。一种可能是用于分泌信号系统的应用。对于这种分析, 在微流控装置中应用纯化配体是一种以精确的时间方式刺激接收单元的替代方法16,17。然而, 在发件人细胞内配体分子的动态生产过程中, 这些方法是不可访问的。采用动态发件人-接收器和光诱导配体表达的方法, 可以进一步解剖从发件人到接收细胞的信号传输。

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胞的 mRNA 半衰期为20分钟。
为了建立稳定的细胞系, 使用了基于 Tol2 transposase 系统的三 cistronic 向量13 (图 1A)。Tol2 表达载体对提高质粒盒的染色体整合效率具有重要意义14。这些载体不仅携带由无人驾驶的促进者驱动的光诱导基因的表达盒, 而且具有光敏蛋白 hGAVPO 和荧光蛋白的表达盒 (iRFP71315或 mCherry)。这种设计使我们能够净化细胞的数量, 有效地将质粒整合到宿主基因组中。
我们建立了光敏发件人细胞, 在蓝光照射下产生 Dll1 配体蛋白 (图 2A)。为此, 采用了基于 Tol2 系统的双 cistronic 矢量 (pAI218)。为检查 Dll1 配体在光照照射下的诱导情况, 在装有图 1B和1C中所示的程控 LED 蓝光源的孵化器中培养细胞。在图 2B和2C中显示了由西方印迹分析检测到的照片诱导的 Dll1 蛋白表达动态的代表性结果。
为了建立响应缺口信号输入的细胞, 我们使用了一个基于 Tol2 系统的双 cistronic 矢量 (pAI177), 在缺口效应基因Hes1的启动子的控制下携带不稳定的荧光素酶报告器和磷酸甘油酸激酶 (PGK) 启动子驱动核-局部红荧光记者 H2B-mCherry。在这种情况下, 荧光记者是有用的两个纯化和单细胞跟踪的时间推移显微镜。
一旦成功建立了可诱导的发件人单元格和接收单元, 就可以通过共同培养这些单元格 (图 2A) 来执行动态发件人接收器检测。在这种检测中, 在蓝光照射下表达凹槽配体蛋白 Dll1 的光敏发件细胞与携带内源 Hes1 振荡器和生物发光 Hes1 记者的不敏感接收细胞共培养 (pHes1-dLuc) (图 2D)。接收细胞内在表达切口受体, 它对相邻细胞所呈现的 Dll1 反应灵敏。
动态发件人-接收方分析的代表性结果显示在图 2E和2F中。为了检测接收细胞具有生物发光记录的动态响应, 我们首先使用了一个带电细胞监测系统, 配备了高灵敏度的光倍增管 (PMT) 用于光子计数和 LED 蓝光光源进行光刺激 (图 1D)。这个系统允许实时记录的生物发光信号在人口水平与预定的光照明。当这两种细胞共培养并暴露于重复光照 (三十年代持续时间, 2.5 小时间隔) 时, 接收细胞的循环响应可在各种混合比率条件下检测 (图 2E)。在此示例中, 我们应用了 Savitzky Golay 过滤 (4 个th顺序和一个13数据点窗口) 来衰减噪声信号。具有重复光照的时间推移显微镜 (2 分钟持续时间, 2.75 小时间隔) 还显示了接收单元在单细胞水平 (图 2F中的灰色线) 和人口水平上的同步响应 (图中的红线2F). 这些数据表明, 发件人细胞中 Dll1 蛋白的循环表达足以同步Hes1相邻接收机细胞的振荡。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57149/57149fig1.jpg"> 图 1: 分析 optogenetic 扰动时细胞响应的策略.(A)用于本协议的代表性质粒向量示意图。这些质粒可根据要求提供。(B)一张 CO2孵化器的照片, 在6井细胞培养板下装有蓝色 LED 设备。(C)通过可编程微型计算机控制 LED 光源电源的电路示意图。固态继电器 (SSR) 被设置为将数字引脚的开/关状态中继到 LED 光源的开/关状态。(D)一种活细胞生物发光监测系统的照片和示意图。请注意, 机动化阶段可以将单向从 PMT 探测器顶部的记录位置移动到 LED 光源顶部的光照位置。此外, 请注意, PMT 探测器可以从好到好, 以监测个别油井的信号。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57149/57149fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57149/57149fig2.jpg"> 图 2: optogenetic 摄动实验的示例.在这些例子中, 使用了小鼠 C2C12 骨骼肌细胞系。(A)动态发件人-接收器检测的示意图。(B、C)用西方印迹分析法分析光致 Dll1 表达的代表性结果。Dll1 蛋白以2.5 小时、2分钟的持续时间和24.7 瓦特/米的强度2的周期光照诱发。(D)发件人和接收单元的共培养系统的荧光图像。(E、F)动态发件人-接收器分析的代表性结果。(E)装有 PMT 的活细胞监测系统记录的接收机响应的时间模式。总单元格数固定为 1.5 x 105单元格, 但发件人和接收方之间的比率由5:1 到1:2 分配。(F)单细胞成像显示, 由周期性光照引起的振荡信息 (2 分钟持续时间、2.75 小时间隔) 从发件人转移到接收单元。使用了 1.5 x 105单元, 其发送者到接收方比率为5:1。根据参考文献10改编。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57149/57149fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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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7.6K 次观看.  Kyoto University. 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。

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

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

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遗传学

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 ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

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

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

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

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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

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

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

通过两个互补细胞同步方案研究细胞周期调控的基因表达

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

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

血栓烷 A2 受体基因 C924T 多态性的研究方法

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

一种基于昆虫蜕皮激素受体的奇异基因开关, 用于对转基因进行有鲁棒性、可逆性和可忽略的泄漏的刻意表达

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

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细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

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植物病原体的遗传操作