这项研究得到了国家卫生研究院 (R01-DE025985) 和美国癌症协会研究学者奖 (RSG-13-383-MPC) 的支持。这项工作还得到了美国农业部 (2015-67015-23268 和 2014-67015-21787) 的赠款和加利福尼亚大学戴维斯分校的新的研究计划赠款的支持。

注意: 此过程中使用的细胞系含有传染性病毒, 要小心, 只在2级 BSL 设施或更高的地方进行。
1. 细胞的制备和维护
<ol><li>培养龙 BCBL-1 细胞 (或其他 KSHV 感染的 PEL 细胞系) 在 RPMI1640 培养基中补充15% 胎牛血清 (FBS) 和1% 青霉素链霉素谷氨酰胺溶液。</li>
	<li>生长在37° c 的细胞与5% 的二氧化碳 (CO2), 确保不断增长和分裂细胞的比率为1:4 每2-4 天。</li>
	<li>每天用光显微镜监测细胞培养, 以确认良好的细胞生长和形态学, 否则建议重新启动细胞培养或相应调整细胞培养条件。</li>
</ol>2. 胸苷同步和溶解诱导
<ol><li>在 10 cm 培养皿中, 平板龙-K-BCBL-1 细胞在新制备的培养基中 (1 x 106 cells/2 介质的 mL)</li>
	<li>加入 200 mm 胸苷 (最终浓度: 2 毫米) 的菜, 混合和孵化为18小时。</li>
	<li>离心机的细胞为5分钟, 在 500 x g. 用无菌1x 磷酸缓冲盐 (PBS) 洗涤细胞, 再次离心, 和重细胞在新准备的媒体。重复共3洗。重在新鲜的准备的媒介。</li>
	<li>允许单元格退出 S 阶段8小时。</li>
	<li>添加胸苷 (最终浓度: 2 毫米), 混合和孵育16小时。</li>
	<li>离心机的细胞为5分钟, 在 500 x g. 用无菌1x 磷酸缓冲盐 (PBS) 洗涤细胞, 再次离心, 和重细胞在新准备的媒体。重复共3洗。重在新鲜的准备的媒介。</li>
	<li>为了诱导病毒活化, 添加12邻十四-phrobol-13-乙酸 (TPA, 最终浓度 20 ng/毫升) 和强力霉素 (DOX, 最终浓度 100 ng/毫升) 的细胞培养, 混合和孵化为 4 h。对于没有 K-的诱导盒的细胞系, 使用 TPA 刺激活化 (最终浓度 20 ng/毫升和丁酸钠 (1 mM))。</li>
	<li>将细胞离心5分钟, 在 500 x g. 用无菌1x 磷酸缓冲盐 (PBS) 冲洗细胞, 再次离心, 并在完整培养基中重细胞。重复共3洗。重在完整的文化媒体。</li>
	<li>允许细胞生长24小时。</li>
</ol>3. 固定和性
注: 在进行前, 确保固定解决方案 (3.7% 甲醛在 DEPC 处理 pbs), 二乙基焦处理的磷酸盐缓冲盐水 (DEPC-pbs), 甘氨酸 DEPC pbs (甘氨酸的最终浓度: 100 毫米) 和 permeabilizating 溶液 (50%丙酮, 50% 甲醇) 准备。通常情况下, 每张幻灯片至少需要50万单元格, 如果需要, 可以进行乘法运算, 特别是在单元格不健康时, 还要包括额外的单元格。
<ol><li>将细胞收集到1.5 毫升的离心管。</li>
	<li>将细胞离心2分钟, 200 x g. 用1毫升无菌 DEPC PBS 冲洗细胞3次。重在1.2 毫升的 DEPC PBS。</li>
	<li>将适当数量的片 (由实验装置确定) 放入6井板的底部。</li>
	<li>吸管200µL 的细胞 DEPC PBS 混合物到每个片, 确保每张幻灯片上至少有50万单元格。允许细胞沉淀2分钟, 并抽出多余的 DEPC PBS 只留下一层薄薄的细胞。在此过程中移除单元格是正常的。 注意: 粉煤灰是有毒的, 穿戴适当的保护。</li>
	<li>小心地添加1毫升的固定解决方案 (3.7% 甲醛在 DEPC PBS), 以每个盖子滑动。允许单元格修复10分钟。</li>
	<li>用 DEPC PBS 清洗片3次。</li>
	<li>小心地添加1毫升的甘氨酸 DEPC PBS (最后浓度的甘氨酸: 100 毫米) 到每个盖子滑动。允许细胞淬火5分钟。</li>
	<li>添加1.5 毫升的 DEPC pbs 和任何地方在一个振动筛5分钟或动摇, 但轻轻地在手1分钟, 吸入过量的 DEPC pbs。注意不要破坏细胞。重复此步骤共3洗。</li>
	<li>小心地将1毫升的 permeabilizing 溶液 (50% 丙酮, 50% 甲醇) 添加到每个盖板上。允许细胞 permeabilize 15 分钟。 注意: 在这一点上, 片可以储存在一个-20 ° c 的冰箱不超过一周的时间。避免冻结或减少在冷冻库中的时间, 因为图像质量会迅速降低。确保在冷冻室中, 细胞保持湿润并覆盖在甲醇/丙酮中, 并确保它们在从冷冻库中取出后, 与 DEPC PBS 适当水化。</li>
	<li>添加1.5 毫升的 DEPC pbs 和任何地方在一个振动筛5分钟或动摇, 但在手轻轻地在1分钟. 吸入过量的 DEPC pbs, 小心不要扰乱细胞。重复此步骤共3洗。</li>
</ol>4. IFA 和 RNA-鱼
注: 关于 RNA 鱼探针标记和准备: 对于959碱基对的 K-含子内含子, 生成31不同的探针分别为20碱基对, 其中含有大约50% 的 GC 含量。探头的工作和储存溶液分别 aliquoted 为100µM 和2.5 µM 浓度。
<ol><li>标签显微镜幻灯片与必要的实验信息或某种可区分的标签。</li>
	<li>用湿纸巾把密封塑料容器的底部排成一行。</li>
	<li>
准备主要抗体溶液 (1.5 µL 的拉娜抗体, 0.75 µL 的 RNA II 抗体, 3 µL 贝克酵母 tRNA, DEPC PBS 30 µL), 并为每片目前的食谱。将30µL 的主抗体溶液放在每个有标记的显微镜幻灯片上。
	<ol>
<li>首先滴定的主要抗体, 并使用最佳的数量。一定要使用纯化的 IgG, 因为血清 (腹水液) 可能含有大量的核糖核酸。</li>
	</ol>
</li>
	<li>将片 (单元格朝向解决方案) 放置在有标记的显微镜幻灯片上, 并将滑块移到容器中, 用湿纸巾。注意不要引入气泡。用塑料包盖住容器的外部。将容器移动到37° c 的恒温箱中1小时。</li>
	<li>准备鱼洗涤缓冲液 (2x 盐水柠檬酸钠缓冲液 (SSC) (最后), 10% 甲酰胺, 90% DEPC dH2O)。</li>
	<li>准备2x 杂交缓冲 (4x SSC, 20% 葡聚糖硫酸盐)。</li>
	<li>准备二次抗体和鱼内含子探针缓冲 (20 µL 2x 杂交缓冲 (最后的 1x Hyb 缓冲), 4 µL 贝克的酵母 tRNA (最后 10%), 4 µL 甲酰胺 (最后 10%), 内含子 K-贸易量探针 (最后 125 nM), 0.8 µg 二级抗体, DEPC dH2高达40µL)。将每张幻灯片所需的食谱相乘。</li>
	<li>在孵育后, 洗涤细胞3次与 DEPC PBS 在一个6井板5分钟每。</li>
	<li>洗涤用鱼洗涤缓冲3次为5分钟每。保持片在洗鱼缓冲液中, 直到完成第二抗体和内含子探针的混合 (步骤 4.7)。</li>
	<li>用于杂交的初级抗体孵化用清洁玻片。</li>
	<li>放置40µL 的二次抗体和鱼内含物探针, 包含溶液在每个玻片上。</li>
	<li>将片与单元格放在幻灯片上, 并将单元侧朝下放在缓冲区顶部, 注意不要引入气泡。</li>
	<li>将幻灯片放入塑料容器中, 底部有一条湿润的纸巾。将塑料容器包装在第一个塑料包装上, 然后用铝箔。</li>
	<li>在37° c 的16-24 小时内用幻灯片孵化容器。</li>
	<li>在孵化后, 小心地从玻璃的边缘滑动片, 并将其放回6个正面朝上的板块, 并在6井盘中洗涤3次的清洗细胞, 每次5分钟。</li>
	<li>用 2x SSC 冲洗细胞2次。</li>
	<li>添加 DAPI (1:1, 000) 到 2x SSC 和允许坐5分钟的室温。</li>
	<li>用 2x SSC 冲洗细胞2次。</li>
	<li>清洁用于杂交的玻片, 并在玻璃片上添加10µL 的安装液。</li>
	<li>将片面向下放置到安装解决方案中。注意不要引入气泡。</li>
	<li>用实验室组织, 轻轻地移除多余的安装解决方案, 注意不要挤压细胞。</li>
	<li>使用指甲油, 应用在片边缘的一个充足的层, 并允许干燥。</li>
	<li>进行荧光显微镜检查。</li>
</ol>5. 3D 荧光显微镜
注意: 虽然协议将随所使用的荧光显微镜系统的类型而异, 但以下步骤将有助于确保获得最佳图像数据以进行定量分析。
<ol><li>预处理固定样品和贴装在防褪色介质中延迟荧光漂白在成像过程中。</li>
	<li>使用高品质的目标 (如 60X 1.42 N. 油浸透镜), 可以捕获整个细胞 (或细胞核) 的视野。</li>
	<li>为每个荧光色通道设置曝光参数 (例如、激发功率、曝光时间), 使用正负控制样本。获取荧光图像。 注: 只包含一个荧光标签的阳性对照样品也应用于确定颜色通道之间的 "串扰" 级别。对于成像固定细胞标本, 荧光激发功率可以减少 (与稍长的曝光时间), 以尽量减少漂白。</li>
	<li>一旦建立了成像协议, 保持相同的曝光参数一致的所有样本的研究, 使图像数据可以得到准确的分析和比较。</li>
	<li>执行采集后图像处理和3D 重建。用 RNA-鱼信号 (红色)、拉娜分子 (绿色) 和核染色质 (适用的地方) 染色 DAPI (蓝色) 识别 K。 注: 细胞细胞核的区域, 在其中 K 和拉娜是共聚的, 因此被认为与病毒转录工厂和复制复合体有关。</li>
</ol>

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本文作者没有金融联系或利益冲突。

该议定书的某些方面可以改变, 以适应不寻常的情况。固定和性缓冲的选择也可以改变。对于固定缓冲器, 甲醛也是有效的, 乙醇可用于性。
推荐用甘氨酸 pbs 猝灭甲醛, 因为它能防止甲醛在没有牛血清白蛋白 (BSA) 的情况下禁用抗体, 但如果片洗得足够彻底, 可以跳过甘氨酸 pbs 的步骤。在协议中, 避免使用牛血清白蛋白作为阻断溶液。在 Izumiya 实验室进行的试验研究显示, rna-鱼信号较弱, 推测是由于 rna 的降解。如果需要阻断主要抗体, 建议使用核糖核酸游离 BSA。从过去的经验, 它被注意到, 如果抗体是具体的, 包括 BSA 是不必要的反应。然而, 建议始终在所有孵化步骤中包括过量的酵母 tRNA, 以防止 RNA 降解。已注意到, 使用 tRNA 作为阻断剂增加了特定的 RNA 鱼信号。
已实现了单元周期同步, 以产生更高效和同步的 KSHV 重新激活。对于细胞周期同步, 羟基或血清饥饿可能是替代方法。已证实, 羟基是有效的使用胸苷阻滞, 虽然孵化与羟基单独重新 KSHV 弱。
这项技术如何适用于其他研究？在 Izumiya 实验室最近的一份出版物中, 它表明定位积极转录 KSHV episomes 和核糖核酸 II, rna 转录的基本酶。然而, 众所周知, 在 KSHV 基因调控中, 有许多共同激活剂、co 抑制和细胞转录因子。定量 rt-pcr 技术已在历史上被用于培养细胞的种群 (重新激活和潜伏的混合物), 量化病毒转录, 并评估对病毒基因表达的影响。使用上述方法, 分析可以缩小到单一的 episomal 水平, 以检查病毒转录。因此, 可以检查其他细胞和病毒酶的时空调控, 以了解它们与 KSHV 活化的关联。还值得一提的是, 由于 rna 内含子区域的瞬态性质依赖于它们的降解, rna 鱼探针将杂交, 并且通常会在活动转录发生的地方定位, 但是如果内含子没有退化立即, 鱼探针可以呈现不总是位于活动转录附近的信号。
目前, 由于其基因组大小和细胞启动子结构的复杂性, 很难研究细胞转录工厂的形成与随后基因表达的关系。在这方面, KSHV episomes 可以是一个理想的工具, 由于其相对较小的基因组大小, 定义的活化机制与明确的 RNA II 聚集形成。利用 KSHV 的操作病毒小染色体, herpesvirology 可以从一个独特的角度对细胞遗传学研究领域作出贡献。

越来越清楚的是, 原子核的时空组织在真核生物中调节微调基因表达中起着重要的作用。大多数组织特异性基因分布在许多染色体上, 需要同步调节, 以协调的方式响应特定的刺激1。细胞构建活性染色质中枢 (乙酰胆碱) 是一种将基因及其顺式调节成分汇集到特定核空间的方法1。
各种研究也表明, 尽管细胞核看起来非常致密和粘性, 但生物活性分子可以通过扩散2快速地穿过细胞核。由于这种短暂的性质, 大多数 DNA 结合蛋白 ' 跳跃 ' 从绑定站点到绑定站点, 感觉他们的方式周围的核空间, 这使得一个高度适应和多才多艺的核2。
尽管细胞核内的生物分子有这种动态行为, 但没有膜的核组织 (但不限于) 核仁、卡哈尔体和早幼粒细胞白血病核体 (PML) 仍然存在。它是通过各种机制, 如串联 DNA 重复 (核仁), rRNA (核仁) 和结构蛋白, 如 coilin (卡哈尔体) 或 pml 蛋白 (pml-NB), 持有这些构造一起3,4,5.这些结构和其他会众, 如转录工厂, 作为脚手架, 不仅增加了所需成分的局部浓度, 而且还调节了蛋白质和核酸的组成, 最终形成有效蜂窝功能的中心站点6。
对研究表观遗传学的研究人员来说, 核结构的形成提供了大量的信息。从病毒学的角度来看, 重新激活潜在感染病毒, 如 KSHV, 极大地改变了细胞核的景观和核酶的分布, 将转录主要从细胞基因转移到病毒基因, 最终生成功能完备的病毒子代7,8。KSHV 如何操纵细胞基因表达机制来促进病毒基因表达？这些信息也可以揭示颞细胞基因调控机制。
和所有其他疱疹一样, KSHV 有两个生命周期称为分解复制和延迟。KSHV 主要驻留在潜伏期阶段, 其中的大多数病毒基因表达是沉默的, 除了潜伏期相关基因9,10。在潜伏期 KSHV 产生潜伏期相关的核抗原 (拉娜), 其中组成结合病毒基因组和系病毒染色质的人类染色体11。由于拉娜与病毒基因组的密切关系, 有可能使用 IFA 和 DAPI 染色和定位的病毒 episomes 与寄主染色质。
为了研究 KSHV 在单个 episome 水平上的重新激活以及与感染细胞中其他病毒 episomes 的联系, 已经建立了一种在原位转录病毒染色质的有效方法。因此, 拉娜和 RNAPII IFA 与内含子 rna-鱼结合, 通过生成 rna 鱼探针, 绑定到内含子区域 (确切的探针序列可以发现在 Izumiya 实验室的最新出版物8) 的 KSHV K-贸易协定-关键的病毒蛋白, 是必要和足够的 KSHV 重新激活-它是可能的, 以确定何处转录实际上发生8,11,12,13,14,15.这种内含子 RNA-鱼技术使研究人员能够直观地看到 mRNA 在被剪接之前被转录到细胞质中, 然后被输出到胞浆16,17。
KSHV 可以重新激活的各种化学刺激, 包括佛波酯类, 如 12 O-Tetradeconoyl-佛波-13 醋酸盐 (TPA) 和组蛋白乙酰抑制剂, 如丁酸钠, 另外 KSHV 可以诱发活化的过度表达病毒转录因子, K-贸易协定19。研究人员成功地提高了 KSHV 激活的效率, 在诱导重新激活18之前同步细胞周期。因此, 对于这些特殊的研究, 细胞是同步使用双胸苷块 (协议描述), 并与 TPA 和强力霉素 (Dox) 孵育短时间。Doxycyline 的使用是因为这些实验中所用的细胞系有一种强力霉素诱导的 k-区域化盒, 它是从 cDNA 中克隆出来的, 不包括 k 区域的内含子区。虽然可以使用仅诱导的 k-KSHV 表达式来重新激活它, 但其他研究人员已经证明, 由于各种不同的生物化学因素, k-贸易协定的表达仅仅证明是一个微弱的活化刺激20。通过结合所有这些, 并通过限制药物孵化到短时间内, 一个强大的, 但不是过度人工 KSHV 的重新激活是实现了成像。
在对拉娜、RNAPII、K-的内含子和 DNA 进行标记后, 采用 widefield 反褶积显微镜进行3D 荧光成像。利用成像软件进行处理后, 可以正确评价活性病毒 episomes 的空间分布。利用这一技术, 可以研究关于形成活性染色质中枢和其他核结构的基本性质的核心问题。具有相同的病毒 episomes 在一个单一的细胞, 处理相同的调控元素可能代表一个独特的研究工具, 以加深对时空基因调控机制的理解。
在不同时间点固定的成像多细胞标本的局限性, 以表征一个固有的动态分子过程是, 微妙或小规模的变化, 荧光分布是不被发现或认为微不足道。这是事实, 除非在罕见的情况下, 每个细胞观察显示同样微妙的变化。因此, 有效的病毒转录和其他核结构的完全时空关系不能用固定成像进行严格的评估。为了解决这些技术难题, 最好的方法是对有标记的病毒 episomes 的活细胞进行图像处理, 并随着时间的推移, 跟踪关键细胞酶的位置。

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functional imaging of viral transcription factories using 3d fluorescence microscopy

众所周知, 基因的时空调控是控制适当基因表达的一个组成部分。因此, 了解在核空间内发生转录的位置和时间, 以及在同一细胞核内感染 episomes 之间的关系是非常宝贵的。在这里, 免疫荧光 (IFA) 和 RNA 鱼已经 combinedto 识别积极转录卡波肉瘤相关的疱疹病毒 (KSHV) episomes。通过染色 KSHV 潜伏期相关的核抗原 (拉娜), 可以找到在细胞核内存在病毒 episomes 的地方。此外, 通过设计 rna 鱼探针来瞄准病毒基因的内含子区域, 这只在生产感染期间表达, 可以找到新生的 rna 转录。利用这种分子探针的组合, 可以可视化大型病毒转录工厂的组装, 分析 KSHV 活化过程中病毒基因表达的空间调节。通过包括抗 rna 聚合酶 ii 抗体染色, 你也可以可视化 rna 聚合酶 ii (RNAPII) 聚集和 KSHV 转录之间的关联在重新激活。

在使用 BCBL-1 细胞的地方, 只有拉娜和 K-区域内 RNA 被染色 (图 1), 才执行了略微删节的协议。这项实验使我们可以检查在哪里积极转录病毒 episomes 是和异质性的反应, KSHV 活化刺激在一个群体的细胞。在图 1中箭头指示的单元格中, 与病毒 episomes 的分布紧密匹配的区域 (与 LANA 标记的) 中的明显的 K-位置荧光, 是接近 KSHV 基因组的活动转录的证据。在同一示例中, 可以观察到图 1中箭头所表示的单元格, 它表现出更弱和弥散的 K-区域荧光, 与 LANA 没有明显的重叠。这说明了一般的发现, 在一个细胞的种群中, 对重新激活刺激的反应程度有很大的变化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56832/56832fig1.jpg" style="font-size: 14px;"> 图 1:可视化病毒 episomes 的活动转录.在 BCBL-1 细胞上进行了 IFA 和 RNA-鱼。BCBL-1 细胞不同步, 但用 TPA 和丁酸钠治疗4小时. 使用 LANA 免疫, 病毒基因组是在绿色的可视化, 而 RNA-鱼的目标是 K-区域内内含子定位为红色的活性转录。细胞染色质染色后固定和性与 DAPI。K-贸易协定内含子和拉娜污渍合并在一起。完整箭头指向已完全重新激活并正在形成 VTFs 的单元格。当箭头指向一个只开始重新激活的单元格时, 如弱 K-位置信号所暗示的那样。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56832/56832fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
其次, 通过对病毒蛋白 K 的表达 (图 2) 的可视化来检测胸苷同步的有效性。在一个双胸苷块进行同步龙 BCBL-1 细胞在 G1/S 相变, 先前描述的协议执行 (没有 LANA 和 RNAPII 染色)。通过检查 K-区域内染色, 很明显的是, 同步细胞对重新激活刺激的反应比非同步的种群多。从先前在 Izumiya 实验室进行的研究 (未显示数据), 验证了 RNAPII 在细胞周期同步后也更频繁地 colocalizes K-环化。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56832/56832fig2.jpg" style="font-size: 14px;"> 图 2:胸苷有效性在龙 K-BCBL-1 细胞上进行了 RNA-鱼的研究。细胞 (由 "2x 你的" 标题表示) 与双胸苷块同步。采用 TPA 和 DOX 的方法对4小时内的非同步细胞进行了处理, 并将其与 RNA 鱼探针杂交。均匀亮红色的细胞不是表达的, 而是死的, 用 DAPI 染色验证 (不显示)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56832/56832fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
作为一个一般的说明, 这是非常重要的染色细胞与 DAPI 时, 执行此协议。亡细胞可以很容易地被核分裂和起泡识别。鉴于大量的诱导刺激和一般细胞培养的做法, 这是常见的一些死细胞被看到。它是重要的总是染色的 DAPI 作为一种主要的方法, 以辨别活细胞和死体细胞。有时, 抗体或 RNA 探针会在凋亡细胞中被捕获并产生信号, 因此从分析中排除这些细胞是很重要的。DAPI 染色可以提供多种附加功能, 例如, 在图 3中, 很明显, 箭头指示的对象实际上是三独立的单元格, 而不是一个。
必须注意的是,图 3和图 4中显示的图像是积的, 从而创建了一个更具点状的图案。通过检查 RNAPII 染色, 可以看到箭头和其他周围细胞所指示的细胞之间的差异。值得一提的是, 由于反褶积, RNAPII 信号比正常的更明显。
使用3D 显微镜和这项技术, 我们可以询问 RNAPII, 拉娜, 和 K 的空间关系。例如, 在图 4中, 环形的 RNAPII 结构可以看到, KSHV 的基因组在边缘上点缀。一般情况下, 拉娜通常 colocalizes 与 RNAPII 在细胞开发转录工厂。然而, 并非所有的拉娜点 colocalize 与 RNAPII, 研究包括 4th维度 (时间) 将是必要的, 以澄清这种表型。重要的是要补充的是, 几个 LANA 点, 不 colocalize 与 K-贸易协定的信号代表 episomal 异质性的反应, 恢复激活刺激甚至在一个单独的细胞 (图 4)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56832/56832fig3.jpg"> 图 3:联合 IFA 和内含子 RNA-鱼.对龙 K-BCBL-1 细胞进行了 IFA 和 RNA-鱼的研究。病毒基因组位于与免疫的拉娜 (绿色), 活跃转录与 RNA-鱼的内含区的 K-区域的 mRNA (黄色), RNAPII (红色) 会众被标记为 IFA, 和细胞最终染色使用 DAPI 图像DNA (蓝色)。龙 K-BCBL-1 细胞同步使用双胸苷块, 然后细胞被激活通过孵化与 TPA 和 DOX 4 小时, 和24小时后, 细胞固定, 化, 并进一步准备成像。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56832/56832fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56832/56832fig4.jpg"> 图 4:病毒转录工厂的3D 可视化.内含子 RNA-鱼使用 k-贸易协定内含子探针 (浅蓝色), 免疫 RNAPII (红色) 和拉娜 (绿色), 和 DAPI DNA 染色 (深蓝色)。积在显微镜上拍摄的 Z 栈的3D 渲染, 在商业成像软件上进行处理和构造 (见材料表)。龙 K-BCBL-1 细胞双胸苷同步, 然后处理的 TPA 和 DOX 4 小时的细胞被处理28小时后重新激活刺激。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56832/Figure.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

病毒转录工厂是分散的结构, 富含细胞 RNA 聚合酶 II, 以增加病毒基因转录在重新激活。本文介绍了一种通过免疫荧光染色和原位RNA 杂交技术在3D 核空间中主动转录病毒染色质位置的方法。

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

functional-imaging-viral-transcription-factories-using-3d

Nanyang Technological University

Research

JoVE Journal

Biology

7.0K 次观看.  University of California, Davis. 病毒转录工厂是分散的结构, 富含细胞 RNA 聚合酶 II, 以增加病毒基因转录在重新激活。本文介绍了一种通过免疫荧光染色和原位RNA 杂交技术在3D 核空间中主动转录病毒染色质位置的方法。

视频: Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

科研

生物学

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

使用荧光显微镜驱动蛋白-1的货物的识别

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...