Name: imaging mycobacterium tuberculosis in mice with reporter enzyme fluorescence
Uploaded: 2018-02-26T14:00:00.000+00:00
Duration: 10 min 6 s
Description: Watch this Scientific Journal Video about Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence at JoVE.com

根据德州 A & M 大学机构动物护理和使用委员会制定的指导方针和条例, 进行了动物研究。可能需要在您的机构中审查和批准来自生物危害安全官员或委员会。
警告: 所有程序都需要 BSL3 控制。人员必须随时佩戴个人防护用品。所有的操作都是在生物安全柜 (BSC) 内进行的, 所有的锐度都在锐化容器中处理。工作表面和平衡计分卡清洗与缓冲苯酚和70% 乙醇之前开始工作和下班后。程序通常在生物安全级别3与结核分枝杆菌进行, 但为了说明和拍摄目的, 提交人在2级生物安全级别上展示了这些程序, 毒性较低的细菌。
1. 菌株和文化条件
注意: 在本研究中使用了m. 结核应变 CDC1551, 但任何结核分枝杆菌菌株都可以以同样的方式使用。
<ol><li>在 OADC 中生长细菌 (7H9 汤补充0.5% 甘油, 10% 油酸葡萄糖复合体, 不含过氧化氢酶, 0.05% Tween-80) 培养基, 其浓度为37摄氏度, 至600 0.5 (~ 0.2 x 107 CFU)。</li>
	<li>稀释 OADC 中的培养 (1:10 稀释的细菌系列)。将细菌的稀释 (105, 106, 107, 108) 在选择性7H11 板上, 以允许 CFU 的测定。</li>
	<li>孵化四周在37°c 或直到殖民地可以准确计数。</li>
	<li>将细菌接种在 8534 x g 处进行离心, 以5分钟获得颗粒。用10毫升生理盐水 (0.9% nacl) 清洗颗粒, 并在15毫升生理盐水 (0.9% 氯化钠) 中重新悬浮颗粒。</li>
</ol>2. 使用麦迪逊室的小鼠气溶胶感染
<ol><li>允许老鼠适应新环境一周。</li>
	<li>在把老鼠装进房间前先称其重量。</li>
	<li>将三根电线插入电源条: 主腔电源、真空泵和空压机, 按顺序排列。</li>
	<li>小心拧开玻璃罐。把玻璃瓶带到生物安全柜, 加入挑战接种悬浮在15毫升盐水 (0.9% NaCl), 以实现 ~ 104 -106 CFU 细菌在肺部。</li>
	<li>在生物安全柜中关闭玻璃罐的盖子。将罐子连接到雾化装置上, 调整垂直不锈钢管, 使下部 (进气) 端大约一英寸, 低于罐内流体的水平。</li>
	<li>加载实验所需的所有动物 (房间可以容纳多达90只老鼠) 进入房间, 并关闭所有闩锁在门上。</li>
	<li>检查主 (室) 空气流量计。确保浮动 (球) 的中心运行约50升/分钟, 测量的左边的刻度。</li>
	<li>在会议厅的控制面板上按 "开始" 按钮。将气流速率通过压缩机空气流量计 (左侧较小的仪表) 设置为4升/分的刻度。目视检查以确保挑战接种正在雾化。</li>
	<li>15分钟后, 当 "控制面板" 前面的红灯亮起, 并有一个发声信号指示运行结束时, 请按 "控制面板" 右下角的 "复位" 按钮重置计时器。</li>
	<li>按住房间门上的小红色按钮释放真空。</li>
	<li>打开房间门, 把动物移走。把老鼠放回笼子里去。</li>
	<li>取下玻璃雾化器罐并放置在密封的、防泄漏的运输容器中。将容器放在生物安全柜内, 将挑战悬浮液丢弃到指定的废物容器中。</li>
	<li>将所用的喷雾器罐放在生化袋内, 并密封袋, 运输到高压釜。</li>
	<li>在感染程序结束时, 用缓冲的苯酚和70% 乙醇喷洒室内, 并允许分室坐10分钟。</li>
	<li>然后, 彻底擦拭所有可访问的内部表面。处理所有受污染的纸巾,等。在生化垃圾桶里用蒸压器把垃圾、废物容器和使用的喷雾器罐。</li>
	<li>把动物放回封存室直到成像时间点。</li>
	<li>在成像当天, 将动物放在辅助容器中传送到成像室。</li>
</ol>3. 动物麻醉
<ol><li>用特制的气体麻醉系统麻醉小鼠与异氟醚。</li>
	<li>每两个木炭过滤器的重量都位于麻醉装置的顶部。</li>
	<li>替换为新的罐子, 如果重量是50克以上的初始重量。</li>
	<li>检查蒸发器单元以确保该过程有足够的异氟醚。</li>
	<li>将鼻锥架置于成像腔内。放置程序所需的鼻锥数, 并用鼻锥阻滞剂封住其余开口。</li>
	<li>打开高压气缸的氧气供应, 并将其设置为 55 psi。</li>
	<li>打开位于麻醉装置前面的疏散泵, 并将其设置为8升/分钟。</li>
	<li>打开位于麻醉装置前面的氧气开关。</li>
	<li>将气体流向麻醉感应室, 将气体流量设置为1.5 升/分. 把煤气流关了。</li>
	<li>将气体流向成像室, 将气体流量设置为0.25 升/分. 把煤气流关了。</li>
	<li>打开异氟醚蒸发器并将其设置为 2-2.5%。根据实验所用动物的数量和重量, 调整异氟醚的水平, 在异氟醚蒸发器上旋转表盘 (2-2.5% 对一只老鼠称20克; 4% 为一只体重300克的豚鼠)。</li>
	<li>把老鼠放进麻醉室, 关上盖子。打开气体流向麻醉感应室。</li>
	<li>把老鼠放在麻醉感应室里 5-10 分钟, 直到完全麻醉。</li>
	<li>一旦老鼠被麻醉, 在眼睛上涂抹一个光学软膏来保护它们。</li>
	<li>将小鼠置于腹侧或胸骨卧床, 使其鼻子放置在鼻锥内, 以方便在成像过程中对小鼠进行麻醉。</li>
</ol>4. 报告酶荧光 (REF) 成像
<ol><li>注射基质 (20 µM, 2.5 µL/克重量) 的腹腔注射入感染和控制小鼠。小鼠在成像室内麻醉下不到1分钟。</li>
	<li>启动成像系统。</li>
	<li>通过单击 "初始化" 来初始化系统。等到温度条变成绿色。 注: 成像室由加热平台组成, 用于在成像时保持动物体温。</li>
	<li>对于成像采集设置, 选择荧光 |在采集控制面板中, 对整个动物或对肺组织、结构和覆盖物进行全光照明。</li>
	<li>设置视场为 B, 单鼠标和灯级至高。</li>
	<li>设置曝光时间为自动, 中等 binning, 2-3 为 f 或停止, 并且励磁过滤器在745毫微米和放射过滤器从780毫微米到840毫微米。</li>
	<li>对于序列设置, 单击序列设置, 选择 9-12 在肺部区域的反光照点。</li>
	<li>点击获取图像获取。</li>
	<li>把鼠标放回到笼子后成像。</li>
	<li>监测小鼠直到完全恢复或牺牲, 以量化 CFU, 如果成像在一个时间点的实验。执行修改后的卡氏评分评分, 以监测小鼠成像后的福祉。</li>
</ol>5. REF 成像分析
<ol><li>打开映像软件。</li>
	<li>通过单击 "浏览" 图标加载图像文件。</li>
	<li>对于光谱分解, 单击工具调色板中的光谱分解, 然后单击 unmix 所需的光谱。点击开始分解。</li>
	<li>对于光学曲面重建, 请单击曲面地形。</li>
	<li>选择背方向, 受皮毛鼠标的左右, 单击 "生成曲面"。</li>
	<li>绘制裁剪图像并为感兴趣的区域设置阈值图像。单击 "完成"。</li>
	<li>对于器官注册, 请转到3D 光学工具下拉菜单中的 "注册" 选项卡。</li>
	<li>在器官图谱中登记感兴趣的器官。</li>
	<li>使用在面板注册工具上/关闭的变换工具调整器官大小或位置以生成的表面形貌。</li>
	<li>转到荧光层析成像 (掠过) 3D 重建, 并选择 "分析" 选项卡. 选择要包含的图像进行分析, 图像类型为规范化的传输荧光 (NTF) 效率, 请单击 "开始"。</li>
	<li>检查 "选择全部" 并在 "数据预览" 选项卡中重建。</li>
	<li>对于荧光量化, 请转到感兴趣的区域 (roi) 工具, 将 ROI 多维数据集添加到感兴趣的机构中, 并调整多维数据集大小以覆盖感兴趣的器官。点击测量 3D ROIs。</li>
	<li>在 "roi 度量" 窗口中, 转到 3D roi 度量值, 选择 "数据类型" 到 "源素和度量单位" 以 pmolM-1cm-1。</li>
	<li>将3D 重建分析的结果保存和/或导出到图形或数据文件中。</li>
</ol>6. CFU 细菌的量化
<ol><li>腹腔注射0.1 毫升戊巴比妥钠 (390 毫克/毫升) 弄死小鼠。</li>
	<li>通过挤压小鼠脚垫来检查踏板反射, 以确保没有反射反应。</li>
	<li>用无菌钳和剪刀将小鼠的肺组织外植体。融汇肺组织在1毫升的1x 磷酸盐缓冲生理盐水 (PBS)。</li>
	<li>在无菌 1x PBS 中进行肺匀浆的10倍串联稀释。</li>
	<li>为电镀, 斑点三整除数20µL 每各自稀释在琼脂板材。</li>
	<li>在37摄氏度的孵化器中孵化板块, 并监测细菌的生长。</li>
	<li>在孵化时间后, 在每一个斑点的殖民地计数, 并表示为 CFU/毫升通过纠正体积和稀释使用以下等式: CFU/毫升 = (C/V) x M 那里 C = 殖民地计数每点, V = 样品容量接种在每个板材 (mL) 和 M = 增殖因素 (被使用的稀释的倒数)。 注: 如果将11个菌落计算在一个点上, 样本体积为0.002 毫升, 样本稀释量为 10-4, 则使用该等式, 菌落计数将计算为 (11/0.02) x 104 = 5.5 x 106 CFU/毫升</li>
</ol>

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当使用像 REF 这样的成像技术时, 有一些关键策略允许生成健壮和一致的数据。光学成像在组织中产生散射光, 这会影响穿透深度, 因为很难捕捉到所有方向发出的光。利用近红外线荧光 (近红外) 基质进行 REF 成像, 在 Querange 中具有激发和发射波长的 700-900 nm, 有利于哺乳动物组织对荧光信号的最小吸收。定制设计的基底是通过将近红外荧光, IRDye 800Cw 到淬火, IRDye QC-1, 通过一个内酰胺环, 允许荧光共振能量转移为基础的淬火构造。IRDye 具有良好的组织穿透和光散射特性, 对哺乳动物没有任何明显的有害影响36, 从血液和器官清除 24 h. 荧光信号显著增加起始4小时后基板管理, 达到最高水平后6小时管理。
在开展大型、复杂的实验前, 应规范细菌感染剂量、感染剂量的管理方式、基质以及影像后感染的时间点。试验性研究可以大大减少成像大量动物的时间和成本, 因为一个标准程序可以优化之前进行的关键实验。细菌负荷应确定的器官/组织的整个身体成像后, 使用反光照和体外肺成像, 利用 epi 照明来验证信号的来源和确定相关的质量与细菌数字存在8。试点研究将提供洞察的阈值检测, 动态范围的技术, 以及确定的最佳实验条件的成像。
与其他荧光和发光策略相比, 使用 REF 成像的主要优点是它具有较高的灵敏度和对自然的m. 结核菌株的成像能力。REF 成像利用催化健壮的酶拉丁, 保存在所有的m. 结核临床分离株和结核复杂菌株。由于拉丁的快速催化速率和宿主细胞对荧光产物的保留, 导致了 REF 成像的极大灵敏度。只要基底可用, 信号就会不断增加, 从而使感染细胞和组织内的信号几乎无限地积聚。与其他成像方法相比, 此提高了 REF 成像的灵敏度, 允许对m. 结核进行特定检测体外和在体内8,23,24, 29。
REF 成像可用于细菌检测, 没有基因修改, 使其直接应用到任何感染模型, 实验动物8,37或人体临床资料29,38.REF 可用于检测和图像范围广泛的病原体39,40, 因为荧光基板可以开发为许多酶的目标以外的拉丁, 如蛋白酶, 激酶, ureases 和β galactosidases。但是, 应仔细考虑目标, 以确保其显示最佳特征的成像。拉丁代表一个良好的模型酶的特点, 将确保成功地应用这一战略。REF 成像提供了一个即时读取的细菌负荷在肺部感染期间, 这大大加快了研究结核病发病机制, 因为细菌数量的测定通常需要三周到六星期, 但即使在更快速生长的生物体这种方法将节省大量的时间。参考文献也可用于鉴别癌症的结核病, 一个关键问题诊断的结节性病变的患者41,42。REF 作为一种新的工具, 以加速翻译结核成像, 甚至可以应用于人类, 可能允许快速预测的治疗结果。

m. 结核的缓慢增长率是结核病快速诊断的一个主要障碍1,2,3。虽然基于区域性的诊断需要几周的时间才能产生结果, 但酸性快速涂片在儿童5中有诊断限制4 , 患者与人体免疫机能丧失病毒6,7有共同感染。光学成像技术最近被认为是替代传统的结核病诊断方法8,9。荧光和生物发光可用于实时10,11,12,13,14的活体动物中的光学图像m. 结核, 15,16,17,18,19。光学成像有助于快速而具体地评估感染与m. 结核20,21,22。
我们概述了使用 REF 对活鼠中的m. 结核进行光学成像的细节。此方法非常特定且敏感23、24 , 与其他光学方法类似, 比其他结核病 (TB) 成像25的方法成本低, 包括计算机断层扫描 (CT)26, 磁性磁共振成像 (MRI)27和 F 氟脱氧葡萄糖正电子发射断层扫描/ct (f-葡萄糖 PET/ct)28。REF 利用自定义的荧光或生物发光基底, 经细菌酶解裂后, 产生荧光产物8,29。因此, 它的优点是不需要重组结核杆菌报告器应变30,31。所述的烦恼基板由由拉丁 (β酶) 水解的β-内酰胺环连接的 fluorochrome 和淬火组成, 自然组成性由结核复合分枝杆菌 8,32. 细菌直接产生的信号, 由于 REF 催化活动, 允许放大的数量级和敏感检测的m. 结核。
本研究所用的 REF 基底在活体动物中具有良好的组织穿透力, 并且由于波长长而减少了背景。有了这个长波长的基底, 就有可能在小鼠 (整个动物)8中, 为近100个菌落形成单位 (CFU)体外和 < 1000 CFU 的m. 结核检测阈值. 33。REF 可作为一个诊断工具的痰, 临床材料, 甚至直接在微内窥镜系统的患者16,32,33,34由于其高灵敏度和特异性。REF 可以应用于任何结核病临床菌株, 因为它使用天然产生的细菌酶, 拉丁, 用于检测目前在所有菌株。这些特点使 REF 成像在临床前结核病研究中具有重要的应用价值, 有助于治疗和疫苗的评估以及发病机制的分析, 但最终也可应用于结核病的诊断。患者.

<table><tbody><tr><td>Isoflurane</td><td>VETONE</td><td>501027</td><td></td></tr><tr><td>CNIR800</td><td>Custom synthesized</td><td></td><td></td></tr><tr><td>Fatal Plus solution</td><td>Vortech Pharmaceutical Ls, Ltd</td><td></td><td></td></tr><tr><td>7H9 Middlebrook broth</td><td>BD</td><td>271310</td><td></td></tr><tr><td>OADC Middlebrook enrichment</td><td>BD</td><td>212351</td><td></td></tr><tr><td>Sporcidin</td><td></td><td>RE-1284F</td><td></td></tr><tr><td>7H11 Middlebrook Agar</td><td>BD</td><td>212203</td><td></td></tr><tr><td>Madison Chamber</td><td></td><td></td><td></td></tr><tr><td>IVIS Spectrum</td><td>Perkin Elmer</td><td>124262</td><td></td></tr><tr><td>XGI-8-gas Anesthesia System</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Living Imaging software</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Transparent nose cones</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>&lt;em&gt;M. tuberculosis&lt;/em&gt; strain CDC1551</td><td>ATCC</td><td></td><td></td></tr><tr><td>Female BALB/C mice, 5-7 weeks</td><td>Jackson Laboratory</td><td></td><td></td></tr></tbody></table>

imaging mycobacterium tuberculosis in mice with reporter enzyme fluorescence

报告酶荧光 (REF) 利用的基底, 是特定的酶存在于目标有机体的成像或检测的荧光或生物发光。我们利用拉丁, 一种由所有结核组成性菌株表达的酶。REF 允许快速定量的细菌在肺部感染的小鼠。同一组小鼠可以在许多时间点成像, 大大降低成本, 更快地列举细菌, 允许在寄主-病原体相互作用方面的新的观察, 增加统计能力, 因为每组更多的动物易于维护.由于自定义荧光共振能量转移 (拉丁) 或荧光基板使用, REF 是非常敏感的由于催化性质的酶记者和具体的。REF 不需要重组菌株, 以确保正常宿主病原体相互作用。我们描述了m. 结核感染的影像, 使用的是最大排放量为 800 nm 的烦恼基底。基底的波长允许哺乳动物敏感的深部组织成像。我们将概述小鼠的气溶胶感染的m. 结核, 麻醉小鼠, 参考基质的管理和光学成像。这种方法已成功地应用于评估宿主-病原体相互作用和抗生素对靶向结核治疗的效果。

图 1A中显示了感染m. 结核和未感染控制鼠标的小鼠的 REF 成像。受感染的小鼠在感染后6周内产生了显著的 (P = 0.0057) 的荧光信号, 与控制基质的管理相对应。在1、2、4、6、15和24后的感染后, 一个典型的时间课程, 研究使用 REF 治疗m. 结核的疗效。荧光强度是通过对肺组织的免疫照射来量化的 (图 1B)。从2周到6周的感染小鼠肺部持续增加的信号表明, REF 能够成功地检测到体内的 m. 结核菌。在以后的时间点 (图 1B) 中, 控制鼠标的背景信号下降可能归因于在6周时间内身体质量和小鼠体积的增加, 从而减少了激发波长的穿透。3D. 在图 2A中, 对感染结核的小鼠的荧光源进行重构。使用相同的激发和串联的发射滤波器, 在鼠标的多个反光照点上获取图像序列。这些图像序列, 然后用于3D 掠过重建的荧光源分布在动物的主题。图 2-d说明了小鼠层析成像与肺器官注册的不同方向 (冠状、矢状和 transaxial)。用于检查重建质量的 NTF 效率图在图 2E中表示。NTF 允许通过使用中性密度过滤器捕获的额外图像, 从反照图像中减去背景光泄漏。使用特定励磁滤镜 (图 2E 测量) 的图像被规范化为用相同的发射过滤器和开放励磁过滤器 (图 2E 模拟) 测量的传输图像, 以给出由基板单独产生的信号。在水平 (图 2F) 和垂直 (图 2G) 配置文件中, 类似的测量和模拟的 NTF 效率配置文件具有近0% 的百分比错误 (图 2E-% 错误)提供了一个良好的质量3D 重建与减少工件和改进的信号定位和灵敏度的证据。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56801/56801fig1.jpg"> 图 1.使用 REF 诊断m. 结核(A) 在2、4和6周的感染后,在体内对未感染和感染m. 结核的小鼠进行成像。颜色条表示荧光信号的强度, 每秒每个 cm2从低 (黄色) 到高 (红色)。(B) 从感染了m. 结核的小鼠身上荧光强度的量化。两个控件 (黑色) 和受感染 (红色) 的荧光值都与每个时间点的标准错误一起表示。结果的重要性由学生t测试确定, p值 < 0.05 被认为是重要的。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56801/56801fig1large.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/56801/56801fig2.jpg"> 图 2. 3D. 在感染结核的小鼠中, 荧光源的重构重建.用不同方向表示的鼠标层析成像;A) 冠状, B) 矢状, C) transaxial 与 D) 肺器官注册。3D 感兴趣的区域 (ROI) 在肺中表示为源测量的红色立方体。(E) 测量和模拟的 NTF 效率图以检查重建质量。比较了实测和模拟 NTF 效率剖面, 提供了良好的3D 重建质量 (类似的测量和模拟 NTF 效率)。F) 水平和 G) 垂直信号剖面, 代表测量 (蓝色) 和模拟 (红色) NTF 效率曲线。水平和垂直红色条表示源位置。颜色条表示荧光信号的强度, 每秒每个 cm2从低 (蓝色) 到高 (红色)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56801/56801fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence at JoVE.com

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

我们描述了使用报告酶荧光 (REF) 感染结核分枝杆菌(m 结核) 的小鼠的光学成像。该协议有助于在临床前动物模型中对m. 结核进行敏感和特定的检测, 以进行发病机制、治疗和疫苗研究。

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

imaging-mycobacterium-tuberculosis-mice-with-reporter-enzyme

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

7.3K 次观看.  Texas A&M Health Science Center. 我们描述了使用报告酶荧光 (REF) 感染结核分枝杆菌(m 结核) 的小鼠的光学成像。该协议有助于在临床前动物模型中对m. 结核进行敏感和特定的检测, 以进行发病机制、治疗和疫苗研究。

视频: Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

遗传学

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

生物工程

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

功能金报告系统测量特定分枝杆菌误调速率

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Reporter Enzyme Fluorescence Imaging for In Vivo Tracking of Bacterial Infection

Source: Sharan, R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56801">Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence.</a> J. Vis. Exp. (2018).
This video demonstrates the reporter enzyme fluorescence (REF) imaging for in vivo detection of Mycobacterium tuberculosis infection. The infected mice are intraperitoneally injected with a substrate for REF imaging. Upon being hydrolyzed by bacterial &#946;-lactamase enzyme, the substrate emits fluorescence, the detection of which aids evaluation of infection.

用于细菌感染体内追踪的报告酶荧光成像

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Fluorescence Molecular Tomography: An Imaging Technique for In Vivo Imaging of Fluorescent Protein-tagged Glioblastoma Xenografts in Mouse Model

Source: Benitez, J. A. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/57448" target="_blank">Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts.</a> J. Vis. Exp. (2018)
In this video, we describe fluorescence molecular tomography or FMT for in vivo imaging of fluorescent protein-tagged glioblastoma xenografts in mouse models. The technique is useful for elucidating the effects of anti-tumor therapeutics at the molecular level.

荧光分子断层扫描:一种在小鼠模型中对荧光蛋白标记的胶质母细胞瘤异种移植物进行体内成像的成像技术

Source: Benitez, J. A. et al., Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts. J. Vis. Exp. (2018)
In this video, we ...

癌症研究

Cancers of the Nervous System

In vivo studies

A Technique to Detect Extrapulmonary Tuberculosis via Superparamagnetic Nanoprobes

Source: Lee, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58227">Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for the detection of extrapulmonary tuberculosis. Superparamagnetic iron oxide (SPIO) nanoprobes, conjugated to Mycobacterium-specific antibodies, are injected into a mouse previously infected with Mycobacterium bovis BCG, labeling the granuloma and providing a better imaging contrast for its detection.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

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