Name: transfecting raw264.7 cells with a luciferase reporter gene
Uploaded: 2015-06-18T14:00:00.000+00:00
Duration: 12 min 58 s
Description: Watch this Scientific Journal Video about Transfecting RAW264.7 Cells with a Luciferase Reporter Gene at JoVE.com

This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.

1.质粒DNA纯化<ol><li>使用根据制造商的协议的大量制备试剂盒提取质粒DNA。重悬的质粒DNA在500μlTE缓冲液中。 </li><li>执行苯酚:氯仿:异戊醇萃取和异丙醇沉淀以除去残留的细菌污染物。 LPS的存在干扰了转9。 <ol><li>加入500微升酚:氯仿:异戊醇（25:24:1，pH 8）中的质粒DNA，并剧烈振荡15秒。酚导致严重的皮肤灼伤和是麻醉剂使烧伤并不总是感觉，直到有严重的损害。通风柜异戊提取和使用警告:执行苯酚:氯仿。 </li></ol></li><li>为5分钟，在RT孵育混合物。离心以13,000 xg离心在室温10分钟。转移350微升上层水相的成一个新的1.5 ml收集管。 </li><li>添加350微升TE缓冲液到包含下层有机相中的管。剧烈摇晃15秒。离心以13,000×g离心5分钟，在RT。转移350微升上层水相，以同为1.5 ml收集管。 </li><li>加入70微升的3M醋酸钠（pH5.2）拌匀。加入700μl100％异丙醇。拌匀，孵育10分钟在室温。离心机以13,000×g离心10 minat 4℃。去除上清。 </li><li>加入500微升75％乙醇，以沉淀。涡旋样品，在4℃混合和离心机在5000×g离心5分钟。去除上清。的DNA是在沉淀中。 </li><li>打开管的盖和空气干燥沉淀在RT 5 - 10分钟。沉淀应成为半透明。加入500微升TE缓冲液以重悬DNA沉淀。热，在50 - 60℃下进行10分钟，以溶解DNA沉淀。 </li><li>测量260nm处的吸光度（A260）和280nm（A280）用分光计。通过用50纳克/微升乘以A260值计算DNA浓度。评估DNA的calculatin质量克A260 / A280比值。 DNA具有较高的质量应该给予1.8的A260 / A280比值 - 2.0。 </li></ol> 2.细胞培养和播<ol><li>培养RAW264.7细胞在DMEM补充有在37℃，5％CO 2培养箱9％胎牛血清（DMEM / 9％FCS）中。在每个通道，通过使用巴氏吸管连续移液分离从培养板上的细胞。通道中的细胞，每2天，和种子1.5 - 2百万个细胞在10cm组织培养物处理的培养皿作为库存。解冻的细胞每隔5的新股 - 6周。 </li><li>在转染当天，种子，每孔200,000个细胞在24孔板中的500微升DMEM / 9％FCS中的体积。孵育细胞在37℃培养箱中4小时。 </li><li>转染的细胞或者与基于脂质的试剂（步骤3.1）或蛋白质/聚胺系试剂（步骤3.2）。 </li></ol> 3.转染<ol><li>基于脂质的转染: <ol><li>热身转染reagen吨至RT在黑暗中。预热的无血清培养基和DMEM / 9％FCS中，以37℃。 </li><li>添加0.5质粒DNA微克至50μl无血清培养基的在1.5ml管中。加入2微升转染试剂与液体的表面之下的枪头。漩涡，持续6秒。 </li><li>离开在黑暗中试剂/ DNA混合物在RT下30分钟。 </li><li>在30分钟温育后，从孔中取出介质，并加入250微升新鲜的DMEM / 9％FCS中的每个孔中。 </li><li> 30分钟后，加入250微升DMEM / 9％FCS中的向试剂/ DNA混合物。通过上下吹打拌匀。 </li><li>添加300微升稀释的混合物的每个孔中。在37℃，5％CO 2培养箱4小时-孵育2。 </li><li>取出转染溶液，加入500微升DMEM / 9％FCS的。在37℃，5％CO 2的细胞刺激前孵化48小时-孵育24。 </li></ol></li><li>蛋白质/聚胺系的转染: <ol><li>热身无血清介质和DMEM / 9％FCS中，以37℃。 </li><li>添加0.75微升转染试剂以18.75微升无血清培养基中。短暂涡旋混合。在室温下孵育5分钟。加入0.5微升1微克/微升DNA导入稀释转染试剂。在室温下孵育10分钟。 </li><li>在10分钟温育后，从24孔板的每个孔中除去培养基并加入250微升新鲜的DMEM / 9％FCS的每个孔中。 </li><li> 10分钟后，加入250微升DMEM / 9％FCS中的入试剂/ DNA混合物。 </li><li>添加270微升稀释混合物到孔中。孵育在37℃，5％CO 2的培养箱中培养2 - 4小时。 </li><li>取出转染溶液，加入500微升DMEM / 9％FCS的。在37℃，5％CO 2培养箱48小时-孵育24。 </li></ol></li></ol> 4.细胞刺激和荧光素酶测定<ol><li>在很好地与250微升新鲜DMEM / 9％FCS的更换介质。 </li><li>加入50微升LPS或LPS + IL-10在所需浓度到孔中。返回板到37℃，5％CO 2培养箱。刺激2 - 6小时。 </li><li>通过抽吸除去刺激溶液，洗净用冰冷的PBS，并添加200μl的1×被动裂解缓冲液中。岩石在室温30分钟。刮和裂解物转移到1.5 ml微量离心管，并通过以20000×g离心在4℃下离心10分钟除去细胞碎片。 </li><li>转移40微升澄清的裂解液（上清液）的成白色，透明底96孔板用于根据制造商的方案的荧光素酶信号的测定。 </li><li>读取整个可见光光谱光度计荧光素酶信号。 </li></ol> 5.数据分析<ol><li>通过从各孔除以萤火虫荧光素酶的各个值由Renilla荧光素酶的值的Renilla比率:计算萤火虫。 </li><li>的处理海肾比深受:除以萤火虫计算倍数变化是的未经处理的很好。 </li></ol>

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作者什么都没有透露。

在这里描述的协议并不仅仅着眼于转染效率，但旨在在效率的细胞的生理状态和保护之间的平衡。具体地讲，我们的过程成功地减少转染试剂的毒性和最大化的荧光素酶信号。 在协议中的一个关键步骤是在细胞的健康。杂草丛生文化不适合的转染作为其生理变化，RAW264.7细胞的一个长的时间内还可以改变单电池10的表型和功能的连续培养。刚解冻的细胞，具有低传代数建议使用用于转染。 另一个重要的考虑因素是转染试剂的选择。基于脂质的转染试剂通常用于研究由于其易用性和可商购。然而，这些试剂引起unintendeD（通常不希望的）在转染细胞11,12改变全局基因表达。为了解决这个问题，将细胞仅孵育与转染试剂的几个小时，而不是典型的O / N或非基于脂质的试剂被选择用于转染。较长的温育时间与转染试剂将提高转染效率，但它也可以是有害的细胞或者引起细胞死亡或细胞活化，这两者都可以与实验设计干涉。 图4A和4B表明，该蛋白质/聚胺基于转染没导致细胞凋亡或坏死，相比未转染细胞。所述基于脂质的转染，即使在短的培养时间，造成细胞死亡的更高的水平。它是相关的所述基于脂质的转染试剂的转染效率较高。然而，当在显微镜下观察转染的细胞，没有显着的形态变化秒（ 图4C）。活化巨噬细胞通常采用一个庞大的形状，但是未转染和转染细胞都没有刺激之前显示激活的迹象，表明它们是在它们的静止状态。此外，转染的细胞的反应相似于LPS和IL-10刺激的未转染细胞（ 图4D）。这些观察共同表明此转染过程不改变细胞的自然行为。 转染和实验性治疗（其余时间）之间的时间也很重要。足够的时间需要为荧光素酶基因的表达和对细胞重新建立自己的休息状态给予;然而，很长的潜伏期可以减少荧光素酶的信号，尤其是当转染效率不高。 这里的程序适用于在我们的实验室中使用的特定荧光素酶报告基因。细微的调整将是东东DED当另一位记者被使用。需要被测试的参数包括不同萤火虫萤光素酶报道:Renilla荧光素酶比，转染和治疗，以及治疗条件之间的休息时间。将50:1的比例的pGL3-IκBζ的:phRL-TK通常使用的，但该比例的范围可以从1:1到100:1。结果发现，从细胞中除去转染溶液和实验治疗开始之间的24小时休息，得到最好的信号。 48小时后，荧光素酶的信号开始下降，并减少或甚至取消治疗组之间的差异。 所描述的转染过程并不限于萤光素酶报告测定。其他的实验设计不需要同源性的群体将受益;例如，荧光显微镜其依赖于从单个细胞的观察。一个缺点，要找到未转染的同行之间的成功转染的细胞，但的是少耗时耗力，可以更期望的好处。在稳定的细胞系是优选的情况下，我们的协议提供一个均值为最初的实验，其中，当正在产生的稳定细胞株实验程序可以优化。

核酸细胞转染在科研多样的应用程序。实例包括（1）的报告基因来研究不同基因元件在基因表达中的作用;（2）蛋白表达质粒过表达感兴趣的蛋白质，和（3）的小干扰RNA下调基因表达。通过操纵特定基因的表达水平和测量这种操作的微分效果，研究人员可以推导出在所选择的生物系统的基因功能。不是所有的转染方法提供相同的转染效率，甚至同一染方法不转染所有类型的细胞同等1。因此，不同的转染方法已被开发，如磷酸钙法，DEAE-葡聚糖，阳离子脂质转染，阳离子非脂质的聚合物的转染，电穿孔，核转染和2,3。 转染巨噬细胞是ESPEcially困难的，由于这一事实，即巨噬细胞是专业的吞噬细胞是给外国的材料，包括来自（甲基）的DNA 4菌非常敏感。外源DNA的引入激活Toll样受体9（TLR9）途径，导致细胞因子的产生和一氧化氮5,6。然后，这些活化巨噬细胞可能是不太适应的治疗，研究人员打算检查。 我们的实验室常规transfects与荧光素酶报告基因的RAW264.7巨噬细胞系，我们已经开发了一个协议，是足够强大的荧光素酶有比信号背景显著高，而且柔情似水的巨噬细胞，以保持其静止状态。转染的细胞的行为由萤火虫荧光素酶报告基因窝藏IκBζ（pGL3-IκBζ）的启动子区进行了评价。 IκBζ表达通过细菌细胞壁成分唇上调opolyssacharide（LPS）的7,8，和由抗炎细胞因子下调，白介素-10（IL-10）8。考虑到井之间转变体中，我们共同转染含有海肾荧光素酶基因（ 例如，phRL-TK）进行归一化目的的对照质粒。描述的协议测试各种参数，包括转染的定时，对转染试剂的类型，转染试剂和质粒DNA的量，以及转染试剂的比率，以质粒DNA后进行了优化。包括在这个协议在两个转染试剂是:（1）一个基于脂质的转染试剂和（2）一种蛋白质/聚胺系转染试剂。

<table><tbody><tr><td>PureLink HiPure Plasmid Maxiprep Kit</td><td>Life Technologies</td><td>K210007</td><td>Any maxiprep kit will work</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol</td><td>Life Technologies</td><td>15593-049</td><td>Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container.</td></tr><tr><td>DMEM</td><td>Thermo Scientific</td><td>SH30243.01</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific</td><td>SH30396.03</td><td>Inactivated at 56 &amp;deg;C water bath for 45 min before use.</td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>31985-070</td><td>Warm to at least room temperature before use.</td></tr><tr><td>XtremeGene HP DBA transfection reagent</td><td>Roche</td><td>6366236001</td><td>Warm to room temperature before use.</td></tr><tr><td>GeneJuice</td><td>EMD Millipore</td><td>70967</td><td>Warm to room temperature before use.</td></tr><tr><td>5x Passive Lysis Buffer</td><td>Promega</td><td>E1941</td><td>30 ml is included in the Dual Luciferase Reporter Assay System</td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr></tbody></table>

transfecting raw264.7 cells with a luciferase reporter gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

图1比较了在RAW264.7两个转染试剂的转染效率。所述基于脂质的试剂通常得到约25％的转染率， ​​而蛋白质/聚胺系的转染导致约5％的效率（ 图1A）。在转染效率的差异，也观察到在转染了的pGL3-IκBζ启动子报告（ 图1B）的RAW264.7细胞的荧光素酶信号。加入LPS到这些转染的细胞的增加的萤火虫萤光素酶信号，所述IκBζ启动子报告的增加转录活性的直接指示。换句话说，结果表明内毒素上调的IκBζ基因，与以前的报告7,8一致的表达。 图2示出了在我们的实验中获得的典型结果，使用基于脂质的转染，例如，从网络获取单个信号值后refly荧光素酶和海肾荧光素酶（ 图2A和2B），萤火虫荧光素酶的信号进行标准化的Renilla荧光素酶信号（ 图2C）。规范化是由于富裕井变化转染效率建议。以确定该处理条件（LPS或LPS + IL-10）改变的记者信号，萤火虫:海肾比（ 即，归一化的信号）的治疗组分别与未处理（未刺激）的样品（ 图2D的比较）。我们的数据表明，治疗RAW264.7细胞用LPS上调所述的pGL3-IκBζ启动子报告的活性，表明LPS增加IκBζ基因的转录水平。在IL-10的存在下，在IκBζ启动子报告有更低的活性，提示IL-10能够抑制IκBζ基因的LPS诱导的转录。蛋白质/聚胺系转染通常给两个萤火虫荧光素酶和海肾萤光素酶的信号低的值。 图3比较了转染和刺激（24小时或48小时）之间的休息时间的长度，并显示了萤光素酶信号随时间而减少。在信号的降低并没有与数据解释干扰时的基于脂质的转染试剂，使用（ 图3A）;诱导LPS和抑制IL-10 48小时的休息后仍在观察。然而，在信号的下降更为显著当蛋白质/聚胺系转染试剂，使用（ 图3B），特别是Renilla荧光素酶的信号。其结果是，经过48小时的休息，没有观察到治疗组之间的差异。 转染的一种不希望有的影响是细胞死亡等细胞凋亡的程度每次转染方法的影响进行了评估。细胞转染，或不是，并进行膜联蛋白V和碘化丙啶（PI）的染色。由这些细胞制备的裂解物用于完整和切割多聚ADP核糖聚合酶（PARP）的蛋白的存在下进行了分析。膜联蛋白V / PI染色的细胞的流式细胞术分析表明，基于脂质的转染略有增加膜联蛋白-V阳性细胞的比例相比，在未转染的细胞中，而蛋白质/聚胺系转染没有（ 图4A） 。同样地，基于脂质的略微转染增加切割的PARP的比例而蛋白质/聚胺系转染没有（ 图4B）。然而，尽管升高的号码凋亡细胞，该细胞的光镜的形态并没有改变（ 图4C）。更重要的是，基于脂质的转染细胞的生物应答保持的那些相同的未转染细胞（ 图4D）的。将细胞用LPS刺激±IL-10和TNF的量^5;分泌到培养上清通过ELISA定量。同时未转染和基于脂质的转染的细胞中响应于LPS作出类似量的TNFα，并通过加入IL10的都被抑制。没有TNFα作出时不被刺激的细胞（ 图4D），表明该细胞仍然染后天真。 <img alt="图1" src="/files/ftp_upload/52807/52807fig1highres.jpg" width="700" /> 图1.基于脂质的转染，得到比蛋白质/聚胺系的转染转染效率较高 。 （A） 的 RAW264.7细胞转染GFP的表达质粒与任一所述基于脂质或蛋白质/聚胺系转染试剂。 GFP信号由流动流式细胞仪后24小时测量。 （ 二）巨噬细胞转染了pGL3-IkBζ启动子报告。 48小时后休息，CELLS分别用LPS刺激6小时。萤火虫荧光素酶的信号是根据制造商的说明进行测量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52807/52807fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52807/52807fig2highres.jpg" width="467" /> 图2.典型的荧光素酶检测数据。巨噬细胞转染TK-海肾 ​​和IkBζ启动子报告。 24小时后剩下的，将细胞用LPS刺激±IL-10的2小时。 （A） 的萤火虫萤光素酶和（B）的Renilla荧光素酶的信号，根据萤光素酶测定法生产商的说明书进行测定。 （C）记者活动是标准化为TK-海肾 ​​信号，并绘制成萤火虫/海肾比例。 （D）的倍数变化是通过将所计算的萤火虫:刺激的样品由未刺激样本的比例海肾<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52807/52807fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52807/52807fig3highres.jpg" width="700" /> 图3.荧光素酶信号的强度是随时间变化的。（A）的基于脂质的转染和（B）的蛋白质/聚胺系转染的细胞静置或者24小时或之前48小时，以LPS刺激±IL-10的2个小时。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52807/52807fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52807/52807fig4highres.jpg" width="700" /> <str翁&gt;图转染对巨噬细胞的影响4.（A）巨噬细胞转染与IkBζ启动子报告。 24小时后休息，细胞死亡评估流式细胞仪采用Annexin-V和碘化丙啶（PI）染色的流量。 （B）的经转染的细胞进行免疫印迹分析的PARP（全长和切割）和GAPDH（上样对照）。带强度然后用成像软件定量。 L =基于脂质的转染，P =蛋白质/聚胺系转染，STP = 0.1μM星形孢菌素。细胞（C）的显微镜图像转染了基于脂质的转染试剂。 （D）转染了基于脂质的转染试剂将细胞用LPS刺激±IL-10的6小时，并在促炎细胞因子TNFα水平用ELISA测定。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52807/52807fig4highres.jpg" target="_blank">请他</a>再查看该图的放大版本。

Watch this Scientific Journal Video about Transfecting RAW264.7 Cells with a Luciferase Reporter Gene at JoVE.com

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

转染RAW264.7细胞用荧光素酶报告基因

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

transfecting-raw2647-cells-with-a-luciferase-reporter-gene

Nanyang Technological University

Research

JoVE Journal

Biology

20.6K 次观看.  Vancouver Costal Health Research Institute. Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells. 

视频: Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

科研

医学

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

癌症研究

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

生物学

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.