Name: an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles
Uploaded: 2015-03-18T13:00:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

我们衷心感谢辛迪N.赵医生的帮助，凝胶电泳，安迪周和迈克尔Grodick的DNA和限制性内切酶的慷慨捐赠。我们非常感谢J.希思教授和D.测机教授慷慨的访问设备和材料。我们感谢卡恩Sorasaenee博士有益的建议。我们感谢玛丽H.唐创建在视频的示意图中使用的插图。资金由强生公司和USC Y86786提供。

注:当与RNA的工作保持一个清洁的工作环境，避免污染DNA酶和RNA酶的酶降解DNA和RNA。确保枪头和管是DNA酶和RNA酶。这也有利于向下擦拭表面的实验室和设备，如移液管，管人等具有净化解决方案。 1. RNA转录与Corrole治疗<ol><li>制备corrole和抑制剂化合物在0.01:[DNA]:的[复杂] 1的摩尔比。 注:在本例中，该比例为4.3 fmol的复杂:0.43皮摩尔DNA，其中所用的DNA模板是从E.西甲基因APET-28C矢量下在T7启动子控制的大肠杆菌 。其它DNA与T7启动子也是有效候选运行转录测定。 <ol><li>溶解放线菌素D，雷公藤，TPFC，和Ga（TPFC）的二甲亚砜（DMSO）中清洁，独立的容器中。获得的一个0.01的最终浓度:[C 1摩尔比omplex]:[DNA]通过使用连续稀释用无核酸酶的水。 <ol><li>溶解1毫克放线菌素D 1.8毫升DMSO。等份加入10μl到单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1/100稀释。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。 注:放线菌素D的最终溶液的浓度为4.3飞摩尔。 </li><li>溶解1毫克雷公藤中0.64毫升DMSO。等份1微升至一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以产生第二1 / 1,000稀释。 注:雷公藤甲素的最终溶液的浓度为4.3飞摩尔。 </li><li>溶解1毫克TPFC 2.9毫升DMSO。等份加入10μl到单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1/100ðilution。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。 注:TPFC的最终溶液的浓度为4.3飞摩尔。 </li><li>溶解1毫克嘎（TPFC）2.6毫升DMSO。等份加入10μl到单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1/100稀释。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。 注:嘎（TPFC）的最终溶液的浓度为4.3飞摩尔。 注意:这些corroles和抑制剂是不溶于水的，必须预先溶解在DMSO中。少量的DMSO（1％以下）的不诱导细胞毒性的细胞（未发表数据）。 </li></ol></li></ol></li><li>开始前的转录反应，单独地准备必要的试剂或从商业供应商购买。 <ol><li>融冰冻结所有Ñ ​​试剂（参照表1冷冻试剂的列表）。 </li><li>允许的10X反应缓冲液和4核糖核苷酸（ATP，CTP，GTP，UTP和）解决方案的时间来混合直到它们完全溶解在溶液中。 </li><li>短暂离心所有试剂，以防止材料的损失在管的边缘。一旦解冻，存储核糖核苷酸在冰上，同时保持10X反应缓冲液在室温下。 </li><li>结合试剂在室温转录反应（参照表2试剂的列表）。 注:在10X反应缓冲的亚精胺可共沉淀模板DNA如果反应组装冰而不是RT。 </li><li>通过轻弹管或移液混合向上和向下轻轻调匀。混合后，短暂离心以在管的底部收集反应混合物中。 </li><li>孵育在37℃下将反应混合物总共2-4小时的。 注:必需的反应时间会有所不同与DNA模板大小和序列。这一步可能需要优化的特定序列。较短的模板，可能需​​要较长的反应时间为高产率的总RNA。 </li><li>从每一个小时，并储存在-20℃下经过每个反应除去器4微升等分试样。根据需要所需的时间点修改。 </li></ol></li></ol> 2. RNA离心柱<ol><li>以下的转录反应，净化用微量兼容层析柱的RNA。 注意:使用的RNA离心柱的纯化的RNA用大于20个碱基的结果在大于80％的回收率44 注:柱色谱法，是纯化的RNA一个通用和方便的方法。其它纯化方法也存在如氯化锂沉淀，苯酚:氯仿萃取，异丙醇沉淀，以及其它可商购的试剂盒。 <ol><li>均匀大力反相悬浮交联葡聚糖基质中的柱缓冲液3列或更多次。通过大幅弹柱做到这一点。 </li><li>除去从柱的顶部上限。会有一些残存的Sephadex矩阵在帽;如果帽填充有基质，更换盖到柱上并重复前面的步骤，直到大部分基质是在列。 </li><li>折断所述塔的底部前端。 注:一些液体可能逃离列。 </li><li>打包的列中。 <ol><li>将列在无菌（RNA酶和DNA酶免费）1.5 ml离心管。 </li><li>离心反应管在台式离心机在1000×g离心1分钟。 </li></ol></li><li>丢弃1.5 ml离心管以从柱中洗脱缓冲液中。 </li><li>立即放置在一个新的无菌的1.5 ml离心管的柱和样品吸取到柱床的中心。用20-50微升的样品，以避免过载的列。 </li><li>离心管的台式离心机在1000 XG1分钟。 注:洗脱液纯化RNA样本。 </li></ol></li></ol> 3.琼脂糖凝胶电泳（1％琼脂糖凝胶）42 注:溴化乙锭荧光在结合DNA和RNA，因此这些生物分子可以通过用0.5微克/毫升溴化乙锭溶液孵育他们用UV光。 <ol><li>通过将5毫克溴化乙锭在10ml水中制备的溴化乙锭（0.5毫克/毫升）的1000倍原液。 </li><li>制备的Tris乙酸EDTA（TAE）运行缓冲液用于凝胶电泳。为了使50×TAE缓冲液中结合的2.0M Tris-乙酸用0.05M乙二胺四乙酸（EDTA）和pH调节至8.5。 </li><li>将10g超纯琼脂糖​​在1L 1X TAE缓冲液和熔化与常规微波炉的琼脂糖制备1％（重量/体积）的琼脂糖凝胶。一旦冷却到50℃，加入1毫升1000倍的溴化乙锭的1％琼脂糖凝胶SOLU灰，通过轻轻摇动或者通过反转在密闭容器中混合，再倒入琼脂糖溶液成凝胶铸造平台，并允许该凝胶凝固在RT。 注:溴化乙锭是一种诱变剂和潜在的致癌物质。戴上手套，小心处理解决方案。对于溴化乙锭的正确的处理程序，请参见: <a href="http://www.sciencelab.com/msds.php?msdsId=9927667">http://www.sciencelab.com/msds.php?msdsId=9927667</a> 。 </li><li>后的凝胶凝固，放置在电泳槽的集凝胶。添加足够的TAE缓冲液，以覆盖凝胶直到井被淹没。检查TAE缓冲液的水平大约为1毫米以上的凝胶的水平。取出梳子。 注意:删除梳井被淹没后，可防止气泡被截留在井。 </li><li>制备RNA样品。结合1微升每种纯化的样品等分试样与1微升凝胶加载缓冲液（或具有1微升10×加样缓冲液和dilut移液器向上和向下微量编到10微升无核酸酶的水）调匀。 注:10×加样缓冲液中含有20％的Ficoll 400，0.1M的Na 2 EDTA，pH值8.0，1.0％SDS，0.25％溴酚蓝。 0.25％二甲苯蓝，也可以加入到加样缓冲液，因为它运行〜50％速度是溴酚蓝，并且可以用于监测非常长的运行是有用的。42 </li><li>通过仔细吹打溶液到每个孔的底部装入每个样品的凝胶。小心不要留下任何气泡或混合井之间的样本。 注:核糖核酸梯也可以用于确定转录物的大小。 </li><li>附加的引线，使得DNA会迁移到朝向正引线的凝胶。设置电压到需要的水平，通常为1至10伏/凝胶厘米。运行凝胶然而长是足够的RNA片段之间显著分离，使用染料的迁移来监测分离的进度。 ñOTE:250 V A23厘米×25厘米的凝胶大约需要1小时，而一个8厘米×8厘米凝胶在150 V大约需要20分钟。小RNA片段具有更好的分辨率，更高的电压运行时。 </li><li>关掉电源时从装载缓冲染料已迁移判断足以使RNA片段之间的分离距离。 </li><li>图像在UV光下的溴化乙锭的RNA会发出荧光。 </li><li>取的图像的图像，并比较来自每个条件的纯化的RNA的荧光强度。 </li></ol>通过紫外 - 可见光谱4. RNA定量<ol><li>放在一个的NanoDrop 2000或类似的机器2微升H 2 O，并测量空白。 </li><li>下一步，放置2微升每种RNA样品的，下面的净化，在分光光度计上并测量紫外 - 可见从200纳米至800纳米的波长范围。 注:一个A 260读数为1.0，相当于核糖核酸约40微克/毫升，和纯RNA具有A 260 / A 280比值为2.1。45 </li><li>在的情况下的吸光度大于1的OD，稀释于无核酸酶水样品，得到的OD小于1。 </li><li>用绘图软件绘制各种样品，并在260 nm的OD比较。 </li></ol>

The authors have nothing to disclose.

该测定表明，添加Ga（TPFC）的抑制RNA的转录可比已知的DNA结合抗癌络合物放线菌素D和雷公藤。嘎（TPFC）（GI 50 = 58.1-154.7μM）的细胞毒性行为可能由于其抑制性能。因为没有转录抑制中观察到TPFC，TPFC的细胞毒性是不是由于RNA的转录抑制，但是由于尚未研究其他手段。 在执行了4转录反应，DNA用放线菌素D，雷公藤，TPFC或Ga（TPFC）处理，在[复]:[模板DNA碱基]为0.01，分别?...

化疗通常涉及广谱细胞毒性剂与不期望的副作用和有限的定位，但与癌症生物学的更多的理解，存在用于抗癌剂不断增加的需求，以更高的癌症靶向功效和副作用较少。1人类癌细胞经常成为依赖单一活化或过表达癌基因的生存。2因此，许多抗癌药物是通过它们抑制RNA的转录的能力进行评估。治疗，阻止这些转化基因的表达是有效的消除癌细胞，并导致细胞死亡。3转化的细胞是在RNA的转录干扰更敏感比是相应的正常细胞。4抗癌药物抑制转录预期选择性地抑制这是必要的癌症细胞生存的癌基因的5因此表达，RNA的转录我nhibition是要找出潜在的抗癌候选药物，并进一步了解其作用机制的有效途径。此协议表明镓（TPFC）抑制RNA的转录顺序相同的化疗药物放线菌素D和雷公藤上;类似的比较，可以使用此协议与其他抗癌候选药物进行。放线菌素D是一种RNA的转录抑制剂通常用于治疗妊娠滋养细胞癌，睾丸癌，肾母细胞瘤，rhabdomyosacoma和尤因氏肉瘤6。放线菌素D被用于癌症治疗近五十年，因为它是第一个被FDA批准在1964.Triptolide是已经在体外和各种肿瘤携带动物模型30年已经研究选择性转录抑制剂。7 corroles的两亲性质的大环赋予比其他类药物显著优点，如小分子或生物第8-14的大环字符允许蜂窝渗透性大于预期对于如此大的分子，它们是大到足以与大分子的表面，如那些蛋白质的相互作用。8 Corroles已知以形成紧密的非共价配合物的生物分子和药物。10除了corrole框架的固有细胞毒性，我们已经表明，磺化corrole充当化学治疗剂与载体分子，特别是DNA的嵌入蒽环类药物阿霉素。当磺化corrole物共施用阿霉素，3倍的增强中阿霉素的IC 50观察到，DU-145细胞。9 corrole框架是稳定的，并具有固有的吸光度和荧光性质是，官能化时，经过独特的吸光度的变化可用于表征。10官能支架的不固有地affec吨的corrole，9-15的光物理性质，但，因为看到带有磺化corrole，选择性地修改所述corrole框架可以基本上改变其生物学特性。16，我们先前评估6 metallocorroles与七人癌细胞系。结果表明，对人体癌细胞的毒性是依赖于特定的金属离子，以及官能团取代。例如，磺化镓corroles经历高的细胞摄取和渗透选择性进入脑转移性前列腺癌细胞（DU-145）的核;同样corrole，虽然它不渗入的其他细胞系的细胞核中，显示出更大的细胞毒性乳腺癌（MDA-MB-231），黑色素瘤（SK-MEL-28），和卵巢（OVCAR-3）的癌细胞比前列腺癌。9 初始基于细胞的试验表明，这些化合物显示出希望作为抗癌治疗剂，其优点菲尔特呃调查行动的机制。转录抑制观察到某些有机金属配合物17-27，我们试图研究这个过程中作为一个可能的机制corrole家庭的细胞毒行为。此转录测定法提供了用于评估转录的抑制，这将导致对这些分子在活细胞中的效应的更详细的信息的简单的，廉价且简便的方法。 这里，镓的转录抑制（Ⅲ）5,10,15-（三）pentafluorophenylcorrole（镓（TPFC））和它的游离碱类似物5,10,15-（三）pentafluorophenylcorrole（TPFC）（ 图1）进行测试。不像一些过渡金属配合物，镓（III）为氧化还原非活性，因此不直接参与的氧化还原类的代谢途径的氧化还原过程。28无论如何，镓（Ⅲ）并不表现出细胞毒特性，并已被研究用于治疗目的。镓是第二个最有前途的金​​属铂之后抗癌治疗，并经历了多次的研究和调查;硝酸盐和氯化物镓盐在防治肝癌，淋巴瘤，膀胱癌等多种疾病的临床试验进行了评估。29-34镓（III），因此理想的抗癌corrole研究。初始数据表明镓（TPFC）和TPFC具有低GI 50，所需的药物浓度，以抑制的最大细胞增殖的50％，与各种癌细胞株（参见图2）;这肯定了关于这两种化合物的进一步的实验的有效性，以确定它们抑制性质。我们比较这些化合物与普通抗癌药物放线菌素D和雷公藤甲素。放线菌素D插层的DNA，抑制RNA的伸长率，并诱导细胞凋亡的某些细胞系在皮摩尔浓度6,35-37雷公藤已经显示出抑制肿瘤生长。它与人XPB / ERCC3，亚基Ø˚F转录因子TFIIH，从而抑制RNA聚合酶II的活性。6-7,38-40 虽然众所周知，corroles表现出细胞毒性性质，存在关于从官能化所产生的不同的机制的信息很少。 RNA转录抑制Corrole将提供他们与生物大分子的相互作用更大的洞察力。已知结合至DNA的其它配合物，如二铑（II，II）配合物，铬（III）络合物，钌（II）多吡啶配合物，铑（Ⅲ）配合物，以及各种其他经受RNA的转录测定法，18- 27致使其与生物大分子相互作用的更深入的了解。这个浅显的和广泛使用的实验也是一个不错的初步测试，以评估特定分子的细胞毒性特性并确定它是否值得进一步的生物测试。所述RNA转录测定法也可进行多种修饰，如varyin克使用的化合物或酶的数量;变的潜伏期，反应时间和采样时间点;并改变所述DNA模板长度和序列，其它感兴趣的变量之间的，从而有可能提供大量的数据。这转录实验也一应俱全，与提供一切必要的反应组分实惠的套装，虽然组件可以购买并单独准备。在这些实验中，我们使用已知具有高产市售的试剂盒。41 为了评估转录的抑制，我们使用两种方法:琼脂糖凝胶电泳和UV-Vis光谱。琼脂糖凝胶电泳是分离，识别和纯化0.5至25-kb的DNA和RNA片段的简单而有效的方法。42 UV-Vis光谱可用于确定浓度和RNA的纯度。43

<table><tbody><tr><td>Actinomycin D</td><td>Sigma-Aldrich</td><td>A1410</td><td>Store at 2-8 &amp;deg;C , protect from light</td></tr><tr><td>Triptolide</td><td>Sigma-Aldrich</td><td>T3652</td><td>Store at 2-8 &amp;deg;C , protect from light</td></tr><tr><td>nuclease-free H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Life Technologies</td><td>AM9938</td><td></td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Life Technologies</td><td>AM1334</td><td>Store at &amp;ndash;20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich</td><td>E7637</td><td>CAUTION: For proper handling procedures of ethidium bromide, please see: http://www.sciencelab.com/msds.php?msdsId=9927667</td></tr><tr><td>Tris Acetate</td><td>Sigma-Aldrich</td><td>T6025</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Life Technologies</td><td>16500-100</td><td></td></tr><tr><td>mini Quick Spin RNA Columns</td><td>Roche Life Science</td><td>11814427001</td><td>Store at 2-8 &amp;deg;C , do not freeze</td></tr><tr><td>1 kb DNA Ladder</td><td>New England Biolabs</td><td>N3232S</td><td>Store at &amp;ndash;20 &amp;deg;C&amp;nbsp;</td></tr></tbody></table>

an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles

化疗通常涉及广谱细胞毒性剂与许多副作用和有限的定位。 Corroles是一类表现出微分细胞抑制和细胞毒特性在特定细胞系中，根据不同的螯合金属和官能团的身份四吡咯大环化合物的。对特定的细胞功能化corroles独特的行为引入靶向化疗的可能性。 许多抗癌药物是通过它们抑制RNA的转录的能力进行评估。这里，我们提出一个一步一步协议用于RNA转录的已知和潜在抑制剂的存在。的转录反应的RNA产物通过凝胶电泳和UV-Vis光谱评价提供潜在的抗癌候选药物的抑制特性的信息，并与修改的测定中，更多关于它们的作用机制。 小已知大约corrole细胞毒性作用的分子机制。在这个实验中，我们考虑两个corrole化合物:镓（Ⅲ）5,10,15-（三）pentafluorophenylcorrole（镓（TPFC））和游离碱类似物5,10,15-（三）pentafluorophenylcorrole（TPFC）。一种RNA转录测定法用于检查corroles的抑制性质。五个转录反应制备:DNA用放线菌素D，雷公藤，镓（TPFC），TPFC在[复]处理:[模板DNA碱基]比为0.01，分别与未处理的对照组。 转录反应4小时后，用琼脂糖凝胶电泳和UV-Vis光谱进行分析。有明显的抑制作用嘎（TPFC），放线菌素D，和雷公藤。 该RNA转录测定法可以被修改通过改变抗癌复杂，DNA或聚合酶的浓度，以提供更详细的机理，或通过培养该DNA聚合酶或与完井XES前RNA转录;这些修改将涉及DNA或酶的抑制机制之间的区别。加入复合后的RNA转录可以被用来测试该络合物是否降解或水解的RNA。该测定也可以用于研究额外的抗癌候选。

RNA转录定性琼脂糖凝胶电泳评定 琼脂糖凝胶电泳用于图像转录的RNA。结合后溴化乙锭荧光（λEM = 605纳米，λ= 前 210纳米，285纳米）的RNA 46允许成像。在凝胶较暗条带对应于更高浓度的RNA。如果放线菌素D，雷公藤，或任corrole复杂抑制RNA的转录，产生的RNA的降低，并且带将出现更轻。使用此概念，相对抑制进行评估。 ...

Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

镓（Ⅲ）5,10,15-（三）pentafluorophenylcorrole和其游离碱类似物显示出低的微摩尔细胞的细胞毒性。这个手稿描述的RNA转录反应中，成像的RNA用溴化乙锭染色的凝胶，并用UV-Vis光谱进行定量的RNA，为了通过corroles评估转录抑制和演示评价抗癌候选属性的一个简单的方法。

一个<em&gt;在体外</em&gt;酶测定来测量转录抑制作用镓（Ⅲ）和H<sub&gt; 3</sub&gt; -5,10,15-三（五氟苯基）corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

an-vitro-enzymatic-assay-to-measure-transcription-inhibition-gallium

Research

JoVE Journal

Bioengineering

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

11.5K 次观看.  California Institute of Technology. 镓（Ⅲ）5,10,15-（三）pentafluorophenylcorrole和其游离碱类似物显示出低的微摩尔细胞的细胞毒性。这个手稿描述的RNA转录反应中，成像的RNA用溴化乙锭染色的凝胶，并用UV-Vis光谱进行定量的RNA，为了通过corroles评估转录抑制和演示评价抗癌候选属性的一个简单的方法。 

视频: An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

发育生物学

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

生物化学

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

体外酶法检测药物陪护与庞贝氏症的反应

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

医学

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

生物工程

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

一个

摘要

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此视频中的章节

体外转录测定及其在药物研究中的应用

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

体外酶法检测药物陪护与庞贝氏症的反应

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

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摘要

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体外转录测定及其在药物研究中的应用

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

体外酶法检测药物陪护与庞贝氏症的反应

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择