Name: quantitative measurement of γ-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms
Uploaded: 2018-01-25T12:30:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

作者感谢中央研究院细胞与有机体生物学研究所的核心设施, 以提供技术支持。这项研究得到了台湾科学技术部的支持 (最多 103-2320-001-016-MY3 至 Y。L.), 生物制药发展的转化创新方案–技术支持平台轴方案 (NP7 至 y.---), 中央研究院 (对 y---)。

1. 荧光报告器信号的测量, 对应于 APP-C99 或 NΔE 的γ-分泌劈裂
注: 有关 CG 和 NG 单元格生成的详细说明, 请参阅以前的出版物20,21。
<ol><li>
种子 NG 或 CG 细胞 (20, 000 细胞/井) 到96井板在最后的体积200μ l/井在生长培养基, 由 Dulbecco 氏改良的鹰培养基 (DMEM) 加上10% 胎牛血清 (FBS), 5 微克/毫升 blasticidin, 250 微克/毫升抗生素 (例如, zeocin) 和200微克/毫升霉 B。
	<ol>
<li>用例计数单元格。</li>
		<li>将板转移到湿润 CO2孵化器, 并在一夜之间在37° c 孵育。</li>
	</ol>
</li>
	<li>从每个井中取出100μ l 的生长培养基。</li>
	<li>添加100μ l/好的生长培养基含有2微克/毫升四环素与0.2% 二甲基亚砜 (亚砜), 2 μ m CL-387,785, 2 μ m n-[n-(35-difluorophenacetyl-丙)]-s-氨 t butylester (DAPT), 或其他相关的 ErbB1/ErbB2 抑制剂。</li>
	<li>在板37° c 的24小时内孵育经治疗的 CG 或 NG 细胞。</li>
	<li>从板中取出150μ l/井生长培养基。</li>
	<li>添加50μ l/井荧光测定试剂 (请参阅材料表)。</li>
	<li>
将板转换为发光微读取器。
	<ol>
<li>在室温下保持板5分钟的温和搅拌。</li>
	</ol>
</li>
	<li>
使用存储在发光微读卡器中的预定义程序确定萤火虫荧光 (FL) 发出的发光。
	<ol>
<li>打开仪器控制软件 (请参阅仪器详细信息的材料表)。</li>
		<li>从 "工具" 菜单中选择 "Luminometry"。<ol>
<li>在 "参数" 设置下, 选择 "无过滤器" 进行发射过滤, 对发射孔径进行 "正常" 刻度, 输入 "1" 计算时间, 点击 "OK" 设置发光读数。</li>
		</ol>
</li>
		<li>在 "工具控件" 选项卡中, 滚动活动协议列表, 并选择预存协议以读取发光。</li>
		<li>在 "实时显示" 选项卡下, 从下拉列表中定义刻度, 并为类型选择 "对数" 或 "线性"。</li>
		<li>在 "温度" 选项卡下, 选择 "关闭" 为板加热。</li>
		<li>单击 "开始" 按钮执行发光读数。</li>
		<li>根据需要, 使用内嵌在控制软件中的 "资源管理器" 将结果导出为电子表格格式。</li>
	</ol>
</li>
</ol>2. γ-分泌活性的定量
<ol><li>
定义一个由 CG 或 NG 细胞所发射的发光信号, 在含有1微克/毫升四环素的生长培养基中处理0.1% 甲基亚砜, 作为100% 相对γ-分泌活性。
	<ol>
<li>通过对不含四环素的生长培养基进行细胞处理, 来估计 CG 或 NG 细胞的背景发光。</li>
	</ol>
</li>
	<li>通过含有1微克/毫升四环素的生长培养基和1μ m CL-387,785 或1μ m 的 DAPT 与由亚砜治疗的 cg 或 ng 细胞 (100% 相对γ-分泌活动) 发出的荧光信号, 使荧光信号从 cg 或 ng 细胞正常化。定义于 2.1)。</li>
</ol>

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作者没有什么可透露的。

在 ad 的 aducanumab 免疫治疗的1b 期试验中, 有希望的结果已经牢固确立了 a 在 ad22发病机制中的关键作用, 并认为反 a 方法仍然是开发抗 ad 药物的可行策略。在这里, 我们描述了以细胞为基础的定量分析γ-分泌催化水解 APP-C99 和 NΔE 使用萤火虫荧光报告系统。APP-C99 和 NΔE 是两个最有研究的γ-分泌基底, 同时代表γ-分泌13的致病和生理活动。我们的发现, 下调 ErbB2 的 CL-387,785 可以优先降低 APP-C99 和分泌 a 的稳态水平, 进一步证实了基片特定的细胞检测γ-分泌在本研究中描述。因此, 当前的协议为发现新型γ-分泌调制器提供了一种有效的方法。
这种γ-分泌基片特定的检测范式的优点是多方面的。例如, 这些特定于基底的γ-分泌分析可以在一个初步的屏幕上使用, 以排除潜在的非选择性γ-分泌抑制剂的进一步发展, 并提供与最小化相关的定量评估潜在的副作用。从这个方法中发现的活性化合物的生物学功效可以在神经元细胞和 AD 动物模型中得到进一步的验证, 如最近的21所示。此外, CG 和 NG 稳定细胞系的均匀性对实验结果的重现性至关重要。重要的是, 四环素诱导的 C99-GV 或 NΔE 的表达, 有效地防止荧光记者表达的饱和, 这是在 C99 或 NΔE 组成表达的其他系统中看到的。这一四环素诱导表达的结果是一个高度可靠和一致的荧光信号, 直接与γ-分泌活动相关, 允许比较的γ-分泌分裂活动之间的两个不同的基质 (APP-C99与 NΔE), 或从实验到实验的高效率的方式。为了获得最佳效果, 应定期监测四环素溶液的活性 (每月最好一次), 以确保最佳的诱导效率。作为一个额外的优势, 这些平台的同质性消除了对萤火虫荧光与本构的Renilla荧光表达式进行规范化的需要。最后, 治疗 CG 和 NG 细胞与1µM avagacestat, 一个已知的缺口保留γ分泌抑制剂, 可以作为一个积极的控制的化验。
使用目前的试验平台的一个可能的告诫是, 在我们的分析中, 分泌基底的过度表达和随后的转录报告的饱和度有可能掩盖抑制活性, 并导致差异抑制效力在不同的化验中23。这种潜在的并发症突出表明, 需要二次化验平台, 以验证的效果, 保留缺口γ-分泌抑制剂, 如最近所示21。这种方法的另一个潜在的限制是, 如果有超过90种不同的膜蛋白被确定为γ-分泌基板, 目前的检测平台不提供γ-分泌基片选择性的完整轮廓。沿着这些线, 保留缺口分裂的抑制可能不是最好的读数与评价γ-分泌抑制剂。应注意的是, avagacestat, 一个缺口保留γ-分泌抑制剂, 未能提高认知24, 而且很可能, 切口抑制可能不是与泛γ-分泌抑制剂的毒性相关的唯一基础,25. 这一新方法确定的命中的真正效用, 可能取决于新发现的缺口保留γ-分泌抑制剂是否具有 avagacestat 中不存在的新的化学实体, 如果这些抑制剂的功效可以使用 AD 的在体内模型进行验证。
最后, 我们认为, 所提出的方法可以大大加快发展 APP-C99-specific γ-分泌抑制剂/调制器的临床应用。无可否认, γ-分泌抑制剂, 无论它们是否有缺口, 可能会引发 AD 患者的毒性, 这将导致不良的耐受和结果如先前建议的26。然而, 随着新的化学工具的引进和改进的验证方案21, γ-分泌应继续是一个诱人的药物靶点, 由于其生物化学的复杂性, 需要更多的前临床研究, 以探讨其在 AD 中的治疗相关性。

聚的淀粉样β (a) 的形式被认为是导致阿尔茨海默氏病 (AD)1患者大脑中神经的主要原因。a 肽是由淀粉样前体蛋白 (APP) 的逐步裂解产生的, 首先是β-分泌, 然后是γ-分泌2。在过去的十年中, 治疗 AD 的治疗方法侧重于预防 a 的生产3。大多数研究都集中在α-分泌活性的增强上, 这可以排除应用的γ-分泌分裂, 从而减少 a 的产生, 或抑制β和/或γ-分泌活动4。不幸的是, 非选择性抑制β或γ-secretases 导致不可避免的副作用, 这是由于干扰功能的其他生理基质的β和γ-分泌5,6。关于γ-分泌抑制剂, 最近的研究报告发现了一些化学和遗传调节剂, 可以调节 a 的生产, 而对必要的γ-分泌介导的加工缺口的影响微不足道的7 ,8,9,10,11,12, 但是, 成功的治疗方法尚未开发。因此, 有必要进一步系统的屏幕, 以发现新的遗传和化学修饰, 可能选择性地调节γ-分泌介导的应用程序处理。
γ-分泌是已知的切割超过90种不同的膜锚定蛋白。在这些基体之中是切口, 它的活动是关键的细胞命运决心和分化在发展期间13。在没有影响缺口处理的情况下, 选择性地调节γ-分泌-催化的应用处理, 对于最小化γ-分泌抑制剂的缺口相关副作用至关重要, 而这种选择性被认为是一个主要因素, 将规定了潜在的γ-分泌调节剂对 AD 慢性治疗的生物学功效。有许多 arylsulfonamide 衍生物, 例如 GSI-953 (begacestat) 和 BMS-708163 (avagacestat), 被发现表现出强有力的和有选择性的抑制γ-分泌14,15。先前的一项研究表明, 使用定量 ELISA 法结合使用斑马鱼16的活体验证, 可以观察应用和切口的γ-分泌裂解的差异抑制。此外, 建立了基于细胞的检测范式, 以解决γ-分泌对 APP 和凹槽1718的潜在基底选择性。使用类似于本文所述的协议, 我们最近发现了一种新的类 (D)-leucinamides, 能调制的γ-分泌分裂的应用程序与缺口备用选择性19。这一集合的研究提供了一个证明的概念, 支持的概念, 基板的选择性/可用性γ-分泌可以受到化学调制和实验验证。然而, 这些化验平台往往依赖相对较低的吞吐量读数和劳动密集型的方法, 可能不符合工业标准的药物发现计划。
我们最近生成了基于细胞的荧光报告基因检测, 可以定量地确定γ-分泌对两个不同基底的催化活性, 即应用 (APP-C99) 的 99-氨基甲酸 C 末端片段和胞外域删除的缺口肽 (NΔE)。APP-C99 和 NΔE 在各种测定方法中被广泛用作γ-分泌的直接基质。为了生成均匀和一致的荧光报告基因检测的γ-分泌分裂的 APP-C99 或 NΔE, 我们产生了一个 HEK 衍生稳定细胞线 (CG) 组成表达 Gal4 启动驱动萤火虫荧光记者 (Gal4-Luc) 和 Gal4/VP16-tagged APP-C99 融合蛋白 (C99-GV)。此外, 我们还生成了另一个 HEK 的稳定细胞系 (NG), 组成表达 Gal4-Luc 和 Gal4/VP16-tagged NΔE (NΔE)。利用这些新的基质特异γ-分泌测定方法, 我们现在提供了一种定量和区分γ-分泌对不同基底 (APP-C99 在 CG 细胞中, 或 NΔE 在 NG 细胞中) 的催化活性。此外, 基板特定的γ-分泌检测的目的是有利于高含量的筛选平台。这些新生成的γ-分泌检测方法可以为发现新的γ-分泌调节剂铺平道路, 通过抑制 a 的产生, 而不引起对下一代治疗的不良抑制, 从而提高认知功能。的广告。

<table><tbody><tr><td>VictorLight luminescence plate reader</td><td>PerkinElmer</td><td>2030-0010</td><td></td></tr><tr><td>Class II, Type 2 Biological Safety Cabinet</td><td>Thermo Scientific</td><td>1300 Series A2</td><td></td></tr><tr><td>CL-387,785</td><td>EMD Calbiochem</td><td>233100-1MG</td><td></td></tr><tr><td>DAPT</td><td>Merck</td><td>565770</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>12100046</td><td>main compnent of growth medium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher</td><td>16000044</td><td></td></tr><tr><td>Blasticidin</td><td>Thermo Fisher</td><td>A1113903</td><td></td></tr><tr><td>Zeocin</td><td>Thermo Fisher</td><td>R25005</td><td></td></tr><tr><td>Hygromycin B</td><td>Gibco</td><td>10687010</td><td></td></tr><tr><td>Hemocytomer</td><td>Sigma-Aldrich</td><td>BR717805 Aldrich</td><td></td></tr><tr><td>96-well microplate</td><td>Nunc</td><td>156545</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma</td><td>T7660</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Steady-Glo luciferase assay reagent</td><td>Promega</td><td>E2510</td><td></td></tr><tr><td>Humidified CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Revco</td><td>Ultima II</td><td></td></tr><tr><td>T-REx293 cell line</td><td>Invitrogen</td><td>R71007</td><td></td></tr><tr><td>Wallac 1420 software version 3.0</td><td>PerkinElmer</td><td></td><td>instrument control software of the luminescence microplate reader</td></tr></tbody></table>

quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms

我们已经开发了一对细胞为基础的记者基因检测, 定量测量γ-分泌分裂的不同基质。本手稿描述的程序, 可用于监测γ-分泌介导的 APP-C99 或缺口, 使用 Gal4 启动器驱动的萤火虫荧光记者系统的裂解。染 HEK293 细胞与 Gal4-driven 荧光报告基因和 Gal4/VP16-tagged C 终端片段 (APP-C99) 建立了稳定的 co-非晶胞蛋白的测定。CG 细胞), 或 Gal4/VP16-tagged 缺口-δ (NΔE;NG 细胞)。利用这些记者并行分析, 我们已经证明, ErbB2 抑制剂, CL-387,785, 可以优先抑制γ分泌分裂的 APP-C99 在 CG 细胞, 但不是 NΔE 在 NG 细胞。CL-387,785 治疗时, CG 和 NG 细胞所表现的差异反应是γ-分泌调节剂的首选特性, 这些反应与 DAPT 诱导的γ-分泌的泛抑制形成了鲜明的对比。我们的研究提供了直接的证据证明γ-分泌活动向不同的基底可以被区分在一个细胞的背景。因此, 这些新的化验方法可能是药物发现的有用工具, 用于改进 AD 疗法。

通过CL-387,785 抑制 ErbB2 可差异促进 APP-C99 的处理γ-分泌不影响缺口解理 为了建立一种能定量测量特定γ - 分泌基质的蛋白水解的细胞分析方法 , 我们通过稳定的联合转染四环素诱导的 c - 终末期 Gal4 / VP16 - tagged , 生成了 HEK293 - derived 的 CG 细胞系。APP-C99 和一个 Gal4 启动驱动萤火虫荧光记者基因。还建立了一个平行 NG 细胞线, 以检测 NΔE 的γ-分泌裂解。在 NG 细胞中, 四环素诱导的 c-端 Gal4/VP16-tagged NΔE 和 Gal4 启动子驱动的荧光报告基因被稳定地与 HEK293 细胞联合转染。APP-C99 在 cg 细胞中的γ-分泌裂解和 NΔE 在 ng 细胞中的γ-分泌裂解, 首先通过处理各种浓度的 DAPT、一种泛γ-分泌抑制剂, 阻止所有γ-分泌基板的分裂 (图 1)。数据还表明, γ-分泌在 CG andNG 细胞中表现出可比的催化作用水平。
为了验证基于基板的γ-分泌分裂的检测方法的有效性, 我们用 ErbB1/2 双抑制剂 CL-387,785 对 CG 和 NG cellstreated 中的发光信号进行了研究, 结果表明它对差异调制APP-C99 和 NΔE 的γ-分泌裂解,21。我们以前确定 CL-387,785 作为一个选择性γ分泌调制器, 由于它干扰蛋白质-蛋白质相互作用之间的 presenilin-1 (PS1) 和 C99 和缺乏干扰的相互作用 PS1 和 NΔE21。在这里, 数据表明, CL-387,785 的治疗有效地阻断了 CG 细胞中 APP-C99 的处理, 而 NG 细胞中 NΔE 的蛋白水解不受影响 (图 2)。这些结果证明了 CG 和 NG 细胞在区分 C99 和 NΔE 分裂之间的有效性, 这种形式有利于高通量筛选平台。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56795/56795fig1.jpg"> 图 1: APP-C99-specific 细胞γ-分泌检测 (CG 细胞).CG 细胞被播种到96井板 (2万细胞/井) 过夜和治疗1μ m 的 CL-387,785 或 DAPT 存在1微克/毫升四环素在37° c 为 24 h。在添加100μ l/井荧光测定试剂后, 从γ-分泌介导的 C99-GV 蛋白水解中获得的发光进行了量化。单独 (0.1% 甲基亚砜) 处理的细胞发出的荧光信号被设置为100% 相对发光。数据显示为平均± SD 从三独立实验。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56795/56795fig1large.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/56795/56795fig2.jpg"> 图 2: 特定于缺口的细胞γ-分泌检测 (NG 细胞).NG 细胞被播种到96井板 (2万细胞/井) 过夜和治疗1μ m 的 CL-387,785 或 DAPT 存在1微克/毫升四环素在37° c 为 24 h。在添加100μ l/井荧光试剂后, 从γ-分泌介导的 NΔE 蛋白水解中获得的发光量被量化。单独 (0.1% 甲基亚砜) 处理的细胞发出的荧光信号被设置为100% 相对发光。数据显示为平均± SD 从三独立实验。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56795/56795fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

我们已经成功地生成了两个基片特定的γ-分泌检测。这两种基于细胞的检测方法都被设计成通过萤火虫荧光记者的输出来量化γ-分泌酶的活性。

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

quantitative-measurement-secretase-mediated-amyloid-precursor-protein

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

6.7K 次观看.  Academia Sinica. 我们已经成功地生成了两个基片特定的γ-分泌检测。这两种基于细胞的检测方法都被设计成通过萤火虫荧光记者的输出来量化γ-分泌酶的活性。

视频: Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

科研

生物化学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

发育生物学

使用纳米荧光素酶报告基因检测定量 DNA 双链断裂修复

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

作者聚焦：开发针对 DNA 损伤反应的新型抗癌疗法

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

使用基于高通量和定量发光的报告基因分析评估 DNA 双链断裂修复活性

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

癌症研究

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 ...

免疫与感染

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 ...

生物学

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

神经科学

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder.

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

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