这项工作由癌症研究协会 (加拿大) 和 CIMS 资助。作者感谢 Samia 博士 Mohand 和吉恩-弗朗索瓦 Beaudoin 的帮助。天使洛佩兹博士 (南澳大利亚大学) 为单克隆 A14。

所描述的体内动物实验是根据一项经批准的议定书和中心 Hospitalier Universitaire de 舍布鲁克伦理学委员会动物实验的道德准则进行的。
1. 抗体肽共轭
注: ChAcNLS 可在任何商业肽制造商或大学附属的多肽综合服务平台上合成。ChAcNLS 的合成可以在参考34中找到。对于程序1和2使用单克隆 7G3, 这是特定的白血病抗原 IL-3Rα 39。
<ol><li>在一个1.7 毫升的离心管, 准备10毫克/毫升 (1 毫克总抗体) 溶液的7G3 在磷酸盐缓冲盐水 (PBS), pH 7.6。</li>
	<li>将磺-当局在二甲基亚砜 (亚砜) 中溶解, 浓度为5-10 毫米。涡流解决方案, 以达到完全溶解的最佳效果。</li>
	<li>添加到包含7G3 溶解磺-当局溶液的管 (通常在 5-20 ul 之间取决于 sulfo-SMCC-to-7G3 所需的摩尔比)。建议对10、25和50到1的 sulfo-SMCC-to-7G3 比率进行测试。室温孵育1小时。</li>
	<li>
在 PBS、pH 值7.0 中使用超滤装置和缓冲交换纯化和浓缩 maleimide-衍生物7G3。离心机在 8000 x g 左右, 约5分钟丢弃, 并填充 PBS, pH 值7.0 和离心机。
	<ol>
<li>执行这7-8 次以完全交换缓冲区。将过滤装置倒在干净的离心管中, 以恢复浓缩7G3 蛋白。离心机为2分钟, 在 1000 x g. 回收量应约0.3 毫升</li>
	</ol>
</li>
	<li>在含有 maleimide 活化7G3 的管中, 增加2倍摩尔的 ChAcNLS 相对于上一步所用的 sulfo-SMCC-to-7G3 比, 并在4摄氏度一夜之间孵化。例如, 如果使用了10到1的 sulfo-SMCC-to-7G3 比率, 则使用20到1的 ChAcNLS-to-7G3 比率。总成交量不应显著改变。 注意: 虽然 ChAcNLS 在共轭过程中不会引起降水, 但这是可能发生的其他肽。在反应过程中观察管内沉淀的迹象, 这很可能是由于多肽是疏水引起的。在这些情况下, 它可能值得重新研究的兴趣肽, 以便设计变化, 以提供增加的亲水性。</li>
	<li>使用超滤装置将 7G3 ChAcNLS 浓缩到所需浓度和 PBS pH 值7.4 的缓冲交换 (见步骤 1.4)。总蛋白回收率应为原起始材料的≥75%。</li>
	<li>使用12% 聚丙烯酰胺凝胶 (提供足够的分离 ChAcNLS 共轭重和轻链从 nonconjugated 链), 执行 SDS 页评估构建的 7G3 ChAcNLS 候选者。混合10微克的 7G3-ChAcNLS 在页面加载缓冲区包含 2 ul β-巯基乙醇和负载入井。</li>
	<li>设置适当的电压 (120 V 为1小时12% 凝胶) 和运行电泳。当考马斯染料前端到达凝胶底部时, 停止电泳。 注意: 不要让考马斯染料前面的凝胶。</li>
	<li>从电泳装置中取出凝胶, 用含有10% 伏/v 的冰乙酸和20% 伏/钒甲醇的脱色溶液冲洗。</li>
	<li>采取凝胶的相片和用标尺和测量距离 (cm) 迁移为标准和7G3 共轭物。计算保留系数 (Rf) 值, 这是迁移距离除以凝胶长度 (从何处将蛋白质加载到考马斯迁移前端)。将分子量 (兆瓦) 与每个蛋白质标准中的射频值进行记录, 并推断7G3 共轭物的大小。</li>
	<li>用1768.5 克/摩尔 (兆瓦 ChAcNLS) 除以7G3 共轭和未修改7G3 之间的差异, 计算每种抗体的肽数量。</li>
	<li>采取考马斯染色凝胶的数字图像, 进行密度分析。在这一点上, 选择一个潜在的候选者可以根据 AC 与最大数量的 ChAcNLS 分子, 不包含重要的聚集物种。</li>
</ol>2. 细胞内聚集评价的共焦显微术
注意: 重要的是要测试的细胞选择性的肽修饰抗体之前, 开发配方与有效载荷的利益。由于达也被证明具有非特异性细胞穿透的倾向, 它值得首先分析细胞内积累和细胞选择性, 然后采取昂贵的有效载荷的开发步骤。因此, 程序1还应修改同种特定无关的控制抗体。对于该协议的其余部分, 我们将使用 10 ChAcNLS 分子每抗体修改 ACs。图 2中描述了包括过程2和3的关键步骤的示意图。
注意: 该协议的这一步骤涉及对多聚甲醛的处理和操作。处理时请遵照制造商的指示操作。
<ol><li>治疗靶 TF-1a 细胞与200毫微米的 7G3-ChAcNLS 包括改良控制抗体。孵育 5 x 106抗原阳性细胞与共轭物为 1 h 在37°c。</li>
	<li>1小时后, 取出上清, 用1毫升3x 的冰冷 PBS 冲洗。添加新鲜的媒体和孵化一个额外的小时在37摄氏度。</li>
	<li>
在增加的小时以后, 去除上清和洗涤3x 在1毫升冰冷 PBS。添加0.5 毫升的 PBS, 含0.25% 胰蛋白酶和乙二胺四乙酸 (EDTA), 37 摄氏度, 3 至30分钟。 注: 在初步试验中, 建议不要胰蛋白酶处理细胞, 以确定交流细胞内积聚的差异。这个协议促进了胰蛋白酶的使用, 我们展示了如何改善不同交流候选者细胞内积累效率的评估。<ol>
<li>通过目视检查所有要分离的单元格, 测试不同的孵化时间。此外, 孵化细胞整除数与生存能力染色解决方案和执行流细胞分析, 如前所述的 40. </li>
		<li>中和胰蛋白酶与1.5 毫升细胞培养 RPMI 1640 media/10%fetal 牛血清。离心细胞在 500 x g 5 分钟, 去除上清, 并且洗涤3x 在0.5 毫升冰冷 PBS。</li>
	</ol>
</li>
	<li>固定细胞在0.5 毫升 pbs 包含1% 多聚甲醛和1% 蔗糖在冰上30分钟洗涤细胞3x 在0.5 毫升冰冷 pbs 并且离心机在 250 x g 在5毫升 pbs 中0.5 个 Permeabilize 细胞 0.15% ml 在冰上包含分钟的海卫 X-100。在冰冷的 PBS 中洗涤细胞并重复离心。</li>
	<li>
悬浮细胞在0.1 毫升 PBS 包含2微克/毫升 (或制造商推荐浓度) 的抗鼠 (同种特异) Fc 次级多克隆抗体共轭到 AlexaFluor 647 在室温下在黑暗中的1小时。
	<ol>
<li>离心机在 250 x g 5 分钟和洗涤细胞3x 在0.5 毫升冰冷 PBS。在0.5 毫升 PBS 中悬浮细胞。 暂停: 单元格可以存储在冰箱中。 注意: 必须选择一个二级抗体, 识别出正确的同种的单克隆的兴趣。AF647 是由于其远红外辐射而选择的, 不干扰碘碘 (PI) 染色的细胞核。</li>
	</ol>
</li>
	<li>添加 PI 到10微克/毫升浓度的容器与细胞。接下来, 将 1 x 105单元格安装到玻璃幻灯片上, 使用安装介质并盖上玻璃盖玻片。</li>
	<li>检查细胞与计划的60X 油浸泡目标 NA 1.42 在反向激光扫描共聚焦显微镜。检测 PI 荧光使用 488 nm 氩激光和光谱扫描棱镜设置为 600-650 nm。为 AF647 荧光使用633毫微米氦氖激光和光谱扫描棱镜设置为 650-700 毫微米。依次收集 PI 和 AF647 的荧光辐射。 注意: 在单元格的评估和比较中使用相同的设置。但是, 与非 trypsinized 单元格相比, 您必须调整 trypsinized 单元格的设置。</li>
	<li>使用显微镜软件获取图像。使用 1024 x 1024 像素的串行水平光学剖面收集图像, 并通过整个细胞厚度在0.5 微米间隔内进行2X 线平均。在最大荧光强度下, 以四连续切片显示图像为堆叠 z 投影。</li>
	<li>用显微镜软件分析细胞。记录共轭物的细胞分布格局。具体来说, 评估细胞质内的细胞内荧光是否被分组, 靠近细胞表面或弥漫和均匀。还要评估每个细胞的相对荧光强度。</li>
</ol>3. 核素交流结构和细胞分馏法评价64铜胞内分娩效率
注意: 程序3和4涉及放射性的处理和操纵。在执行这些步骤之前, 研究人员应该已经批准了他们的家庭机构的辐射安全局批准的安全培训和协议。
注: 对于程序3和 4, 我们使用单克隆 A14, 这是特定的浸润性膀胱癌抗原 IL-5Rα41。此外, 由于64Cu 的短半衰期, 所有实验都是时间敏感的。一般而言, 最好不要等待过去1周来执行体外实验, 而不超过72小时用于体内研究。
<ol><li>在1.7 毫升管悬浮 22 '-(7-(2-(25-dioxopyrrolidin-1-基) 氧)-2-乙醛基)-14, 7-triazonane-14-己) diacetic 酸 (不-NHS) 在亚砜到浓度10毫米。</li>
	<li>反应50倍摩尔过量的不-nhs 与 A14 (2 毫克起始材料) 在0.1 米碳酸氢钠在 pH 8.6 为 1 h 在室温下的容积≤0.5 毫升 (最后的非 NHS 卷不应超过 2% v/v 的总体积)。</li>
	<li>净化和缓冲交换 NOTA-A14 使用一个超滤装置在 PBS, pH 值 7.6 (见步骤 1.4)。</li>
	<li>对于后续的 ChAcNLS 连接, 请执行步骤 1.2-1.6。</li>
	<li>反应 NOTA-A14-ChAcNLS (250 微克批次) 与 100 megabecquerel (MBq) 64CuCl2在 0.1 M 碳酸氢钠, pH 5.5 为 1 h 在37°c ≤0.1 毫升。64铜是在 CIMS 42上的 TR PET 回旋加速器上生产的。</li>
	<li>将64Cu-A14-ChAcNLS 集中在 PBS pH 值7.4 中, 使用超滤装置达到所需浓度 (见步骤 1.4)。</li>
	<li>
用色谱条和 0.1 M, pH 值5.5 柠檬酸钠 (ITLC 淋洗) 作为溶剂, 确定64Cu-A14-ChAcNLS 的 radiolabeling 效率 (ITLC)。将反应溶液的 0.5 ul 整除应用于 ITLC 条。执行 autoradiograph, 获取数字图像。
	<ol>
<li>执行密度测量以获得绑定和自由64Cu 的比例。如果可用64Cu 内容为 > 5%, 请返回上一步并执行其他净化操作。</li>
		<li>此外, 使用考马斯染色进行 SDS 页来评估聚合。执行第二个 SDS 页, 然后 autoradiograph 的凝胶, 以确定是否有核素的聚合物种。</li>
	</ol>
</li>
	<li>
亲和结合质量检查点: 孵育64Cu-A14 共轭在增加浓度 (0-100 nM) 以重复与 1 x 106目标细胞每毫升。要估计非特异性绑定, 请将64Cu-A14 与细胞的重复样本混合在100倍摩尔超过未标记 A14 的情况下。
	<ol>
<li>在冰上孵化1小时后, 在伽玛计数器中冲洗细胞和计数总束缚抗体样本和总非特异性束缚抗体样本。图特定绑定 (总绑定抗体-非特异性绑定抗体) 反对无活性自由64Cu-A14 (nM) 在化验中使用。通过使用图形软件进行非线性回归确定离解常数 (Kd)。</li>
	</ol>
</li>
	<li>对于64cu 细胞积累研究: 以 100 nM 的64铜标记的 A14 共轭物治疗细胞 1 h, 6 h, 24 h 在37°c, 如前所述33。包括其他参数, 如在未修改的 A14 存在的情况下进行治疗, 以阻止 IL-5Rα地点和4摄氏度, 以阻断受体介导的内部化。</li>
	<li>在定义的时间点, 如前述步骤 2.3-2.3.2, 添加0.5 毫升的 PBS, 含有0.25% 胰蛋白酶和 EDTA 在37°c, 其次是中和和洗涤。</li>
	<li>在 1% NP-40, 10 毫米三 pH 7.5, 10 毫米氯化钠, 3 毫米氯化镁2缓冲器的等离子膜裂解缓冲液中孵化细胞为10分钟. 离心机在 90 x g 的等离子膜裂解细胞为5分钟。</li>
	<li>取出上清液并放置在新鲜的管子中。这代表细胞质分数。在冰冷的 PBS 中洗涤原子核 3x, 并将洗涤液加入胞浆部分。</li>
	<li>确保含有核和细胞质馏分的瓶子密封。然后将它们放在校准为64Cu 的伽玛计数器中, 以便将原始计数转换为 MBq。</li>
	<li>
通过对 Lamin、限制性核蛋白和 Rab7, 对全细胞裂解液、核和细胞质馏分进行了丰富的细胞质蛋白分析, 确定核分离的质量。
	<ol>
<li>将 5 x 106单元格与 500 ul 的无线电免疫沉淀检测 (马国贤) 缓冲器和在3.12、1% 和 2% NP-40 的步骤4% 中的等离子膜裂解缓冲液治疗。此外, 在 500 ul 马国贤缓冲液中分离出的细胞核, 以获得分离的核蛋白。</li>
		<li>用4x 的冷丙酮样品体积, 沉淀分离的核、细胞质和全细胞蛋白, 60 分钟-20 摄氏度。离心机在 1.3万 x g 的10分钟和醒酒上清, 小心不要去除蛋白颗粒。添加 100 ul 的 PBS, 以溶解蛋白质。</li>
		<li>将10微克的蛋白质装入10% 页凝胶的每个井中。按照 1.7-1.9 的描述进行电泳。执行以前在 refeference 43中描述的西方印迹传输。</li>
		<li>找出最符合 40 kDa 的阶梯标记。用这个标记把膜切成两半。用 Lamin A/C 特异抗体探测含有较高兆瓦蛋白的印迹。用7特异性抗体探测膜与下兆瓦蛋白。西方污点是一个众所周知的技术, 并没有在本议定书中描述。</li>
	</ol>
</li>
</ol>4. 肿瘤靶向的 PET 成像评价
注: 在小鼠体内植入肿瘤细胞以产生异位移植的技术是众所周知的, 大多数实验室都有为其肿瘤系统量身定做的内部协议。因此, 议定书没有涵盖这一点。异种移植模型将非肥胖糖尿病/重症合并 immunodeficient (点头/免疫缺陷) 小鼠, IL-5Rα-positive 浸润性膀胱肿瘤 HT-1376 和 HT-B9。IL-5Rα-positive 浸润性膀胱肿瘤细胞包括 > 66% 的总细胞在异种移植。相比之下, 仅有11% 的 IL-5Rα-positive HT-B9 细胞包含在发育中的移植性41中。因此, 该模型提供了一个很好的例子, 以评估交流肿瘤靶向两个肿瘤, 代表可预见的病人肿瘤异质性。
<ol><li>在含有≥ 4 (点头/免疫缺陷) 小鼠的组中, 向尾静脉注入20-30 微克 (6-9 MBq) 64Cu-A14-ChAcNLS、 64Cu-A14-NLS 和64Cu-A14。</li>
	<li>在同一天放置一个圆柱形幻影 (24.8 毫升), 其中包含 5 MBq 的64铜, 将每秒的放射性计数转换为每克组织的注射剂量 (%ID/克)。</li>
	<li>等待48小时, 然后麻醉老鼠放置在一个感应腔内, 1.5 升/分钟的氧流与2% 异氟醚。在植入 PET 扫描仪前, 将无菌眼膏应用于鼠眼。</li>
	<li>将鼠标迅速转移到一个宠物扫描仪表中的头俯卧位置与鼻锥的异氟醚。定位呼吸和直肠探头, 并监测温度和呼吸率, 以确保在实验中保持生理条件, 如前所述的44。</li>
	<li>使用常规采样模式设置和 250-650 凯文的能量窗口开始获取 PET 数据。通常, 每只鼠标45分钟的静态扫描足以为高质量的 PET 图像的重建和生产提供足够的重合事件。</li>
	<li>在64铜交流扫描之后, 从扫描仪中取出鼠标并将其放回笼子中。允许鼠标在返回动物设施之前充分恢复。</li>
	<li>利用三维最大似然期望最大化算法的20次迭代获得重建图像。</li>
	<li>
使用酰胺软件对肿瘤和视觉上应评的器官进行兴趣区 (ROI) 分析。
	<ol>
<li>绘制 ROIs 获取定量%ID/g 数据手动使用3D 手绘工具设置, 并仔细描绘肿瘤或相邻大腿肌肉。ROI 中每个像素的计数将从上一步骤4.2 转换为%ID/g。</li>
	</ol>
</li>
	<li>比较64Cu-A14、 64Cu-A14-ChAcNLS、 64Cu-A14-NLS 图像以进行肿瘤对比, 以及定量 ROI%ID/g 和肿瘤到背景比率。</li>
</ol>

作者没有什么可透露的

系统地提供抗癌药物的主要目标是增加肿瘤部位的积聚, 并在癌细胞中吸收, 并减少健康组织中有害的副作用。交流靶向肿瘤细胞提供分子有效载荷是一种非常有前景的治疗和检测肿瘤的方法。然而, endosome 和下游溶酶体退化所引起的疗效仍然是一个重要的挑战。虽然该协议利用 ChAcNLS 肽作为下一代 ACs 的一个例子, 但所描述的程序可以适应任何被开发的多肽修饰交流, 目的是增加细胞内交付的负载?...

抗体共轭 (ACs) 是生物成熟成为一个转化类的有效药物改善癌症治疗和检测肿瘤。ACs 由与分子有效载荷 (如放射性同位素、小分子和生物毒素) 共轭的单克隆抗体组成, 能够将这些有效载荷传递给具有精致靶抗原亲和性和特异性的癌细胞。因此 ACs 有可能显著减少非特异性毒性, 并增加肿瘤部位的有效载荷活动。治疗, ACs 运输细胞毒性小分子 (通常称为抗体-药物共轭) 已被批准处理乳腺癌和霍奇金淋巴瘤谁已经失败常规治疗1,2. 此外, 运输放射性同位素 (通常称为 radioimmunoconjugates) 的 ACs 也在开发中。通过交流传输放射性同位素进行成像, 确认前列腺癌转移3。随着更多的治疗 acs 提交批准4, 乐观是高的未来 acs, 以改善癌症护理5。
然而, 在运送化疗或放射性同位素时, ACs 很难有效地在靶细胞内积聚这些有效载荷。在许多情况下, 这方面显著地有助于 ACs 无法提供持久无疾病生存或高对比度肿瘤成像6,7。一般而言, 一旦 ACs 结合其目标抗原, 它们就会通过称为受体介导的吞的过程内部化。然后, ACs 被包裹在内涵体内并被贩运到溶酶体中, 用于降解和有效载荷释放 8. 细胞内的贩运过程给 ACs 带来了挑战, 以达到高有效载荷特异性和抗靶向癌细胞的功效。例如, 许多抗原, 如 Her2 (治疗交流曲妥珠单抗-emtansine 靶) 可以在前 30分钟9 中回收多达85% 的绑定抗体。此外, 一旦降解发生, 释放的化疗和放射性同位素可以通过增加表达和/或活动的膜相关传输蛋白10,11积极出口。溶酶体的降解也阻碍了新的生物有效载荷的传递, 如治疗酵素和寡核苷酸, 可以被停用12,13。从本质上讲, 癌细胞是高效的废除细胞内积累的有效载荷由 ACs 提供。
该协议描述了如何实现 ACs-耦合到病毒衍生肽的概念, 特别是为了逃避 endosome 诱捕和本地化到细胞核。这种复杂的操作宿主细胞系统, 这并不奇怪, 病毒衍生蛋白和肽作为潜在的生物的发展早已根深蒂固的治疗研究14。为了有效地进入宿主细胞, 在数以百万年的时间里, 病毒进化来获得一种特殊的蛋白质集合, 能够利用正常的生理哺乳动物细胞系统。对于通过受体介导的吞内化的病毒, 他们也面临着逃避贩运到溶酶体的挑战, 那里的蛋白酶局部浓度的冲击可能会对生存有问题。一种具有良好特征的病毒衍生肽, 用于药物的传递, 以逃避 endosome 诱捕是人体免疫机能丧失病毒 transactivator 转录 (达) 蛋白15。达能通过传感低 pH 值来逃避 endosome 诱捕, 在这种情况下, 蛋白质构象的变化发生, 使达能够插入和扰乱细胞膜膜16。这就产生了能进入细胞质的达-负载共轭物。与此协议相关的第二个病毒操作元素是用于将治疗基因和药物传递给核的方法17。病毒已经进化到成功地操纵宿主细胞机械, 通过核孔复合体通过核膜的进展。蜂窝大分子包含 (或结合含有的蛋白质) 核定位信号 (NLSs), 以绑定到核转运蛋白 (例如karyopherins α和β), 通过 NPC 提供必要的运动。病毒已经开发出含有 NLS 序列的蛋白质, 使它们能够利用宿主细胞传输蛋白来传送到细胞核18。
许多 ACs 以前已经功能性与达-和 NLS 衍生肽, 并测试他们的能力积累在癌细胞和靶向肿瘤19,20,21,22 ,23,24,25,26,27,28,29,30 (表 1)。提供细胞毒性有效载荷的研究表明, 通过病毒衍生肽进行修饰的 acs 能够显著增加细胞积累、细胞毒性和肿瘤死亡, 超过未修改的 ACs 22,26。这个新的交流类的一个共同特点是他们的建设。通常情况下, 多肽含有一个末端半胱氨酸, 提供游离巯基。抗体首先反应的是 noncleavable 双交联剂, 其中包含N-琥珀 (NHS) 和 maleimide 组在相反的一端。NHS 酯在单克隆抗体上与主要胺反应形成酰胺键。与自由 maleimide 组的反应, 然后反应与巯基组对肽形成一个硫酯键, 从而连接肽和单克隆。虽然同型交联剂已使用 28, 但 heterobifunctional 交联剂更常用于构建病毒衍生肽-ACs22,23,26, 31,32。本议定书特别使用交联 sulfosuccinimidyl 4-(n-马来) 环己烷-1-羧酸酯 (磺当局), 以便于使用, 因为它被用于经批准的抗体-药物共轭曲妥珠单抗-emtansine, 在许多病毒衍生肽-ACs 8,22,23,26,31,32。月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 是初步确定共轭效率和半定量的多肽每单克隆的主要方法。共焦显微术使用荧光标记的二次抗体特定于单克隆, 通常是初步评价病毒衍生肽修饰 ACs 细胞内分布特性的方法。迄今为止, 放射性同位素是由病毒衍生的肽修饰 ACs 提供的主要有效载荷。放射性同位素是有利的, 因为细胞中的放射性可以很容易地通过伽玛计数来量化。此外, 被翻译成老鼠的人类癌症模型的 ACs 为研究人员提供了评估肿瘤靶向性的能力, 例如单光子发射计算机断层扫描和正电子发射层析成像 (PET)。23,32,33. 一般而言, 研究人员主要使用的构造和验证测试方法为在初始开发阶段对病毒衍生肽进行了改进的 ACs 提供了很好的评估, 以便有效地进入和交付靶细胞内的有效载荷和靶向肿瘤。
达-和 NLS 修饰的 ACs 已经照亮了进一步改善肿瘤细胞和肿瘤内有效载荷的关键领域。关于 NLS 修改的 ACs, 细胞内积累的效率可以是适度的23,31,34。细胞内积聚效率低下是由持续的细胞膜诱捕引起的。在体内肿瘤靶向也可以减少与达-和 NLS 修改 ACs。有效序列的达和 NLS 含有几个正电荷残留物。当附加到抗体时, 总阳离子电荷可以显著增加35。因此, 经和 NLS 改良的 ACs 增加了对健康组织的吸收, 并增加了快速的血液清除量。
我们的小组开发了一个复合化合物组成的胆酸与 NLS (ChAcNLS;图 1)。ChAcNLS 修饰的 acs 能够增加所提供的放射性同位素的胞内积累和改善肿瘤靶向性, 与 NLS 修改和传统 ACs 33,34。胆酸的机制是由选择 nonenveloped 病毒的能力所激发的, 不能依靠膜融合来利用胆酸通过神经酰胺的形成来触发 endosome 逃逸。例如, 猪肠道病毒新兵胆酸激活 sphingomyelinase, 催化鞘磷脂水解成神经酰胺 36, 37, 38. 这破坏细胞膜膜, 并允许病毒逃逸。因此, 胆酸是另一个病毒衍生成分, 补充 NLS。
随着这一领域的向前推进和未来的进步发生在负载交付由 ACs 修改与病毒衍生肽, 这是一个适当的时间来提供视觉示范其生物化学和功能特性的初步发展。在这里, 我们描述我们的协议, 初步评估病毒衍生肽修饰 ACs 的有效而简单的确定细胞内积累和肿瘤靶向在早期发展。我们使用商业上可用的抗体7G3 和 A14 作为示例模型系统。步骤1描述了使用 SDS 页作为一种方法, 允许对构造的 ACs 进行 "转/不走" 决策。程序2描述了一种使用 trypsinization 的方法, 用于改善交流细胞内分布和积累的可视化。步骤3描述了一种改进细胞内分馏的方法, 以准确确定核定位。在这个过程中, 我们利用负载64Cu (t1/2 = 12.7 h), 因为它易受细胞外流, 是正电子发射器10。因此, 程序4描述了在体内肿瘤靶向特征的 PET 成像, 以可视化肿瘤摄取相对于背景 (即nontarget 健康组织), 并确定该例交流是否可以具体和有效地靶向肿瘤。这些方法足以为研究人员开发的 ACs 修改与病毒衍生肽, 以确定候选者进一步提高。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>Thermo Scientific</td>
			<td>22122</td>
			<td>There are many homo- and hetero-bifunctional maleimide crosslinkers to choose from.</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 mL Centrifugal Filters</td>
			<td>EMD Millipore</td>
			<td>UFC505096</td>
			<td>There are pack sizes of 8, 24, and 96. Choose according to your needs.</td>
		</tr>
		<tr>
			<td>Precision Plus Protein Kaleidoscope Standards</td>
			<td>BioRad</td>
			<td>1610375EDU</td>
			<td>Mulicolor recombinant proteins from 10-250 kDa.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200056</td>
			<td>100 or 500 mL volumes to choose from.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td>Thermo Scientific</td>
			<td>A-21235</td>
			<td>1-10 &#956;g/mL recommended</td>
		</tr>
		<tr>
			<td>NOTA-NHS</td>
			<td>CheMatech</td>
			<td>C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamin A/C antibody (N-18)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6215</td>
			<td></td>
		</tr>
		<tr>
			<td>Rab7 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376362</td>
			<td></td>
		</tr>
		<tr>
			<td>A14 mAb</td>
			<td>BD Biosciences</td>
			<td>555902</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4x)</td>
			<td>Thermo Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TF-1a cells</td>
			<td>ATCC</td>
			<td>ATCC CRL-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>ATCC</td>
			<td>ATCC 30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis and extraction buffer</td>
			<td>Thermo Scientific</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>AMIDE medical imaging software</td>
			<td>available at amide.sourceforge.net</td>
			<td></td>
			<td>Completely free download</td>
		</tr>
		<tr>
			<td>FluoView FV1000 Confocal Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoview Software</td>
			<td>Olympus</td>
			<td></td>
			<td>www.olympus-lifescience.com</td>
		</tr>
		<tr>
			<td>ITLC strips</td>
			<td>Biodex</td>
			<td>150-771</td>
			<td></td>
		</tr>
	</tbody>
</table>

initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting

用病毒衍生肽修饰的抗体共轭物 (acs) 是一种潜在强大的肿瘤细胞传递剂, 用于癌症治疗和影像学的分子有效载荷, 原因是细胞积累超过了目前的 ACs。在早期交流体外开发过程中, 荧光技术和 radioimmunoassays 对确定细胞内定位、积累效率和靶细胞特异性都有足够的作用。目前, 对于制备细胞以评价交流细胞内聚集和定位的标准化方法尚无共识。对使用病毒衍生肽进行修饰的 ACs 的初步测试是至关重要的, 特别是如果已经建立了几个候选者。通过荧光法测定细胞内的细胞积累, 可影响其表面的背景信号, 使堆积物的解释复杂化。对于 radioimmunoassays, 通常被处理的细胞被分割, 并测量不同细胞间的放射性。然而, 细胞裂解从细胞到细胞不同, 通常细胞核和细胞质隔间没有充分的隔离。这可能会产生关于负载传递属性的误导性数据。核素病毒衍生肽修饰 ACs 在肿瘤荷载小鼠中的静脉注射和核素显像是确定肿瘤靶向和有效载荷传递特性的有力方法, 在体内阶段发展.然而, 这是一个相对较新的进展, 很少有团体以这种方式评估病毒衍生的肽修饰 ACs。我们描述治疗细胞的处理, 以更准确地评估病毒衍生的肽修饰交流积累时, 使用共焦显微镜和 radioimmunoassays。具体地说, 一种 trypsinizing 单元格移除单元格表面绑定 ACs 的方法。我们还提供了一种改善细胞分馏的方法。最后, 本协议提供了一个体内方法, 使用正电子发射断层扫描 (PET) 评估肿瘤小鼠的初始肿瘤靶向性。我们使用放射性同位素64Cu (t1/2 = 12.7 h) 作为本协议中的一个示例负载。

在程序1中, 用磺当局作为交联剂 ChAcNLS 改性7G3 的结构是非常可靠的。通常, 当加载到12% 凝胶和分析的 SDS 页, 这导致可区分逐步增加兆瓦比例增加 sulfo-SMCC-to-7G3 比率使用, 并允许重和轻链单独评估 ChAcNLS共轭 (图 3)。7G3 反应在 10, 20, 25 和50到 1 sulfo-SMCC-to-7G3 比率跟随射频测量和兆瓦推断结果在 3, 7, 10 和 20 ChAcNLS 分子每7G3。通过密度测定, 7G3 3-10 ChAcNLS, 单体的百?...

Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

病毒衍生肽与抗体结合 (ACs) 是一种获得动力的方法, 由于可能提供的分子负载增加肿瘤细胞积累。利用常用的方法来评估肽共轭、AC 和有效载荷的胞内积累和肿瘤靶向, 这项协议帮助研究人员在关键的初步发展阶段。

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

initial-evaluation-antibody-conjugates-modified-with-viral-derived

Research

JoVE Journal

Bioengineering

7.6K 次观看.  Université de Sherbrooke. 病毒衍生肽与抗体结合 (ACs) 是一种获得动力的方法, 由于可能提供的分子负载增加肿瘤细胞积累。利用常用的方法来评估肽共轭、AC 和有效载荷的胞内积累和肿瘤靶向, 这项协议帮助研究人员在关键的初步发展阶段。

视频: Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

固定化酶和稳定的疏水盐改性的Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

科研

生物工程

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...