This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1.准备细胞培养<ol><li>购买人细胞的市售股票。 注:此协议利用HCT116结直肠癌和初级人肾小管上皮细胞（HRPTEC）。 </li><li>使对HCT116的培养500毫升培养基:Dulbecco改良的Eagle培养基（DMEM）中补充有7.5％胎牛血清（FBS）和1％青霉素/链霉素（P / S）/高葡萄糖培养基。 </li><li>作出HRPTEC的培养500毫升培养基:补充有5％FBS，1％的上皮细胞生长添加物上皮细胞培养基和1％P / S。 </li><li>制成500毫升之1×磷酸盐缓冲盐水（PBS）中的:140 mM氯化钠，3毫米氯化钾，10毫的 Na 2 HPO 4，15mM的KH 2 PO 4。调节pH至7.4，并通过高压灭菌121℃40分钟灭菌。 </li></ol> 2.细胞培养启动<ol><li>小心吸从80-90％汇合盘介质，而不会干扰Ť他的细胞。 </li><li>每培养皿等分试样3-5毫升1×PBS中，每个烧瓶轻轻摇动以覆盖培养皿的表面面积，并小心地吸出1×PBS中。重复第二1X PBS洗。 </li><li>等分试样的3ml的0.05％胰蛋白酶 - 乙二胺四乙酸（EDTA）到各培养皿并轻轻摇动以均匀涂层用胰蛋白酶-EDTA细胞。孵育在37℃下2-3分钟。 </li><li>转移分离的细胞入15ml离心管中并离心以4,000 xg离心90秒。 </li><li>在1毫升完全DMEM中悬浮细胞沉淀。 </li><li>算使用血球细胞。计算需要多少无热原和聚苯乙烯百毫米细胞培养皿以达到每平方厘米大约2,500-5,000细胞的初始接种密度。 </li><li>在步骤在37℃水浴中30-45分钟1.2或1.3制成预暖完整细胞培养基。 </li><li>净化培养基瓶和细胞小瓶，用70％的乙醇。从这点向前所有操作要无菌条件下进行。 </li><li>等分试样6毫升完全DMEM成使用在步骤2.6中提到的接种密度内冷冻细胞的整个体积需要作为100多MM细胞培养皿。 </li><li>传送在步骤2.6到每个100mm的培养皿计算细胞悬浮液的体积。手动摇动每个板到板的表面上均匀地分布的细胞。 </li><li>放置在孵化器中播种百毫米细胞培养皿于37℃在5％CO 2气氛中 。允许的下一步骤之前细胞孵育至少24小时。 </li></ol> 3.继代培养<ol><li>查看在显微镜下各细胞培养皿以确定细胞汇合的（％细胞覆盖培养皿的面积）的比例。返回细胞的孵化器和预温完全培养基和1x PBS。继续进行下一步骤，如果所述细胞是80-100％汇合。 </li><li>小心吸出用过的培养基，而不会干扰细胞在每个培养皿中。 </li><li>每培养皿等分试样3-5毫升1×PBS中，每个烧瓶轻轻摇动以覆盖培养皿的表面面积，并小心地吸出1×PBS中。重复第二1X PBS洗。 </li><li>等分试样3毫升0.05％胰蛋白酶-EDTA的到各培养皿并轻轻摇动以均匀涂层用胰蛋白酶-EDTA细胞。孵育在37℃下2-3分钟。 </li><li>在此孵化，分装在规定的10 ml完全培养基到尽可能多的培养皿获得每平方厘米2 2,500-5,000细胞的细胞密度。 </li><li>当细胞已经从板取下，迅速加入3毫升1×定义胰蛋白酶抑制剂和轻轻摇动培养皿1分钟，以确保所有的胰蛋白酶-EDTA的已中和。 </li><li>转移分离的细胞进入无菌15ml离心管中，放在一边。添加3毫升1×PBS中的菜，以收集任何残留的细胞，并传送到相同的15ml离心管中。 </li><li>通过离心150 XG颗粒细胞5分钟。 </li><li>吸出上清液，重悬细胞沉淀在3毫升完全培养基。 </li><li>算使用血球细胞和种子含有15 ml完全培养基，得到每平方厘米2,500-5,000细胞的细胞密度150毫米培养皿。使用两个150毫米每个帽结合试验。 注:通过液体介质氧扩散到细胞是依赖于量36。它建议保持实验之间保持一致的介质的体积是否使用在悬浮粘附细胞或细胞。媒体可以预调节（在工作站孵育如讨论讨论24小时），以避免在扩散这些变异。 </li><li>放置在孵化器中播种培养皿于37℃在5％CO 2气氛中 。允许在进行下一步骤之前，细胞孵育至少24小时。 </li></ol> 4. Physioxic曝光<ol><li>查看在显微镜下的细胞。对于BES吨的结果，确保细胞是一个24小时曝光70-80％汇合，50-60％汇合为一个48小时的曝光，而对于72小时曝光40-50％汇合。 </li><li>放置细胞进入所需时间（24，48，或72小时取决于实验）缺氧工作站。 </li><li>设置工作站到适当physioxia。 注:例如，3％O 2的设置将5％ 的 CO 2和92％N 2陪同。 注:如果动态范围内的氧气波动具体时间表，节目时间表所希望的过到仪器或手动调节在所需的时间间隔中的氧设置。 </li><li>湿度设定为60％。该工作站的气氛非常干燥尽管如此，和媒体将在很长的实验（&gt; 48小时）显着蒸发。保持在细胞培养烧瓶介质储备在工作站内（使介质与工作站中平衡），以补充介质在细胞培养DIS他是。 注意:任何解决方案，将与之前的裂解（例如PBS和胰蛋白酶-EDTA）的细胞相互作用应预调节24小时的缺氧工作站内使用前，使溶解氧与大气中的氧气36达到平衡。 </li><li>保持工作站，直到裂解细胞内。 </li></ol> 5.准备缓冲器，用于帽结合测定<ol><li>制备1×Tris-缓冲盐水（TBS）。 <ol><li>在800毫升的dh 2 O中，溶解8.76克NaCl和6.05克Tris碱。 </li><li>使溶液以使用1M的HCl的7.4的最终pH值。 </li><li>使终体积到使用DH 2 O 1升</li></ol></li><li>制备非变性裂解缓冲液。制备裂解缓冲帽结合测定当天。作出额外的裂解液洗空白琼脂糖珠和γ氨基苯基M 7 GTP琼脂糖C10联珠（步骤7.9和7.13）。 <ol><li> 7毫升的dh 2 O，加160微升5米NAC升，160微升的1M的Tris-HCl pH 7.4的40微升的200mM的NaF，40微升的1M MgCl 2的，和40微升1 1mM原钒酸钠。 </li><li>添加40微升的Igepal至7 ml溶液。切吸管尖，以帮助找回的Igepal，因为它是非常粘稠。 </li><li>吹打向上和向下，或涡流混合，7 ml溶液，直到所有的Igepal已经解散。 </li><li>升高溶液至8毫升的最终体积，并保持在冰上。 </li></ol></li><li>准备4X十二烷基硫酸钠（SDS）-PAGE样品缓冲液。 <ol><li>在一个烧杯中，将结合16毫升的1M的Tris-HCl pH 6.8的，12.64毫升甘油，和8毫升二硫苏糖醇（DTT）。 </li><li>添加3.2克SDS和0.16g溴酚蓝。允许粉末通过用磁力搅拌棒混合充分溶解。 </li><li>分装成的1.5 ml离心管中并储存在-20°C。 </li></ol></li></ol> 6.细胞裂解<ol><li>制备非变性裂解缓冲液，并保持在冰上日的日Ë帽结合试验。 </li><li>预暖1×PBS中和胰蛋白酶在37℃水浴中30分钟。 </li><li>正在执行等分试样980微升裂解缓冲液成每个帽结合测定1.5ml微量离心管中。 </li><li>添加4-（2-氨基乙基）苯磺酰氟盐酸盐的10微升和10微升100×蛋白酶抑制剂鸡尾酒的到980微升裂解缓冲液中。置于冰上。 </li><li>丢弃以150毫米的菜媒体在缺氧工作站内的废弃物容器中。 </li><li>清洗细胞3-4毫升温1X PBS，并放弃所有的液体。 </li><li> 1毫升温暖0.05％胰蛋白酶-EDTA添加到每个培养皿让坐在2分钟，或直至细胞不再附着到板上。 </li><li>使用移液管，一个板的细胞转移到1.5ml微量离心管中。根据需要，使细胞的每个板被转移到一个单独的1.5 ml离心管重复。 </li><li>离心1.5 ml离心管在6000 XG 90秒ŤØ颗粒细胞。 </li><li>吸用吸管胰蛋白酶，而不会干扰沉淀。如果颗粒被干扰，重新离心1.5 ml离心管在6000×g离心90秒。 </li><li>轻轻吹打200微升PBS的入管刚好沉淀上述要用温水1X PBS细胞。重新离心如果粒料被干扰。 </li><li>吸出PBS，而不会干扰沉淀。 </li><li>从1毫升吸管将500μl制备的裂解缓冲溶液到第一蜂窝沉淀。完全由上下吹打悬浮颗粒。 </li><li>结合重悬的细胞与所述第二细胞沉淀并通过上下抽吸悬浮所述第二沉淀。直到所有的粒料已经被组合和再悬浮每个样品重复此过程。 </li><li>结合裂解物与在1.5ml微量离心管中剩余的500μl的裂解缓冲液中。 </li><li>从缺氧工作站删除样本。 注:加入裂解缓冲液之后，将LL后续步骤如缺氧工作站被设定为37℃及以下的步骤将被执行的冷（4℃或冰上）在环境空气中进行。 </li><li>裂解用轻轻搅动在4℃下旋转样品1.5-2小时的细胞。 </li><li>离心裂解的细胞以12,000 xg离心15分钟，在4℃以除去细胞碎片。 </li><li>转移裂解物到新的1.5 ml离心管并弃沉淀。 </li><li>保留上清的一部分（5-10％），以用作将来Western印迹分析全细胞裂解物的输入控制。 </li></ol> 7.帽结合测定<ol><li>对于每个样品，转移空白琼脂糖珠控制浆料的50微升和50微升γ氨基苯基-间7 GTP琼脂糖C10联珠浆液以分离1.5ml微量管中。用剪刀取出吸移管的尖端，以便于珠浆的汇编。 </li><li>颗粒珠bý离心在500 xg离心浆液30秒。 </li><li>小心地取出上清，500微升TBS重悬珠。 </li><li>重复步骤7.2和7.3。沉淀小珠，在500×g离心30秒，并去除上清。 </li><li>从步骤6.19传送包含裂解物上清液到包含空白琼脂糖珠的1.5 ml离心管。 注:空白琼脂糖珠充当信息预先步骤，以从非特异性地与胎圈交互的裂解物除去蛋白质。 </li><li>孵育在4℃下10分钟，温和搅拌。 </li><li>通过在500 xg离心离心30秒沉淀空白琼脂糖珠。 </li><li>转移裂解物到含有γ氨基苯基-间7 GTP琼脂糖C10联珠的1.5 ml离心管。 </li><li>通过再悬浮于500μl裂解缓冲液的珠子，通过在500 xg离心离心30秒造粒的珠，并丢弃superna洗空白琼脂糖珠重要的。重复此四次总共五个洗涤。 </li><li>重悬在1×SDS-PAGE样品缓冲液空白琼脂糖珠，并在95℃下沸腾的珠90秒。储存在-20℃下以供将来Western印迹分析，观察感兴趣的蛋白质是否结合非特异性到胎圈。 </li><li>孵育轻轻摇动的γ氨基苯基-间7 GTP琼脂糖C10联珠1小时的溶胞产物在4℃下以捕获帽结合蛋白。 </li><li>通过在500 xg离心离心30秒沉淀γ氨基苯基-间7 GTP琼脂糖C10联的珠。弃去上清液。 </li><li>通过重复步骤7.9洗γ氨基苯基-间7 GTP琼脂糖C10联的珠。 </li><li>重悬在600微升裂解缓冲液的珠子。 </li><li>添加GTP到1mM的终浓度。 </li><li>孵育γ氨基苯基-间7 GTP琼脂糖C10联珠+ 1mM的GTP轻轻搅动在4℃下1小时。注:此将离解非特异性具有m 7 GTP相互作用的蛋白质（ 即，也与非甲基GTP交互）。 </li><li>通过在500 xg离心离心30秒沉淀γ氨基苯基-间7 GTP琼脂糖C10联的珠。 </li><li>将上清转移至新的1.5 ml离心管，加入200微升4×SDS-PAGE样品缓冲液（50mM的Tris-HCl，100mM的DTT，2％SDS，0.1％溴酚蓝和10％甘油）中的。储存在-20℃下对GTP对照样品供将来免疫印迹分析37。重复步骤7.9洗珠。 </li><li>重悬在1×SDS-PAGE样品缓冲液（50mM的Tris-HCl，100mM的DTT，2％SDS，0.1％溴酚蓝和10％甘油）中，并煮沸珠粒在95℃下90秒。储存在-20℃第m 7 GTP结合的级分以供将来免疫印迹分析37。 </li></ol>

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The authors declare that they have no competing financial interests.

在暴露于生理氧条件的人细胞帽结合蛋白质的分析可允许的新颖氧 - 调节翻译起始因子的鉴定。这些因素为mRNA或其它帽相关蛋白的5&#39;帽的亲和性可以通过其关联的强度被测量到m 7 GTP联的琼脂糖珠。这种技术的一个需要注意的是它测量蛋白裂解后的帽结合的潜力，但它是该保持蛋白质 - 蛋白质相互作用和翻译后后修饰非变性条件下进行。几个蛋白 - 蛋白相互作用介导的eIF4E家族的5&#39;帽的结合。该4E-BP结合并阻断其与eIF4G相互作用和防止43S-开始前38复杂的招聘压抑eIF4E的。所述的eIF4E / 4E-BP复杂保持结合到5&#39;表达帽，从转录阻止任何引发，并能用作eIF4E的抑制以m 7 GTP亲和柱的标记。相反，eIF4E的与eIF4G对M 7 GTP列的相互作用可能是积极的翻译起始的标志。翻译后修饰是调节的翻译起始因子的活性的另一种方法。例如，通过抑制Mnk1的eIF4E的磷酸化可以增加其亲合力的5&#39;的mRNA帽39。由干扰素刺激基因15（ISG15）编码的蛋白质改性剂是增加在经由病原体感染40响应于干扰素诱导eIF4E2的帽结合活性的PTM。该ISG15基因包含在其启动子表明，该系统能够在低氧气调节eIF4E2缺氧反应元件。很少是关于翻译后修饰可如何在生理相关低氧条件改变帽结合蛋白的活性已知的。这种技术，随后通过质谱分析可以揭示新颖氧 - 调节和生理有关翻译后修饰第在介导的翻译起始因子的活性。 与一个m 7 GTP的帽类似物捕获的另一种方法是免疫沉淀的已知帽结合蛋白如eIF4E的或eIF4G并执行质谱来识别相关联的蛋白质。这一战略的局限性在于帽结合蛋白往往有替代细胞的作用，如内应力颗粒41或帽独立的翻译1,4。因此，利用M 7 GTP帽类似物是隔离帽结合蛋白，研究新的上限相关的蛋白质，特别是当最好的，最可靠的方法。利用一个m 7 GTP的帽类似物的一个限制是某些帽结合蛋白如清除剂脱帽酶DCPS不仅与第m 7 GTP的mRNA帽相互作用，而且还需要用于结合42的第二基材。此外，脱帽酶在一般情况下，如DCP1 / DCP2复杂，可以HYDRolyze M 7 GTP，有效降低树脂产能。因此，一些帽结合蛋白可以在此分析丢失。要绕过这些告诫，耐酶的mRNA帽类似物可以使用。两个非水解帽类似物，单核苷酸M 7 GpCH2pp或二核苷酸米7的GpCH2ppA已经显示捕捉DCPS，DCP1和DCP2 43。这些mRNA的帽类似物是不可商购，但必须从头化学合成。这个协议的另一个限制是，只有帽结合经由"捎带"的蛋白质或蛋白质与帽结合蛋白相互作用将被捕获。而帽依赖性翻译起始是起始的主要形式，帽非依赖性机制在细胞应激1的条件尤其普遍。然而，在该技术在翻译起始6-8,24领域出现的趋势，确定新型应激诱导因素的上下文和PArticipate在帽依赖性开始是研究的一个大有前途的领域。 在这个协议中要考虑的另一个因素是在媒体的氧。在液体培养基中培养的细胞提供了细胞和大气之间的屏障。它已经显示，液体介质可以采取多达24个小时与大气气体组成36达到平衡。因此，细胞必须在24小时中所需的氧气供应裂解前培养，或媒体可如果需要更短的孵育进行预调节。预调节包括保持在缺氧工作站的介质中的无菌培养烧瓶中，在至少24小时，允许气体交换。引入到缺氧工作站内的细胞的任何氧化溶液可能迅速改变细胞信号途径，例如是在该协议被检查的帽依赖性翻译起始。由于在细胞中的氧传感途径的敏感性，任何解决方案，将与之前的裂解例如PBS和胰蛋白酶-EDTA细胞相互作用也必须预调节至少24小时。裂解和裂解后的洗涤缓冲液，必须使用冷，并在37℃缺氧工作站，因此不预调节。虽然一些氧依赖性修饰蛋白质可能是活性裂解后，需要的冷缓冲液以最小化的酶活性和蛋白质 - 蛋白质或蛋白质-RNA复合物的"混洗"，它可能导致假象。的修改本协议，以增加严格可能是预先条件裂解和裂解后的洗涤液24小时，然后在使用前在工作站内培养他们在冰上。媒体和缓冲液不应该保持在缺氧工作站为三天以上，因为这些解决方案会集中，由于水的蒸发。对于长期的研究（&gt; 3天），种子细胞的初始确认金额必须仔细考虑。随着细胞分裂和盘的表面面积变得拥挤，其它应力，例如媒体酸化可能影响帽结合蛋白的活性。在实验的最后一天，将细胞应当具有80-90％铺满。 有此协议的四个关键步骤:，细胞的使用量，洗涤珠保持在缺氧工作站，直到裂解细胞，和非甲基化的GTP控制。 1）有几种方法来维持在低氧细胞。大多数的这些包括小室可以容纳仅细胞培养皿，但是任何操作或细胞裂解，必须在环境空气中的腔室外部执行。氧传感机械部件，如缺氧诱导因子被激活或在一个或两个分35的事失活。因此，如前面提到的，在一个缺氧工作站裂解细胞是至关重要的，以确保与相应的氧可用性相关联的帽结合蛋白的活性。 2）细胞数量在此协议（二150毫米菜）建议是足够的强M 7 GTP相互作用者如eIF4E的家庭或eIF4F复杂（eIF4A和eIF4G）的成员。多个小区，至少五个150毫米菜，可以要求检测具有短暂的相互作用或仅通过eIF4F第m 7 GTP的mRNA帽准蛋白质。 3）与细胞裂解物孵育他们后洗珠可在严格的和不严格的方式来执行。这里介绍的协议提供了，其中的裂解缓冲液用于洗涤的珠五次严格的选择。如果较弱相互作用蛋白质帽结合蛋白是感兴趣，可以执行较少的洗涤液并利用Tris缓冲生理盐水代替裂解缓冲液。 4）虽然是重要的预清除与未缀合的琼脂糖细胞裂解物以除去非特异性地与胎圈相互作用的蛋白质，部分帽结合蛋白可以不是真正的mRNA帽结构区分，甲基化的GTP（M7 GTP）和非甲基化的GTP。一个这样的蛋白质是eIF4E的同源eIF4E3 44。与GTP洗脱珠粒会撞出也识别非甲基GTP，这可能是感兴趣的任何蛋白质。识别两个M 7 GTP和GTP尚未蛋白质的生物相关性被完全阐明。这是通过涉及eIF4E3（这是与一个保守的帽结合结构域的善意帽结合蛋白）相对eIF4E与eIF4E2蛋白，其执行从非甲基GTP识别M 7 GTP的少数出版物强调。 这种技术所提出，可以为有针对性的方法来识别感兴趣M 7 GTP相互作用物或通过分析通过质谱M 7 GTP洗脱广泛的办法进行。其他一些技术可以按照physioxic曝光（协议4）分析帽结合的活动，如亚细胞分离，免疫，免疫共沉淀，一第二多聚核糖体分析。平移控制正在成为一个平等的贡献者基因表达的转录。利用这种技术来研究帽结合复合物的活性和组合物将棚上帽依赖性翻译起始如何响应于低氧调节光。

平移控制正在成为基因表达的转录调节同样重要的一步，尤其是在细胞应激1期。翻译调控的一个重点是在（M 7 GTP）5&#39;的mRNA 2帽引发的限速步骤，其中的蛋白质合成的第一个步骤涉及真核细胞起始因子4E（eIF4E的）的结合到7-甲基。 eIF4E的是一个名为eIF4F三聚体复杂，包括eIF4A，一个RNA解旋酶和eIF4G，对其他翻译因子和核糖体40S 3招聘要求支架蛋白的一部分。在正常生理条件下，绝大多数的mRNA经由帽依赖性机制翻译，但在细胞应激期间人类mRNA的约10％含有5&#39;非编码区，可能允许不依赖帽的翻译intiation 1,4。帽依赖性翻译在历史上同义词OU中与eIF4F，但是，eIF4F的特定压力变化已经成为一个热门话题5-8。 各种细胞应激引起雷帕霉素通过复杂1（mTORC1的）的哺乳动物目标受到抑制eIF4E的活动。这种激酶变得在压力下，这导致在其目标之一的增加的活性受损，所述4E结合蛋白（4E-BP）。非磷酸化4E-BP结合eIF4E与阻断其与eIF4G引起帽依赖性翻译9,10的压制交往的能力。有趣的是，一个名为eIF4E2（或4EHP）的eIF4E的同源物具有4E-BP 11低得多的亲和力，或许允许它逃避压力介导的镇压。事实上，最初表征为翻译阻遏由于其缺乏与eIF4G 12相互作用，eIF4E2发起数百包含在其3&#39;端非编码区的RNA缺氧反应元件的mRNA的低氧应激6,13-在翻译。这种激活我s至与eIF4G3，RNA结合蛋白基序4，和低氧诱导因子（HIF）2α构成低氧eIF4F复杂，或eIF4Fħ6,13-相互作用来实现的。如正常条件下的阻遏，eIF4E2结合与GIGYF2和ZNF598 14。这些复合物，在部分地通过琼脂糖联M 7 GTP的亲和树脂识别。这种传统方法15在翻译领域的标准，是最好的，最常用的技术，在下拉隔离帽结合复合物和体外结合试验16-19。作为帽依赖性翻译机器正在成为灵活性和适应性与间变化部分6-8,13，这种方法是一个强大的工具，迅速查明参与应激反应小说帽结合蛋白。此外，eIF4F的变化可能有广泛影响几个真核模型系统出现使用eIF4E2同源的应激反应等如拟南芥 20， 裂殖酵母 21， 果蝇 22和线虫 23。 有证据表明，在eIF4F变化可能不严格限于强调的条件，但参与正常生理学24。氧气供给到组织中（在毛细管两端）或组织中（通过微电极测定）从2-6％在脑中25而变化，在肺26 3-12％，在肠道27，在4％3.5-6％肝脏28，在肾脏29 7-12％，在肌肉30 4％，在骨髓中31 6-7％。细胞线粒体含有比1.3％的氧气少32。这些值比周围的空气，其中细胞常规培养更接近缺氧。这表明什么以前被认为是特定缺氧细胞过程可以是在生理环境相关。有趣的是，eIF4F和eIF4F ^ h </suP&gt;积极参与不同池或mRNA的类的翻译起始于暴露于生理氧或"physioxia"24几个不同的人细胞系。低氧也推动适当的胎儿发育33和电池通常具有较高的增殖率，寿命长，少的DNA损伤和physioxia 34较少的一般应激反应。因此，eIF4F H是可能在生理条件下选择的基因的表达的关键因素。 这里，我们提供了一个协议，以在固定的生理氧条件下或在这是可能的更具代表性的组织微环境的动态波动范围培养的细胞。这种方法的一个优点是，细胞在缺氧工作站内裂解。它是不是经常不清楚如何从缺氧细胞培养细胞裂解转变在其他协议执行。细胞常常首先从一个小缺氧孵化器中删除是前的裂解，但是这种暴露于氧气可能影响生化途径以氧气的细胞反应是快速（一个或两个分）35。某些帽结合蛋白需要用第二碱相互作用或能水解第m 7 GTP，因此一些帽交互件可在纯化过程被遗漏。琼脂糖联以酶促抗帽类似物可以在该协议被取代。探索通过这里描述的方法的活性和eIF4F H和eIF4F的其它变型的组合物将在该细胞中的生理条件或胁迫应答利用复杂的基因表达的机械线索。

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
			<td>Antibiotic component of DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
			<td>Hypoxia workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100</td>
			<td>SDS</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>56508</td>
			<td>Na3VO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td>Component of buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</td>
		</tr>
	</tbody>
</table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

在一个m 7 GTP亲和柱的响应eIF4E与eIF4E2氧帽结合力分析 图1和2表示响应于氧的波动在两个人细胞系中两个主要的帽结合蛋白的典型M 7 GTP的亲和纯化的蛋白质印迹:原代人肾小管上皮F中igure 1和大肠癌细胞（HRPTEC）（ HCT116）在图2中。细胞维持在所指示的氧气供应24小时，以确保在液体介质中的溶解的氧已与缺氧工作站内的空气平衡。输入车道（IN）表示的全细胞裂解物的10％的M 7 GTP联琼脂糖珠加入到捕获帽结合蛋白之前。此输入车道用作基线测量富集的在第m 7 GTP下拉靶蛋白。对GTP车道是从m 7 GTP珠子用1mM GTP分别洗脱的洗脱物。这个步骤允许检测还识别非甲基化的GTP蛋白。帽结合蛋白eIF4E与eIF4E2识别M 7 GTP的具体，但它们的同系物eIF4E3（此处未示出）还结合非甲基GTP。 eIF4E与eIF4E2向5&#39;的mRNA帽结构（M 7 GTP）结合的能力可以用m 7相对于在输入车道的强度对GTP柱在其条带的强度相比较（总的可用池）测定。 这种量化可以通过测量蛋白质带的像素强度与图像分析软件三次生物学重复如ImageJ的进行。结果表示为的密度单位（RDU）±均值的标准误差相对的装置。原始值之前正常化从实验和对照样品由未配对双尾学生t检验进行比较。 P &lt;0.05为差异有统计学显著。这代表实验表明，这两种细胞系对他们的eIF4E与eIF4E2使用氧气的不同范围。 图1显示了在HRPTEC仅eIF4E的结合强至m 7 GTP在8％O 2（ 图1A），这两个eIF4E与eIF4E2被显著具有m 7 GTP从3-5％ 的 O 2（ 图1B - C）相关联，而且只有eIF4E2在1％ 的 O 2（ 图1D）强烈结合到m 7 GTP。在图2中 ，在HCT116细胞中，这些帽结合蛋白的氧依赖性用法不同的是:唯一的eIF4E显著结合到m 7 GTP的在12％ 的 O 2（ 图2A），这两个帽结合蛋白显著结合到m 7 GTP在5-8％O 2的范围（ 图2B - C）中 ，并且仅eIF4E2在3％ 的 O 2（ 图2D）显著结合到m 7 GTP。从GTP的M 7 GTP列没有洗脱表明，eIF4E与eIF4E2特定于M 7 GTP和不承认非甲基化的GTP。条形图代表使用ImageJ三次生物学重复量化。我们的结果表明，两个主要的帽结合蛋白，eIF4E与eIF4E2，在其与mRNA M 7 GTP的帽结构结合在氧依赖性的能力有所不同。 根据以前的文献，这两种蛋白质发起mRNAs的独特类别的翻译，这种转变在帽结合活性可能导致产生的，以适应在氧气供应的变化不同蛋白质组。这种技术可以用于表征与该5&#39;的mRNA帽相互作用的新型翻译起始因子（或帽结合蛋白）通过有针对性的免疫印迹法或一种广泛的做法，如一个m 7 GTP洗脱的质谱分析。 <img alt="图1" src="/files/ftp_upload/55112/55112fig1.jpg" /> 图1:3-5％O 2 的HRPTEC physioxia期间eIF4E与eIF4E2活动重叠 。使用M 7在人肾小管上皮细胞的GTP珠粒捕获测定（HRPTEC）裂解物暴露于（A）的8％，（B）的5％，（C）的3％或（D）的1％ 的 O 2 24小时。 GTP，GTP洗来衡量米7的GTP特异性; M 7 GTP，GTP后洗净势必米7的GTP珠蛋白。代表性的Western印迹显示，并从至少三个独立实验的数据用ImageJ的量化和表示为相对密度单位（RDU）的相对于全细胞裂解液的10％输入（IN）。 * p &lt;0.05（非配对双尾学生t检验）被认为是在帽结合级分（M 7 GTP）到IN比较eIF4E的或eIF4E2的富集时显著变化。数据，平均值±三个独立实验的平均值（SEM）的标准误差。这项研究最初发表在生物化学杂志。 Timpano专门S.和Uniacke，在生理氧气培养J.人体细胞利用两个帽结合蛋白招募不同的mRNA翻译。 2016年; 291（20）:10772-82 24。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55112/55112fig1large.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/55112/55112fig2.jpg" /> 图2. eIF4E与eIF4E2活动从5-8 HCT116 physioxia期间重叠％O 2。使用M 7 GTP珠在暴露于（A）中的12％，（B）8％，（C）的5％或（D）的3％ 的 O 2 24小时HCT116人结肠直肠癌裂解物捕获测定。 GTP，GTP洗来衡量米7的GTP特异性; M 7 GTP，GTP后洗净势必米7的GTP珠蛋白。代表性的Western印迹显示，并从至少三个独立实验的数据用ImageJ的量化和表示为相对密度单位（RDU）相对于全细胞裂解液的10％输入（IN）。 * p &lt;0.05（非配对双尾学生t检验）被认为是在帽结合级分（M 7 GTP）到IN比较eIF4E的或eIF4E2的富集时显著变化。数据，平均值±三个独立实验的平均值（SEM）的标准误差。这项研究最初发表在生物化学杂志。 TIMP肛门，S和Uniacke，在生理氧气培养J.人体细胞利用两个帽结合蛋白招募不同的mRNA翻译。 2016年; 291（20）:10772-82 24。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55112/55112fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

analysis-cap-binding-proteins-human-cells-exposed-to-physiological

Nanyang Technological University

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JoVE Journal

Genetics

7.7K 次观看.  University of Guelph. Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

视频: Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...