这项工作是由以色列科学基金会[资助1721至1712年，1368年至1312年，以及1444年至1416年（到AP）的支持。 AP隶属于Brettler中心和David R.布鲁姆中心，药学院，耶路撒冷希伯来大学。

1.结扎兴趣的基因到载体阻遏网站<ol><li>获得诱导载体如的pcDNA5 / FRT / TO或pcDNA4 / TO。 </li><li>通过在硅片 DNA分析软件4分析在该还设有在所选择的载体的多克隆位点的基因的中间任何潜在易感限制性位点感兴趣的基因。 </li><li>使用标准的重叠PCR技术4中 ，侧翼具有两个选择限制性内切酶识别位点（即未在感兴趣的基因中发现）感兴趣的基因，第一起始密码子前插入，并在终止密码子之后的第二个。 </li><li>消化这两个矢量和与酶"工作温度一小时，然后加入一单位小牛肠磷酸酶（CIP）的限制性内切酶（在步骤1.2选择），以仅30分钟的载体反应，为了避免自插入结扎。 </li><li>加载消化的DNA上样于1％琼脂糖凝胶并电泳9分离裂解的片段。 </li><li>使用UV光，切带刀片凝胶的含有载体的所需的段的段，并插入到被连接。 </li><li>提取从琼脂糖凝胶片的DNA区段（使用市售的DNA提取试剂盒），并在260nm处测量其由分光光度计最终浓度。 </li><li>结扎感兴趣的基因导入用T4连接酶，在室温下进行20分钟的诱导选择的载体。为了增加用该载体插入结扎的概率，使用3:插入物1摩尔比载体。对于控制，准备连接反应只包含载体。 </li><li>对于细菌转化，增加单独载体，载体+基因的10毫微克到50微升主管大肠杆菌 。在冰上孵育管30分钟，随后通过45秒孵育在42℃。立即放回在冰上两分钟和转化的细菌转移到500微升LB培养基。在220rpm搅拌下于37℃孵育一小时。 </li><li>对于成功转化的细菌的选择，板100微升在含有合适的抗生素准备的LB琼脂平板上的每个反应（如在1.9中所述）的（ 例如 ，使用100微克/毫升氨苄青霉素时pCDNA4 / TR）。 </li><li>允许在37℃生长过夜。板可以通过在塑料石蜡膜紧紧包裹它们被储存在4℃下长达两周。 </li><li>解除用10微升末端的单个菌落，并允许过夜在3mL LB +抗生素培养基，在37℃搅拌下在220rpm生长。用感兴趣的DNA的细菌可以冷冻（-20℃），用于通过离心LB和细菌在11000 xg离心并吸出上清液短期存储。长期储存要求在-80℃冷冻细菌甘油股票。 </li><li>提取DNA THROUGH迷你准备和在260nm处5衡量分光光度计终浓度。 </li><li>测序纯化结构4证实目的基因的成功插入。可替代地，通过使用电泳分离切割段和评价它们的存在限制性内切酶和长度构建体的诊断消化也可以利用该端部4。 </li></ol> 2.培养细胞系表达四面体<ol><li>获得细胞培养系窝藏掺入其基因组DNA的四环素阻遏质粒。本文的协议将使用人胚肾293T细胞（HEK-293T）窝藏pcDNA6 / TR质粒作为例子写入。 </li><li>种子细胞在100mm组织培养板用Dulbecco改良的Eagles培养基（DMEM），补充有10％FBS，1％青霉素 - 链霉素，2mM的L-谷氨酰胺，和25mM HEPES，pH值7.3（这里:完全DMEM）中孵育ö在37 / N＃176; C和5％ 的 CO 2。与细胞的所有工作都应该在无菌条件下的生物罩来完成。 </li><li>为了保持四环素阻遏物基因的表达，吸出培养基并用补充有5微克/毫升杀稻瘟完整的DMEM更换。孵育细胞在37℃和5％的CO 2。 </li><li>一旦细胞达到80 - 90％汇合，吸出介质并轻轻用DPBS洗涤细胞两次（没有钙和镁）加热到37℃。 </li><li>为了轻轻提起细胞，而不会引起机械损伤，孵育培养在含有0.05％胰蛋白酶的DMEM温热至37℃1.5分钟。 </li><li>方框用等体积的完全DMEM中的胰蛋白酶作用。轻轻移液器上下，以解除细胞，并将它们转移到无菌管。 </li><li>离心细胞，在200×g离心5分钟。 </li><li>吸出上清液并用1mL完整的DMEM更换。吸取与细胞的溶液上下直到没有细胞团块可以看出。算，并计算使用血球细胞的数目。 </li><li>种子1 - 2×10 6个细胞在含有12毫升完全DMEM中以5微克/毫升杀稻瘟100mm组织培养皿。分裂细胞一周两次，因为它们达到90％汇合。 </li></ol> 3.转染利益的质粒转染细胞<ol><li>使用分裂细胞的相同的方法，用0.9毫升全DMEM制备的12孔板如上所述，转移细胞的孔，足以使50％汇合（〜200,000个细胞），并在CO 2在37℃下孵育过夜孵化器。 </li><li>与pcDNA4制备转染混合物/ TO含有目的基因，在惰性质粒以使总DNA的量，以1微克（如果需要），3微升脂质转染试剂和DMEM使最终体积为100μL。作为不同的蛋白质在不同的效率表示要使用的质粒DNA的最佳量变化很大。对于选rophysiological实验，包括绿色荧光蛋白在转染鸡尾酒哺乳动物表达质粒，以可视化，成功地经受转染4个细胞。 </li><li>孵育在室温转染混合物30分钟。 </li><li>转染细胞通过在12孔板滴加移液转染混合物到铺板的细胞并剧烈摇动平板，以确保DNA和转染试剂的均匀分散体。细胞应为〜在转染时80％汇合。 </li><li>孵育转染细胞O / N在37℃和5％的CO 2。 </li><li>确认细胞已成功由下述早晨在紫外灯观察EGFP的荧光信号，如果进行电转染。 </li></ol> 4.聚-D-赖氨酸（PDL）电镀细胞盖玻片/韦尔斯<ol><li>通过用70％异丙醇的乙醇溶液浇熄消毒12毫米盖玻片。干燥并放置一个单coverslIP在电生理记录一个24孔板的每个孔中。 </li><li>吸取0.1的溶液 - 0.2毫克/毫升的PDL溶液到钙成像腔室的每个盖玻片或很好。 </li><li>允许在37℃下在5％CO 2的培养箱中静置30分钟。 </li><li>用细胞培养级双蒸水（DDW）三次洗涤，每次洗涤之间进行抽吸。继最后洗净，晾透，让坐在在室温。 </li><li>为电，填充每个孔含有一个PDL涂覆有500微升全DMEM盖玻片加热到37℃。 </li><li>执行如上所述分裂转染细胞的方法相同的方法。重悬的细胞胰蛋白酶处理之后和堵塞，转印80μL的细胞（或约30,000个细胞）的每个PDL涂覆盖玻片为电记录的中心后。让细胞沉降至少1.5小时，或在5％ 的 CO 2培养箱中于37℃孵育过夜。 </li><li>对于细胞的钙成像转移和点20μL（约20,000个细胞）给每个PDL涂钙成像中心好。让细胞沉降至少30分钟，并添加180微升全DMEM到每个孔中。 </li></ol> 5.诱导基因表达<ol><li>根据制造商的说明制备的1毫克/毫升的强力霉素原液在DDW。保持原液于4℃避光长达3周。对于长期储存保持在-20°C多西环素原液。 </li><li>制备新鲜2微克/毫升（电），或3微克/毫升（钙成像）强力霉素溶液完整的DMEM，并温热至37℃。 </li><li>用于电生理记录，吸管的2微克/毫升的强力霉素溶液以每500μL以及使1微克/毫升的强力霉素的最终浓度。对于钙成像，加100微升3微克/毫升强力霉素解决方案，以每口井做出最后concentra1微克/毫升的强力霉素和灰。写下感应小时。 </li><li>孵育的时间为诱导，在37℃和所需量的5％的CO 2。 </li></ol> 6.蛋白表达的校准时间表<ol><li>通过钙成像5，10校准蛋白的表达<ol><li>制备林格氏溶液（140mM的氯化钠，2.5mM的氯化钾，1.8mM的氯化钙 ，2毫硫酸镁 ，20mM的HEPES，pH值调至7.4，用NaOH）并暖至37℃。 </li><li>准备在林格氏溶液饱和通道激动剂的浓度。因为激动剂的量当加入到含有细胞和细胞外溶液中的钙成像室中稀释，双激动剂的所需最终浓度的量。这也使得在每个孔中的关键控制的浴溶液的量。 </li><li>制备D中的非离子型的F-127的20％W / V的溶液MSO。加热直到非离子的F-127溶于该溶液至40℃。存储在RT非离子的F-127溶液，使用前它温热至40℃。非离子型的F-127用于帮助分散的荧光离子指标如的Fura-2在水溶液中的乙酰氧基甲基（AM）酯。 </li><li>抽吸培养基并用的Fura-2AM加载溶液更换（林格氏溶液补充有2 - 3微米的Fura-2AM，0.02毫克/毫升的非离子型的F-127和10mM D-葡萄糖）。 </li><li>在室温下孵育在黑暗中60分钟。 </li><li>吸的Fura-2AM的解决方案和洗涤细胞林格氏+ 10毫米D-葡萄糖溶液除去细胞外染料。重复此步骤两次。 </li><li>留在每个200微升林格氏+ 10mM的D-葡萄糖溶液以及孵育在黑暗中在RT 30分钟。 </li><li>放置在显微镜鼻子上面的一块舞台室，并与支架固定。 </li><li>打开灯，照相机和显微镜，并选择的Fura-2滤光片在510纳米WAV检测发射elength。 </li><li>调整所需的放大倍率和重点细胞在选定的领域。 </li><li>在黑暗中，减去背景光并设置曝光时间。 </li><li>在的Fura-2装载的细胞的通过激励他们在340和380纳米的波长所需的速率，记录荧光响应设置图象采样。三百八十分之三百四十〇信号的比率是细胞内Ca 2+浓度为TRPV1活性的指标，因此也。 </li><li>在200μL的林格氏液加2微米辣椒素通过移液以评价TRPV1的表达水平来创建为1μM辣椒素的最终饱和浓度。 </li><li>根据诱导时间分析TRPV1响应并确定协议的目的蛋白质。 </li></ol></li><li>使用膜片钳技术4中 ，11的全细胞组态调节异源系统蛋白质表达。 <ol><li>准备ŧ他浴（mM计）140 NaCl的，2.3氯化钾，2- 硫酸镁 ，5 HEPES，和5 2-（N-吗啉代）乙磺酸（MES），的溶液调节至pH 7.4，用NaOH。 </li><li>制备在细胞外溶液饱和通道激动剂的浓度。 </li><li> 4MΩ - 用内径为1.10毫米（ID）和火抛光到2的电阻上拉玻璃电极。 </li><li>准备（MM）130氯化钾，氯化钠4的吸管溶液2 硫酸镁 ，氯化钙0.5 2，1 EGTA和10 HEPES调节pH值至7.2 KOH。过滤器在使用前吸管的解决方案。每次实验之前，一个单一的，火抛光的玻璃吸管被回通过在吸管溶液浸没的尖端为至少一分钟填充，然后用熔融的和拉伸的尖端与填充有相同的移液管溶液的注射器填充。 </li><li>轻轻抬起与铺板的细胞的单个PDL涂覆盖玻片从井到填充有在RT约2mL槽液35毫米的塑料陪替氏培养皿中。一张EP下观察I-荧光显微镜。 </li><li>定位EGFP阳性细胞，与啶显微镜观察，对所制备的盖玻片并移动到可视域的中心。 </li><li>附加填充吸管的显微的吸移管支架，并注入正压少量进入系统，以避免胞内吸移管溶液的污染。它降低到刚刚高于GFP - 阳性细胞，检查适当性。 </li><li>作为吸管接触细胞，应用以创建玻璃吸管和细胞膜之间GΩ的密封负压少量。 </li><li>吸的短，尖脉冲，打破细胞膜，使吸移管以与所述细胞的细胞内内容物接触。 </li><li>开关放大器模式为全细胞，并降低贝塞尔滤波器和输出增益为1 - 5 kHz和0.5α，分别为。 </li><li>记录到的饱和电流响应通道激动剂的浓度使用电压斜坡或无间隙协议。 </li><li>由于表达效率不同的蛋白质之间切换，重复使用不同的感应长度乘以录音。 </li></ol></li><li>确定理想的诱导时间为单声道录音4。 <ol><li>制备（毫摩尔）150钠葡糖酸盐，15的NaCl，5 EGTA，和10 HEPES，调节至pH 7.4，用NaOH中的溶液，如移液管溶液。对于槽液中，使用步骤6.2.1解决方案。 </li><li>使用ID0.86毫米玻璃吸管火抛光到10的阻力，重复整个过程的细胞 - 12MΩ，并设置保持电位为-40 mV的。 </li><li>上创建如前所述和破裂膜的膜形成密封。 </li><li>使用显微操作，解除与膜补片从小区离开吸管。 </li><li>含膜膜片吸管直接旁边的灌注体系中的地位。 </li><li>低位的t他贝塞尔滤波器和输出增益为2 - 5 kHz和10α，分别为。 </li><li>灌注饱和激动剂浓度到膜修补，评估如果补丁根据响应中包含一个或多个通道。 </li><li>分析成功地记录针对施加到细胞中的诱导时间单通道补丁的数量。根据所期望的结果，调整诱导时间。 </li></ol></li></ol>

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作者什么都没有透露。

转染蛋白表达和研究一种广泛使用的协议，与许多不同的变化，以提高表达的一致性和稳定性。瞬时转染试剂提供一种简单，易于使用的协议，其中感兴趣的细胞和蛋白质可在数小时内从转染时进行分析以过夜。不幸的是，当分析模式需要蛋白的表达，如电2单声道录音的水平一致这种做法是不可预知的。可替代地，四环素阻遏表达系统下的稳定细胞株已开发提供了一种方法，以控制感兴趣12的蛋白质的表达水平。这种方法提供了与引入四环素衍生补充剂，诸如多西环素的培养所有细胞的一致表达。虽然这允许蛋白水平的一致性和精确的控制，叔他说去建立一个稳定的细胞系的时间和精力，不便和不利的研究各种各样的蛋白质8时。 这里介绍的协议提供了中间的路线的解决提供了当前转协议。通过瞬时转可控启动下一个基因插入具有一个春节阻遏细胞，我们可以同时避免冗长而复杂的程序，实现可控制的表达。在这里，我们使用具有钙成像和电分析的非选择性阳离子通道的TRPV1。我们发现，提供了理想的诱导时间为单通道分析此特定蛋白质的受控表达为2 - 3小时温育1微克/毫升的强力霉素（ 图1 - 3）。在更大范围内，这种技术也可以适用于任何感兴趣的蛋白质。该协议还提供了一个解决方案，以控制表达式时，LAR不同的蛋白质或蛋白质序列的突变的戈范围是感兴趣和创建对于每个变体的稳定细胞系是不切实际的。重要的是，虽然结果在这里代表了适用于TRPV1表达的最佳时间和浓度，每个基因都有其单独的和具体的翻译和贩运的步伐，从而每种蛋白质的标定和表达时间将有很大的不同13。 尽管该技术提供了一个方便的解决方案，以控制的表达，但也不是没有局限性。只有市售的细胞系窝藏四环素系统可与该系统中，如果这些特定细胞系是不适于分析的类型是要执行它给出了一个问题地使用。相比稳定转线另一缺陷是，不是所有的细胞进行转染，并且因此并不是所有的细胞表达目的蛋白质。总体而言，我们提供给控制蛋白E的便捷方式XPRESSION在可应用到多种生物医学研究的异源系统。

异源表达系统是最广泛使用的技术来研究细胞功能1众多之一。其低的内源性蛋白图谱，最少的维护要求，可靠的增长，并采取和表达外源DNA的能力已使细胞系，如人类胚胎肾（HEK293）和中国仓鼠卵巢（CHO）几乎是必不可少的，以生物学研究2，3。采用异种系统的研究领域包括膜蛋白，细胞内信号和酶活性。以下外源DNA的转染进入细胞内，许多不同形式的分析可以被执行，包括电，比例钙成像，免疫印迹等 4，5。 由于广泛的潜在应用为异源表达系统中，许多各色核苷酸的试剂和产品已经发展到利用这些细胞和它们的质量6。外国DNA暂时或永久地融入细胞研究外源蛋白的DNA递送系统已成为生物学研究最流行和最有用的工具之一。更具体而言，瞬时转染的DNA导入细胞被广泛地用作一种简单，直接的过程，需要相对少的时间和材料。另外，该经过转染的细胞的成功率很高7。这种技术是非常可靠的，当用一个标记基因诸如绿色荧光蛋白（GFP）组合，并且可用于许多不同的技术，如钙成像和电5。不幸的是，虽然，瞬时表达的DNA导入宿主细胞带有一些重大的缺陷，而不是在每个细胞中的表达水平是不可靠的最少。质粒DNA的拷贝数采取ū每个细胞p是不可控的，因此个别实验之间的表达式差别很大2。当任一试图复制生理条件下，或在执行精确的数据收集技术这个问题变得显著。 作为解决上面提到的，稳定转染的协议已经被设计的复杂，其中感兴趣的基因可以被插入到可诱导启动子，的严密控制下的细胞的基因组，如四环素阻遏表达系统，确保单该质粒的拷贝整合到每个细胞的基因组和转录机制诱导后仅表示，例如，在多西环素的存在。虽然这解决了不一致蛋白表达水平的障碍，该方法失去瞬时转染的快速和相对简单的协议的便利性。建立一个稳定的细胞系发生在whic至少几周高电平单必须校准杀灭曲线由特定抗生素设定为保持蛋白质表达并确保载体整合并巧妙地选择和生长的细胞集落。总的来说，这需要显著更多的时间和精力用较低的成功率8。 在这里，我们引入两个流行的转染方案的长处绘制，以提供一个简单而有效的方法来控制在任何可诱导的细胞系的表达水平的中间的协议。同时保持与可诱导的tet系统细胞，我们瞬时转染我们感兴趣的基因，瞬时受体电位阳离子通道亚家族V部件1（TRPV1），连接到可与阻遏系统同源结合的载体。以这种方式，可以将基因导入细胞，而不开始表达。只有加入强力霉素不基因开始表达，让我们来校准蛋白质expr的水平根据在生理条件所观察到的技术或水平分裂国家。我们的协议还避免了产生稳定表达的细胞系繁琐的并发症。我们首先表示从钙成像TRPV1激活的变化水平未诱导通过4小时感应以及如何在细胞内钙水平的上升密切相关的。然后，我们重复在膜片钳技术的全细胞组态的协议，表示随感应时间的增加的电流。最后，我们提出单通道电记录的例子，并且表明，基于蛋白质的个别单位寻找精确的数据收集时该技术是用于控制的表达特别有用。通过我们的协议中，我们提供了以控制在异源系统蛋白质表达的便利方式，同时避免冗长的细胞培养的并发症，因此提供了一种控制实验和提供莫之间的条件重新复制的结果。

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			<td height="42" style="height:42px;width:216px;">Owl&#8482; EasyCast&#8482; B1A Mini Gel Electrophoresis System</td>
			<td style="width:153px;">ThermoScientific Fisher</td>
			<td style="width:119px;">B1A</td>
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			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">R710-07</td>
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			<td height="19" style="height:19px;width:216px;">DMEM (1X), liquid (high glucose)</td>
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			<td style="width:119px;">41965-039</td>
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			<td height="21" style="height:21px;width:216px;">HindIII-HF&#174;</td>
			<td style="width:153px;">NEB</td>
			<td style="width:119px;">R3104S</td>
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			<td height="21" style="height:21px;width:216px;">ApaI</td>
			<td style="width:153px;">NEB</td>
			<td style="width:119px;">R0114S</td>
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			<td height="21" style="height:21px;width:216px;">CutSmart&#174; Buffer</td>
			<td style="width:153px;">NEB</td>
			<td style="width:119px;">B7204S</td>
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			<td height="42" style="height:42px;width:216px;">pcDNA&#8482;6/TR&#160; Mammalian Expression Vector</td>
			<td style="width:153px;">ThermoScientific Fisher</td>
			<td style="width:119px;">V102520</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fetal Bovine Serum, qualified, E.U.-approved, South America origin</td>
			<td style="width:153px;">Gibco</td>
			<td align="right" style="width:119px;">10270106</td>
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			<td height="21" style="height:21px;width:216px;">HEPES Buffer Solution (1 M)</td>
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			<td style="width:119px;">03-025-1B</td>
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			<td style="width:153px;">Biological Industries</td>
			<td style="width:119px;">03-031-1B</td>
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			<td style="width:153px;">Biological Industries</td>
			<td style="width:119px;">03-022-1B</td>
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			<td height="42" style="height:42px;width:216px;">Heracell&#8482; 150i CO2 Incubator</td>
			<td style="width:153px;">ThermoScientific Fisher</td>
			<td align="right" style="width:119px;">51026406</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MSC-Advantage&#8482; Class II Biological Safety Cabinet</td>
			<td style="width:153px;">ThermoScientific Fisher</td>
			<td align="right" style="width:119px;">51025411</td>
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			<td height="21" style="height:21px;width:216px;">Blasticidine S hydrochloride</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">15205-25MG</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Saline Modified, without calcium chloride and magnesium chloride</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537-500ML</td>
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			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:153px;">Gibco</td>
			<td align="right" style="width:119px;">25300054</td>
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			<td height="63" style="height:63px;width:216px;">DOUBLE NEUBAUER RULED METALLIZED HEMACYTOMETER</td>
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			<td align="right" style="width:119px;">31000</td>
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			<td align="right" style="width:119px;">31985070</td>
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			<td height="42" style="height:42px;width:216px;">glass coverslips, #1 thickness, 12mm diameter round</td>
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			<td align="right" style="width:119px;">354210</td>
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			<td height="21" style="height:21px;width:216px;">Water, Cell Culture Grade</td>
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			<td style="width:119px;">03-055-1A</td>
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			<td height="21" style="height:21px;width:216px;">Doxycycline hyclate</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-1G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fura-2, AM ester</td>
			<td style="width:153px;">Biotium</td>
			<td style="width:119px;">BTM-50034</td>
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			<td height="21" style="height:21px;width:216px;">Pluronic&#174; F-127</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2443-250G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#181;-Slide 8 Well</td>
			<td style="width:153px;">ibidi</td>
			<td align="right" style="width:119px;">80826</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(E)-Capsaicin</td>
			<td style="width:153px;">Tocris</td>
			<td align="right" style="width:119px;">462</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus IX70 Fluorescence Microscope</td>
			<td style="width:153px;">Olympus</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lambda DG-4 Wavelength Switcher</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">n.a</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EXi Blue Fluorescence Microscopy Camera</td>
			<td style="width:153px;">QImaging</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaFluor Fluorescence Ratio Imaging Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Walled Borosilicate Tubing</td>
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			<td style="width:119px;">B150-110-7.5HP</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard Walled Borosilicate Tubing</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">B150-86-7.5HP</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide anhydrous</td>
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			<td align="right" style="width:119px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 micropipette puller</td>
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			<td style="width:119px;">P-1000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MF-900 Microforge</td>
			<td style="width:153px;">NARISHIGE</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ValveBank perfusion sysytem</td>
			<td style="width:153px;">AutoMate Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digidata&#174; 1440A Low-noise Data Acquisition System</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axopatch 200B Amplifier</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pCLAMP 10.6 Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micromanipulator</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">MP-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

controllable ion channel expression through inducible transient transfection

转染，外来核酸递送入细胞，是在蛋白质的研究的强有力的工具。通过这种方法，离子通道可通过电生理分析，生化表征，突变研究，以及它们对细胞过程的影响进行调查。瞬时转染提供了一个简单协议，其中蛋白质在几个小时内变得可用于分析到几天。虽然这种方法提供了一个相对简单的和时间有效的协议，关键部件中的一个被校准感兴趣的适合于分析生理相关水平或水平的基因的表达。为此目的，能够提供以控制感兴趣的基因的表达的能力的许多不同的方法已经出现。数的稳定细胞转染方案提供了一种方法以永久引入目的基因到细胞基因组中的四环素控制的转录的调控下TIONAL激活。虽然这种技术产生可靠的表达水平，每个感兴趣基因需要熟练工作包括杀灭曲线的校准，细胞集落的选择，以及整体更多的资源在几个星期。这里，我们提出使用该瞬时受体电位阳离子通道亚家族V部件1（TRPV1）基因的瞬时转染在可诱导系统来表达以受控的方式是在离子通道分析所必需的蛋白质的有效方法的协议。我们表明，使用这种技术，我们能够与具有单个转染每种类型的数据收集所需的控制信道的水平进行钙成像，全细胞，和单信道分析。总体而言，这提供了可用于研究离子通道的结构和功能可复制的技术。

快速创建一个诱导型表达模式，我们利用HEK293细胞的表达四环素阻遏蛋白（TR）（ 例如，T-REX-293），并包含在CMV启动子和多克隆位点之间的四环素操纵子序列（TETO）载体（ 例如，pcDNA4 / TO）。当转染入TREX-293细胞，在pcDNA4 / TO感兴趣的基因的表达被抑制，如TR结合TETO。添加多西环素的培养基阻止TR-TETO相互作用，从而允许转染的蛋白的表达。为了控制鼠TRPV1（rTRPV1）的表达，我们先插入此通道的基因插入pcDNA4 /使用上述协议载体。其次，T-REX-293细胞转染与rTRPV1- pcDNA4 /根据该协议的第3点的步骤质粒。转染后，rTRPV1表达（诱导的多西环素的存在下孵育细胞1μ克/毫升）1 - 4小时。重要的是，不同的多西环素的浓度可用于实现期望的表达率。我们使用电生理和钙成像方法通过施加其激动剂辣椒素来评估在每个时间点rTRPV1表达水平。 如图 1所示，使用活细胞钙成像，之后辣椒素应用细胞内钙水平与较长诱导时间逐渐增加。未诱导的细胞的活化，可能是因为在对TR活性的基底泄漏，质粒的过表达，或在细胞中的培养基成分残留四环素。接下来，我们将用毫伏-80之间的电压斜坡至+80 mV的膜片钳技术的全细胞记录配置从细胞中的电流。 图 2显示了较高的电流振幅具有较长诱导时间获得。在4小时的诱导电流幅度的饱和是consiste核苷酸在钙响应看到的饱和度（ 图1）。最后，我们在膜片钳技术的外面向外配置分析rTRPV1表达水平。之一的在研究离子通道结构 - 功能是达到适合于单通道分析表达水平的主要困难。根据已发表的酉rTRPV1电导4中 ，记录电流幅值用于确定在切下的补丁的信道数。 如图 3中 ，在比例补丁增大到强力霉素温育时间的信道数。感应的一小时后，我们无法检测到任何通道活性（0总分8）。然而，在二维和三个小时的时间点，我们的成功率相似记录单声道的活动。值得注意的是，同时在两小时内最补丁没有反应，在三小时内最补丁显示单到多通道活性。 3小时诱导之后记录多个频道的机率很高，因此最佳的诱导时间以记录当前从单个信道是所提出的条件下，两到三个小时。一起这些结果表明，离子通道的表达可被严格控制，并使用该协议瞬时转染后调节。 <img alt="图1" src="/files/ftp_upload/55370/55370fig1.jpg" /> 图1.根据诱导时间TRPV1激活增加的响应，通过钙成像可视化。 （A）T-REX-293细胞瞬时表达rTRPV1，前（&#39;基础&#39;）和辣椒素（2μM）申请后的伪彩色图像。白线代表30微米。比例尺为细胞内钙水平。 （二）在处理转染的T-REX-293细胞内钙水平的时间的变化如图所示在答:每个图代表50辣椒素敏感的细胞的平均值。注意相对于诱导时间在辣椒素反应的逐步增加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55370/55370fig1large.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/55370/55370fig2.jpg" /> 图2. TRPV1电流随着在全细胞膜片钳记录增加诱导时间的结果。 （一）从T-REX-293全细胞记录瞬时在-40 mV的控股潜力与rTRPV1转。细胞用强力霉素（1微克/毫升）在指定的时间诱导，然后暴露于辣椒素（&#39;第&#39;; 1μM;红色条）。显示的是6个有代表性的踪迹 - 11个独立的录音。 （B）的平均值/代表全细胞振幅分散散点图诱发如图A.统计组之间的人显着性测定用ANOVA和多重比较，其中***表示对≤0.001和NS-没有统计学显著（每组6 - 11细胞）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55370/55370fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55370/55370fig3.jpg" /> 图3.单通道膜片在TRPV1录音的成功率是诱导时间有关。 （一）从T-REX-293之外出瞬时记录在+50 mV的钳制电压与rTRPV1转。细胞用强力霉素（1微克/毫升）在指定的时间诱导暴露于辣椒素（&#39;第&#39;; 1μM;红色条）。显示的是6个有代表性的踪迹 - 9个独立的录音。 （B）的平均较通道在切下的补丁作为数/分散点图在组间A.统计意义示出测定用ANOVA和多重比较，其中***表示对≤0.001和NS-没有统计学显著（每组6 - 9细胞）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55370/55370fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Controllable Ion Channel Expression through Inducible Transient Transfection at JoVE.com

Controllable Ion Channel Expression through Inducible Transient Transfection

通过异源表达系统研究离子通道已成为生物医学研究核心技术。在这个手稿，我们提出了一个时间有效的方法通过诱导型启动子的控制下进行瞬时转染来实现紧密控制的离子通道的表达。

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

controllable-ion-channel-expression-through-inducible-transient

Nanyang Technological University

Research

JoVE Journal

Genetics

9.3K 次观看.  The Hebrew University of Jerusalem (HUJI). 通过异源表达系统研究离子通道已成为生物医学研究核心技术。在这个手稿，我们提出了一个时间有效的方法通过诱导型启动子的控制下进行瞬时转染来实现紧密控制的离子通道的表达。 

视频: Controllable Ion Channel Expression through Inducible Transient Transfection

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

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

风土通过概念葡萄果实代谢组学和转录组解释的

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Inducible and Reversible Dominant-negative DN Protein Inhibition

Here we present a protocol to develop a dominant-negative inducible system, in which any protein can be conditionally inactivated by reversibly overexpressing a dominant-negative mutant version of it.

诱导和可逆显性阴性 (dn) 蛋白抑制

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...