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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D., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+small-molecule+modulators+of+inward+rectifier+potassium+channels.">High-throughput screening for small-molecule modulators of inward rectifier potassium channels.</a> J Vis Exp. (71), e4209 (2013).</li></ol>

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...

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。

<table><tbody><tr><td>pcDNA&amp;trade;4/TO Mammalian Expression Vector</td><td>ThermoScientific Fisher</td><td>V102020</td><td></td></tr><tr><td>pcDNA&amp;trade;5/TO Mammalian Expression Vector</td><td>ThermoScientific Fisher</td><td>V103320</td><td></td></tr><tr><td>PureLink Quick PCR Purification Kit</td><td>Invitrogen</td><td>K310001</td><td></td></tr><tr><td>Swift&amp;trade;&amp;nbsp;MaxPro Thermal Cycler</td><td>Esco&amp;nbsp;</td><td>n.a</td><td></td></tr><tr><td>Restriction Enzymes</td><td>ThermoScientific Fisher</td><td>ER0501</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>Invitrogen</td><td>K210012</td><td></td></tr><tr><td>NanoDrop 2000c</td><td>ThermoScientific Fisher</td><td>ND-2000C</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>ThermoScientific Fisher</td><td>EL0011</td><td></td></tr><tr><td>One Shot&amp;reg; TOP10 Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>ThermoScientific Fisher</td><td>C404006&amp;nbsp;</td><td></td></tr><tr><td>Ampicillin (Sodium), USP Grade</td><td>Gold Bio</td><td>A-301-5</td><td></td></tr><tr><td>Tryptone for microbiology</td><td>Merck</td><td>6.19305E+13</td><td></td></tr><tr><td>Yeast Extract</td><td>BD worldwide</td><td>212750</td><td></td></tr><tr><td>SIF6000R Incubated Shaker</td><td>LAB COMPANION</td><td>45H118</td><td></td></tr><tr><td>NucleoSpin&amp;reg;plasmid</td><td>Macherey Nagel</td><td>740588.25</td><td></td></tr><tr><td>MS 300V Power Supply</td><td>Major Science</td><td>MP-300V</td><td></td></tr><tr><td>Owl&amp;trade; EasyCast&amp;trade; B1A Mini Gel Electrophoresis System</td><td>ThermoScientific Fisher</td><td>B1A</td><td></td></tr><tr><td>T-REx&amp;trade;-293 cell line</td><td>Invitrogen</td><td>R710-07</td><td></td></tr><tr><td>DMEM (1x), liquid (high glucose)</td><td>Gibco</td><td>41965-039</td><td></td></tr><tr><td>HindIII-HF&amp;reg;</td><td>NEB</td><td>R3104S</td><td></td></tr><tr><td>ApaI</td><td>NEB</td><td>R0114S</td><td></td></tr><tr><td>CutSmart&amp;reg; Buffer</td><td>NEB</td><td>B7204S</td><td></td></tr><tr><td>pcDNA&amp;trade;6/TR&amp;nbsp; Mammalian Expression Vector</td><td>ThermoScientific Fisher</td><td>V102520</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, E.U.-approved, South America origin</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>Biological Industries</td><td>03-025-1B</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Biological Industries</td><td>03-031-1B</td><td></td></tr><tr><td>L-Alanyl-L-Glutamine (Stable Glutamine) (200 mM)</td><td>Biological Industries</td><td>03-022-1B</td><td></td></tr><tr><td>Heracell&amp;trade; 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>ThermoScientific Fisher</td><td>51026406</td><td></td></tr><tr><td>MSC-Advantage&amp;trade; Class II Biological Safety Cabinet</td><td>ThermoScientific Fisher</td><td>51025411</td><td></td></tr><tr><td>Blasticidine S hydrochloride</td><td>Sigma-Aldrich</td><td>15205-25MG</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline (DPBS) Modified, without calcium chloride and magnesium chloride</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>Double Neubauer Ruled Metallized Hemacytometer</td><td>Hausser Scientific</td><td>31000</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>TransIT&amp;reg;-LT1 Transfection Reagent</td><td>Mirus</td><td>MIR 2300</td><td></td></tr><tr><td>glass coverslips, #1 thickness, 12 mm diameter round</td><td>Knittel Glass</td><td>GG-12</td><td></td></tr><tr><td>BioCoat&amp;trade; Poly-D-Lysine (PDL)</td><td>Corning</td><td>354210</td><td></td></tr><tr><td>Water, Cell Culture Grade</td><td>Biological Industries</td><td>03-055-1A</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-1G</td><td></td></tr><tr><td>Fura-2, AM ester</td><td>Biotium</td><td>BTM-50034</td><td></td></tr><tr><td>Pluronic&amp;reg; F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td></td></tr><tr><td>&amp;micro;-Slide 8 Well</td><td>ibidi</td><td>80826</td><td></td></tr><tr><td>(E)-Capsaicin</td><td>Tocris</td><td>462</td><td></td></tr><tr><td>Olympus IX70 Fluorescence Microscope</td><td>Olympus</td><td>n.a</td><td></td></tr><tr><td>Lambda DG-4 Wavelength Switcher</td><td>Sutter Instruments</td><td>n.a</td><td></td></tr><tr><td>EXi Blue Fluorescence Microscopy Camera</td><td>QImaging</td><td>n.a</td><td></td></tr><tr><td>MetaFluor Fluorescence Ratio Imaging Software</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>Thin Walled Borosilicate Tubing</td><td>Sutter Instruments</td><td>B150-110-7.5HP</td><td></td></tr><tr><td>Standard Walled Borosilicate Tubing</td><td>Sutter Instruments</td><td>B150-86-7.5HP</td><td></td></tr><tr><td>Dimethyl sulfoxide anhydrous</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>P1000 micropipette puller</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>MF-900 Microforge</td><td>NARISHIGE</td><td>n.a</td><td></td></tr><tr><td>ValveBank perfusion sysytem</td><td>AutoMate Scientific</td><td></td><td></td></tr><tr><td>Digidata&amp;reg; 1440A Low-noise Data Acquisition System</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>Axopatch 200B Amplifier</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>pCLAMP 10.6 Software</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>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（rT...

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

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

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

一种基于昆虫蜕皮激素受体的奇异基因开关, 用于对转基因进行有鲁棒性、可逆性和可忽略的泄漏的刻意表达

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

科研

遗传学

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

免疫与感染

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides.
This video describes the basics behind chemical mediated transfections and introduces some of the most commonly-used reagents, including charged lipids, polymers, and calcium phosphate. Each step is described from the preparation of cells for transfection through analysis of transfection efficiency. Additionally, the applications section of this video-article describes the use of electroporation and a biolistic transfection as alternative methods for introducing nucleic acid into mammalian cells. It also describes an advanced use of transfection where co-transfection of interfering RNA and DNA are introduced as a way to down-regulate a naturally occurring protein while at the same time producing a mutant variant of it within the same cell.

Transfection: Inserting Genetic Materials into Mammalian Cells

细胞转染介绍

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

教育

JoVE Science Education

基础生物学

细胞分子生物学中的基本方法

Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System

Source: Migneault, F. et al, <a href="https://www.jove.com/v/60654">Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells.</a> J. Vis. Exp. (2020).
This video describes the method of regulating gene expression in cultured cells by transfection with a plasmid containing a gene of interest and a regulatory plasmid. The expression of this plasmid can be controlled by tetracycline or its synthetic analogs, making this system a useful model to study regulatable gene expression.

诱导型 Tet-Off 调节系统:一种使用 Tetracycline-off 系统调节培养细胞中基因表达的体外方法

Source: Migneault, F. et al, Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in ...