这项工作得到加拿大自然科学与工程研究理事会的支持。

获得感兴趣的基因<ol><li> 设计合成基因。 <ol><li>优化大肠杆菌表达的基因序列。 </li><li>从序列（AvrII，NdeI，EcoRI和XbaI）中除去任何有问题的限制性位点。 </li><li>纳入克隆的限制性位点建议使用5&#39;-NdeI位点和3&#39;-EcoRI位点。必要时可以使用其他网站;指选择的质粒的多克隆区（ 图S1 -S4 ）。如果需要，包括用于Western印迹检测的5&#39;或3&#39;编码标签。 </li><li>商业命令设计的基因。 </li><li>根据制造商的说明书，使用钝性克隆试剂盒将基因平滑克隆到钝性载体中;设计引物扩增基因（然后进行到步骤1.2.2）;或者添加另外的5&#39;和3&#39;末端直接克隆到pMGX质粒中，并进行到步骤2e代表性的结果来自钝性克隆感兴趣的基因。 </li></ol></li><li> 扩增所需基因（来自设计和优化的合成基因或含有所需基因的模板DNA）。 <ol><li>设计具有克隆限制性位点的引物;建议使用5&#39;-NdeI位点和3&#39;-EcoRI位点。必要时可以使用其他网站;指选择的质粒的多克隆区（ 图S1 -S4 ）。如果需要，包括用于Western印迹检测的5&#39;或3&#39;编码标签。 </li><li> PCR扩增所需基因15 。 </li><li>通过琼脂糖凝胶电泳分析PCR。 </li><li>如果存在非特异性扩增，则通过凝胶提取来清除扩增的基因。如果没有，请使用酶清洁套件。 </li><li>通过检查吸光度，用分光光度计定量DNAe在260nm;用洗脱缓冲液17空白。 </li></ol></li></ol> 2.将感兴趣的基因克隆到多基因表达系统载体中，pMGX 18 <ol><li> 用NdeI和EcoRI限制性消化获得的目的基因和所需载体pMGX。 注意:如果获得大量的再循环质粒，则允许NdeI反应在添加EcoRI之前进行1小时。 <ol><li>使用含有0.5-1.5μgDNA的40μL消化反应和1μL每种酶与4μL适当的10x缓冲液。 </li><li>对于NdeI和EcoRI消化，使用EcoRI缓冲液，首先加入NdeI内切核酸酶。在37°C消化1小时。然后加入EcoRI内切核酸酶，并使消化进行一个小时。 NdeI对接近DNA末端的裂解敏感，所以EcoRI的初步消化可以防止NdeI的有效消化。 </li></ol></li><li> ELECTR对琼脂糖凝胶进行限制性消化（对于1 kb基因，在110V下使用0.7％琼脂糖凝胶55分钟;选择不同基因大小的琼脂糖凝胶的百分比，参见参考文献16 。 </li><li>使用干净的手术刀或剃刀刀片将切口插入物和载体条带放置在1.5 mL管中。 </li><li>使用凝胶提取试剂盒根据制造商的方案从琼脂糖凝胶中提取DNA。 </li><li> 使用3:1插入片段向量比例将感兴趣的基因连接到pMGX载体中;建立消化的pMGX阴性结扎，无需插入。 <ol><li>设置20μL连接反应，其中含有1μLT4 DNA连接酶，0.15-0.5μg载体DNA（〜5μL）和适量的插入物，基于3:1插入物载体比例和插入基因; pMGX骨架的大小在5,312和5,504 bp之间。包括一个负面控制反应，包含一切but插入的基因。确保载体DNA的量在阴性对照连接和载体加插入连接反应中相当。 </li></ol></li><li> 将连接反应转化入XL1-Blue化学感受态大肠杆菌细胞和连接质粒pMGX- yfg1 （含有感兴趣的基因1），pMGX- yfg2 （含有感兴趣的基因2）... pMGX- yfgn （含有感兴趣的基因n）。使用无菌技术（生物安全柜内或火焰下）。 <ol><li>将化学感受态XL1-Blue 大肠杆菌的 100μL等分试样在冰上解冻5分钟，然后加入5μL连接反应。在冰上孵育30分钟。 </li><li>在保持在42℃的水浴中将细胞热休克45秒，然后加入200μL冷LB。在冰上孵育2分钟。 </li><li>在37℃，220rpm下摇动细胞1小时，然后将板100μL铺展到含有正常的LB-琼脂平板优选选择标记（氨苄青霉素或卡那霉素）。 </li></ol></li><li> 筛选阳性转化体的克隆。 <ol><li>比较阴性对照和结扎板上的菌落数。需要大于1:2的菌落计数比。如果阴性对照板上存在大量的菌落，请返回步骤2.1并查看该记录。 </li><li>从插入用于筛选的不同基因（1n）的每个连接反应中选择4-8个单独的菌落（取决于阴性对照:连接比率），并接种4mL LB和适当的抗生素过夜培养物/个体菌落。在37℃和220rpm下振荡培养过夜。 </li><li>使用质粒DNA分离试剂盒根据制造商的方案分离质粒DNA。 </li><li>设置20μLNdeI + EcoRI消化反应，含有150-500 ng的DNA和1μL的每种酶，2μL适当的10倍缓冲液。在情况下，在感兴趣的基因将在限制性位点之间的同一时间添加NdeI和EcoRI。建议使用空的pMGX载体的阴性对照。在37℃的水浴中消化2小时。 </li></ol></li></ol> 3.将基因2插入到含有基因1的pMGX载体中，pMGX- yfg1 <ol><li> 用AvrII限制性消化pMGX- yfg1并用小牛肠磷酸酶（CIP）处理。 <ol><li>使用含有0.5-1.5μg载体DNA（〜5-10μL分离的DNA）和1μLAvrII的40μL消化反应物与4μL适当的10x缓冲液。在37℃下消化1.5小时，然后加入1.5μLCIP。在37°C下放置另外30分钟。 </li></ol></li><li> 用AvrII和XbaI限制性消化pMGX- yfg2以释放感兴趣的基因。 <ol><li>使用含有0.5-1.5微克DNA和1微升的40μL消化反应; L每个酶用4μL适当的10倍缓冲液。消化2小时37°C。 </li></ol></li><li>电泳限制性消化在合适的百分比（0.7％）琼脂糖凝胶上，并使用干净的手术刀/剃刀刀片切除插入物和载体条带（参见步骤2.2-2.3）。 </li><li>使用凝胶提取试剂盒根据制造商的方案提取DNA，并定量DNA 17 。 </li><li>使用3:1插入到载体比例将基因2引导到pMGX- yfg1中 。设置消化的pMGX- yfg1的阴性结扎，无需额外插入。在步骤2.5中设置如上。 </li><li>将5μL连接反应转变成XL1-Blue化学感受态大肠杆菌细胞，连接质粒pMGX- yfg1,2 （含有感兴趣的基因1和2）和阴性pMGX- yfg1对照，如步骤2.6所示。 </li><li>比较阴性对照和连接板上的菌落数。菌落计数比g需要1:2以上。如果阴性对照板上存在大量的菌落，请返回到步骤3.1并检查CIP处理。 </li><li>从连接反应中选择4-8个单独的菌落（取决于阴性对照:连接比例），并接种4mL LB加上每个菌落合适的抗生素，并在37℃和220rpm下生长过夜。 </li><li>使用质粒DNA分离试剂盒根据制造商的方案分离质粒DNA并定量DNA 17 。 </li><li> 通过用EcoRI进行pMGX- yfg1,2的限制性消化，筛选第二基因的有效插入。 <ol><li>使用含有150-500ng DNA的20μL消化反应和1μLEcoRI酶与2μLEcoRI 10x缓冲液。消化2小时37°C。 </li></ol></li><li>在适当的百分比琼脂糖凝胶上电泳限制性消化;寻找一个对应的乐队s到基因2的大小（参见步骤2.2）。基因可以以不期望的反向取向插入载体中。 </li></ol> 4.将第三个基因添加到含有基因1和2的pMGX载体中，pMGX- yfg1,2 <ol><li>使用AvrII限制性消化pMGX- yfg1,2并用CIP处理，如步骤3.1所示。 </li><li>使用AvrII和XbaI限制性消化pMGX- yfg3 ，如步骤3.2所示。 </li><li>在适当的百分比琼脂糖凝胶上电泳限制性消化，并使用干净的手术刀/剃刀刀片切除插入物和载体条带;请参阅步骤2.2-2.3。 </li><li>使用凝胶提取试剂盒从琼脂糖凝胶中提取DNA，并定量DNA 17 。 </li><li>使用3:1插入到载体比例将基因3引导到pMGX- yfg1,2中，并建立消化的pMGX- yfg1,2的阴性结合，而不需要另外的插入物，如步骤3.5所示。 </li><li>将5μL的连接反应转化为XL1-Blue化学感受态大肠杆菌细胞，连接的质粒pMGX- yfg1,2,3 （含有感兴趣的基因1,2和3）和阴性pMGX- yfg1,2对照，如步骤2.6所示。 </li><li>比较阴性对照和连接板上的菌落数。如果阴性对照板上存在大量的菌落，请返回到步骤4.1并检查CIP处理。 </li><li>从连接反应中选择4-8个单独的菌落（取决于阴性对照:连接比例），并接种4mL LB加上每个菌落合适的抗生素;在37℃和220rpm下生长过夜。 </li><li>使用质粒DNA分离试剂盒根据制造商的方案分离质粒DNA。 </li><li>通过用EcoRI进行pMGX- yfg1,2,3的限制性消化，筛选第三种基因的有效插入，如步骤3.10所示。 </li><li>在适当的百分比琼脂糖凝胶上电泳限制性消化;看对应于基因2和基因3的大小的条带（参见步骤2.2）。注意:基因3可以以不期望的反向取向插入载体。如果基因2和3的大小相同，则必须选择另一个适当的限制性消化位点进行筛选。 注意:根据需要重复每个新基因。 </li></ol> 5.使用多基因表达系统生产感兴趣的蛋白质，并通过Western Blotting评估生产<ol><li> 将含有所有感兴趣的基因的阳性克隆转化为化学感受态的蛋白质生产大肠杆菌如BL21-（λDE3）。 <ol><li>将化学感受态的BL21-（λDE3） 大肠杆菌的 100μL等分试样在冰上解冻5分钟，然后加入1μL的阳性克隆质粒DNA;在冰上孵育30分钟。 </li><li>在42℃的水浴中将细胞热休克45秒，然后加入200μL冷LB。在冰上孵育2分钟。 </li><li>在37℃和220rpm下摇动细胞1小时，然后将板100μL铺板到含有适当选择标记（氨苄青霉素或卡那霉素）的LB-琼脂平板上。 </li></ol></li><li> 通过异丙基-β-D-1-硫代吡喃半乳糖苷（IPTG）诱导表达蛋白质。 <ol><li>从B21-（λDE3）转化板中选择一个分离的菌落，接种4 mL LB加合适的抗生素;生长过夜，在37℃和220rpm下摇动。 </li><li>使用1mL过夜培养物接种100mL LB加适当的抗生素培养物。 </li><li>在37℃下以220rpm摇动生长至OD 600为0.6。 </li><li>用100μMIPTG诱导培养，并在25℃和220rpm下生长15小时。 </li><li>取1 mL培养液，以13,000 xg离心1 min;丢弃上清液。 </li></ol></li><li>按照制造商的说明使用裂解液溶解细胞并预处理可溶性细胞裂解物的蛋白质印迹，以确定所有蛋白质是否成功生产19 。 </li></ol>

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作者没有什么可以披露的。

多个基因的共表达越来越重要，特别是在复杂的多基因代谢途径3,4,5的表征和重建中。 pMGX系统在大肠杆菌常规6,7,8中进行多基因共表达，并可以由不同的研究人员访问。在本研究中，显示了使用pMGX-A的单个质粒系统同时产生了5种目的蛋白质。目前可获得的许多共表达系统仅允许插入两个基因，产生双顺反子质粒例如pET Duet载体12 。如果需要添加?...

多重蛋白质的共表达越来越重要，特别是在合成生物学应用中，其中必须表达多个功能模块1 ;在研究蛋白质 - 蛋白质复合物时，其中表达和功能通常需要共表达2,3 ;并且在表征和利用生物合成途径时，其中通路中的每个基因必须表达为4,5,6,7,8。已经开发出许多用于共表达的系统，特别是在宿主生物体大肠杆菌中 ，实验室重组蛋白表达的工作马9 。例如，具有不同选择标记的多个质粒可用于使用大量不同的ex<html>压迫矢量10,11 。用于多重蛋白表达的单个质粒系统已经使用多个启动子来控制每个基因的表达10,12 ;合成操纵子，其中多个基因编码在单个转录物2,13上;或者在一些情况下是编码最终被蛋白水解处理的多肽的单个基因，产生所需的期望的蛋白质14 。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55187/55187fig1.jpg" /> 图1:显示多顺反子载体构建的pMGX工作流程。 pMGX系统为可诱导T7启动子控制下的合成操纵子的构建提供了一种灵活的，易于使用的策略。e.com/files/ftp_upload/55187/55187fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 在该手稿中，描述了在诱导型T7RNA聚合酶（ 图1 ）的控制下使用高效系统来构建多基因合成操纵子。该系统允许许多基因从一个质粒同时表达。它基于质粒系统，原来称为pKH22，已被成功应用于许多不同的应用6,7,8 。在这里，扩增该质粒组包括四个相关载体:pMGX-A，缺少任何C-或N-末端标签的表达载体和氨苄青霉素抗性标记; pMGX-hisA，编码N-末端六组氨酸标签和氨苄青霉素抗性标记的表达载体; pMGX-K，表达载体l检测任何C-或N-末端标签和卡那霉素抗性标记物;和pMGX-hisK，编码N-末端六组氨酸标签的表达载体和卡那霉素抗性标记。在本研究中，使用pMGX系统（特别是pMGX-A）产生含有五个基因的多顺反子载体的方法与大肠杆菌中每种单独蛋白质的成功产生一起被证明。

<table><tbody><tr><td>&lt;strong&gt;Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alkaline Phosphatase, Calf Intestinal (CIP)</td><td>New England Biolabs</td><td>M0290S</td><td></td></tr><tr><td>AvrII</td><td>New England Biolabs</td><td>R0174S</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td></td></tr><tr><td>NdeI</td><td>New England Biolabs</td><td>R0111S</td><td></td></tr><tr><td>XbaI</td><td>New England Biolabs</td><td>R0145S</td><td></td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent Technologies</td><td>600677</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 kb DNA ladder</td><td>New England Biolabs</td><td>N3232L</td><td></td></tr><tr><td>4-20% Mini-PROTEAN TGX Stain-Free Protein Gels</td><td>Bio-Rad</td><td>456-8095</td><td></td></tr><tr><td>50x TAE</td><td>Fisher Thermo Scientific</td><td>BP1332-4</td><td></td></tr><tr><td>Agar</td><td>Fisher Thermo Scientific</td><td>BP1423-500</td><td></td></tr><tr><td>Agarose</td><td>Fisher Thermo Scientific</td><td>BP160-500</td><td></td></tr><tr><td>Ampicilin</td><td>Sigma-Alrich</td><td>A9518-5G</td><td></td></tr><tr><td>BL21 (DE3) chemically comeptent cells</td><td></td><td></td><td>Comeptent cell prepared in house</td></tr><tr><td>B-PER Bacterial Protein Extraction Reagent</td><td>Fisher Thermo Scientific</td><td>PI78243</td><td></td></tr><tr><td>dNTP mix</td><td>Agilent Technologies</td><td></td><td>Supplied with polymerase</td></tr><tr><td>Gel Extraction Kit</td><td>Omega</td><td>D2500-02</td><td>E.Z.N.A Gel Extraction, supplied by VWR Cat 3: CA101318-972</td></tr><tr><td>Glycine</td><td>Fisher Thermo Scientific</td><td>BP381-1</td><td></td></tr><tr><td>His Tag Antibody [HRP], mAb, Mouse</td><td>GenScript</td><td>A00612</td><td></td></tr><tr><td>Immobilon Western Chemiluminescent HRP Substrate</td><td>EMD Millipore</td><td>WBKLS0100</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Alrich</td><td>15502-10G</td><td></td></tr><tr><td>LB</td><td>Fisher Thermo Scientific</td><td>BP1426-500</td><td></td></tr><tr><td>Methanol</td><td>Fisher Thermo Scientific</td><td>A411-20</td><td></td></tr><tr><td>Pasteurized instant skim milk powder</td><td>Local grocery store</td><td></td><td>No-name grocery store milk is adequate</td></tr><tr><td>Nitrocellulose membrane</td><td>Amersham Protran (GE Healthcare Life Sciences)</td><td>10600007</td><td>Membrane PT 0.45 &amp;micro;m 200 mm x 4 m, supplied by VWR Cat #: CA10061-086</td></tr><tr><td>Plasmid DNA Isolation Kit</td><td>Omega</td><td>D6943-02</td><td>E.Z.N.A Plasmid DNA MiniKit I, supplied by VWR Cat #: CA101318-898</td></tr><tr><td>pMGX</td><td>Boddy Lab</td><td></td><td>Request from the Boddy Lab Contact cboddy@uottawa.ca</td></tr><tr><td>Primers</td><td>Intergrated DNA Technologies</td><td></td><td>Design primers as needed for desired gene</td></tr><tr><td>Synthetic Gene</td><td>Life Technologies</td><td></td><td>Design and optimize as needed</td></tr><tr><td>Thick Blot Filter Paper</td><td>Bio-Rad</td><td>1703932</td><td></td></tr><tr><td>Tris base</td><td>BioShop</td><td>TRS001.1</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Alrich</td><td>P9416-50ML</td><td></td></tr><tr><td>XL1-Blue chemically competent cells</td><td></td><td></td><td>Comeptent cell prepared in house</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioSpectrometer</td><td>Eppendorf</td><td>RK-83600-07</td><td></td></tr><tr><td>Gel box - PAGE</td><td>Bio-Rad</td><td>1658005</td><td>Mini-PROTEIN Tetra Vertical Electrophoresis Cell</td></tr><tr><td>Gel Imager</td><td>Alpha Innotech</td><td></td><td>AlphaImager EC</td></tr><tr><td>Incubator-oven</td><td>Fisher Thermo Scientific</td><td>11-690-650D</td><td>Isotemp</td></tr><tr><td>Incubator-shaker</td><td>Fisher Thermo Scientific</td><td>SHKE6000-7</td><td>MaxQ 6000</td></tr><tr><td>Personna Razors</td><td>Fisher Thermo Scientific</td><td>S04615</td><td></td></tr><tr><td>Power Pack</td><td>Bio-Rad</td><td>S65533Q</td><td>FB300</td></tr><tr><td>Transilluminator</td><td>VWR International</td><td>M-10E,6W</td><td></td></tr><tr><td>Thermocylcer</td><td>Eppendorf</td><td>Z316091</td><td>Mastercycler Personal, supplied by Sigma</td></tr><tr><td>UV Face-Shield</td><td></td><td>18-999-4542</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Thermo Scientific</td><td>15-460-2SQ</td><td></td></tr><tr><td>Western Transfer Apparatus</td><td>Bio-Rad</td><td>1703935</td><td>Mini-Trans Blot Cell</td></tr></tbody></table>

inducible t7 rna polymerase-mediated multigene expression system, pmgx

多重蛋白质的共表达对于合成生物学，研究蛋白质 - 蛋白质复合物以及表征和利用生物合成途径来说日益重要。在该手稿中，描述了在可诱导T7RNA聚合酶的控制下使用高效系统来构建多基因合成操纵子。该系统允许许多基因从一个质粒同时表达。这里，一组四个相关载体pMGX-A，pMGX-hisA，pMGX-K和pMGX-hisK与氨苄青霉素或卡那霉素抗性选择性标记（A和K）和具有或缺少N-末端六组氨酸标签（他）被披露。使用该载体系统构建合成操纵子的详细方案与相应的数据一起提供，显示含有五个基因的基于pMGX的系统可以容易地构建并用于在大肠杆菌中产生所有五种编码的蛋白质。这个系统em和协议使研究人员能够在大肠杆菌中常规表达复杂的多组分模块和途径。

在这项研究中，目标是共同表达来自单个质粒的五种蛋白质。编码N-或C-末端六组氨酸标签的五密码子优化的合成基因片段商购。通过PCR扩增合成基因，并单独克隆到PCR平片载体中并进行测序。为了产生多顺反子质粒，首先将5个感兴趣的基因克隆到合适的pMGX质粒pMGX-A中。 图2显示除了用NdeI + EcoRI消化的pMGX-A之外的PCRBlunt- yfg1和PCRBlunt-yfg2（参见?...

Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

该研究描述了使用pMGX质粒系统在大肠杆菌中使用单个质粒的多个基因的T7介导的共表达的方法。

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 ...

inducible-t7-rna-polymerase-mediated-multigene-expression-system-pmgx

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

13.2K 次观看.  University of Ottawa. 该研究描述了使用pMGX质粒系统在大肠杆菌中使用单个质粒的多个基因的T7介导的共表达的方法。 

视频: Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

科研

环境科学

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

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 ...

遗传学

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 ...

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 ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

生物化学

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

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

生物工程

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

生物学

Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model

Source: Hubner, E. K. et al.&#160; <a href="https://jove.unicartagenaproxy.elogim.com/v/56613">Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a> J. Vis. Exp. (2018)
This video demonstrates the tetracycline, or Tet-On, gene expression system in a mouse model. In the presence of doxycycline, a tetracycline derivative, the Tet-On system consisting of reverse tetracycline-controlled transactivator protein becomes activated and expresses the gene of interest.

诱导型 Tet-On 基于调节系统的体内基因表达:用于小鼠模型中靶基因转录激活的四环素诱导基因表达系统

Source: Hubner, E. K. et al.&#160; Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein ...