我们要感谢麦克拉扬森在得克萨斯理工大学健康科学中心，拉伯克，TX和Mladen Yovchev在美国匹兹堡大学医学中心，匹兹堡，宾夕法尼亚州的慷慨提供3.1的pCDNA MYC-他的（A）和HEF1α含子质粒。研报本出版物中由儿童健康和国立卫生研究院人类发展的尤尼斯·肯尼迪·施莱佛国立研究所在获奖数量R01HD037109（以CCM）的支持。额外的支持是从劳拉·布什研究所妇女健康（以CCM和PNG）。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。 作者宣称，他们有没有竞争的财务权益。

1. 在质粒硅片设计的重叠引物和生成注意：该步骤的目的是要组装构建体的完整核苷酸序列和设计以被用来产生与用于遗传克隆重叠端片段的引物。 <ol><li>设计来表示最终的质粒的连续核苷酸序列。 </li><li>获得或列出将被用作PCR中的模板的实际的质粒和DNA片段。 注：DNA片段不容易获得 - 如标签和促销员不同的组合 - 可以订购作为单个或多个合成的双链DNA片段（SDNA）。这些片段是可商购的成本相对较低，并且可高达2 kb的长度（ 见表材料 ）。 </li><li>分组装在步骤1.1成适合于PCR的DNA片段的构建体的连续核苷酸序列。 Confir米，该片段匹配可获得的质粒和SDNA片段。避免DNA片段小于200 nt的小。 </li><li>访问底漆生成工具（ 见表设备 ）。选择“设置首选项”菜单。通过单击“更改吉布森大会设置”弹出窗口中的“CHANGE PREFS”选项卡上选择适当的设置。 也可以在不使用底漆生成工具的执行引物设计：注。然而，使用的引物生成工具的简化了工艺。 </li><li>首先选择“建立结构”菜单中建​​立结构。插入拆分的DNA片段中的引物生成工具顺序地从5&#39;到3&#39;末端的。 注意：用作质粒骨架的DNA片段可方便地分成两片。在最终的PCR产物的最后一个片段的末端5&#39;该第一片段和3的端部“在一个连续的DN被链接在一起片段表示载体骨架。 </li><li>粘贴表示FASTA格式的载体DNA（核苷酸序列的基于文本的表示）插入到“输入向量或插入片段”弹出窗口的5&#39;端的第一个DNA片段。命名该DNA片段。选择合适的方式来获得DNA片段，无论是作为PCR，RE文摘或合成。点击“继续”选项卡上。 </li><li>如果有必要在最终的构建体的连接处增加额外的核苷酸/限制性位点使用中的“添加的插入片段的装配”窗口提供的“正转或启底漆间隔基”的空格。仅该DNA片段之一和不添加额外的核苷酸/限制性位点，以这两种片段。点击“DONE”选项卡。 </li><li>重复所有的片段直至该构建完成。选择“视图引物”菜单，查看引物序列。 </li><li>重复所有的结构（向量backbones和插入），或用于DNA片段是不同的。 </li></ol> 2.扩增的DNA片段通过PCR使用热启动校对DNA聚合酶（2×Master Mix中） 注意：目标到这一步，以获得足够的DNA使用PCR的装配反应。 <ol><li>购买设计在先前步骤如脱盐产品和在可能的最小规模的PCR引物。它们稀释至10μM的在水或TE中（10mM的Tris-HCl，pH值7.9，1mM EDTA）中。 </li><li>购买DNA片段不容易获得的SDNA片段。 </li><li>稀释所有的DNA片段，包括SDNA片段将被用作用于PCR的至1ng /μl的在水中的模板。 </li><li>装配PCR反应在室温下进行。简要地说，用2.5微升（从10μM的储备液）的引物对的每一个各自的引物，1微升（1纳克/微升原液）的模板DNA片段，25μ的升热启动校对DNA聚合酶（DNA聚合酶; 2个主结构; 见表材料 ）和19微升水。混合管轻轻轻拂，并进行了简短的离心收集液滴。 </li><li>根据用于该DNA聚合酶的建议在同样大小的分开的管中的DNA片段的同时扩增。执行25 - 28个PCR循环或确定产生足够的DNA产量的周期数。 </li><li>运行PCR反应体积（5微升）在一个标准的琼脂糖凝胶电泳溴化乙锭染色（0.2微克/毫升终浓度）的10％。确保代表的PCR产物的单一DNA带是可见的。确定使用DNA分子量标准DNA片段的大小和相对量。如果需要的话，重复的PCR获得的DNA片段的足够量。 </li></ol> PCR产物的3 DPNI消解注意：该步骤的目的是DIGEST残留质粒DNA模板，从第2节DPNI将消化质粒DNA，只有当它的发生质粒DNA 大坝+细菌菌株生长的甲基化，这样的项目完成报告。因此，不与DPNI处理，如果质粒DNA将被用作在GA反应的DNA片段。 <ol><li>加入2微升DPNI限制酶的45微升第2节中孵育所产生的PCR产物在37℃下1小时。直到需要与第4或冻结继续在-20℃。 </li></ol> 4.净化和DNA片段的浓度超过DNA纯化磁珠注意：该步骤的目的是为了纯化和浓缩，可以使用在第2和第3其它PCR纯化方法获得的PCR产物为好。 <ol><li>平衡的DNA纯化磁性珠粒至室温。重新暂停的珠子通过短暂的震荡。 </li><li>转移的PCR预消化无线第DPNI到1.5ml管中，并添加81微升DNA纯化磁性珠粒到每个管中。在室温下孵育该混合物10分钟。 </li><li>放置在磁集电极管约2分钟。丢弃使用移液管的清亮液体。用200μl80％乙醇30秒洗两次。 </li><li>允许粒料干燥。保持管的盖子打开时，与定位在磁收集器管。 </li><li>重悬的干燥珠在10微升10毫摩尔Tris盐酸，pH值8.0。在室温下孵育2分钟。简要地旋在管的底部收集液体。 </li><li>定位在磁性集电极2分钟的管子。除去8.5 - 10微升澄清溶液，并将其放置在一个新的预标记的试管中。确定由紫外光谱的DNA片段的浓度（ 见表设备 ）。 </li></ol>该产品在E. 5.大会克隆反应与转型大肠杆菌 &lt;P类=“j​​ove_content”&gt;注：客观上这一步是计算3：1的比例插入：向量和进行组装反应。 <ol><li>使用至少100纳克表示载体骨架或DNA片段携带选择标记的DNA片段。计算出3倍摩尔过量为将要用作插入的DNA片段。 </li><li>转换的摩尔过量的NG需要每一个特定的插入。 注：方便的方式做计算是使用基于Web的应用程序（ 见表设备 ）。 </li><li>混合的DNA片段的计算量在PCR管中，调节音量，以10微升。加入10微升的GA预混（2 ​​倍， 见表材料 ）。孵育在50℃下1小时为4组装在反应 - 6片段或15分钟为2-3的片段装配在PCR热循环仪。 </li><li>进行组装产品在主管E.转型大肠杆菌或冻结的产品在-20℃，直到需要。 </li><li>按照附带的化学或电感受态细胞的转化过程。通常情况下，使用2微升每个转化反应组装反应。 </li><li>转换完成后，铺在补充有合适的选择性抗生素的琼脂板上转化的细胞。 </li></ol> 6.质粒隔离，酶切和测序注意：该步骤的目的是为了隔离从大肠杆菌质粒DNA 菌 ，然后验证构建体通过限制性消化和测序。 <ol><li>传播几个单菌落迷你棉片和质粒隔离。使用2-5毫升隔夜LB液体培养基辅以适当的抗生素。通过以下所中所使用的微型制备试剂盒中描述的方法分离出相应的质粒。 </li><li>确定使用分光得到的量和质粒的浓度光度计。 </li><li>用一种或几种限制性内切酶来〜0.5微克纯化的质粒进行限制性消化。使用限制性内切酶消化提供特性鲜明图案的DNA结构。 </li><li>确认DNA克隆成功通过使用特定的或标准的引物的DNA测序。为准确分析测序数据。 </li></ol>

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成功使用遗传克隆应当前始终有一个仔细设计的完整构建体（ 图2和图3）的。 由底漆生成工具设计引物序列的仔细核查，也极力推荐。在不使用的引物生成工具的可产生引物的GA。然而，使用该工具是高度推荐的，因为它简化了工艺。通常，引物对的GA克隆必须具有两个功能上不同的序列。第一序列是DNA片段特异性的，并且允许使用PCR片段的扩增。第二序列与相邻的片段，这是必要的GA组件重叠。一个典型的DNA片段的特异序列是长18-22个核苷酸。的DNA片段用于扩增相同的DNA特定序列必须具有相似的解链温度和GC含量。重叠序列应该至少为15核苷酸长度的至少48℃的熔化温度。大于4的DNA片段的装配将需要重叠序列为至少20个核苷酸。更长的重叠将使产生更正确组装的DNA片段退火的提高的特异性。建议避免被扭曲他们的GC或AT开发重叠序列的内容，因为倾斜的序列可能会损害DNA正确的装配序列。 我们也建议使用作为阴性对照，其不应该产生任何抗药性的细菌菌落，只对应于一个GA反应的载体骨架中的DNA片段。可替代地，包括了“插入”的DNA片段中的一个可从GA反应，这也应导致无抗药性的细菌菌落被删去。之所以没有菌落生长会缺少相邻DNA片段重叠的端部，这将呈现并发症的组装ETE质粒。 在协议中所描述的，重叠的，因为所用的DNA片段的数目（ 图3）的25个核苷酸序列来产生引物组使用。引物生成工具网站的建议（ 见表设备 ）是用至少20个核苷酸的重叠序列装配4 - 6的DNA片段。另外，更长的重叠序列可确保DNA链的适当互补（参见图1）增加的准确组装的产品数量。 目前，几个可用的系统无缝克隆。然而，这些系统仍然采用限制性内切酶（ 即金门克隆18）。其他人使用基于痘苗病毒DNA聚合酶和单链DNA结合蛋白来自相同生物源19的酶的专有共混物。这两个系统都是由肖尔相比限于GA重叠序列器长度。因为较短的重叠序列可能无法提供足够的特异性以相邻的DNA片段的退火工序中，使超过23的DNA片段有问题的正确装配。这些缺点是不存在于遗传系统。 的DNA片段被扩增的尺寸也应考虑以不超过该大小通过PCR（ 即，小于8千碱基长）可靠地扩增。即使在过去10年中的DNA聚合酶的功能的改进，大的DNA片段将用较少的效率和准确度被放大。如果需要的话，更大的DNA片段可能来自其他来源的替代PCR方法， 例如可以得到，由质粒分离和适当的DNA酶切。具体地讲，在当前的手稿中描述的协议，我们合理使用的PCR为基础的可用的DNA片段的大小，这比所有少5 KBP。如果有识别用琼脂糖凝胶电泳多个PCR产物，所希望的片段大小的凝胶纯化，使用任何可用的分子生物学技术或合适的试剂盒推荐的。在当前的协议的耐热性DNA聚合酶被用于（ 见表材料 ）。但是任何DNA聚合酶提供高保真和产量将是适合于本协议中使用。如果在协议说明以外的不同高保真DNA聚合酶是可用的，如在相应手册中所述使用设置的条件。在当前的协议，化学感受态大肠杆菌细胞被用于与该遗传试剂盒中提供。另外，化学或电主管E.大肠杆菌菌株，如DH5α或DH10B中都可以使用。 装配克隆2X预混液很容易用最少的动手时间使用。但是，准确的移液是NE，因为小体积的要求EDED并加以混合。良好的分子生物学技术，需要在所有的时间以及行使。 对GA克隆提供了无限的可能性的DNA片段，质粒和载体，这是超过3千碱基的大小的结构。此外，它在合成生物学领域的广泛影响，因为它允许合成和装配，例如，一个完整的细菌（ 丝状支原体 ）的基因组或酵母（酿酒酵母）的染色体20,21。该技术也适用于常规克隆需要生成无缝结构。 总之，GA克隆提供快速，可靠和灵活的替代传统的DNA克隆过程。

常规的DNA克隆方法依赖于使用限制性内切酶切割的DNA和DNA连接酶来连接DNA片段一起。含有不同DNA片段的定制表达构建体的产生是一个连续的过程，其中包括DNA的切割与一个和/或多个限制性内切酶和DNA片段通过连接的后续插入。这个过程的主要缺点是，合适的限制性酶对DNA片段中的一个可能是难以确定（ 即，可能有几个切割位点）描绘感兴趣不可能的全长蛋白质的成功的DNA克隆。因此，代定制表达下的有效的细胞类型特异性启动子的转录调控定制蛋白质标记构建需要非常仔细的设计。这也是一个时间和劳动消耗的技术。最近，几个报告中所述的方法来组装MULTIP在同时在任一个或两个步骤的反应的连续序列，而无需使用限制的文件不同的合成的DNA片段切酶1-3。一步法克隆反应（不包括所有的准备步骤），取决于所使用的混合DNA核酸外切酶，DNA聚合酶，DNA连接酶2,3和DNA片段的重叠末端（ 图1）的。由于没有使用限制酶的，任何尺寸和序列组合物（不包括高度重复序列）的DNA片段，可以稠合在一起以无缝结构。最近，一种商品试剂盒（吉布森组件;乔治亚州）用于一步法克隆反应变得可用。该套件可在定制发起人和蛋白质标记单个向量的任何DNA片段的快速，经济高效的装配。 用于表达在哺乳动物细胞培养物的模型的外源蛋白质的广泛使用的质粒表达载体下的转录章往往ulation病毒巨细胞病毒（CMV）或猿猴病毒40（SV40）启动子。这些病毒启动子提供一种在大多数的哺乳动物细胞培养的基础模型的外源蛋白质的健壮瞬时表达。然而，新一代的细胞系稳定表达外源蛋白质往往是因为CMV或SV40发起人的建立过程中4,5转录沉默不成功。此外，SV40和CMV病毒启动子将不会充分地促进外源蛋白质在细胞中来自淋巴谱系或胚胎干细胞6,7的表达。解决病毒启动子的固有限制是使用强组成的非病毒启动子8-10。人来源的一个良好表征强组成的非病毒启动子是延伸因子1α（HEF1α）启动子（HEF1α参与氨酰tRNA向核糖体11对GTP依赖关联的催化）。然而，是含有HEF1α启动子的表达载体不是作为广泛使用含有质粒的病毒启动子，特别是那些还含有3×FLAG在感兴趣的蛋白的氨基末端。 在64000兆瓦裂解刺激因子蛋白（CSTF-64）是参与大多数的mRNA 12,13，包括复制依赖性组蛋白的mRNA 14,15的3&#39;端的处理。 CSTF-64表达在所有体组织12。其RNA识别基序结合的切割和聚腺苷酸化位点16的下游新生转录，GU富含RNA序列。此CSTF-64到前mRNA的结合促进了新生转录物的有效核酸内切裂解。 这里，一个协议描述了使用该DNA片段的PCR扩增，一个吉布森组件克隆试剂盒（其包括化学感受细菌细胞）以产生3XFLAG标记米定制载体乌斯CSTF-64或突变CSTF-64至HEF1α启动子1的表达在其氨基末端。

<table border="0" cellpadding="0" cellspacing="0" width="553">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:196px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:196px;">Synthetic double-stranded DNA fragment (sDNA)</td>
			<td style="width:71px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td>We used gBlocks, which can be up to 2 kb in length. However, there are many commercial sources of synthetic DNA available.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:196px;">DNA purification magnetic beads</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td>Agencourt AMPure XP - PCR Purification system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:196px;">Plasmid Isolation Mini Kit</td>
			<td style="width:71px;">Omega Bio-Tek Inc</td>
			<td style="width:119px;">D6942-01</td>
			<td>E.Z.N.A. Plasmid Mini Kit I</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:196px;">Gibson Assembly Cloning Kit</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;">E5510S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">DNA polymerase&#160;</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;">M0494S</td>
			<td>Q5 Hot Start High-Fidelity 2X Master Mix</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:196px;">DpnI</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;">R0176S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:196px;">NheI</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;">R3131S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:196px;">NotI</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;">R3189S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:196px;">HindIII</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;">R3104S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:196px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Spectrophotometer</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>NanoDrop device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">EditSeq</td>
			<td style="width:71px;">DNASTAR</td>
			<td style="width:119px;"></td>
			<td>Part of Lasergene Core Suite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">SeqBuilder</td>
			<td style="width:71px;">DNASTAR</td>
			<td style="width:119px;"></td>
			<td>Part of Lasergene Core Suite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Word</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>Part of&#160; Microsoft Office</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">NEBuilder</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;"></td>
			<td>primer generation tool: http://nebuilder.neb.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">NEBioCalculator</td>
			<td style="width:71px;">New England BioLabs</td>
			<td style="width:119px;"></td>
			<td>ligation calculator: http://nebiocalculator.neb.com/#!/ligation</td>
		</tr>
	</tbody>
</table>

generation of plasmid vectors expressing flag-tagged proteins under the regulation of human elongation factor-1&#945; promoter using gibson assembly

吉布森组件（GA）的克隆提供了一种快速，可靠和灵活的替代常规的DNA的克隆方法。我们使用遗传算法以创建定制的质粒的小鼠胚胎干细胞的外源基因（mESCs）的表达。的SV40或人巨细胞病毒启动子的控制下的外源基因表达转染到mESCs后迅速减弱。所述的治疗药本减弱表达是使用人延伸因子-1α（HEF1α）启动子来驱动基因表达。是包含HEF1α质粒载体没有被广泛作为SV40-或含CMV质粒，特别是那些还含有N-末端3XFLAG-标签。此处所描述的协议是一个快速的方法来创建的质粒表达FLAG标记CSTF-64和CSTF-64下HEF1α启动子的表现的调节突变。 GA使用的共混物的DNA外切核酸酶，DNA聚合酶和DNA连接酶，使重叠的可能的DNA片段的末端的克隆。基于模板的DNA，我们有可用的，我们设计了构建体被组装成一个单一的序列。我们的设计中使用的四种DNA片段：3.1的pCDNA载体骨架，HEF1α启动子部分1，HEF1α启动子部分2（其包含购买作为双链合成DNA片段3XFLAG标签），以及任一CSTF-64或特定CSTF-64突变体。这些片段的序列被上传到的引物生成工具设计适当的PCR引物，用于产生DNA片段。 PCR后的DNA片段混合用含有选择性标记和遗传克隆反应的载体装配。从个别转化的细菌菌落的质粒中分离得到。质粒的初始画面通过限制性消化完成，随后测序。总之，GA使我们能够在五天创建定制的质粒的基因表达，包括构建屏幕和验证。

其次，该协议的工作流程示于图2A中 。我们想克隆CSTF-64和突变CSTF-64蛋白下HEF1α启动子（ 图2B和图3）的表现的调控融合至3XFLAG标签。包含质粒HEF1α其次3XFLAG标签是不提供给我们。但是，下面的质粒是可供选择：3.1的pCDNA MYC-他（A;从麦克拉詹森一份厚礼），HEF1α含质粒（从姆拉登Yovchev一份厚礼）和鼠标CSTF-64质粒12（ 图3）。使用核苷酸和文本编辑应用程序（ 见表设备的图3）的构建体（多个）对整个序列进行装配。随后，序列（S）被分裂在四个方便件（ 图2B，红色块和图3）相对应的可用质粒DNA。放大Primers用底漆生成工具（ 见表设备 ）与4约束的设计- 6片段与25 nt的最小重叠，成立了“更改吉布森大会设置”弹出窗口。的NheI和NotI限制性位点被包括在所述引物设计用于识别正确组装质粒的目的。 NheI位点位于之间3.1的pCDNA和HEF1α启动子的5&#39;端的引物序列。 NotI位点位于终止密码子的CSTF-64和3.1的pCDNA载体骨架（UGA）之后。在同时消化这两种酶的DNA片段，由HEF1α启动子，3XFLAG和CSTF-64或突变与CSTF-64将被释放（见下文和图2B）。引物被勒令在尽可能小的规模和脱盐。含3XFLAG标记的DNA片段的HEF1α启动子的第二部分（490碱基对， 图2B，图3）被购买作为单个SDNA片段（SEE 表材料 ）。在装配反应中所用的DNA片段，使用DNA聚合酶（ 见表材料 ）扩增。 HEF1α启动第1部分，HEF1α启动第2部分，全长和突变CSTF-64的DNA片段，在一个单独的管同时扩增为28个周期（ 图4A），继DNA聚合酶的供应商的建议（ 见表材料对于每个循环变性为7秒，在98℃，退火45秒，在55℃，伸长90秒，在72℃）。最初，所述的pCDNA 3.1骨干扩增22个循环（使用相同的条件如上述除外的伸长时，将其设置为3分钟，在72℃）。然而，所得到的DNA产率是不充分的在装配反应（ 图4A）被使用。因此，一个附加的扩增以获得足够的DNA。 PCR产物获得从质粒模板编辑必须DPNI限制性酶消化以除去质粒DNA，否则会污染所得到的组件的反应产物和会产生假阳性药物抗性的细菌菌落。因此，将PCR产物进行消化，进行DPNI限制性酶，从坝+ E其中切割甲基化和半甲基化的质粒DNA中分离大肠杆菌菌株。使用合成的DNA片段得到的PCR产物，作为模板不需要与DPNI因为化学合成的DNA被消化不含有甲基化或半甲基化碱基。 的DNA片段进行纯化，并浓缩过的DNA纯化磁性珠粒（ 见表材料 ）作为在协议步骤4的PCR为3.1的pCDNA载体骨架合并一起描述和DNA纯化磁性珠粒的用量作了相应的调整。该DNA产率，测定使用分光光度计（ 表设备1和表 ）。装配反应为CSTF-64和突变CSTF-64构建体组装在冰（ 表1）。有3倍摩尔过量视为“插入”DNA片段的使用（ 表1，图3）。该混合的DNA片段的最终体积调节至10微升的水和10μl装配主混合物（2×）溶液中加入的。将反应物​​混合并温育在50℃下搅拌1小时。阳性对照反应，根据联大试剂盒说明书的建议还组装与CSTF-64和突变CSTF-64的反应同时培养。所建议的协议，2微升的各装配反应的转化在化学感受态大肠杆菌提供与装配克隆试剂盒（ 见表材料 ）。所述进行转化为在试剂盒中所描述手册。阳性克隆，在氨苄青霉素琼脂/ LB板上选择。每各装配反应6个菌落，随机选择要传播。质粒DNA使用质粒分离mini试剂盒（ 见表材料 ）分离， 在用限制性构建体芯片上的消化酶NheI和NotI导致两种片段与4590碱基对，3032碱基对CSTF-64和4590碱基对大小， 2711碱基对突变CSTF-64（ 图2B和图4B）。消化与限制性酶HindIII和NotI导致三个片段与以下尺寸：5,872 bp的1005基点和745基点（CSTF-64）和5872 bp的1005基点和424基点（突变CSTF-64， 图2B和图4C）。事实上，分 ​​离的质粒的消化所显示的预期特性的图案（ 图4B，C）。需要注意的是通过用HindⅢ消化所述CSTF-64突变体的和NotI产生的424 bp的DNA片段图4C在质粒上被弱染色，由于其尺寸小。 2出6分离的质粒被送往测序。我们测序HEF1α启动子，和CSTF-64或突变CSTF-64份的构建体，以确认不存在缺失，插入或取代。我们强烈建议由此产生的DNA结构或任何基于PCR的协议序列。测序结果表明每个测序质粒中的一个中包含的HEF1α，3XFLAG标签和CSTF-64或突变CSTF-64的区域中的预期的序列。每个其它质粒具有相应的DNA片段的扩增过程中引入的点突变。含CSTF-64在小鼠胚胎干细胞中的质粒的表达，产生的外源蛋白质丰富量可比野生型表达17。 <img alt="图1" src="/files/ftp_upload/52235/52235fig1highres.jpg" width="450" /> 图吉布森组装机构的一示意图。DNA片段重叠的两端分别等温组装在一个单一的连续的序列。重叠端的第一是由5&#39;核酸外切酶，这是逐渐热灭活咀嚼回来。因此，重叠的端部不同的DNA片段，将等温退火。 DNA聚合酶将填补国内空白和耐热DNA连接酶ligates的刻痕。 <a href="https://www.jove.com/files/ftp_upload/52235/52235fig1highres.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/52235/52235fig2highres.jpg" width="650" /> 该协议的图2（A）流程图说明装配克隆。质粒（B）表示，PGA-CSTF-64使用GA部件生成红色-在装配反应中使用的DNA片段：的pCDNA3.1; HEF1α启动子部分1（hEF1a - 1）; HEF1α启动第2部分（hEF1a - 2）订购的合成DNA;鼠标CSTF-64（mCstF-64）。蓝色 - 开放阅读框。紫 - 病毒和非病毒启动子。绿色-裂解和多聚腺苷酸化地区<a href="https://www.jove.com/files/ftp_upload/52235/52235fig2highres.jpg" target="_blank">请点击这里查看此图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52235/52235fig3highres.jpg" width="650" /> 图吉布森装配CSTF-64质粒3.设计。黑色方框代表的DNA片段可用来设计一个单一的吉布森装配CSTF-64 在硅片序列。接着，该序列被分为4的DNA片段，它通过PCR扩增。需要注意的是，由于3XFLAG标签序列被设计成一个SDNA连同HEF1αPR的小尺寸<html> omoter第2部分。 <a href="https://www.jove.com/files/ftp_upload/52235/52235fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52235/52235fig4highres.jpg" width="650" /> 图4的PCR中的克隆的反应和所得到的质粒的代表限制性酶消化所用的DNA片段 （A） 的代表性的PCR使用热启动高保真2X主混合物用于在装配反应中使用的DNA片段：（B）的代表性HEF1α，全长CSTF-64，3.1的pCDNA构造（PGA-CSTF-64）和HEF1α，突变CSTF-64，3.1的pCDNA构造（PGA-mutCstF-64）用NheⅠ消化和NotI质粒。 （C）的相同质粒如乙用HindIII消化和NotI酶。“_blank”&gt;点击此处查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> DNA片段的名称</td><td>预期大小（bp）的</td><td>浓度。 （毫微克/微升） </td><td>稀释（纳克/微升） </td><td>微升用于GA CSTF-64从稀释</td><td>微升用于GA mutCstF-64，从稀</td><td>摩尔比（INS：VEC） </td></tr><tr><td> 3.1的pCDNA（矢量） </td><td> 4618 </td><td> 158 </td><td>未稀释</td><td> 1 </td><td> 1 </td><td></td></tr><tr><td> hEF1_启动第1部分</td><td> 825 </td><td> 213 </td><td> 75 </td><td> 1 </td><td> 1 </td><td> 3：1的</td></tr><tr><td> hEF1_启动第2部分为CSTF-64 </td><td> 516 </td><td> 229 </td><td> 50 </td><td> 1 </td><td></td><td> 3：1的</td></tr><tr><td> CSTF-64 </td><td> 1796 </td><td> 161 </td><td>未稀释</td><td> 1 </td><td></td><td> 3：1的</td></tr><tr><td> hEF1_子部分2突变CSTF-64 </td><td> 516 </td><td> 199 </td><td> 50 </td><td></td><td> 1 </td><td> 3：1的</td></tr><tr><td>突变CSTF-64 </td><td> 1448 </td><td> 201 </td><td> 171 </td><td></td><td> 1 </td><td> 3：1的</td></tr></tbody></table> 表1产率的DNA片段上的磁珠，稀释后的浓度，并设置了装配反应。

Watch this Scientific Journal Video about Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1Î± Promoter Using Gibson Assembly at JoVE.com

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

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Research

JoVE Journal

Biology

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

36.7K 次观看.  Texas Tech University Health Sciences Center. Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning. 

视频: Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

终结者使用PGR-蓝质粒和金门议会的快速验证

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

CAPRRESI：通过质粒恢复和限制酶位点插入的嵌合体装配

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...