这项工作得到了墨西哥国家科学委员会，墨西哥国家科学委员会（ACACYT），墨西哥国家科学院（154833）和墨西哥国立自治大学的支持。作者希望感谢VíctorGonzález，Rosa I.Santamaría，Patricia Bustos和SoledadJuárez的行政和技术咨询。

CAPRRESI协议注意: 图1表示总体CAPRRESI协议。该技术基于计算机设计和随后构建期望的嵌合体。 <ol><li> 选择克隆质粒pUC18或pUC19。 <ol><li>选择最适合技术需求的pUC载体。 注意:两个载体具有相同的序列，但多克隆位点的方向除外。这对于寡核苷酸的设计和PCR质粒破坏是重要的。 </li></ol></li><li> 在野生型基因和pUC18 / 19序列的计算机分析中。 <ol><li>获取野生型基因的DNA序列，并将其保存在FASTA格式文件（ 例如， gene.fas）中。 注意:pUC18 / 19载体的序列包含在pUC.fas文件中（ 图1 ，步骤1）。 </li><li>确定哪个限制enz通过运行REsearch.pl脚本，ymes不切割野生型基因和pUC18 / 19序列。或者，使用在线工具执行限制酶位点搜索（ 图1 ，步骤2）。 <ol><li>在终端中键入以下内容: perl REsearch.pl基因 perl REsearch.pl pUC.fas 注意:这些命令将创建以下输出文件:genes_lin.fas，genes_re.fas，pUC_lin.fas和pUC_re.fas。 </li><li>选择适当的限制酶，通过比较genes_re.fas和pUC.fas文件，将野生型基因克隆到选定的pUC18 / 19载体的多克隆位点。选择插入物相对于pUC18 / 19载体的氨苄青霉素抗性基因（ ampR ）的方向。 </li></ol></li><li>设计正向和反向寡核苷酸以扩增野生型基因（外部寡核苷酸）的编码区。在每个5&#39;端包含适当的限制酶位点的寡核苷酸;这将定义插入方向。 <ol><li>在正向寡核苷酸中包含功能性核糖体结合位点。 </li><li>在载体内插入相同方向的两个野生型基因。 </li></ol></li></ol></li><li> 选择嵌合体装配和质粒破坏的断裂区域。 <ol><li>通过运行translate.pl脚本获取野生型和ampR基因的氨基酸序列（ 图1 ，步骤3）。 <ol><li>在终端中键入以下内容: perl translate.pl基因 perl translate.pl ampR.fas 注意:此脚本将创建以下输出文件:genes_aa.fas和ampR_aa.fas。 </li></ol></li><li>将两个野生型氨基酸序列与适当的软件（ 如 MUSCLE）全局对齐9 。 </li><li>为嵌合体选择所需的断裂区域（3-6个氨基酸）基于序列比对。将它们保存为FASTA格式的文件（ 例如 ，区域 .fas）。 </li><li>找到编码所选氨基酸的两个野生型基因的DNA序列。 </li></ol></li><li> 含有限制酶位点的同义词DNA序列的计算机生成。 <ol><li>获得编码每个氨基酸序列拉伸的所有同义词DNA序列，选择为断裂区（ 图1 ，步骤4）;这些序列存储在regions.fas文件中。 <ol><li>在终端中键入以下内容: perl synonym.pl regions.fas 注意:此脚本将创建regions_syn.fas输出文件。 </li></ol></li><li>搜索同义词DNA序列上发现的限制酶位点。 <ol><li>在终端中键入以下内容: perl REsynonym.pl regions_syn.fas 注意:此脚本将创建regions_syn-re.fas输出文件。 </li></ol></li><li>选择一种在野生型基因的同义词序列之间共享的限制酶;该网站将用于通过限制酶消化装配嵌合体。验证选择的限制酶既不切割野生型基因也不切割pUC18 / 19载体序列。 <ol><li>比较在genes_re.fas，pUC_re.fas和regions_syn-re.fas文件中找到的限制酶。 </li></ol></li><li> 在两个野生型基因的相应位点处，将原始DNA替代为同义词DNA序列。将同义词DNA序列添加到野生型序列文件（genes.fas）中。 </li><li>获取基因中包含的基因的氨基酸序列。文件（ perl translate.pl genes.fas ）。 </li><li>对齐从野生型和同义词DNA序列翻译的氨基酸序列。验证两个序列是否相同。 </li><li>用pU上存在的ampR基因重复本节的所有步骤C18 / 19载体。 注意:目标是将ampR基因（文件ampR.fas）分解成两个互补的部分。选择用于破坏ampR基因的限制酶应与用于嵌合体装配的限制酶不同。 </li></ol></li><li> 将两种野生型基因独立克隆到选择的pUC18 / 19载体中（图1，步骤3）。 <ol><li>使用质粒DNA纯化试剂盒纯化所选择的pUC18 / 19质粒DNA。 <ol><li>例如，用pUC18质粒转化大肠杆菌 DH5α，并在37℃下在固体LB-0.3mg / mL氨苄青霉素（Amp）上生长转化体过夜。挑取转化体集落，接种新的LB-0.3mg / mL Amp板，并在37℃下生长过夜。 </li><li>取部分先前的培养物，并在液体LB-0.3mg / mL Amp中在37℃下生长6-8h。使用试剂盒提取质粒DNA。 </li></ol></li><li> PCR使用approp从整个基因组DNA扩增野生型基因对抗外部寡核苷酸。如果可能，请使用高保真DNA聚合酶。选择最大化产量的DNA聚合酶。根据制造商的指导和寡核苷酸的解链温度进行PCR。 <ol><li>根据DNA聚合酶，扩增子大小，模板和寡核苷酸，进行PCR循环。例如，为了扩增rpoD DNA，制备50μL的反应物:5μL的10×缓冲液， 2μL的 50mM MgSO 4，1μL的 10mM dNTP混合物，2μL的每个10μM寡核苷酸（表2），2 μL模板DNA（ 大肠杆菌 DH5α总DNA），35.8μL超纯水和0.2μL高保真DNA聚合酶。进行以下PCR循环:94℃1分钟进行初始变性，然后进行30个循环的扩增（94℃至30℃，变性30秒，寡核苷酸退火30秒，68℃退火2分钟）扩展）。 </li><li>溶解1.2克通过在微波炉中加热将它们在100mL双蒸水中进行琼脂糖。组装凝胶托盘并梳理，并将其放入水平凝胶浇铸机。用熔化的琼脂糖溶液填充凝胶托盘，使其凝固。取下梳子。 </li><li>将凝胶放入电泳室。用1x Tris-acetate-EDTA缓冲液填充室。将50μLPCR产物载入凝胶孔。电泳样品（ 例如 ，在110V下1小时）。 </li><li>电泳后，将缓慢振荡的含有100μL1mg / mL溴化乙锭溶液的100mL双蒸水中的凝胶染色10分钟。在缓慢摇动下，用双蒸水洗涤凝胶10分钟;使用水平摇床。 注意:溴化乙锭是一种有毒化合物;戴防护手套和棉花实验室外套。 </li><li>使用试剂盒从凝胶的相应条带纯化野生型基因DNA。通过将染色的凝胶置于UV室中可视化条带（推荐波长:300nm）。保持凝胶的暴露至少。使用DNA纯化试剂盒，并按照制造商的说明进行操作。 注意:紫外线辐射是危险的;戴防护罩，眼镜和棉花实验室外套。 </li></ol></li><li>根据制造商的说明书，用限制性内切酶纯化野生型基因和pUC18 / 19质粒DNA。尽可能使用快速消化酶。例如，在10μL超纯水，2μL10X缓冲液， 1μLKpn I限制酶和1μLXba I限制酶中消化6μLpUC18 DNA（300 ng /μL）。在37℃下保持反应30分钟。 <ol><li>根据制造商的指南禁用限制酶。例如，将80℃下的双消化反应Kpn I- Xba I灭活5分钟。 </li></ol></li><li>混合插入:体积比例的载体DNAo为3:1。按照制造商的说明进行连接反应。尽可能使用快速连接酶（在25℃下保持10分钟）。 注意:通常，使用试剂盒纯化后的DNA浓度足以进行消化和连接反应。例如，加入6μL的rpoD DNA（ KpnI - XbaI ，150ng /μL），3μL的pUC18 DNA（ KpnI - XbaI ，150ng /μL），10μL的2x缓冲液，1μL的超纯水和1μLT4 DNA连接酶。在25℃保持连接反应10分钟。 </li><li>用2-4μL的连接反应转化大肠杆菌 DH5α，如补充材料所述。将转化的细胞平板化成固体LB / Amp / X-gal / IPTG平板。将板在37℃下过夜。 </li><li>选择白色的转化体大肠杆菌菌落，并在新的LB / Amp平板上条纹。将板在37℃下过夜/ LI&gt; <li>进行菌落PCR以选择用于测序反应的候选物，如补充材料中所述。从候选物中提取质粒DNA。或者，使用用于克隆插入片段的相同限制酶的消化候选质粒DNA。 </li><li>具有测序服务提供商的序列候选结构10 。使用DNA汇编器11 （ 图1 ，步骤5） 以计算机方式组装序列。 </li></ol></li><li> 通过PCR进行质粒破坏，然后进行限制酶消化。 <ol><li>分别设计用于野生型和ampR基因的断裂区域的硅片正向和反向寡核苷酸。将其相应的同义词DNA序列（在步骤1.4中获得）替换为野生型片段。 注意:该同义词DNA序列含有限制酶位点。正向和反向寡核苷酸在t处重叠他限制酶位点。寡核苷酸应在21-27个核苷酸的范围内，以胞嘧啶或鸟嘌呤结束，并含有限制酶位点。正向寡核苷酸与模板DNA上的靶区域具有相同的序列。反向寡核苷酸是模板DNA上靶区域的相反互补序列。 </li><li>使用两个野生型基因构建作为PCR反应的DNA模板（ 例如， pUC18 rpoD和pUC18 sigA ）。获得每个结构的两个互补部分（pUC18 rpoDσ1 -σ2，pUC18 rpoDσ3 -σ4，pUC18 sigAσ1 -σ2和pUC18 sigAσ3 -σ4）（ 图1 ，步骤6）。 </li><li>将DNA样品加入琼脂糖凝胶1-1.2％[w / v]。通过电泳（ 例如 110V，1小时）将PCR产物与DNA模板分离。或者，用Dpn I消化PCR反应以破坏DNA模板。 注意: DpnI酶仅识别甲基化DNA序列（5&#39;-GATC-3&#39;）。 <ol><li>使用DNA纯化试剂盒纯化样品。按照制造商的指导。 </li></ol></li><li>根据制造商的指示，用合适的限制性酶将所有互补DNA片段双重消化。尽可能使用快速消化限制酶。例如，使用Afl II和Spe I使用制造商的方案双重消化pUC18rpoDσ1-σ2DNA片段。将消化反应在37℃下保持15分钟。 </li><li>根据制造商的指南禁用限制酶。例如，将Afl II- Spe I在80℃下灭活20分钟。 </li></ol></li><li> 使用DNA连接和细菌转化的质粒回收。 <ol><li>将合适的DNA片段以1:1的比例混合以组装所需的嵌合基因（ 图1）1，步骤7）。 注意:以这种方式，恢复ampR基因的完整性，产生功能性蛋白质。 <ol><li>例如，混合4μLpUC18 rpoDσ1 - σ2DNA （用Afl II- Spe I消化，150ng /μL）， 4μLpUC18 sigAσ3 -σ4DNA（ Afl II- Spe I，150ng /μL） 10μL2x缓冲液，2μL超纯水和1μLT4DNA连接酶;试剂盒纯化后的DNA浓度足以消化和随后的连接。将连接反应在25℃保持10分钟。 </li></ol></li><li>用3-5μL的嵌合体装配连接反应（补充材料）转化大肠杆菌 DH5α。将LB / Amp / X-gal / IPTG板中的转化体细胞在37℃下培养过夜一夜。 </li><li>选择白色的转化体大肠杆菌菌落，并在新的LB / Amp平板上条纹。在37分钟过夜C。 </li><li>进行菌落PCR以选择候选物进行测序反应，如补充材料中所述。通过进行电泳（在步骤2.3.2.1中描述）可视化1-1.2％（w / v）琼脂糖凝胶中的条带。 </li><li>从积极的候选人培养文化。使用质粒DNA纯化试剂盒从其中提取质粒DNA。 </li></ol></li></ol>序列候选嵌合结构<ol><li>通过遵循序列服务提供商的指导，确保质粒DNA的质量对于测序反应是足够好的。使用纯化工具提取DNA。例如，在序列服务提供商的互联网页面上，请特别注意样品推荐的DNA浓度。使用DNA定量仪器获取样品的浓度。 </li><li>具有测序服务提供商的序列候选嵌合构建体10 。 </li><li>组合se<html>使用电脑 DNA汇编器11进行排序读取。 </li><li>使用序列比对工具9,12对齐组装和使用硅片设计的嵌合序列。验证嵌合基因是否融合成功。 </li></ol>做准备注意:涉及活细胞的所有步骤都应在本生灯开启的干净的层流罩内进行。 <ol><li> 生产大肠杆菌 DH5α感受态细胞。 <ol><li>为了生产大肠杆菌感受态细胞，遵循已公布的方案13或参见补充材料 。或者，使用市售的感受态细胞。 </li></ol></li><li> 转化大肠杆菌 DH5α感受态细胞。 <ol><li>对于大肠杆菌培养物的化学转化，遵循已公布的方案class ="xref"&gt; 13或查看补充材料 。或者，使用电穿孔进行细菌转化。如果使用市售的感受态细胞，请遵循制造商的指导。 </li></ol></li><li> 从大肠杆菌 DH5α中纯化基因组和质粒DNA。 <ol><li>按照补充材料中所述 ，使用市售的试剂盒或以下公开的方案进行核酸提取13 。 </li></ol></li><li> 进行菌落PCR。 <ol><li>使用该技术鉴定候选转化体集落用于随后的测序反应。 注意:此方法不代表序列完整性的最终验证。 补充材料中有详细的说明。 </li></ol></li><li> 准备解决方案 <ol><li>看到<strong&gt;表1有关溶液制备的更多信息。 </li></ol></li><li> 下载CAPRRESI协议所需的脚本和序列文件。 <ol><li>下载含有所需物种完整基因组序列的基因库文件，使用基因组浏览器提取所选基因的DNA序列，并以fasta文件格式保存。下载CAPRRESI协议所需的Perl脚本。将脚本和序列文件存储在同一目录中。 </li></ol></li></ol>

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作者宣称他们没有竞争的经济利益。

CAPRRESI被设计为PRM 2的替代品。原来的PRM是一种强大的技术;它允许沿所选基因的任何部分融合DNA序列。对于PRM，野生型基因应该被克隆到相同的质粒中。之后，在质粒内发现野生型和抗生素抗性基因内设计寡核苷酸。通过使用平端，高保真DNA聚合酶和先前设计的寡核苷酸的PCR实现质粒破坏。互补PCR产物的连接组装嵌合基因并恢复抗生素抗性盒，导致功能质粒恢复。 PRM使得能够在...

嵌合基因组装已被广泛用于分子生物学以阐明蛋白质功能和/或用于生物技术目的。存在融合基因的不同方法，如重叠PCR产物扩增1 ，质粒回收2 ，同源重组3 ，CRISPR-Cas9系统4 ，定位重组5和吉布森组装6 。这些都提供不同的技术优势;例如，重叠PCR设计的灵活性，质粒恢复期间的体内选择构建体 ，或CRISPR-Cas9和Gibson系统的高效率。另一方面，在执行这些方法时可能会出现一些困难;例如，前两种方法依赖于平头DNA片段，并且这些类型的产品的连接在技术上可能与stic结扎结扎。位点定向重组可以在原始样品上留下痕迹的额外的DNA序列（疤痕），如CrelxP系统5 。除了目标部位之外，CRISPR-Cas9有时可以修改其他基因组区域。 在这里，我们通过质粒回收和限制性内切酶位点插入（CAPRRESI）介绍嵌合体装配，这是一种融合蛋白质编码基因的方案，将基因质粒恢复方法（PRM）与限制酶位点插入同义词DNA序列，增强连接效率。为了确保氨基酸序列的完整性，将限制酶位点插入同义词DNA序列片段。 CAPPRESI的优点之一是可以使用普通的实验室试剂/工具（ 如酶，感受态细胞，溶液和热循环仪）进行，并且可以快速测定结果（使用适当的酶时）。依靠限制从同义词DNA序列出现的酶位点可以限制目标蛋白内部精确融合点的选择。在这种情况下，应使用重叠的寡核苷酸融合靶基因，并将限制酶位点插入到载体的抗性基因上。 CAPRRESI由七个简单步骤组成（ 图1 ）:1）选择克隆载体pUC18或pUC19 6 ; 2）待融合的野生型序列的计算机分析; 3）嵌合体装配和质粒破坏的断裂区域的选择; 4） 计算机生成含有限制酶位点的同义词DNA序列; 5）将野生型基因独立克隆到所选择的质粒中; 6）通过PCR进行质粒破坏，然后进行限制酶消化;和7）使用DNA连接和细菌转化的质粒回收。应该验证这种技术产生的嵌合基因测序。 pUC18 / 19载体提供克隆和嵌合体装配的技术优势，如小尺寸（ 即 2,686碱基对），高拷贝数，有利的测序反应特征和商业可用性7 。在这里，使用大肠杆菌宿主来组装和处理嵌合体，因为细菌培养物便宜且生长快。鉴于此，将需要随后将融合片段克隆到最终靶质粒中（ 例如，在哺乳动物细胞中的细菌或pCMV中的pRK415的表达载体）。 测试CAPRRESI融合两个主要的σ因子基因: 大肠杆菌D和根瘤菌（Rhizobium etlisigA） 。原始sigma因子是负责转录起始的RNA聚合酶亚基，它们由四个结构域组成（ 即 σ1，σ2，σ3和σ4） 8 。氨基酸序列长度由rpoD和sigA编码的蛋白质的h分别为613和685。 RpoD和SigA共享48％的身份（98％的覆盖率）。这些主要的σ因子在区域σ2和σ3之间分成两个互补的片段。根据该设计组装两个嵌合基因:嵌合体01（RpoDσ1-σ2+SigAσ3-σ4）和嵌合体02（SigAσ1-σ2+RpoDσ3-σ4）。通过测序验证DNA融合产物。

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Platinum Taq DNA polymerase High Fidelity</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">11304011</td>
			<td style="width:355px;">Produces a mix of blunt/3&#8217;-A overhang ended PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High pure plasmid isolation kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:115px;">11754785001</td>
			<td>Used for all plasmid purification reactions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High pure PCR product purification kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:115px;">11732676001</td>
			<td>Used for all PCR purification reactions from agarose gels</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">AflII restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R0520L</td>
			<td>Recognizes sequence 5&#8217;-CTTAAG-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">KpnI-HF restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R3142L</td>
			<td>Recognizes sequence 5&#8217;-GGTACC-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SpeI-HF restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R3133L</td>
			<td>Recognizes sequence 5&#8217;-ACTAGT-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">XbaI restriction enzyme</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">R0145L</td>
			<td>Recognizes sequence 5&#8217;-TCTAGA-3&#8217; and cuts at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ampicillin sodium salt</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">A0166-5G</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nalidixic acid</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">N8878-5G</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Yeast Extract</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">Y1625-1KG</td>
			<td>Bacterial cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Casein peptone</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">70171-500G</td>
			<td>Bacterial cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">S9888-1KG</td>
			<td>Sodium chloride</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agar</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:115px;">05040-1KG</td>
			<td>Bacterial cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">XbaI restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD0684</td>
			<td>Fast digest XbaI enzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KpnI restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD0524</td>
			<td>Fast digest KpnI enzyme</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Quick Ligation Kit</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:115px;">M2200S</td>
			<td>Fast DNA ligation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AflII restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD0834</td>
			<td>Fast digest AflII enzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SpeI restriction enzyme</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:115px;">FD1253</td>
			<td>Fast digest SpeI enzyme</td>
		</tr>
	</tbody>
</table>

caprresi: chimera assembly by plasmid recovery and restriction enzyme site insertion

在这里，我们通过质粒恢复和限制性酶切位点插入（CAPRRESI）提供嵌合体装配。 CAPRRESI受益于原始质粒回收方法的许多优点，并引入限制酶消化以缓解DNA连接反应（嵌合体装配所需）。对于该协议，用户将野生型基因克隆到同一质粒（pUC18或pUC19）中。 在嵌套嵌合体的氨基酸序列区域的计算机选择之后，用户获得编码它们的所有同义词DNA序列。 Ad hoc脚本使用户能够确定所有同义词DNA序列。在此步骤之后，另一个Perl脚本在所有同义词DNA序列上搜索限制酶位点。该计算机分析也使用在pUC18 / 19质粒上发现的氨苄青霉素抗性基因（ ampR ）进行。用户在同义词区域内设计寡核苷酸，通过PCR破坏野生型和ampR基因。在obt之后调整和纯化互补DNA片段，进行限制酶消化。通过连接适当的互补DNA片段来实现嵌合体装配。为CAPRRESI选择了pUC18 / 19载体，因为它们具有技术优势，如小尺寸（2,686碱基对），高拷贝数，有利的测序反应特征和商业可用性。用于嵌合体装配的限制酶的使用消除了产生平端产物的DNA聚合酶的需要。 CAPRRESI是一种快速且低成本的融合蛋白质编码基因的方法。

图1描绘了CAPRRESI。使用该方法，通过交换两个细菌原始sigma因子（ 即大肠杆菌 RpoD和R.etli SigA）的结构域来组装两个嵌合基因。使用GenBank基因组文件NC_000913和NC_007761的Artemis Genome浏览器14分别获得rpoD和sigA基因的DNA序列。从NCBI服务器的核苷酸数据库获得pUC18载体的DNA序列。通过运行REsearch.pl Perl脚本对DNA序列进行限制酶位点?...

Watch this Scientific Journal Video about CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion at JoVE.com

CAPRRESI：通过质粒恢复和限制性酶位点插入进行嵌合体组装 |生物学 |视频

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

在这里，我们通过质粒恢复和限制性酶切位点插入（CAPRRESI）提供嵌合体装配，这是基于将限制酶位点插入同义词DNA序列和功能质粒恢复的方案。该协议是一种快速和低成本的融合蛋白质编码基因的方法。

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 ...

caprresi-chimera-assembly-plasmid-recovery-restriction-enzyme-site

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SCIENTISTS IN ACTION

11.5K 次观看.  Universidad Nacional Autónoma de México. 在这里，我们通过质粒恢复和限制性酶切位点插入（CAPRRESI）提供嵌合体装配，这是基于将限制酶位点插入同义词DNA序列和功能质粒恢复的方案。该协议是一种快速和低成本的融合蛋白质编码基因的方法。 

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

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

特定地点的重组酶（SSR）技术的原则

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

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 ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

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 ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...