这项工作得到了西华师范大学提供的博士生科研启动基金（编号 20E059）的财政支持。

1. PRRSV 基因模板的制备
<ol><li>在 1 mL RNA 提取试剂中解冻病毒原液（参见 材料表）。</li>
	<li>加入 0.2 mL 氯仿并充分混合。孵育 3 分钟。</li>
	<li>在 4 °C 下以 12,000 × g 离心混合物 15 分钟。 注：混合物分为三个相，即无色水相、间相和红色苯酚-氯仿相。</li>
	<li>吸出无色水相并将其转移到新管中。</li>
	<li>将水相与 0.5 mL 异丙醇充分混合，并在 4 °C 下孵育 10 分钟。</li>
	<li>在 4 °C 下以 12,000 × g 离心 10 分钟。 注：试管底部有白色 RNA 沉淀物。</li>
	<li>使用微量移液器丢弃试管的上清液。</li>
	<li>加入 1 mL 75% 乙醇以重悬沉淀并短暂涡旋。</li>
	<li>在 4 °C 下以 7,500 × g 离心 5 分钟。 使用微量移液器丢弃试管的上清液。</li>
	<li>将 RNA 干燥 5 分钟，加入 20-50 μL 不含 RNase 的水以重悬 RNA，并充分混合。</li>
	<li>继续进行逆转录;设置反应以进行逆转录，如 表 1 所示。 注：为确保逆转录成功，请使用高质量的 RNA 模板。</li>
	<li>使用所得的 cDNA 进行 PCR 或将其储存在 -20 °C。</li>
</ol>2. PCR 引物设计
<ol><li>设计正向引物<ol><li>打开软件并选择 New DNA File（新建 DNA 文件）。</li>
		<li>将 PRRSV 基因序列 （GenBank： FJ548852.1） 从 NCBI 粘贴到软件中。单击 OK 生成序列文件。</li>
		<li>分析序列并标记片段连接。设计片段的正向特异性序列引物。在大多数情况下，优选的熔解温度 （Tm） 在 55 °C 和 62 °C 之间，GC 含量为 40-60%。</li>
		<li>单击 Primers 并选择 Add Primer。</li>
		<li>粘贴特异性序列，并将载体的重叠序列添加到特异性序列引物的前 5' 核苷酸中。在每个连接附近，选择 20-40 bp 作为两个相邻片段之间的重叠区域。</li>
		<li>命名包含突出端和特定序列的正向引物（参见 补充图 S1）。</li>
		<li>单击 Add Primer to Template（将引物添加到模板）。</li>
	</ol></li>
	<li>设计反向引物<ol><li>在软件中分析序列并标记片段连接。设计片段的反向特异性序列引物。</li>
		<li>单击 Primers 并选择 Add Primer。</li>
		<li>粘贴特异性序列，并将限制性位点添加到特异性序列引物的前 5' 核苷酸上。 注：除多个克隆位点外，插入片段和载体中必须不存在添加的限制性位点。</li>
		<li>将 20-40 bp 的载体突出端序列添加到限制性位点的前 5' 核苷酸中。</li>
		<li>命名包含突出端和特定序列的反向引物（参见 补充图 S1）。</li>
		<li>单击 Add Primer to Template（将引物添加到模板）。</li>
	</ol></li>
</ol>3. PCR 扩增片段
<ol><li>设置六个单独的 PCR 反应（表 2）：对于片段 1，使用引物 P1 和 P2（参见 补充图 S2）;对于片段 2，使用引物 P3 和 P4（参见 补充图 S2）;对于片段 3，使用引物 P5 和 P6（参见 补充图 S2）;对于片段 4，使用引物 P7 和 P8（参见 补充图 S2）;对于片段 5，使用引物 P9 和 P10;并使用引物 P11 和 P12 扩增片段 6（参见 补充图 S2）。 注：使用前解冻、混合并短暂离心每种组分。</li>
	<li>使用 表 2 中的三步方案运行 PCR。</li>
	<li>向 5 μL PCR 产物中加入 1 μL 6x DNA 上样缓冲液，混合并短暂离心内容物。</li>
	<li>使用 1% 琼脂糖凝胶电泳分析样品。</li>
</ol>4. PCR 片段的纯化
注：使用凝胶提取试剂盒从凝胶中纯化 PCR 产物（参见 材料表）对于载体构建很重要。
<ol><li>向 45 μL PCR 产物中加入 9 μL 6x DNA 上样缓冲液，混合并短暂离心内容物。</li>
	<li>进行 1% 琼脂糖凝胶/goldview 电泳以分离 DNA 片段。 注：请勿重复使用 TAE 电泳缓冲液，因为其 pH 值会影响 DNA 片段的回收率。</li>
	<li>条带充分分离后，使用锋利的手术刀小心地切除 DNA 条带。 注：通过修剪掉多余的琼脂糖来最小化凝胶切片的大小。</li>
	<li>在干净的 1.5 mL 微量离心管中称取凝胶切片，向凝胶切片中加入等体积的结合缓冲液（例如，0.3 mL 至 0.3 g 切片），并在 60 °C 下孵育 7 分钟。</li>
	<li>将微型柱插入 2 mL 收集管中。将步骤 4.4 中的 700 μL DNA/琼脂糖溶液添加到迷你柱中。</li>
	<li>在室温下以 10,000 × g 离心 1 分钟。丢弃滤液并重复使用收集管。</li>
	<li>加入 300 μL 结合缓冲液，并在室温下以 13,000 × g 离心 1 分钟。丢弃滤液并重复使用收集管。</li>
	<li>加入 700 μL 洗涤缓冲液，并在室温下以 13,000 × g 离心 1 分钟。丢弃滤液并重复使用收集管。</li>
	<li>以最大速度旋转空迷你柱 2 分钟以干燥柱基质。将微型柱放入干净的 1.5 mL 微量离心管中。</li>
	<li>将 20 μL 去离子水直接添加到柱膜中心，并在室温下静置 2 分钟。以 13,000 × g 的速度离心 1 分钟。</li>
	<li>将 DNA 储存在 -20 °C。</li>
</ol>5. 线性化向量的制备
注意：制备质粒后，可以使用选定的酶对其进行切割。长消化或双酶消化对于确保所有 DNA 的消化至关重要。这将减少后续实验中假阳性克隆的数量。
<ol><li>pVAX1 线性化
	<ol><li>在室温下按所示顺序制备反应混合物（表 3）。 注：应添加水的体积以保持指示的总反应体积。在这里，用反应混合物中的酶消化 1 μg 质粒。根据质粒浓度，可以调节反应混合物中质粒的体积。</li>
		<li>轻轻混合;然后向下旋转。在 37 °C 下在加热块或水恒温器中孵育 60 分钟。</li>
		<li>使用凝胶提取试剂盒进行类似于第 4 节中 PCR 片段的纯化进行凝胶纯化（参见 材料表）。</li>
	</ol></li>
	<li>pVAX1-F1 线性化 注：pVAX1-F1 是从第一轮 pVAX1（NdeI 和 HindIII）和片段 1 重组中获得的载体。pVAX1-F2 是从 pVAX1-F1 （NdeI） 和片段 2 重组的轮次中获得的载体。pVAX1-F3 是从 pVAX1-F2 （NdeI） 和片段 3 重组的轮次中获得的载体。pVAX1-F4 是从 pVAX1-F3 （NdeI） 和片段 4 重组的回合中获得的载体。pVAX1-F5 是从 pVAX1-F4 轮（EcoRV 和 NtoI）和片段 5 重组中获得的载体。<ol><li>对于每种载体，按照所示顺序在室温下分别制备反应混合物（表 3）。</li>
		<li>轻轻混合;然后向下旋转。在 37 °C 下在加热块或水恒温器中孵育 60 分钟。</li>
		<li>使用凝胶提取试剂盒进行类似于第 4 节中 PCR 片段的纯化进行凝胶纯化（参见 材料表）。</li>
	</ol></li>
</ol>6. 亚克隆到新载体
注：使用 50-200 ng 载体和插入片段时，可以获得良好的克隆效率。
<ol><li>设置 ExonArt 无缝克隆和组装反应（表 4）。使用灭菌的去离子 H2O 将总反应体积调节至 10 μL 并混合。</li>
	<li>将反应物在热循环仪中于 50 °C 孵育 15-60 分钟。 将样品储存在冰上。 注意：将孵育时间延长至 60 分钟可以提高组装效率。</li>
	<li>在冰上解冻 DH5α 化学感受态细胞。</li>
	<li>向感受态细胞中加入 10 μL 组装产物;然后，通过上下吹打轻轻混匀。</li>
	<li>将混合物放在冰上 30 分钟。</li>
	<li>在 45 °C 下热休克 45 秒，然后将试管转移到冰上 3 分钟。</li>
	<li>向试管中加入 900 μL SOC 培养基。</li>
	<li>在 37 °C 振荡培养箱中以 225 rpm 摇动试管 1 小时。</li>
	<li>将转化反应物以 6,000 × g 离心 2 分钟。弃去上清液，将细胞重悬于 100 μL 新鲜 SOC 培养基中。</li>
	<li>将转化的细胞悬液铺在含有 50 μg/mL 卡那霉素的单独 LB 板上。</li>
	<li>将所有板在 37 °C 下孵育过夜。 从每个实验板中挑选单独的分离菌落。</li>
</ol>7. 分析转化体
<ol><li>将 8 个菌落挑取到 20 μL 含有 50 μg/mL 卡那霉素的 LB 培养基中。</li>
	<li>设置菌落 PCR 反应并运行 PCR（表 5）。 注：对于片段 1 的菌落 PCR 反应，使用引物 P1 和 P2（参见 补充文件 1）;对于片段 2，使用引物 P3 和 P4（参见 补充文件 1）;对于片段 3，使用引物 P5 和 P6（参见 补充文件 1）;对于片段 4，使用引物 P7 和 P8（参见 补充图 S2）;对于片段 5，使用引物 P9 和 P10;并使用引物 P11 和 P12 检测片段 6（参见 补充图 S2）。</li>
	<li>向 5 μL PCR 产物中加入 1 μL 6x DNA 上样缓冲液，混合并短暂离心内容物。</li>
	<li>使用 1% 琼脂糖凝胶电泳分析结果。</li>
	<li>在 5 mL 含有 50 μg/mL 卡那霉素的 LB 培养基中选择一个阳性菌落，并在 37 °C 下生长过夜。</li>
	<li>根据制造商的说明，使用质粒 DNA 迷你试剂盒（参见 材料表）获得质粒。</li>
	<li>使用 1% 琼脂糖凝胶电泳可视化结果。</li>
</ol>

在 pVAX1 表达载体中克隆全长 PRRSV 基因

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+infectious+clone+of+HuN4-F112,+an+attenuated+live+vaccine+strain+of+porcine+reproductive+and+respiratory+syndrome+virus+high+copy+plasmid.">Generation of an infectious clone of HuN4-F112, an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus high copy plasmid.</a> Virol J. 8, 410 (2011).</li><li>Ni, Y. Y., Huang, Y. W., Cao, D. J., Opriessnig, T., Meng, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+DNA-launched+infectious+clone+for+a+highly+pneumovirulent+strain+of+type+2+porcine+reproductive+and+respiratory+syndrome+virus:+identification+and+in+vitro+and+in+vivo+characterization+of+a+large+spontaneous+deletion+in+the+nsp2+region.">Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region.</a> Virus Res. 160 (1-2), 264-273 (2011).</li></ol>

作者声明没有利益冲突。

Gibson 组装技术是一种基于 体外 重组的分子克隆方法，用于组装 DNA 片段8。该方法能够在单管等温反应中将多个 DNA 片段组装成一个环状质粒。然而，Gibson Assembly 技术的障碍之一是从 cDNA 获取长片段。由于多种原因，长片段难以准确扩增。例如，引物在长时间延伸时更容易错配，cDNA 质量可能不佳，或者富含 GC 的区域会阻止 DNA 聚合。当使用 PCR 无法从 cDNA 中轻易获得全长...

分子克隆是构建基于 DNA 的原核细胞和真核细胞表达实验工具的重要技术，是实验生物学中非常重要的组成部分。分子克隆涉及四个过程：获取插入片段 DNA、将插入片段连接到适当的载体中、将重组载体转化成大肠杆菌（大肠杆菌）以及鉴定阳性克隆1。到目前为止，已经采用了多种方法通过使用限制性内切酶 2,3 和 PCR 介导的重组 4,5,6 来连接 DNA 分子。同源重组，称为无缝克隆技术，是一组克隆方法，允许将一个或多个 DNA 片段不依赖于序列且无疤痕地插入载体中。该技术包括不依赖序列和连接的克隆 （SLIC）、无缝连接克隆提取物 （SLiCE）、In-Fusion 和 Gibson 组装。它采用核酸外切酶降解插入片段的一条链，利用载体生成内聚末端，并通过体内修复或体外重组通过形成磷酸二酯键将插入片段共价连接到载体上。将单个插入片段连接到任何序列的载体而没有任何疤痕的能力非常有吸引力。此外，该技术能够按预定顺序连接 5-10 个片段，而不受序列限制。
作为众多重组 DNA 技术之一，Gibson 组装技术是目前最有效的克隆方法 7,8，是一种强大而优雅的基于核酸外切酶的方法，可无缝组装一个或多个线性化 DNA 片段。Gibson 组装反应是在等温条件下使用三种酶的混合物进行的，即 5' 核酸外切酶、高保真聚合酶和热稳定 DNA 连接酶。由 5'-3' 核酸外切酶产生的单链 3' 突出端有助于在一端共享互补性的片段的退火。高保真聚合酶通过添加 dNTP 有效填充退火单链区域的间隙，热稳定的 DNA 连接酶密封缺口以形成关节 DNA 分子8。因此，该技术方法已被广泛用于基因表达载体的构建。
猪繁殖与呼吸综合征 （PRRS） 是一种病毒性疾病，可导致PRRSV 在任何 9 岁时引起猪的生殖障碍和呼吸衰竭。该综合征表现为发热、厌食、肺炎、嗜睡、抑郁和呼吸窘迫。此外，在一些流行病中观察到临床症状，包括耳朵的红色/蓝色变色。作为动脉病毒家族的一员，PRRSV 通过直接接触和交换液体（包括尿液、初乳和唾液）广泛传播到猪肉生产国。由于 PRRSV 在美国的传播，根据 580 万头母猪和 1.1 亿头猪的育种规模，猪肉行业的总经济损失估计约为每年 6.64 亿美元10,11。动植物卫生检验局报告显示，49.8% 的未接种疫苗的猪在血清12 中存在 PRRSV，受感染猪体内低水平的 PRRSV 通过唾液、鼻腔分泌物、尿液和粪便排泄13。已经实施了多种策略来控制 PRRSV 传播 14,15,16。除了消除程序以创建完全病毒阴性的群体或改善生物安全和管理外，接种疫苗是控制 PRRS 的有效手段。
PRRSV 是一种包膜、单链、正义 RNA 病毒，长度约为 15 kb。PRRSV 基因组由至少 10 个开放阅读框 （ORF）、一个短的 5' 非翻译区 （5' UTR） 和 3' 末端的 poly （A） 尾部组成（图 1A）17。负链 RNA 病毒的基因组是非传染性的，而正链 RNA 病毒的基因组是传染性的。生成病毒后代18 的 RNA 和 DNA 转染有两种主要策略。然而，克隆与 RNA 基因组相对应的全长片段对于感染性克隆的构建至关重要。由于 PRRSV 基因组的长而复杂，无法通过 PCR 一次轻松获得全长基因组。此外，尽管人工合成 PRRSV 基因是一种有效的解决方案，但长片段的合成通常很昂贵。因此，为了获得 PRRSV 全长表达载体，我们尝试通过多个插入片段同源重组方法创建它19,20。不幸的是，我们无法获得全长基因表达载体。因此，在本研究中，我们在反向引物中添加了合适的限制性位点，并通过几轮同源重组反应成功获得了 pVAX1-PRRSV 表达载体。此外，该方法还可以实现靶基因的缺失或突变，并有效地将大量 DNA 片段连接到表达载体上。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 kb plus DNA Ladder</td>
			<td>Tiangen Biochemical Technology (Beijing) Co., Ltd</td>
			<td>MD113-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Universal Green PCR Master Mix</td>
			<td>Rong Wei Gene Biotechnology Co., Ltd</td>
			<td>A303-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Microcentrifuge</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FRESCO17</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean Bench</td>
			<td>Sujing Antai Air Technology&#160; Co., Ltd</td>
			<td>VD-650-U</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Electrophoresis Equipment</td>
			<td>Cleaver Scientific&#160; Co., Ltd</td>
			<td>170905117</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Loading Buffer (6x)</td>
			<td>Biosharp Biotechnology Co., Ltd</td>
			<td>BL532A</td>
			<td></td>
		</tr>
		<tr>
			<td>E. Z. N. A. Gel Extraction kit</td>
			<td>Omega Bio-Tek Co., Ltd</td>
			<td>D2500-01</td>
			<td></td>
		</tr>
		<tr>
			<td>E.Z.N.A. Plasmid DNA Mini Kit I</td>
			<td>Omega Bio-Tek Co., Ltd</td>
			<td>D6943-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-heating Standing-temperature Cultivator</td>
			<td>Shanghai Hengyi Scientific Instrument Co., Ltd</td>
			<td>DHP-9082</td>
			<td></td>
		</tr>
		<tr>
			<td>ExonArt Seamless Cloning and Assembly kit</td>
			<td>Rong Wei Gene Biotechnology Co., Ltd</td>
			<td>A101-02</td>
			<td></td>
		</tr>
		<tr>
			<td>ExonScript RT SuperMix with dsDNase</td>
			<td>Rong Wei Gene Biotechnology Co., Ltd</td>
			<td>A502-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest Eco321 (EcoRV)</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0303</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest HindIII</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0504</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest NheI</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0974</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest NotI</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>FD0596</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Doc XR</td>
			<td>Bio-Rad Laboratories Co., Ltd</td>
			<td>721BR07925</td>
			<td></td>
		</tr>
		<tr>
			<td>Goldview Nucleic Acid Gel Stain</td>
			<td>Shanghai Yubo Biotechnology Co., Ltd</td>
			<td>YB10201ES03</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice Maker Machine</td>
			<td>Shanghai Bilang Instrument Manufacturing Co., Ltd</td>
			<td>FMB100</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Platinum SuperFi II DNA Polymerase</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>12361010</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar Plate (Kanamycin)</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>B530113-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>LB sterile liquid medium (Kanamycin)</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>B540113-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettors</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave Oven</td>
			<td>Panasonic Electric (China) Co., Ltd</td>
			<td>NN-GM333W</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shakers</td>
			<td>Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd</td>
			<td>ZHWY-2102C</td>
			<td></td>
		</tr>
		<tr>
			<td>PRRSV virus</td>
			<td>Sichuan Agricultural University</td>
			<td>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>SnapGene</td>
			<td>GSL Biotech, LLC</td>
			<td>v5.1</td>
			<td>To design primers</td>
		</tr>
		<tr>
			<td>T100 PCR Gradient Thermal Cycler</td>
			<td>Bio-Rad Laboratories&#160; Co., Ltd</td>
			<td>T100 Thermal Cycler</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>B040123-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific&#160; Co., Ltd</td>
			<td>15596026</td>
			<td>RNA extraction reagent</td>
		</tr>
	</tbody>
</table>

a seamless cloning approach for porcine reproductive and respiratory syndrome virus expression vector construction

基因表达载体的构建是实验生物学实验室工作的重要组成部分。随着 Gibson Assembly 等技术的进步，载体构建变得相对简单和高效。然而，当猪繁殖与呼吸综合征病毒 （PRRSV） 的全长基因组不能轻易地通过 cDNA 的单个聚合酶链反应 （PCR） 扩增，或者难以在 体外通过多个插入片段的同源重组获得全长基因表达载体时，目前的 Gibson Assembly 技术无法实现这一目标。
因此，我们的目标是将 PRRSV 基因组分成几个片段，并将适当的限制性位点引入反向引物以获得 PCR 扩增片段。通过同源重组技术将以前的 DNA 片段连接到载体中后，新载体获得了限制性内切酶切割位点。因此，我们可以使用新添加的酶切割位点对载体进行线性化，并在上游 DNA 片段的下游引入下一个 DNA 片段。
上游 DNA 片段 3' 端引入的限制性内切酶切割位点将被消除，新的切割位点将被引入下游 DNA 片段的 3' 端。通过这种方式，我们可以将 DNA 片段一一连接到载体上。该方法适用于成功构建 PRRSV 表达载体，是将大量片段组装到表达载体中的有效方法。

在本文中，我们提出了一种 体外 重组系统，该系统使用反向引物通过连续引入的限制性位点组装和修复重叠的 DNA 分子（图 1B）。该系统是一种简单高效的程序，包括制备线性载体和含有 PCR 引入的突出端的插入片段，引物具有适当的 5' 延伸序列和限制性位点; 体外 单一等温反应和重组产物向合适的宿主细菌的标准化学转化;以及选择阳性菌落并获得用于下一?...

作者聚焦：探索全长 DNA 片段的克隆技术

在这里，我们提出了一种通过在插入片段的 3' 末端引入合适的限制性位点来获得 pVAX1-PRRSV 表达载体的方案。我们可以通过同源重组技术将载体线性化，并将 DNA 片段一一连接到载体上。

猪繁殖与呼吸综合征病毒表达载体构建的无缝克隆方法

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson ...

该方案演示了冗长而复杂的基因表达载体的构建。当 cDNA 单次 PCR 无法获得全长 DNA 片段，或体外多次插入同源重组无法获得全长基因表达载体时，该方法适用。目前，使用多插入片段连接克隆来获得全长 DNA 片段，以便克隆到合适的载体中。该方法可实现靶基因的缺失或突变，并高效地将大量 DNA 片段连接到表达载体上。

a-seamless-cloning-approach-for-porcine-reproductive-respiratory

Research

JoVE Journal

Biology

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction

445 次观看.  China West Normal University. 该方案演示了冗长而复杂的基因表达载体的构建。当 cDNA 单次 PCR 无法获得全长 DNA 片段，或体外多次插入同源重组无法获得全长基因表达载体时，该方法适用。目前，使用多插入片段连接克隆来获得全长 DNA 片段，以便克隆到合适的载体中。该方法可实现靶基因的缺失或突变，并高效地将大量 DNA 片段连接到表达载体上。

视频: 作者聚焦：探索全长 DNA 片段的克隆技术

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

牛痘病毒感染及病毒基因表达的时空分析:第1部分

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

科研

生物学

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

牛痘病毒感染及病毒基因表达的时空分析:第2部分

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

牛痘病毒感染及病毒基因表达的时空分析:第3部分

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究:在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...