感谢国立台湾海洋大学海洋生物学研究所宏博士提供3种质粒结构。这项研究得到了科学和技术部 (大多数) 的赠款 106-2311-B-197-001 的支持。

1. 筹备工作
<ol><li>昆虫细胞培养<ol>
<li>准备50毫升细胞培养基。为此, 添加500µL 抗生素 (两性霉素 B = 0.25 µg/毫升, 青霉素 = 100 单位/毫升, 链霉素 = 100 µg/毫升) 和5毫升的热灭活胎牛血清在无血清细胞培养培养基 (不含血清或抗生素)。 注意:使用前, 在水浴中将胎牛血清加热65摄氏度, 30 分钟。</li>
		<li>维护斜纹 frugiperda (鳞翅目: 夜蛾科), sf9 昆虫细胞。通过摇动烧瓶并在光镜下检查, 从25厘米2细胞培养瓶中分离出约80% 的细胞。然后, 将细胞悬浮液的50% 转移到新的25厘米2细胞培养瓶中, 并允许细胞在室温下附着15分钟。用5毫升新鲜细胞培养培养基取代培养基, 在28摄氏度的孵化器中生长。通道细胞每2到3天取决于细胞的生长。</li>
	</ol>
</li>
	<li>细胞转染用质粒的制备<ol>
<li>将目标 DNA 片段 (例如, 毒蛾MNPV (LyxyMNPV) iap3基因及其删除构造) 插入 pDHsp/V5-His, 方法是基于 PCR 的克隆法11。利用 pcr 主配比 (2X) 和 pDhsp-F2/Op-IE2R 底漆集, 对转化后菌落的生长进行了菌落 pcr 检查。通过商业测序服务确认质粒序列。 注意:表 1列出了用于 PCR 的引物和相应的构造。</li>
		<li>培养单序列细菌菌落, 其中包含上述质粒结构 (步骤 1.2) 在200毫升 LB 培养基含有选定的抗生素 (50 µg/毫升), 分别。</li>
		<li>根据制造商的说明12, 从培养的大肠杆菌中提取质粒。</li>
	</ol>
</li>
	<li>用1.5 毫升细胞培养培养基和8.5 毫升无血清细胞培养培养基制备电镀培养基。</li>
	<li>放线菌 D (ActD) 细胞培养培养基, 加入1.5 µL ActD (1 毫克/毫升) 到10毫升细胞培养培养基 (最终浓度 = 150 ng/毫升)。存储在4摄氏度。</li>
</ol>2. 蛋白质瞬时表达
<ol><li>细胞播种<ol>
<li>通过摇动培养瓶来收割 sf9 细胞, 将细胞悬浮液倒进50毫升管, 并通过 P10 吸管将10µL 转移到 hemocytometer。在光显微镜下计数细胞数。</li>
		<li>板材 3 x 105 sf9 细胞入每个井在24井板材为15分钟在室温下。用0.5 毫升电镀介质替换介质。</li>
	</ol>
</li>
	<li>质粒的转染<ol>
<li>稀释细胞转染试剂: 稀释8µL 细胞转染试剂100µL 无血清细胞培养培养基, 并用涡流混合1秒。</li>
		<li>添加2µg 质粒 DNA (pDHsp70-Ac-P35/V5-His 或 pDHsp70-Ly-IAP3/V5-His 或 pDHsp70-Ly-IAP3-BIR/V5-His 或 pDHsp70-Ly-IAP3-RING/V5-His) 到100µL 的无血清细胞培养培养基和混合涡流 1 s (图 2)。</li>
		<li>将稀释质粒 DNA 与稀释细胞转染试剂 (210 µL) 相结合, 涡流 1 s. 室温孵育30分钟。</li>
		<li>添加210µL DNA 转染试剂混合物滴入细胞由 P1000 吸管。孵育28°c 为5小时。</li>
		<li>用 P1000 吸管取代电镀培养基, 用0.5 毫升的细胞培养培养基。用胶带封住24井板, 在28摄氏度孵化细胞16小时。</li>
	</ol>
</li>
	<li>热休克转染细胞: 把盘子放在42摄氏度的水浴中 (漂浮在水面上)。热30分钟, 并返回24井板28°c 孵化器。</li>
	<li>蛋白质表达的检测<ol>
<li>1小时或5小时热休克后, 用0.5 毫升 1x PBS 缓冲液冲洗细胞, 简要三次。 注: 将1毫升 10x pbs 缓冲器添加到9毫升灭菌 ddH2O, 将 10x pbs 缓冲器稀释为 1x pbs 缓冲器。</li>
		<li>溶解细胞与40µL 的 1x SDS 加载染料吹打上下。 注: 通过混合30µL 1x PBS 缓冲器和10µL 4x sds 样品缓冲器稀释 4x SDS 加载染料。</li>
		<li>在热块中将蛋白质样品加热98摄氏度, 10 分钟, 向下旋转1分钟, 然后放入冰上进行印迹测定。</li>
		<li>杂交印迹法 按照 Eslami 和 Lujan13中的西方印迹检测程序进行操作。运行 SDS 页凝胶14: 一凝胶受考马斯蓝染色 (加载控制, 以检查每个井中的蛋白质样品数量是否相等) 和其他受西方印迹检测, 根据 Eslami 和 Lujan13.</li>
		<li>检测 V5-tagged 融合蛋白与兔 anti-V5 抗体 (5 毫克/毫升) (1:5000 稀释 TBST 缓冲工作浓度1µg/毫升) 和山羊抗兔 IgG-辣根过氧化物酶 (HRP) 共轭 (0.8 毫克/毫升) (1:10000 稀释 TBST 缓冲工作浓度0.08 µg/毫升)。 注意:将聚丙烯酰胺的百分比调整为 17.5%, 当蛋白质分子量为 < 17 kDa 时。</li>
	</ol>
</li>
</ol>3. 抗凋亡活性测定
<ol><li>基因诱导细胞凋亡: 重复上述过程从2.1 到2.3。共染1µg 的 pDHsp/D-弹性蛋白/旗-他质粒 dna (含凋亡诱导基因) 与1µg 的质粒 dna [pDHsp70/V5-His 向量 (阴性对照), pDHsp70-Ac-P35/V5-His (阳性对照), pDHsp70-Ly-IAP3/V5-His, pDHsp70-Ly-IAP3-BIR/V5-His或 pDHsp70-Ly-IAP3-RING/V5-His, 分别.]。在5小时后热休克治疗, 进行细胞活力检测 (图 2)。</li>
	<li>化学诱导细胞凋亡: 重复上述步骤从2.1 到2.3。在步骤2.1.2 中, 板 1 x 106 sf9 单元格放入6井板中的每个井中。染4µg 质粒 DNA [pDHsp70/V5-His 矢量 (阴性对照)、pDHsp70-Ac-P35/V5-His (阳性对照)、pDHsp70-Ly-IAP3/V5-His、pDHsp70-Ly-IAP3-BIR/V5-His 或 pDHsp70-Ly-IAP3-RING/V5-His 分别.]。在5小时后热休克, 治疗 sf9 细胞2mL 的 ActD 细胞培养培养基16小时, 并进行细胞活力检测 (图 2)。 注意:6井板一井盖的最小容积为1毫升。</li>
	<li>执行上述抗凋亡活性试验, 包括3.1 和 3.2 triplicates。</li>
</ol>4. 细胞活性测定
<ol><li>通过添加1毫升 1x PBS 缓冲液, 1 分钟3次, 冲洗处理过的细胞。并用重悬细胞由吹打1毫升 1x PBS 缓冲器包含0.04% 台盼蓝和染色3分钟室温。将细胞悬浮液转移到1.5 毫升微细。 注意:通过混合9毫升 1x PBS 缓冲器和1毫升0.4% 台盼蓝溶液稀释0.4% 台盼蓝溶液。</li>
	<li>将10µL 台盼蓝染色细胞悬浮液转移到 hemocytometer, 用 P10 吸管, 并在光显微镜下计数存活完好的细胞。</li>
	<li>统计分析<ol>
<li>计算所记录的数据, 并以此作为 S.D. 所有计数的方法。</li>
		<li>使用学生的双尾 t 测试与 Microsoft Excel 一起分析数据。将统计重要数据定义为P值 < 0.05。</li>
	</ol>
</li>
</ol>

<ol>
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作者声明他们没有竞争的财政利益。

基于热休克的 pDHsp/V5-His/sf9 细胞系统的概念在1994年的8中首先被克莱克et描述。杆状病毒基因启动子 (IE1) 和 果蝇hsp70 的比较表明 hsp70 在蚊子细胞10中的效率更高.此外, 由于热休克诱导, 在热休克治疗后, 可以精确控制蛋白质表达的时间。该系统随后应用于虾和 nucleopolyhedrovirus (NPV) 蛋白的蛋白质功能测定9,<sup cl...

两种蛋白表达系统通常用于生产蛋白质: 原核生物蛋白表达系统 (大肠杆菌基因表达系统) 和真核细胞蛋白表达系统.一种流行的真核细胞蛋白表达系统是杆状病毒表达载体系统 (BEVS)1。杆状首次被用作全球农业和森林害虫的生物控制剂。在过去的几十年中, 杆状被开发为蛋白质表达载体的生物技术工具。杆状的基因组由双链循环 DNA 和封装 nucleocapsids2组成。到目前为止, 已有七十八多个杆状病毒分离序列3。根据寄主昆虫细胞杆状病毒基因表达的时间级联, 基因转录可分为四个颞叶栅, 包括即刻早、延迟早、晚期和非常晚的基因 4.
BEVSs 被指定为非常晚基因促进者 (即, 多面体或 p10 启动子) 被用来驱动目标基因, 而重组杆状病毒是由同源重组产生的。重组杆状病毒在昆虫细胞中的外蛋白表达与转化后修饰的哺乳动物蛋白相似 (适合于糖蛋白的生成)。因此, 杆状病毒已被广泛使用5,6,7。然而, 一个限制是在昆虫细胞中存在着不同的 n-糖基化通路7。
因此, 建立了一种新的杆状病毒表达系统--瞬态基因表达系统。该系统在昆虫细胞中杆状病毒直接早期促进剂 (ie-1 启动子) 驱动下表达国外基因.通过使用该系统, 可以在ie-1启动子的控制下立即表达目标蛋白, 同时修改昆虫细胞中的 n-糖基化通路, 从而得到更好的 n-链寡糖7。此外, 杆状病毒直接早期基因由宿主细胞 RNA 聚合酶 II 转录, 不需要任何病毒因子激活 4.因此, 在短时间内, 外来蛋白可以在昆虫细胞中表达。目前, 瞬态基因表达系统是在杆状病毒体外细胞培养系统中进行蛋白质功能测定的重要技术之一。该系统可用于分析杆状病毒或外来蛋白质的功能。一个商业上可用的瞬态基因表达系统是基于直接早期基因 (IE) 促进者的Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) (OpIE2 和 OpIE1)。
然而, 当奥佩启动子的瞬态基因表达系统使用8,9,10时, 昆虫细胞外基因的表达水平仍然是一个问题。因此, 根据果蝇热休克蛋白 70 (hsp70) 基因8,9的启动子, 构建了另一个瞬时基因表达系统。在昆虫细胞的热休克诱导下, hsp70 的启动子比杆状病毒 IE 启动子更有效 10.在该系统中, 目标基因在果蝇热休克 70 (Dhsp70) 启动子的驱动下表达。利用 PCR 克隆方法可以很容易地将外来基因复制到瞬态基因表达质粒中。同时, 通过热休克诱导, 可以对基因表达的时机进行控制。
在本报告中, 我们遵循的方法和表达三不同截断的杆状病毒基因 (细胞凋亡3, iap3 从 毒蛾 MNPV) 使用热休克为基础的瞬态蛋白表达系统和进一步应用这些表达蛋白的抗凋亡活性分析。该系统既可以快速表达国外蛋白, 又可进一步应用于 sf9 细胞蛋白质抗凋亡活性的评价, 同时也具有应用于其他蛋白质活性测定的潜力。

<table>
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic, 100X</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>for insect cell culture</td>
		</tr>
		<tr>
			<td>Certified Foetal Bovine Serum</td>
			<td>Bioind</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf-900 II SFM</td>
			<td>Thermo Fisher</td>
			<td>10902096</td>
			<td>serum-free cell culture medium</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Nunc, Thermo Fisher</td>
			<td>156340</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscopy</td>
			<td>WHITED</td>
			<td>WHITED WI-400</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC HIT Competent Cell</td>
			<td>Bioman</td>
			<td>RH618-J80</td>
			<td>Escherichia coli (DH5&#945;)</td>
		</tr>
		<tr>
			<td>L.B. Broth (Miller)</td>
			<td>Bioman</td>
			<td>LBL407</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, Bacteriological Grade</td>
			<td>Bioman</td>
			<td>AGR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>ant-zn-1</td>
			<td>selection antibiotic</td>
		</tr>
		<tr>
			<td>PCR Master Mix (2X)</td>
			<td>ThermoFisher</td>
			<td>K0171</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneaid Midi Plasmid Kit (Endotoxin Free)</td>
			<td>Geneaid</td>
			<td>PIE25</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinomycin D</td>
			<td>SIGMA</td>
			<td>A9415</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50 mL centrifuge tubes</td>
			<td>SIGMA</td>
			<td>CLS430829-500EA</td>
			<td>50 mL tubes</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Gizmo Supply Co</td>
			<td>B-CNT-SLDE-V2</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Multidish</td>
			<td>Nunc, Thermo Fisher</td>
			<td>142475</td>
			<td>24-well plate</td>
		</tr>
		<tr>
			<td>Cellfectin II Reagent</td>
			<td>Thermo Fisher</td>
			<td>10362100</td>
			<td>cell transfectin reagent</td>
		</tr>
		<tr>
			<td>PBS-Phosphate-Buffered Saline (10X) pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#215;SDS Loading Dye</td>
			<td>Bioman</td>
			<td>P1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (PVDF Blotting Membranes)</td>
			<td>Merck Milipore</td>
			<td>IPVH00010</td>
			<td>PVDF membranes</td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Cell system</td>
			<td>BIO-RED</td>
			<td>1703930</td>
			<td>Blotting device</td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>SIGMA</td>
			<td>6226-79-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-V5</td>
			<td>SIGMA</td>
			<td>V8137</td>
			<td>rabbit anti-V5 antibody</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG-horseradish peroxidase (HRP)</td>
			<td>Jackson</td>
			<td>111-035-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck</td>
			<td>817072</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well Multidish</td>
			<td>Nunc, Thermo Fisher</td>
			<td>145380</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4 % trypan blue solution</td>
			<td>AMRESCO</td>
			<td>K940-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 pipetman</td>
			<td>Gilson</td>
			<td>F144802</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipetman</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Symbio</td>
			<td>PPS7</td>
			<td>24 well tape ( 19 mm&#215;36 M)</td>
		</tr>
	</tbody>
</table>

transient expression of foreign genes in insect cells (sf9) for protein functional assay

瞬态基因表达系统是在杆状病毒体外细胞培养系统中进行蛋白质功能分析的重要技术之一。该系统是在瞬态表达质粒中杆状病毒启动子控制下表达国外基因的一种方法。此外, 该系统可应用于杆状病毒本身或外来蛋白质的功能检测。以Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) 的直接早期基因启动子为基础, 建立了最广泛、最商业化的瞬时基因表达系统。然而, 在昆虫细胞中发现外来基因的表达水平较低。因此, 构建了一种用于改善蛋白质表达的瞬态基因表达系统。在该系统中, 构建了重组质粒, 以包含在果蝇热休克 70 (Dhsp70) 启动子控制下的目标序列。本协议介绍了这种基于热休克的 pDHsp/V5-His (V5 表位与6组氨酸)/斜纹 frugiperda细胞 (sf9 细胞) 系统的应用;该系统不仅用于基因表达, 而且可用于评价昆虫细胞中候选蛋白的抗凋亡活性。此外, 该系统可以转染一个重组质粒或联合转染两个潜在的功能拮抗重组粒的昆虫细胞。该协议证明了该系统的有效性, 并提供了该技术的实际应用实例。

基于热休克的 sf9斜纹夜蛾pDHsp/V5-His/细胞 (frugiperda 细胞) 系统, LyxyMNPV 中 Ly-IAP3 的全长和其他两个截断 (和环域) 抗原在 sf9 细胞中。pDHsp/V5-His 含有一个果蝇热休克蛋白启动子, 它通过使用细胞转录因子和翻译系统 (图 1)8 , 在42摄氏度的温度下驱动下游基因表达.,9,11。整...

Watch this Scientific Journal Video about Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay at JoVE.com

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

本协议描述了一种热休克诱导蛋白表达系统 (pDHsp/V5-His/sf9 细胞系统), 可用于表达外来蛋白或评价潜在外来蛋白的抗凋亡活性及其截断的氨基酸昆虫细胞中的酸。

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

transient-expression-foreign-genes-insect-cells-sf9-for-protein

Research

JoVE Journal

Genetics

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

12.2K 次观看.  National Ilan University. 本协议描述了一种热休克诱导蛋白表达系统 (pDHsp/V5-His/sf9 细胞系统), 可用于表达外来蛋白或评价潜在外来蛋白的抗凋亡活性及其截断的氨基酸昆虫细胞中的酸。

视频: Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

Controllable Ion Channel Expression through Inducible Transient Transfection

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

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

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

科研

遗传学

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

nerii 蚜生物培养和基因表达的维持--植物-昆虫相互作用的非模型系统

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...