作者承认中国国家重点研究开发计划 (2016YFC1200500)、中国国家自然科学基金 (81672054 和 81371846)、自然研究团队计划的资助。广东省科学基金 (2014A030312016) 和广州科技计划 (201508020263)。我们感激地承认教授乔纳森 (科罗拉多州立大学) 提供 pUCA 和 p7NS1-GFP 质粒和批判阅读这篇手稿。

所有协议均由南方医科大学伦理委员会批准. 
 1. 设计人工内含子 注意: 在这项工作中使用的人工内含子长度为 65 bp, 序列为5和 #39;-GTAAGAGTCGATCGACGCGTTACTAACTGGTACCTCTTCTTTTTTTTTTGATATCCTGCAG-3 和 #39;. 
<ol> <li> 将 hpa i 限制站点放在人工内含子的两端, 使 hpa i 消化可以从质粒中释放内含子 (见 图 1 A ). </li>
	<li> 将两个 Xba i 限制在内含子中的站点, 以便 Xba i 消化可以用于插入小 RNA 表达序列. </li>
	<li> 从 dna 制造商订购人工内含子的全长 dna 序列的化学合成。合成的 DNA 是从克隆成一个标准的 pUC19 质粒. 
	注: 人工内含子的基本成分包括几个一致的核苷酸元素, 包括5和 #39; 供体剪接点 (DS) 与序列 GUAAGAGU, 一个保守的分支点序列 (BPS) 与序列 UACUAAC, 聚嘧啶(PPT) 与序列 UCUUC (U) 10 和一个3和 #39;-受体剪接位址 (AS) 与序列 GAUAUCCUGCAG ( 图 1 a )。在 DS 和 BPS 之间引入了两个 Xba I 限制站点, 可用于插入小 RNA 表达式序列。从哺乳动物和蚊子细胞中有效拼接这个人工内含子的能力已经被确认为 14 . </li>
</ol> 2。设计一种人工子小 RNA 表达式卡带 注意: 对于内生 miRNA, 在 DS 和 BPS 之间插入编码前体 miRNA 或 miRNA 海绵的序列。以 miRNA 为例, 从 Ae. 伊蚊 aal-let-7 中选择了一种内源性前兆, 构建了 miRNA 过度表达和击倒的重组病毒载体。根据先前描述的方法 15 设计了一个 anti-let-7 海绵。为了打击幼虫中的靶基因, 我们设计了特定的人工 miRNA (amiRNA) 或短发夹 RNA (shRNA) 序列, 使用在线软件 16 , 17 ;作为一个例子, V atp 酶亚基 A mRNA (加入 no。AY864912) 在本研究中被选为靶基因。amiRNA 将被称为 amiVTP 以后。 补充表 1 中描述了 pre-miRNA、海绵、amiRNA 和 shRNA 的所有序列详细信息. 
<ol> <li> 订购前体 rna、miRNA 海绵和 shRNA 的全长 cDNA 序列的化学合成, 并使用 Xba 我在 DNA 制造商的两端限制站点. </li>
	<li> 在接收到有序的 dna 后, 在 1000万 x g 上旋转含有 dna 块的管子1分钟, 以确保所有干燥的 dna 都在试管的底部. </li>
	<li> 重 dnasei 水中的干 DNA, 以达到 10 ng/和 #181 的最终浓度. </li>
	<li> 将质粒主干和 DNA 序列与 Xba I 在37和 #176; C 表示1小时, 然后 dephosphorylate 5 和 #39; 根据制造商和 #39 的规定, 用小牛肠碱性磷酸酶 (鼓掌) 的线性化质粒骨架的末端.</li>
	<li> 在65和 #176 中加热60分钟以禁用鼓掌; C. </li>
	<li> 运行1.0% 琼脂糖凝胶和切割线性骨架。然后根据制造商和 #39 的说明, 用凝胶萃取试剂盒从凝胶切片中提取 DNA. </li>
	<li> 将 dna 片段结扎到 backboneby T4 dna 连接 (5 U/和 #181; L) 在22和 #176; C 为 1 h. </li>
	<li> 将步骤2.7 中的结扎反应的产品转换为合格的 大肠杆菌 ( 大肠杆菌 ) 细胞, 并将含有氨苄青霉素的性汤 (LB) 琼脂板 (浓度为100和 #181; 克/毫升). 
	<ol> <li> 通过热休克方法 18 执行转换, 并使用适当的 大肠杆菌 应变。例如, 使用 大肠杆菌 应变 DH5 和 #945; 用于热休克转换. </li>
	</ol> </li> <li> 从步骤2.8 中选取单个菌落, 并在含有氨苄西林的3毫升培养基培养基中孵育 (浓度为100和 #181; g/毫升) </li>
	<li> 根据制造商和 #39 的说明, 使用 mini-prep 试剂盒从细菌培养物中提取质粒 DNA。将样品送到 DNA 测序公司. 
	注: 对于内源性 miRNA, 在两个 Xba I 站点之间插入编码前体 miRNA 或 miRNA 海绵的序列。所有 miRNA 表达式或海绵结构必须小于 400 bp (10% 的病毒基因组长度), 由于包装限制的病毒 19 . </li>
</ol> 3。通过将 miRNA 或 shRNA 表达式磁带复制到质粒主干中来生成 rAeaDV 结构 注意: pUCA 是一种感染性克隆, 包含 pUCA 基因组的 AaeDV 质粒 (3981 nt) 19 ,Prof. 乔纳森提供了亲切的, 并已在前面详细介绍了 11 。p7NS1-DsRed 表达非结构蛋白 1 (NS1)-红色荧光蛋白 (DsRed) 融合蛋白从 p7 启动子, 并在其他地方详细描述了 14 。一个人特有的 miR-941-1 前体 20 和它的海绵, 一个炒 shRNA (CGACGACTATCGTGCAATTTCAAGAG AATTGCACGATAGTCGTCG), 也亚到 AaeDV 作为一个负控制. 
<ol> <li> 结扎将 pre-miRNA、海绵、shRNA 或 amiVTP 表达式卡带到 Hpa I 站点中 AaeDV NS1-coding 区域的 pUCA 中, 如协议步骤2.4 至2.7 中所述. </li>
	<li> 将 DNA 转化为 Stbl3 的能力 大肠杆菌 单元格, 并按照步骤2.8 至2.10 所述选择一个正克隆. </li>
	<li> 下面, 根据制造商和 #39 的说明, 使用最大的准备套件从细菌细胞中提取 rAaeDV 质粒. 
	注: Stbl3 或 Stbl4 大肠杆菌 细胞应用于 rAaeDV DNA 转化, 以尽量减少不受欢迎的 ITR 重组。最大的准备应用于提取 rAaeDV 质粒, 以获得大量的 DNA (和 #62; 100 和 #181; g)。在生成病毒之前, rAaeDV 质粒的序列完整性应始终通过 DNA 测序 21 进行分析. </li>
</ol> 4。染 C6/36 细胞与 rAaeDV 质粒 <ol> <li> 在罗斯威尔公园纪念研究所 (RPMI) 1640 培养基中含有10% 胎牛血清 (FBS) 和1% 青霉素/链霉素在28和 #176; C 孵化器. </li>
	<li> 在0天, 在十 25 cm 中的单元格中, 2 培养烧瓶 18-20 h, 然后在90-95% 稀释中分裂1:2 汇合细胞. 
	注: 在1天, 细胞应达到90-95% 汇合. </li>
	<li> 在1天, 染 rAaeDV 质粒的系列 22 的单元格. 
	<ol> <li> 在600和 #181 中稀释10和 #181; 在离心管中 RPMI-1640 培养基中的 g DNA, 然后轻轻地混合。同时, 准备600和 #181; RPMI-1640 培养基和 25 #181; 转染试剂每转染的 l. </li>
		<li> 室温孵育5-10 分钟 (RT)。然后, 在 1640-质粒 DNA 混合物中添加625和 #181; 1640 转染试剂混合物中的 L. </li>
		<li> 在 RT 中孵育此混合物20-30 分钟。在转染前, 用2毫升的 RPMI-1640 培养基冲洗两次细胞. </li>
		<li> 在 RT 孵育 (步骤 4.3.3) 后, 将 the1640-transfection 试剂 DNA 质粒混合物添加到 25 cm 2 培养烧瓶中, 并在28和 #176 中孵育6小时; C. </li>
		<li> 在大约 6 h post-transfection, 删除转染培养基, 用5毫升的 RPMI-1640 培养基冲洗两次细胞, 再加入 RPMI-1640 培养基和 10% FBS. 
		注: rAaeDV 显示在 Ae. 伊蚊 幼虫中感染率高 (95%); 然而, 根据我们的经验, 在将近50% 的感染幼虫中, 感染仅限于主要感染部位 (如: 如 、肛门乳突和刚毛细胞), 而不发布 23 。因此, 如果有必要感染幼虫在多个组织, 一个有缺陷的表达荧光蛋白的重组病毒应合并, 以使感染点的可视化。例如, 要产生有缺陷的重组病毒表达 DsRed, p7NS1-DsRed 应 co-transfected 与辅助质粒进入 C6/36 细胞根据上述程序 (cotransfection 浓度比应为 2:1). </li>
	</ol> </li> </ol> 5。从转染的 C6/36 细胞中收获 rAaeDV 病毒 <ol> <li> 导入后5天内捕获细胞。用5毫升的玻璃滴管将盘子里的细胞取出并悬浮, 用培养基移上下。将所有细胞悬浮液转移至无菌15毫升管. </li>
	<li> 冻结在-80 和 #176; c 或在干冰/乙醇浴中30分钟, 然后在37和 #176 解冻; c. 涡流1分钟重复冻结和解冻过程3次. </li>
	<li> 离心样品在 4800 x g 为20分钟在4和 #176; c. 收集上清液并丢弃颗粒。然后, 通过0.22 和 #181; 我一次性注射器过滤器的上清。分和存储最终纯化的病毒股票在-80 和 #176; C. 
	注意: 避免重复的冻融循环. </li>
</ol> 6。量化 rAaeDV <ol> <li> 准备 DNA 标准。线性所有 rAaeDV 质粒与 Hpa I, 和纯化的消化产品与凝胶提取试剂盒 24 。
	<li> 根据前面描述的方法构建绝对定量标准曲线并测量病毒的效价 25 . </li>
</ol> 7。蚊子转导 <ol> <li> 洗涤 100 1 st 幼虫 Ae. 伊蚊三次使用去离子水, 然后将幼虫转移到含有95毫升去离子水的烧杯中。添加5毫升的 rAaeDV 库存 (最终浓度 1.00 x 10 10 拷贝/毫升). </li>
	<li> 在28和 #176 孵育24小时; C、用去离子水冲洗幼虫三次, 然后将幼虫移回盘子并定时进食. </li>
	<li> 监视 qPCR 或西印迹幼虫中靶基因表达的水平 (代表结果如图 3 和 4 ). 
	注意: 荧光信号检测方法可用于更精确地隔离感染幼虫。例如, 为了检测感染了表达 DsRed 的重组病毒的幼虫的荧光信号, 幼虫可以放在凹滑片上, 每8小时在倒置荧光显微镜下观察, 直到2天感染。一旦检测到荧光幼虫, 它们就应该被分离成单独的塑料测试杯, 以便随后的连续观察。使用此方法可以识别多组织感染的幼虫. </li>
</ol>

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作者声明他们没有竞争的金融利益。

重要的是要克服限制 rAaeDV 建设的两个关键障碍。第一是生产有缺陷的重组病毒。据报道, MDV 可以作为一个载体来表达适当大小的外来基因, 如蝎虫特异 neurotoxins20 和 GFP 蛋白;然而 , 无论建设策略 , 一旦码的 MDV 入或替换为外源基因 , 病毒不能独立形成 , 除非帮助的质粒是 cotransfected 提供缺少的不可缺少的病毒蛋白。有缺陷的病毒是无法参与二次传播, 这发生在体内蚊子与缺陷的基因组。我?...

蚊子传播的疾病, 如疟疾、登革热、zika 发烧和黄热病, 是主要的国际公共卫生问题, 继续占全球传染病负担的很大一部分1,2。传统的杀虫剂, 已被用于响应的载体, 是一个主要组成部分的可持续的综合防治蚊子传播疾病的控制战略。然而, 这种战略被证明是相对无效或不可取的, 由于相关的负面环境影响和蚊子的抗药性的演变3,4。由于这些原因, 迫切需要采取替代控制蚊子的方法, 利用转基因方法生产无菌的雄性蚊子, 并释放抗病菌蚊子, 这是有希望的新的控制战略。为了开发有效的新的控制方法, 如安全有效的体内基因传递, 需要对蚊子基因功能进行综合分析。
直接微注射质粒 DNA, 双链 rna (dsRNA) 或小干扰 rna (siRNA) 是最常用的体内基因传递方法的蚊子。事实上, 转基因蚊子的生产仍然依赖于一个胚胎微注射的过程5,6,7。然而, 微注射有几个局限性。首先, 这项技术要求严格, 涉及复杂的程序。其次, 注射引起对胚胎、幼虫、蛹和成虫的物理侮辱, 直接影响到目标生物体的生存能力。第三, 由于大多数生活在水生栖息地, 并具有特有的蠕动运动, 难以固定蚊子幼虫进行微注射。第四, 1st-2nd幼虫的大小为 10-20 倍, 小于 4th和老幼虫, 前者的角质层更细腻。这些特性使得对 1st-2nd幼虫的处理与在较旧的阶段相比难以操作。相结合, 这些因素有助于减少幼虫的后存活率 (约 5%), 与成人相比8。Viral-based 的分娩系统已经开发, 以克服相关的细胞外和细胞内屏障。这些系统具有易于操作、高转导效率、长期和强健的表达水平以及在体内产生持久性效果的能力。因此, 利用病毒、慢和腺的基因传递系统已被广泛应用于 inmammalian 细胞系和模型种。辛德毕斯病毒表达系统曾用于分析成年蚊子的基因功能;然而, 除了生物安全问题, 注射仍然是病毒感染的必要过程9。虽然, 在 dsRNA 溶液中浸泡幼虫的口服已被报告为一种可行的分娩方法, 但不适用于小 RNA 功能分析10。因此, 有效的病毒传递方法仍然必须为蚊子开发。
蚊子 densoviruses (MDVs) 是Parvoviridae的Densovirinae亚科的一部分, 除了一个属于Brevidensovirus11。MDV 病毒是 non-enveloped, 由单 DNA (ssDNA) 基因组和体衣壳 (直径 20 nm) 组成。病毒基因组的大小约为 4 kb, 并在宿主细胞的细胞核内复制。MDVs 在环境中相对稳定, 对蚊子具有高特异性的狭窄寄主范围。这些病毒有可能传播和保持自然在蚊子的人口, 并可以侵入这些昆虫的几乎所有器官和组织, 包括中肠, 氏小管, 脂肪体, 肌组织, 神经元和唾液腺12。
完整的 MDV 基因组可以亚成质粒载体产生 plasmid-based 感染性克隆;当这些克隆被送进蚊子细胞, 病毒基因组从质粒载体被提取, 并且传染性的病毒微粒被生产。由于 MDV 有一个小的 ssDNA 基因组, 感染性克隆很容易构建, 病毒基因组可以很容易地操作11,13。这些特性使 MDV 成为研究蚊子生物学的有价值的试剂。然而, 由于几乎所有的病毒基因组序列对病毒增殖是必不可少的, 通过替换或插入外来基因而产生的重组病毒会导致病毒包装和/或复制能力的丧失, 从而造成障碍为 MDVs 的发展作为基因交付传染媒介。在这里, 我们报告使用人工子小 rna 表达策略, 开发一个无的 rAaeDV在体内rna 传递系统, 具有方便的质粒结构的优势和维护的功能病毒, 可以产生稳定和长期的表达在宿主细胞不需要野生型病毒。此外, 这种方法可以方便幼虫的转导。
本研究介绍了以下步骤的协议: 1) rAaeDVs 编码子小 RNA 表达盒的设计 2) 利用 C6/36 包装细胞株生产重组病毒颗粒, 3) 无细胞定量分析rAaeDV 基因组复制数字, 和 4) 感染的Ae. 伊蚊幼虫直接引入病毒进入水体的幼虫栖息地。总的来说, 这项工作表明, 特定的小 rna 或靶基因可以表达或被打倒在蚊子幼虫使用发达的 MDV 分娩系统。

<table><tbody><tr><td>Centrifuge machine</td><td>Thermo Scientific</td><td>75004260</td><td></td></tr><tr><td>Centrifuge System</td><td>Beckman Coulter</td><td>363118</td><td></td></tr><tr><td>GeneJET Gel Extraction Kit</td><td>Thermo Scientific</td><td>K0691</td><td></td></tr><tr><td>E.Z.N.A. Plasmid Midi Kit</td><td>Omega Biotech</td><td>D6904</td><td></td></tr><tr><td>Purified Agar</td><td>OXOID</td><td>LP0028A</td><td></td></tr><tr><td>FastAP Thermosensitive Alkaline Phosphatase (1 U/&amp;micro;L)</td><td>Thermo Scientific</td><td>EF0651</td><td></td></tr><tr><td>FastDigest &lt;em&gt;HpaI&lt;/em&gt;</td><td>Thermo Scientific</td><td>FD1034</td><td></td></tr><tr><td>FastDigest &lt;em&gt;XbaI&lt;/em&gt;</td><td>Thermo Scientific</td><td>FD0684</td><td></td></tr><tr><td>T4 DNA Ligase (5 U/&amp;micro;L)</td><td>Thermo Scientific</td><td>EL0011</td><td></td></tr><tr><td>TransStbl3 Chemically Competent Cell</td><td>Beijing Transgen Biotech</td><td>CD521-01</td><td></td></tr><tr><td>Ampicillin&amp;nbsp;</td><td>Beijing Tiangen Biotech</td><td>RT501</td><td></td></tr><tr><td>Proteinase K</td><td>Promega</td><td>MC5008</td><td></td></tr><tr><td>SuperReal PreMix Plus (SYBR Green)</td><td>Beijing Tiangen Biotech</td><td>FP205</td><td></td></tr></tbody></table>

use of a recombinant mosquito densovirus as a gene delivery vector for the functional analysis of genes in mosquito larvae

体内微注射是最常用的基因转移技术, 用于分析单个蚊子的基因功能。然而, 这种方法需要一个更严格的技术要求的操作, 并涉及复杂的程序, 特别是当用于幼虫由于其体积小, 相对薄和脆弱的角质层, 和高死亡率, 这限制了它的应用。相比之下, 基因传递的病毒载体已经被开发来克服细胞外和细胞内屏障。这些系统具有操作简单, 基因转导效率高, 基因表达的长期维持, 以及在体内产生持久性效应的能力。蚊子 densoviruses (MDVs) 是特定于蚊子的、小单的 DNA 病毒, 能有效地将外来核酸转化为蚊子细胞;然而, 替换或插入外国基因来制造重组病毒通常会导致包装和/或复制能力的丧失, 这是这些病毒作为传递媒介发展的障碍。
在此, 我们报告使用人工子小 RNA 表达策略, 开发无重组的伊蚊蚊核 (AaeDV)体内交付系统。详细的程序的建设, 包装和定量分析的 rAaeDV 载体, 并为幼虫感染描述。
本研究首次论证了开发无重组 MDV 微 RNA (miRNA) 表达系统的可行性, 从而为蚊子基因的功能分析提供了有力的工具, 为病毒 paratransgenesis 在控制蚊媒疾病中的应用。我们证明, 白纹伊蚊1st幼虫可以通过将病毒引入幼虫孳生地的水体中而容易有效地感染, 而开发的 rAaeDVs 可以用来过度或击倒特定靶基因在幼虫中的表达, 为蚊子基因的功能分析提供了工具。

rAaeDV 结构的策略 产生了一个缺陷的 rAaeDV 载体来表达蚊子幼虫中的 DsRed 基因。所产生的质粒包含一个 NS1-DsRed 融合蛋白盒与 VP 蛋白删除 (图 1a)。设计的 rAaeDV 质粒包含 miRNA、miRNA 海绵、shRNA 和 amiRNA 的表达结构 (如图 1B所示)。例如, 过度 aal-let-7 在蚊子幼虫中生成了一个 rAaeDV 向量。所产?...

Watch this Scientific Journal Video about Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae at JoVE.com

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

我们报告使用人工子小 RNA 表达策略, 开发一个无重组的伊蚊蚊核 (AaeDV)体内交付系统。详细介绍了 rAaeDV 载体的构建、包装和定量分析以及幼虫感染的具体步骤。

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

use-recombinant-mosquito-densovirus-as-gene-delivery-vector-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.7K 次观看.  Southern Medical University. 我们报告使用人工子小 RNA 表达策略, 开发一个无重组的伊蚊蚊核 (AaeDV)体内交付系统。详细介绍了 rAaeDV 载体的构建、包装和定量分析以及幼虫感染的具体步骤。

视频: Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

科研

免疫与感染

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统：可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

女答：埃及伊蚊蚊的dsRNA注入

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

生物学

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成蚊（冈比亚按蚊的RNAi实验议定书）

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

环境科学

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa.
In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

遗传学

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

使用埃及伊蚊的高分辨率熔融分析进行CRISPR/Cas9诱变后的Indel检测

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi的触发传送到<em&gt;冈比亚按蚊</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

摘要

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此视频中的章节

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

甲病毒转导系统：可视化，以免蚊虫感染的工具

女答：埃及伊蚊蚊的dsRNA注入

成蚊（冈比亚按蚊的RNAi实验议定书）

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

使用埃及伊蚊的高分辨率熔融分析进行CRISPR/Cas9诱变后的Indel检测

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

RNAi的触发传送到

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

摘要

探索更多视频

此视频中的章节

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

甲病毒转导系统：可视化，以免蚊虫感染的工具

女答：埃及伊蚊蚊的dsRNA注入

成蚊（冈比亚按蚊的RNAi实验议定书）

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

使用埃及伊蚊的高分辨率熔融分析进行CRISPR/Cas9诱变后的Indel检测

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

RNAi的触发传送到

对成年 冈比亚按蚊 的有效口服RNA干扰（RNAi）给药

对成年冈比亚按蚊的有效口服RNA干扰（RNAi）给药