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。我们的结果验证了一个子表达策略, 提供了一个新的范例, 可以克服 MDVs 缺陷基因组的限制, 使外来基因有效地传递到蚊子。
第二是由病毒衣壳的大小所强加的重组病毒基因组的苛刻长度限制。为有效包装, 基因组大小不能超过4400核苷酸 (110% 的长度的 MDVs 基因组)。子 miRNA 表达盒 (包括 miRNA 表达盒和人工内含子) 不超过 400 nt, 可引入 AaeDV 基因组。虽然重组病毒基因组的长度仍然适用于包装, 这一交付系统有改进的余地。由于基因组长度的限制, 难以表达无缺陷子表达策略的基因。
重要的是要确保 C6/36 细胞是健康的, 使成功的转染和包装的 rAaeDV。然而, 在这里使用的阳离子脂质体试剂和商业昆虫细胞特异性转染试剂, 不提高 C6/36 细胞转染的效率 (40-60%)。因此, 为了收获 high-titer 重组病毒, 必须根据先前描述的生长特性14, 在转染后5天内维持细胞。此外, 为成功的幼虫感染, 它是必要的幼虫与传染性病毒颗粒接触 24-36 h, 以确保高感染率23。
最后, 本文所描述的子表达策略是第一个克服了缺陷 MDV 产生的障碍。这一战略使生产的无重组 MDVs, 可以过度或慈 rna 和目标基因的蚊子。该技术为蚊子基因的功能分析提供了一个工具, 不使用复杂的幼虫微注射程序, 具有明显的商业应用。进一步的研究将扩大应用描述的表达策略, 以调查蚊子生物学和 paratransgenesis 登革热病毒的控制。

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。

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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。所产生的质粒包含在病毒 NS1 外显子中的子 pre-aal-let-7 盒 (图 1B)。在病毒 NS1 外显子中含有子 aal-let-7 海绵表达式盒的 rAaeDV 向量被生成以在蚊子幼虫中击倒 aal-let-7 表达式 (图 1B和图 2b)。在蚊子幼虫 (图 2C) 中, 分别使用 rAaeDV shRNA 向量和含有子 shRNA 和 pre-amiRNA 表达式卡带的 rAaeDV amiRNA 载体。
基于用线性回归分析生成的标准曲线计算 rAaeDV 滴 y-axis 表示标准 DNA 分子浓度的 Log10 值, x 轴表示 CT 值。CT 数字 (x 轴) 和 Log10 (浓度) 值 (y-axis) 显示了良好的相关性 (R2 = 0.9988), 并满足公式 y =-0.288x + 13.87 (图 3)。使用线性方程 (表 1) 计算样本的 rAaeDV 滴。使用协议中描述的方法 (步骤 6.2和表 1), rAaeDV 的高滴 (4 mL、#62; 7.65 x 1010粒子/ml) 被捕获。重组病毒 (VrepUCA-7、VrepUCA-7s、VrepUCA-amiVTP、VrepUCA-shVTP、VrepUCA941-1、VrepUCA-941-1s 和 VrepUCA-shct) 是由染相应的感染克隆 pUCA-7、pUCA-7s、pUCA amiVTP、pUCA941-1、pUCA941-1s、pUCA-shct 成 C6/36 细胞 (节3、4和 5)。
rAaeDV 向量的 miRNA 表达和击倒效率的确定 在本例中, 100 1st龄幼虫的处理量相等 (1.00 x 1010粒子/毫升) 的 rAaeDV 含有子 pre-let-7 表达载体, 子 let-7 海绵表达载体, 人类特有pre-miR-941-1 表达载体或 miR-941-1 海绵表达载体通过将病毒添加到幼虫栖息地水体中。感染后五天, aal-let-7 的表达由 qPCR (图 4, n = 3 决定;补充表 2)。let-7 的水平, 显示为平均± SD 的三生物复制在幼虫, 大大增加或减少的 pre-let-7-expressing rAaeDV (表达2843.31 ± 80.72%, p 和 #60; 0.0001, t 测试) 和let-7-sponge-expressing rAaeDV (downregulated 74.78 ± 1.60%, p 和 #60; 0.0001, t 检验), 分别。
监测 rAaeDV 载体的 V atp 酶 mRNA 击倒效率 共有 100 1st龄幼虫被同等数量 (1.00 x 1010粒子/毫升) 的子 amiRNA 表达 rAaeDV, 子 shRNA 表达 rAaeDV, 人类特有的 pre-miR-941-1-expressing rAaeDV orscrambleshRNA-通过将病毒添加到幼虫栖息地的水体中来表达 rAaeDV。感染后五天, qPCR (图 5, n = 3;补充表 2)。以 amiRNA 表达的 rAaeDV (downregulated 43.01 ±6.37%, p = 0.0011, t 检验) 和子 shRNA-表达 rAaeDV (downregulated 72.88 ±), 在幼虫中显示为三生物复制的平均± SD 的 V atp 酶水平大大减少了。5.74%, p = 0.0001, t 测试)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56121/56121fig1.jpg"> 
图 1: 重组 AaeDV 质粒的示意图组织.(A) 三七总皂苷和 pVP 病毒促进剂分别驱动 NS1/NS2 基因和 VP 基因的表达。在 p7NS1-DsRed 质粒中, DsRed 基因与 NS1 基因融合。(B) pUCA-7、pUCA-7s、pUCA-941-1、pUCA-941-1s、pUCA-shct、pUCA-shVTP 和 pUCA-amiVTP 包含人工内含子, 包括 aal-let-7、aal-let-7 海绵、has-miR-941-1、has-miR-941-1 海绵、炒 shRNA、V atp 酶 shRNA 和人工miRNA, 分别被克隆到HpaI 站点的 NS1 基因。人工内含子是由剪接施主 (DS) 和受体位点 (AS) ﬂanked 显示的, 包含有一个分枝域 (BrP)、一聚嘧啶 (PPT) 和 pre-miRNA。miRNA 海绵或人工 miRNA 序列插入内的内含子之间的5剪接网站和 BrP.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56121/56121fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56121/56121fig2.jpg"> 
图 2: 生物人工子 rna (miRNA) 和生成 miRNA 海绵和人工 rna 的策略.(a) 子 miRNA 在 NS1 的前体信使 RNA (体) 内 co-transcribed, 它将由 pNS1 启动子驱动, 并通过 RNA 拼接从体中分割出来。虽然显被结扎, 形成一个成熟的信使 RNA (mRNA) 的 NS1 蛋白质合成, 拼接内含子与 pre-miRNA 进一步加工成成熟的 miRNA 切块。(B) 生成 anti-let-7 和 anti-miR-941-1 构造的策略。分别用 aal-let-7 和 has-miR-941-1 对 anti-let-7 和 anti-miR-941-1 序列进行对准。给出了种子区与 anti-miRNA 序列和尾区的完全匹配。let-7 和 miR-941-1 海绵含有三重复反义构造 (红色字母), 可以分别绑定到 aal-let-7 和 has-miR-941-1。(C) 用于本研究的 miRNA-based 人工 rna 和 shRNA 的序列和预测前体结构。成熟的人工 rna 和 shRNA 以红色显示, 其相关靶基因序列呈蓝色。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56121/56121fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56121/56121fig3.jpg"> 
图 3: rAaeDV 滴定的标准曲线和计算的让步ntrations 的 MDVs. (A) 实时 qPCR 执行的 pUCA 标准被稀释了10倍的放大率, 而 CT值是在 ABI 7500 上被测量为x。根据步骤6.2 和6.3 计算复制号, 并使用以下公式: 复制编号 (y) =-0.288x + 10.87;R2 = 0.9988。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56121/56121fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56121/56121fig4.jpg"> 
图 4: 重组 MDV 介导的 aal-let-7 表达和击倒在Ae. 伊蚊的幼虫. (A) 对含有 aal-let-7 表达式盒 (左面板) 的重组病毒感染后幼虫内源性 miRNA 表达的分析。(B) anti-let-7 结构的效率是在幼虫 (右面板) 中确定的。has-miR-941-1 和 has-941-1 海绵 (阴性对照) 表达和 downregulated 成熟的 let-7 rna, 分别与对照组的基底水平相比较。miRNA 丰度被规范化为 5S rRNA, 数据显示为三生物复制的平均± SD。T 检验是在 diﬀerent 水平的意义上进行的。星号表明受感染的和相应的模拟处理的幼虫 (四星号, p 和 #60; 0.0001) 之间有统计学意义的差异。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56121/56121fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56121/56121fig5.jpg"> 
图 5: 在含有 shRNA 和 amiRNA 盒的重组病毒感染后的 V 型 atp 酶 mRNA 表达的分析.mRNA 丰度被正常化为 aalrpS7。误差线表示 2−ΔΔCT值在Ae. 伊蚊幼虫中的标准偏差, real-time rt-pcr (**、p 和 #60; 0.01; **、p 和 #60; 0.001)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56121/56121fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/56121/56121table1.jpg"> 
表 1:重组 MDVCT由 qPCR 确定的值对应于x公式中的值, 用于计算yvalue每个1µL 样本中的病毒基因组拷贝数是由函数幂 (10, y) 获得的, 它最终乘以 40, 将值转换为病毒悬浮病毒的浓度。
<img alt="Supplemental Table 1" src="/files/ftp_upload/56121/56121suptable1.jpg"> 
补充表 1: miRNA 前体的序列。
<img alt="Supplemental Table 2" src="/files/ftp_upload/56121/56121suptable2.jpg"> 
补充表 2: qPCR 数据。

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

Nanyang Technological University

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

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

科研

发育生物学

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...