作者希望感谢斯蒂芬克鲁斯dysfusion的cDNA，DYS抗体和W 1118苍蝇。

1。胚胎收集<ol><li>设立了25笼° C使用2-4日龄瓦特1118 flies.Grape汁板被改变白天每隔一小时同步前1-2天期间收集的鸡蛋收集</li><li>收集胚胎为1小时25 ° C </li><li>切一块长方形的葡萄汁琼脂，用刀片轻轻切在中间，一条线留在琼脂</li><li>使用一个金属探针胚胎从葡萄汁板转移到您的葡萄汁琼脂片，线在胚胎长在45度角，在琼脂中轴线的直线。获取胚胎约50直线。确保所有的胚胎点在同一方向，指向行结束后。 </li><li>剪下一块长方形的双节棍磁带，并将其放置到18x18mm盖玻片。 </li><li>拿起双节棍带盖玻片上的胚胎</li><li>将载玻片盖玻片，干燥空气中约10分钟的胚胎。 </li><li>盖一层薄薄的700卤烃油的胚胎，现在的准备注射。 </li></ol> 2。拉针拉玻璃毛细管微量注射针。从世界精密仪器（型号的Pul - 1），我们使用一针的车夫。针拉马程序规范是：热量：1，延迟4。 3。灌装针头回到负荷针使用的Eppendorf Microloader提示安装的Eppendorf P20移液器，通常负载1.5-2μL的双链RNA 4。打破针<ol><li>才打破针，放到幻灯片盖玻片。 </li><li>准备幻灯片，在幻灯片中的油下降</li><li> Picospritzer三picopump转到调整空气压力</li><li>打破对盖玻片边缘的充满针，创建一个尖点。 </li><li>当针尖被打破，踩脚踏Picospritzer三picopump，一些dsRNA的会泄漏出来针尖。 </li><li>取出盖玻片的幻灯片。 </li><li>下在油价下跌前的准备幻灯片中插入针。 </li><li> Picospritzer三picopump调整设置100 - 200pl双链RNA，当你脚踏一步。双链RNA可以被看作是一个小液滴。 </li></ol> 5。注塑<ol><li>针尖到同一焦平面第一胚胚胎后部和偏离中心注入胚胎蛋的长度在30-50％左右。 </li><li>之后Picospritzer三picopump脚踏一步，会出现少量的dsRNA在注射部位的小圆点。 </li></ol> 6。表型分析<ol><li>注射后，放入有盖湿盒内（塑料培养皿中）在​​18 ° C的幻灯片，直到胚胎发育所需的萌芽阶段。 </li><li>使用探针删除双面胶带的胚胎，并将其推到盖玻片边缘。 洗掉的幻灯片，在尼龙网收集瓶底部使用庚烷的胚胎，与PBT洗3次。 与PBT Dechorionate胚胎在50％漂白水5分钟，洗3次。 胚胎从收集瓶转移到一块尼龙网，然后浸入固定液（5毫升庚烷，4.5毫升PBS，0.5毫升37％的甲醛）释放胚胎的尼龙网。修复胚胎在固定液为30min。 用甲醇Devitellinize胚胎，胚胎转移到Eppendorf管中，并使用标准协议，免疫染色的胚胎。 </li><li>胚胎也可以发展到适当的幼虫阶段。 </li><li>准备幻灯片，在中间的卤烃700油下降</li><li>一个幼虫的幻灯片，把顶部盖玻片</li><li>亮视野显微镜下观察幼虫。 </li></ol> 7。代表性的成果： 的dsRNA的一个例子击倒dysfusion Drosophlia胚胎（DYS）基因是在图1所示。绿色荧光蛋白的双链RNA注射的胚胎是阴性对照。 DYS是一个BHLH PAS气管融合细胞表达的转录因子。 GFP -双链RNA注射胚胎正常显示气管的融合，DYS蛋白融合细胞（图1A）。另一方面，DYS -双链RNA注射的胚胎失败的分支融合，DYS蛋白融合细胞（图1B）不存在。当双链RNA注射胚胎发育到幼虫阶段，注入DYS - dsRNA的幼虫呈分支融合失败（Fig.1D）相比，GFP -双链RNA注入了阴性对照（图1C）。这些结果表明，有效的击倒的内源性DYS基因表达的双链RNA注射到果蝇胚胎<img src="/files/ftp_upload/2477/2477fig1.jpg" alt="图1" /> 图1。DYS功能去除DYS - RNAi技术揭示了气管融合ð烹调。注射使用GFP -双链RNA（阴性对照）或DYS - dsRNA的胚胚胎（W 1118）和气管缺陷检测。 （一）第一阶段16胚胎注射GFP - dsRNA的α- DYS和单克隆抗体2A12污渍气管管腔的染色。集中反映的是横向干线，其中有融合。箭头指向侧主干DYS积极的融合细胞。 （二）第一阶段16胚胎注射双链RNA DYS -α- DYS和单克隆抗体2A12的染色。胚胎表明缺乏横向主干融合（星号表示在控制胚胎的横向主干融合的正常位置）。没有DYS -阳性细胞进行观察，表明DYS - RNA有效地取消DYS蛋白。 （C到D）二龄幼虫与GFP -双链RNA或DYS - dsRNA的气管缺陷的明场显微镜研究。 （三）融合的地点是在GFP -双链RNA注射控制幼虫的星号表示。 （四）横向干线失败保险丝在DYS - RNA注入幼虫，（*）表示横向主干融合的正常位置。

<ol>
	<li>St Johnston, D., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+pattern+and+polarity+in+the+Drosophila+embryo.">The origin of pattern and polarity in the Drosophila embryo.</a> Cell. 68, 201-219 (1992).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+gene+manipulation+in+Drosophila+melanogaster.">Emerging technologies for gene manipulation in Drosophila melanogaster.</a> Nat. Rev. Genet. 6, 167-178 (2005).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95, 1017-1026 (1998).</li><li>Misquitta, L., Paterson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+gene+function+in+Drosophila+by+RNA+interference+(RNA-i):+a+role+for+nautilus+in+embryonic+somatic+muscle+formation.">Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 1451-1456 (1999).</li><li>Horowitz, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis.">Branching morphogenesis.</a> Circ. Res. 103, 784-795 (2008).</li><li>Hogan, B. L., Kolodziej, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis:+molecular+mechanisms+of+tubulogenesis.">Organogenesis: molecular mechanisms of tubulogenesis.</a> Nat. Rev. Genet. 3, 513-523 (2002).</li><li>Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis+of+the+Drosophila+tracheal+system.">Branching morphogenesis of the Drosophila tracheal system.</a> Annu. Rev. Cell Dev. Biol. 19, 623-647 (2003).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfusion+transcriptional+control+of+Drosophila+tracheal+migration,+adhesion,+and+fusion.">Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion.</a> Mol. Cell. Biol. 26, 6547-6556 (2006).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+dysfusion+basic+helix-loop-helix+(bHLH)-PAS+gene+controls+tracheal+fusion+and+levels+of+the+trachealess+bHLH-PAS+protein.">The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein.</a> Mol. Cell. Biol. 23, 5625-5637 (2003).</li></ol>

双链RNA注射方法在座使一个非常敏感和快速的分析，在果蝇的基因功能的气管发展。这种方法有可能被应用到其他组织和器官的发育的基因功能分析。

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<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
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<td>Picospritzer III picopump</td>
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<td>Parker Precision Fluidics</td>
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rnai interference by dsrna injection into drosophila embryos

遗传筛查是为获得到复杂的生物过程的见解1最有力的方法之一。多年来许多改进和基因操纵的工具已成为在 果蝇 2 。后不久，3 Frie和梅洛的初步发现，双链RNA可以用来击倒的单个基因的线虫，RNA干扰（RNAi）是提供了强大的反向基因的方法来分析果蝇的器官发育基因的功能活动4，5。 许多器官，包括肺，肾，肝，和血管系统，是由支管网络，运输至关重要的液体或气体6，7。 果蝇气管形成的分析提供了一个极好的模型系统研究等8管状器官的形态发生。伯克利果蝇基因组计划已经发现了数百个，气管系统中表达的基因。要研究管形成的分子和细胞机制，面临的挑战是了解这些基因的作用在气管发展。在这里，我们描述了详细的双链RNA注射到果蝇胚胎敲除个别基因的表达方法。我们成功地撞倒内源性dysfusion（DYS）基因表达的dsRNA注射。 DYS是BHLH - PAS气管融合细胞表达的蛋白质，是气管的分支融合9，10所必需的。 DYS - RNA干扰完全消除DYS表达，并导致气管融合缺陷。这种相对简单的方法提供了一种工具来识别tissure 在果蝇的器官发育requried的基因。

Watch this Scientific Journal Video about RNAi Interference by dsRNA Injection into Drosophila Embryos at JoVE.com

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA干扰已被证明非常有效的分析基因功能<em&gt;果蝇</em&gt;气管发展。说明了一个详细的协议，由江实验室用于注入飞胚胎击倒基因表达的dsRNA。这种技术的组织和器官的发育需要筛选基因的潜力<em&gt;果蝇</em&gt;。

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

rnai-interference-by-dsrna-injection-into-drosophila-embryos

Research

JoVE Journal

Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K 次观看.  Oakland University. RNA干扰已被证明非常有效的分析基因功能果蝇气管发展。说明了一个详细的协议，由江实验室用于注入飞胚胎击倒基因表达的dsRNA。这种技术的组织和器官的发育需要筛选基因的潜力果蝇。

视频: RNAi Interference by dsRNA Injection into Drosophila Embryos

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 ...

科研

生物学

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

基因转移到老年人的鸡胚<em&gt;前OVO</em&gt;电穿孔

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

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.

Methods Article

RNAi的干扰双链RNA注射到果蝇胚胎