在显微注射前准备： <ol><li>血喂蚊子：从周一至周三上周五饲料女性注射。要在同一个星期的周四和周五，饲料女性在周一注入。 </li><li>准备使用方案2和等渗缓冲液（见材料）的石英针。 </li><li> “铺设管”的胚胎是普通果蝇文化小瓶。湿脱脂棉在底部，一个覆盖它的过滤纸的湿磁盘。 </li><li>准备贴双面胶的一端塑料盖滑。修剪磁带覆盖滑，因此，它在盖玻片边缘结束。 </li><li>干燥油准备。 </li><li>准备一个用等渗缓冲液的培养皿中孵化的胚胎转移。 </li></ol>设立被迫铺设： <ol><li>收集与使用抽吸6-10血液美联储女性，他们转移到棉花和滤纸用等渗缓冲湿果蝇文化小瓶。 </li><li>然后把蚊子放回在黑暗的insectery条件和奠定为1小时15分钟的鸡蛋。 </li><li>让成年人飞入笼，并与胚胎取出滤纸盘。 </li><li>要排队鸡蛋，根据解剖范围。鸡蛋串，并用细的画笔（紫貂，0000）收集，转移到一个3MM的Whatman纸用等渗缓冲液中浸泡的准备平方米。保持所有的时间湿纸。不要让鸡蛋得到干燥。取出用刷子exochorion，有利于更好地坚持鸡蛋到磁带。 </li><li>使用罚款forcep第5号或罚款的画笔，拿起暗灰色的胚胎，并安排3MM滤纸湿用等渗缓冲平方米。线从20-30胚胎。所有胚胎必须在相同的方向，注射后极。前年底的胚胎是比后部略宽。然后，用3MM的Whatman纸的条纹，干燥过滤器内衬蛋线两侧用力挤压，不碰蛋鸡蛋。 </li><li>要转移的鸡蛋，反转的幻灯片，其中包含的双面胶带（医药），并轻轻按对鸡蛋。后结束的鸡蛋都必须非常接近边缘的双面胶带。湿润的纸，这是关键的，如果是太湿，他们不会坚持。我从5-10秒的胚胎干燥，但干燥时间取决于从潮湿和显微注射室的温度。 </li><li> 干燥鸡蛋的关键是：有点太多和胚胎不会孵化。没有干燥的DNA是不会成胚胎。 </li><li>封面，干涸的卤烃油胚胎，以防止进一步的干燥。 </li></ol>微量注射胚胎： 最重要的是良好的注射针的质量（见注）。 <ol><li>填充使用microloader（＃5242 956.003 Eppendorf公司）的DNA溶液注入一针。注射soliution很少是必要的，1〜2毫升。 </li><li>连接针的Eppendorf Transjector，控制喷油时间和压力，以及背压。 × 10的放大倍率和显微（徕卡）（莱卡）在移动阶段使用显微镜进行显微注射​​。如果有必要，提出显微镜阶段可能准备堆放4-5显微镜幻灯片。将盖玻片上提出阶段进行胚胎。胚胎注入150 °角;渗透应在后极。注射，我把针固定和移动显微镜的阶段。当针头是新的，提示是密封的，通常在第一次注射打破。我总是工作驱逐石油在每次注射之间的DNA的小液滴所需的压力。 </li><li>我注射时间为0.2 - 0.9秒和1000百帕时，针头是新的。我不同的压力和时间，直到小液滴被认为是来自针入油。背压需要100左右。由于针尖得到磨损，减少注射压力，以保持注射量低（约300百帕）。在此之后，针头过大，是杀死大部分的胚胎。 </li><li>注射后，取出用纸巾石油和盖用等渗缓冲液的培养皿中的胚胎和地方的幻灯片。放置在insectory的菜肴，让他们那里，直到胚胎孵化。他们开始孵化后2-6天。幼虫转移到蒸馏水中，与地面鱼类食品的捏。 </li></ol>注释使用EndoFree质粒套件的DNA注射 ：注射质粒DNA溶液分离。构建质粒和辅助质粒正确的浓度混合，用异丙醇沉淀。在注射缓冲区resuspending前用70％乙醇洗涤沉淀。注射前，干净的DNA使用Millex - GV列。 Izotonic缓冲区： <table border="1" cellpadding="8"><tbody><tr><td> 1.68 M氯化钠 - 88.3毫升 1.68 M氯化钠 - 2.9米 1 M羟乙基-10.7毫升 1.12米氯化钙2 - 2.2毫升 DH 2 O - 896毫升</td><td>或</td><td> 5 M氯化钠 - 29.67毫升 1个M氯化钾 - 4.87毫升 1 M羟乙基 - 10.7毫升 1.12米氯化钙2 - 2.2毫升 DH 2 O - 952.56毫升</td></tr></tbody></table>调整pH至7.2

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Injection Quartz needles</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>&nbsp;</td>
<td>O.D.- 1.0 mm, I.D.-0.70, 10 cm length, are prepared by drawing capillary tubing into a fine 3-8 um with a shaft of approximately 100-300 mm micropipette puller. </td>
</tr>
<tr>
<td>Quartz puller</td>
<td>Tool</td>
<td>Sutter Instrument Co</td>
<td>P-2000</td>
<td>Set-up: Heat-275, Fil-3, Vel-38, Del-250, Pull-141</td>
</tr>
<tr>
<td>EndoFree plasmid kit</td>
<td>&nbsp;</td>
<td>Quiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millex-GV column</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>SLGV RO4 NL</td>
<td>for DNA cleaning</td>
</tr>
<tr>
<td>Construct DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.5 mg/ml </td>
</tr>
<tr>
<td>Helper plasmid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.3 mg/ml </td>
</tr>
<tr>
<td>Injection solution</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1x solution: 5mM KCl, 0.1mM sodium phosphate, ph 6. 8</td>
</tr>
<tr>
<td>Isotonic buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>see recipe in the protocol section</td>
</tr>
<tr>
<td>Dessication oil</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Halocarbon oil 700:Halocarbon oil 27(1:1). Mix well. Prepare in advance of microinjection.</td>
</tr>
<tr>
<td>Blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to feed mosquitos</td>
</tr>
<tr>
<td>Anopheles stephensi</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mosquitos, male and female.</td>
</tr>
<tr>
<td>Drosophila culture vial</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for egg laying</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with isotonic buffer, for embryo hatching</td>
</tr>
<tr>
<td>Fine paintbrush</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Sable # 0000</td>
</tr>
<tr>
<td>3MM Whatman paper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Forceps #5</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>double sided tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microloader</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Transjector</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>with moving stage and micromanipulator, set at 10x magnification </td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

injection of an. stephensi embryos to generate malaria-resistant mosquitoes

引入外源基因的蚊子的基因组需要针对特定​​物种的利益显微注射技术。这个视频协议演示由詹姆斯实验室用于microinject到按蚊改造的蚊子的产生胚胎的DNA结构的方法。都说明，为准备显微注射针，收集和准备胚胎和执行显微注射​​技术。

Watch this Scientific Journal Video about Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes at JoVE.com

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

按蚊的蚊子是疟疾居住在印度和整个亚洲的载体。本视频演示了执行这个品种与转基因，这将赋予抵抗疟疾的蚊子microinjections技术。在此视频中演示的方法大多是适用于其他蚊种的显微注射技术。

注射的。 stephensi胚胎生成抗药性疟疾的蚊子

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

injection-an-stephensi-embryos-to-generate-malaria-resistant

Research

JoVE Journal

Biology

12.9K 次观看.  University of California, Irvine (UCI). 按蚊的蚊子是疟疾居住在印度和整个亚洲的载体。本视频演示了执行这个品种与转基因，这将赋予抵抗疟疾的蚊子microinjections技术。在此视频中演示的方法大多是适用于其他蚊种的显微注射技术。

视频: Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

埃及斑蚊胚胎显微注射获得转基因蚊子

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

科研

生物学

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

E16大鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

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 ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

强迫流涎法分析蚊虫的载体能力

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

A Blood-Free Diet to Rear Anopheline Mosquitoes

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable production of mosquitoes is currently inhibited by the supply of fresh vertebrate blood. Alternatives to blood are required to promote efficient control strategies for malaria and other vector borne diseases that are transmitted by blood feeding insects. With this in mind, artificial liquid diets were formulated as substitutes for fresh vertebrate blood. Herein we report a blood-free artificial liquid diet that delivers feeding rates similar to blood and mimics the physiological effects of a fresh vertebrate blood meal. The diet induces ovarian and egg maturation of Anopheles mosquitoes and also produces good larval survival and development of functional adults. The formulated blood-free liquid diet is an important advance towards sustainable mosquito breeding in captivity and will reduce the maintenance costs of mosquito colonies and eliminate the need for fresh vertebrate blood.

A protocol is presented for formulation of a blood-free artificial diet to feed Anopheles mosquitoes in captivity. This diet has a similar performance to vertebrate blood and triggers oogenesis and egg maturation and produces viable adult progeny.

后尾性异草蚊子的无血饮食

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable ...