这项工作是由G.哈罗德和莱拉华马瑟斯奖HL和美国国立卫生研究院资助（R24 GM080527），以RESCEJ是一个NRSA博士后研究员（NIH F32GM9037222），目前由指导式研究科学家开发奖由国家支持老年研究所（K01AG04435）。我们要感谢评审的有益的意见。

1，制备质粒DNA，针头和注射的菜<ol><li>准备使用高速马克西和Midi试剂盒的质粒DNA，并收集DNA，以2.5毫克/毫升用乙醇沉淀。将DNA沉淀应溶解于无核酸酶的去离子水。存储在10-20微升等份的DNA于-20℃。 （可用矢量列表，请参阅补充表1） </li><li>用琼脂糖构建一个低谷举行的胚胎在100×15毫米的培养皿使注射的菜。 <ol><li>将一个75×50显微镜载玻片成100×15毫米的培养皿以一定的角度。倒入约50ml的2％的琼脂糖熔化水润介质24到培养皿中。或者，先倒入琼脂糖入菜，并飘起了"显微注射模具的鱼"在上面。 </li><li>经过琼脂糖凝固，取出载玻片或模具。如果使用载玻片上，切掉多余的琼脂糖用剃刀BLA德成墙。 水润凝中等入菜，并立即使用它，或它在4℃保存备用。注意:如果储存在4℃下使用之间注射菜肴可重复使用。 </li></ol></li><li>拉从高硼硅玻璃毛细管针，灯丝使用下列条件的微量拉马:加热525，拉75，速度100，时间50商店针按成泥的狭长地带在培养皿中，使他们手持平行于培养皿的底部。 </li><li>制备注射溶液，结合3微升2.5毫克/毫升的质粒DNA溶液与2微升10％酚红。涡旋该溶液，随后又旋转它10分钟，在最大速度下在微量离心以除去可能堵塞针的任何颗粒。 </li><li>在解剖显微镜下，夹针从尖端几毫米用钳子来创建一个小口。注意:在打开的适当大小有一定的灵活性荷兰国际集团，因为压力可以在步骤3.3进行调整，以适应这种变化。 </li><li>放置在培养皿，以便它保持在针与所述尖端向下的竖直位置。由液体吸取入针的后端装入大约0.5微升注射溶液的针。允许毛细管作用拉动溶液进针的尖端。 </li></ol> 2，准备胚胎注射<ol><li>每天扫描息肉产蛋水润 AEP的菌种。收集这些息肉和将它们搁置在一个单独的培养皿。白天观察这些息肉每隔几个小时监测卵形成的进展。当鸡蛋突破外胚层和坐在缩回 ​​外胚层细胞环，他们已经准备好注射25。把这些息肉有几个水润有睾丸注射，以便受精前至少1小时的菜。 水润&lt;/ em&gt;的雄性将精子释放没有任何特殊的操作。 </li><li>如果女性被发现在AEP菌落与完全成形的鸡蛋，这是大约400微米的直径25，或1至8细胞期胚胎中，也收集这些用于注射。立即注入已经开始切割胚胎。注意:这是不可能的，告诉完全成形的鸡蛋和单细胞受精卵的区别，因而这些应孵育注射前男性。 </li><li>注射前，除去大部分的亲组织的上方和下方用手术刀以＃15刀片的胚胎。只留下胚胎用一小片体柱的附着，所使用，如果必要的操纵胚胎用钳子。注意:被解剖远离胚胎将重新生成，并应保存，以确保女性保持水润群体的组织。 </li><li>使用巴斯德吸管，将胚胎注入培养皿，安排它们平行的壁琼脂糖低谷。 </li></ol> 3，显微注射质粒DNA的<ol><li>安装在磁性底座，坐在铁板的微量。装入注射保持器，其是保持在针上的控制杆显微操作的微量的一部分。安装操纵杆操纵到铁板上，带有磁性的立场。放置在右侧解剖显微镜的这整个设置和注射夹持位置，以使得它是在观察视野中可见。 </li><li>将注入培养皿用解剖显微镜下的胚胎，定向成使得琼脂糖槽的垂直壁是到左边。将针头插入注射座，降低针尖入菜的水润媒体。 </li><li>慢慢转动顺时针方向注射器的旋钮，直到矿物油填充针和注射液源源不断的顶部观察退出针头插入液联中等。如果流过强，转动旋钮，逆时针降低压力。实行此步骤，以定期获得合适的压力，这取决于针开口的尺寸（ 即，注射针是如何被修剪的步骤1.5）。注意:如果流太强，胚胎不会注入生存。 </li><li>同时，通过解剖显微镜观察，将注入培养皿，使得第一胚胎是在视场的中心。移动针，以便它正在触摸的胚胎。用显微刺穿用针胚胎。注意:在琼脂糖槽的垂直壁将保持胚胎从一旁通过​​针推。 </li><li>使注射液流入胚胎1或2秒，然后迅速取出针。如果胚胎中有多个小区，分别注入各小区。使用镊子重新定向胚胎对准下一个单元与针的尖端。 </li><li>第一安莉芳后o为喷射，移动盘，使下胚胎是在注射位置，并重复该过程;继续下去，直到所有的胚胎被注入。 </li></ol> 4，培养和胚胎孵化<ol><li>将所有注入胚胎与几个水润有睾丸一盘（这是为了确保所有的胚胎受精）。孵化的胚胎在18℃培养箱中培养，而他们继续胚胎。 </li><li>一旦角质层阶段为止，将各注入胚胎到一个24孔板中填充有水润介质的单个井。 </li><li>孵育注入胚2周在黑暗中在18℃。 </li><li>在2周潜伏期后，检查解剖显微镜下每个注射胚胎。注意，在其中角质层期胚胎已经从小块父母组织的分离的和亲本组织已再生的任何情况。立即删除该再生性息肉，使其不英里staken一个新的幼体。 </li><li>移动注射胚胎的菜下水族灯在室温。每天检查板和收集幼体。新的幼体将白色的，因为水润息肉的典型的粉红色的颜色来自于他们所吃的丰年 。 </li><li>观察荧光显微镜下的幼体来检测转基因表达。 </li></ol>

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The authors have nothing to disclose

水润常规无性繁殖，但需要环境刺激，开始产生配子。这些刺激是不明确定义为最水润物种可能与应变的应变。 à显著障碍产生转基因水润的是获得定期的胚胎，因为它可能很难在实验室环境诱导水润成为性。 AEP的菌株25，但是，产生配子容易在实验室中，这是已经被使用至今用于制备转基因品系的唯一菌株。用于诱导配子生产中最常用的方法是通过饮食操纵?...

水润已用于研究再生，格局的形成，以及干细胞大约有250岁2 水润有由三个细胞系一个简单的身体构造:外胚层上皮，内胚层上皮，间质。所述管状体由外胚层和内胚层上皮细胞系，其中每一个是单电池层而形成的。所有的上皮细胞在体内的列是有丝分裂。当上皮细胞被移动到末端3，头部（嘴和触须）在的反口端的口端部或支脚（基底光盘），它们阻止在细胞周期的G2期和改变细胞命运4。间质细胞系的细胞位于上皮细胞之间的间隙内。此系由多能干细胞存在于身体塔5的外胚层上皮细胞层的支持。间质干细胞产生3躯体大公升型（神经，腺体细胞，nematocytes）和生殖细胞6,7。 由于刺丝胞动物门中的一员，该姐妹团所有两侧对称， 水润可以在多细胞动物中共享的基本生物过程揭示。直到最近，这些努力受到缺乏可靠的方法对基因功能的扰动阻碍。然而，随着转基因方法1的发展，我们现在能够采取水润充分利用，以更好地了解常见的多细胞动物，例如干细胞的功能，再生和构图的基本机制。转基因水润线通过注射的质粒DNA导入胚胎，这导致随机整合和表达嵌合在刚孵化的主要频率的确定。在一个特定的血统与统一表达式A线可通过无性繁殖来确定。以无性繁殖transg的能力ENIC 水润线是在大多数动物模型，这只能通过有性生殖传播的优点。此外，转基因细胞可以容易地在活体内由于动物的透明度和缺乏内源性荧光蛋白8的跟踪。 在由于第一转基因水润线七年作了1，如线已被用于各种各样的应用。在不同类型的细胞的荧光蛋白的表达使得有可能跟踪细胞运动，观察变化，细胞形态，并且追踪细胞命运都在野生型的条件和化学扰动1,5,9-12之后。此外，不同的荧光蛋白在各种细胞系表达允许特定细胞群的FAC​​S分离。这种技术已被用于干细胞特异性的mRNA和谱系特异性小RNA 13,14测序。而启动子两水润肌动蛋白基因之一得到了最广泛的应用，一些细胞类型特异性启动子已经确定，并用于驱动绿色荧光蛋白的表达在转基因水润 9,11,15,16。在未来，细胞类型特异性启动子将允许对任何特定的细胞类型的观测和收集。此外，转基因的方法已成功地用于定义WNT3子17的顺式作用调节元件。 转基因方法水润的发展提供了强有力的方法来通过异位表达，表达和击倒测试基因的功能。转基因动物已表达荧光标记的蛋白，以检查这两个功能和细胞定位18-20。此外，RNA发夹的绿色荧光蛋白转基因的3&#39;UTR的表达导致靶基因21,22击倒。在这些方法中的GFP是必需的，以确定并创立了转基因株系的追踪期间的转基因组织。但是，很可能在一些情况下，绿色荧光蛋白分子会干扰标记的蛋白质的功能。最近的研究表明， 水螅基因可以被布置在一个操纵子结构， 即，多顺反子转录物制成，然后由反式剪接前导另外分离和分开23翻译。通过将编码蛋白质或操纵子和荧光蛋白基因的下游位置的上游位置的RNA发夹的基因，可以追踪转基因组织，而无需标签编码的蛋白质或RNA发夹的基因。此方法已被用于表达与的DsRed2的操纵子结构的RNA发夹结构，以实现基因敲低14。

<table><tbody><tr><td>AEP Hydra Strain</td><td></td><td></td><td>Email: Celina Juliano at celina.juliano@yale.edu or Hiroshi Shimizu bubuchin2006@yahoo.co.jp</td></tr><tr><td>High Speed Maxi Kit</td><td>Qiagen</td><td>12662</td><td></td></tr><tr><td>100 x 15 mm Petri Dishes</td><td>BD Falcon</td><td>351029</td><td></td></tr><tr><td>75 x 50 mm Glass Microscope Slide</td><td>Sigma</td><td>CLS294775X50</td><td></td></tr><tr><td>Microinjection Fish Mold</td><td>IBI Scientific</td><td>FM-600</td><td></td></tr><tr><td>Borosilicate Glass with Filament</td><td>Sutter Instrument</td><td>BF100-50-10</td><td>O.D.: 1.0 mm, I.D.: 0.50 mm, 10 cm length</td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>P3532</td><td></td></tr><tr><td>Jewelers Forceps, Durmont #5</td><td>Sigma</td><td>F6521</td><td></td></tr><tr><td>Scalpel Blade #15</td><td>Fisher</td><td>50-822-421</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M8410-500ML</td><td></td></tr><tr><td>Microinjector</td><td>Narishige</td><td>IM-9B</td><td></td></tr><tr><td>Magnetic Stand</td><td>Narishige</td><td>GJ-1</td><td></td></tr><tr><td>Iron Plate</td><td>Narishige</td><td>IP</td><td></td></tr><tr><td>Joystick Micromanipulator</td><td>Narishige</td><td>MN-151</td><td></td></tr></tbody></table>

generation of transgenic hydra by embryo microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

建立转基因水润线饲料的转基因幼体，每2-3天用卤虫无节幼体 。稚龟孵出有时后不要吃了两天。一些新的幼体永远不会吃的，因此将无法生存。如果幼体是转基因无论是在外胚层（ 图1A）或内胚层上皮组织，让动物芽和收集有最转基因组织（ 图1B，C）的花蕾。继续与新的芽为此，直至转基因系建立与转基因中任一的外胚层（ 图1D）<...

Watch this Scientific Journal Video about Generation of Transgenic Hydra by Embryo Microinjection at JoVE.com

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

转基因的产生<em&gt;水润</em&gt;通过胚胎显微注射

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

generation-of-transgenic-hydra-by-embryo-microinjection

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Generation of Transgenic Hydra by Embryo Microinjection

14.0K 次观看.  Yale University School of Medicine. Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi. 

视频: Generation of Transgenic Hydra by Embryo Microinjection

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

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 ...

生物学

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

遗传学

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

果蝇中 phiC31 整合酶介导的胚胎转基因

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

作者聚焦:优化胚胎显微注射以促进果蝇的转基因

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

胚胎显微注射用于果蝇转基因

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

转基因的产生

摘要

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此视频中的章节

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

埃及斑蚊胚胎显微注射获得转基因蚊子

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

无神经的一代和长期维持