Name: in vivo microinjection and electroporation of mouse testis
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Description: Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

我们感谢生殖医学和男科在明斯特大学的中心吉特Westernstroeer教学睾丸显微注射。此外，我们感谢厄休拉Antkewitz和佩特拉Reckling技术援助。我们感谢德国研究基金会（DFG），用于支撑该作品（WE2458 / 10-1）。 &#160;

所有进行的动物实验已获得当地伦理委员会（Landesamt献给Landwirtschaft，Lebensmittelsicherheit UND Fischerei，梅克伦堡 - 前波莫瑞州，德国）。 1质粒制备<ol><li>质粒制备，使用质粒纯化试剂盒（参见材料表）或类似方法与内毒素去除缓冲液，使得能够避免动物的免疫反应。按照说明书的指示。采用DDH 2 O稀释质粒的解决方案。 </li><li>通过旋转的DNA溶液以最大速度（20,000 XG）消除碎片。然后收集上清液。 </li><li>确定质粒浓度用分光光度计（见材料）。 </li><li>调整质粒浓度DDH 2 O的1-3微克（DNA）/微升。注:低浓度会降低转染效率，而高浓度可能会导致太粘注射液。此外，该转染效率依赖于质粒的大小，并进行单独测试。 </li><li>在注射前，制备溶液混合以例如40微升质粒连同5微升PBS（10×）和5μl坚牢绿（0.5％）。坚牢绿需要跟踪注射过程。最好用薄壁PCR管或封口膜。注:根据睾丸的大小，20〜50微升的体积将需要为每个睾丸。 </li></ol> 2，准备显微注射移液器的<ol><li>用硼硅毛细管（见材料表）编制显微注射移液器。 </li><li>拉玻璃毛细管垂直毛细管拉马（见材料表）。 </li><li>通过轻轻地绑扎打破毛细管末端用钳子。尖端通常在50-80微米的直径和1厘米左右长的。尽量避免太长，因为这些是不够坚硬穿透组织技巧。 </li><li>最后，沙通过使用微量倒角（见材料表）在30°尖rpen以45°角。 </li><li>加载显微注射移液管的注射溶液混合（见质粒的制备）。将溶液放入玻璃毛细管的背面用小注射器。注意:尽量避免气泡而加载，否则它们将被注入到生精小管沿与质粒溶液，因而会降低导电性，以及可能导致组织损伤。 </li></ol> 3，麻醉和手术<ol><li>通过使用无菌的材料（ 即，注射器，针，外科器械等）进行无菌条件下操作。这将减少动物的感染，并确保良好的存活率。 </li><li>用雄性老鼠电在6-8周的时代。 </li><li>初始化术后镇痛治疗，提供了鼠标止痛药加在手术前一天的饮用水。这马屁URES的情况下，极有可能手术后hypodipsia（减少水的摄入量）的超前镇痛。为此，露出饮用水例如儿科布洛芬悬浮液（20毫克/毫升）。本药膳饮水也应在一天之内和两个复苏相同浓度的供给。这意味着，每日剂量为7.5毫克/千克，有效期为5毫升/ð饮用水和体重25克为8岁的C57BL / 6J星期。这镇痛方案保证了基本的缓解疼痛，并伴随着高福利26加速复苏。 </li><li>用于制备麻醉工作手术当天溶液，混合10％氯胺酮和2％赛拉嗪的比例为1:1（见材料表）。 </li><li>麻醉动物，适用0.25毫升/ 100毫克体重麻醉剂溶液皮下注射（氯胺酮=0.125克/公斤;赛拉嗪= 0.025克/千克）。使用骨盆和肢体之间的注射部位以防止小鼠的器官损害和伤害。通常，10分钟，需要直到鼠标深度麻醉。 </li><li>涵盖鼠标眼睛兽医药膏，以防止麻醉过程中干燥。 </li><li>测试深麻醉逮捕，这是完全没有反应明显。为此，只需夹住动物的脚趾。 </li><li>要启动手术，取出腹腔头发用电动剃须刀或类似的消毒与sterilium或类似的操作区域。 </li><li>做腹部切口在腹部中心的包皮腺的正上方。在那个地方，首先拉扯皮肤路程，进行约8-14毫米的小的横向皮肤切口。然后，继续沿腹部肌肉层。 注意:病变应尽可能小，以减少伤害的动物。 </li><li>拉起腹部脂肪垫小心露出附睾丸，将其放在事先准备好的防水一次性纸悬垂只是旁边的切口部位（ 图2A）。 </li><li>使用双筒望远镜到fIND的输出管的注入目标。动脉导管传出被确定为睾丸和附睾的细槽的交界处。它位于邻近突出睾丸动脉。动脉导管几乎运行在45°到动脉的角度和明显进入附睾头部。注:根据鼠标的年龄，动脉导管通常是埋藏在脂肪组织中。 </li><li>用细镊子清除这个脂肪组织的输出管。在这之后，放置在无菌纸带的释放导管，以确保管道的清晰的可视性不损害环境。 </li></ol> 4，显微注射和DNA应用<ol><li>连接显微注射吸管，其被装载用质粒溶液的显微/喷射器单元。 </li><li>放置平行于输出管与尖端指向睾丸（ 图2B）的显微注射针。 </li><li>用细镊子和剥离管在显微注射吸管。确保在毛细管被保持平行于管道而拉过来。 注意:此过程比通过移动针的穿透显微血管更方便。 </li><li>小心地引导所述针朝向睾丸和停止仅仅因为它直接穿透白膜（ 图2C）的下方睾丸网。 注意:在越轨睾丸网的情况下，将质粒溶液会漏入间隙空间。这通常会导致转染的曲细精管的故障。 </li><li>用于注射的质粒溶液的利用微量注射器具有以下设置:圆周率:100百帕斯卡以下，Ti:0.2秒，和pc:0百帕（ 图2D）。 </li><li>监控通过观察睾丸填充状态与绿色的帮助下，整个注射过程。注意使睾丸仅填充至其与复量的三分之二asmid解决方案（ 图2E）。 注意:如果注射量超标，睾丸组织可能受到伤害。 </li></ol> 5，电穿孔睾丸<ol><li>为了实现有效的电，浸泡镊子电极在PBS（1X）。这可确保足够的电导。 </li><li>顺利地挤在湿电极（ 图2F）之间的睾丸。测量与所述电穿孔仪的电阻。 </li><li>适用于当前的睾丸。执行八方形脉冲，在40 V在4个不同的睾丸部位以50毫秒的脉冲时间950毫秒的间隔时间恒定的持续时间。 </li></ol> 6，伤口愈合和手术后<ol><li>当电穿孔完成后，将睾丸放回原来的位置。 </li><li>缝的内肌肉层用手术用缝合线（见材料）。 </li><li>通过采用缝合夹（见材料表）合皮肤。拉扯皮肤机智ħ镊子。请务必排除肌层。放置剪辑，每个在不超过5mm的距离。 注:由于手术缝合线可以通过鼠标来吞食，缝合剪辑青睐封闭患处皮肤。 </li><li>让鼠标从麻醉中恢复无菌纸巾放置在无菌空鼠笼，这是锻炼在37℃的温水垫。该垫应确保的二分之一的笼产生的热梯度的变暖。对于动物这使得能够搜索的个体，最舒适的区域，由于温度变化，在保持架。此外，覆盖了鼠标的无菌纸巾的片材，使得应力可进一步减小。注:由于鼠标的机身表面采用非同寻常的高相对于体量，与较大的动物相比，热的支持是对啮齿动物的成功恢复特别重要的意义。 </li><li>当操作的动物已经完全觉醒，传送I吨进一步恢复到一个完全配备新的鼠笼。不要把动物恢复到旧笼或一组老鼠​​。确保轿厢是作为清洁和消毒，尽可能防止伤口感染。监测整个恢复过程。要传输的鼠标背部的动物护理设施，等到它已经完全康复，并出现正常的行为。 注:其他per-和手术后的战略是通过普里切特，康宁KR 等 6所讨论。 </li></ol>

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貂米氏，亚历山大Sobczak和约阿希姆米韦策尔受聘于生殖生物学研究所，莱布尼茨学会家畜生态学（FBN），Dummerstorf，德国宣布，他们已经没有竞争的经济利益。

研究在生殖生物学领域，特别是在男性生育能力和精子发生不可避免的区域依赖于生物体。为了检查睾丸功能，没有足够的细胞培养/ 体外系统已经建立能够反射从二倍体精原细胞为单倍体成熟精子1,2的所有关键的形态变化的。因此，转基因动物的产生往往是必要的，在男性生殖生物学这样一个有价值的工具。为此，有相当多的转基因，基因敲除和基因敲除小鼠已建立?...

哺乳动物精子发生被认为是自我更新的干细胞连续地进行有丝分裂，减数分裂和分化，以发育成成熟的单倍体精子的一个复杂的过程。这些形态变化是由不同类型的细胞编排和尽管深刻的尝试，但仍然不可能在细胞培养1,2模仿这些过程。因此，在精子发生到现在为止的研究依赖于生物体的体内模型。在一般情况下，基因功能研究通常是基于转基因动物。然而，产生和维持这种动物模型是费时，高成本的并且相当复杂的。这归因于需要长期饲养过程中，用于生成和维持该转基因在世代。另外，通过转基因或基因敲除方法的整个有机体的基因操作容易，当靶向克造成生理损伤埃内斯在多个地区， 例如基本功能外睾丸或全身。 此外，一些瞬时转染方法与一些重要的缺点有关。例如，病毒介导的基因转移的典型缺点是免疫反应及其他安全法规的可能的挑衅，而脂质体3和微粒轰击4可能会损坏组织和被限定在一定的细胞深度足够的转染效率。 相反，电穿孔（EP）作为瞬时转染的另一种常见的方法，好像构成一个有前途的技术，为使在体内转染的和一致的体内基因分析。在一般情况下，EP被称为动态现象，它取决于当地的跨膜电压与纳秒内从而介导孔。这些差距可以维持毫秒，足以吨Ø授予访问权限的DNA，RNA或小分子5。当所施加的电压太高，EP的通常是短暂的字符是由于产热和感应太全面透化与所述小区5的随后的不可逆的损伤的抵消。 这里，我们表明，电穿孔是一种有效的和经济的转染系统，其能够被用于遗传睾丸转化为了阐明体内睾丸基因特征。本文介绍质粒制备，注射通过输出管和小鼠睾丸的后续电。这个过程可以是选择实现小鼠睾丸体内的生精小管的快速，特异性和高效的转染为了研究精子发生的过程的手段。

<table><tbody><tr><td>Centrifuge</td><td>Sigma</td><td>1-15 PK</td><td></td></tr><tr><td>Spectrophotometer</td><td>Nanodrop</td><td>ND-1000</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-10</td><td>vertical capillary puller</td></tr><tr><td>Microinjection capillaries</td><td>Clark</td><td>GC100-10</td><td>borosilicate standard wall</td></tr><tr><td>Micropipette beveler</td><td>Bachofer</td><td>Typ 462</td><td>rotating disk beveler</td></tr><tr><td>Electroporator</td><td>Nepagene</td><td>CUY 21EDIT</td><td>square wave electroporator</td></tr><tr><td>Tweezer electrodes</td><td>Nepagene</td><td>CUY 650P5</td><td>5 mm &amp;Oslash; disk electrodes</td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>Cold light source</td><td>Zeiss</td><td>KL 1500LCD</td><td></td></tr><tr><td>Microinjection pump</td><td>Eppendorf</td><td>Femtojet</td><td></td></tr><tr><td>Micromanipulator</td><td>homemade</td><td></td><td>XYZ cross table</td></tr><tr><td>Surgical instruments</td><td>FST</td><td></td><td>scissors, forceps, needle holder</td></tr><tr><td>Fine forceps</td><td>FST</td><td>Dumont #5, #7</td><td>efferent ducts preparation</td></tr><tr><td>Michel clip applying stapler</td><td>FST</td><td>Michel, 12029-12</td><td></td></tr><tr><td>Michel suture clips</td><td>FST</td><td>Michel, 12040-01</td><td>7.5 x 1.75 mm</td></tr><tr><td>Surgical suture</td><td>Catgut, Markneukirchen, Germany</td><td>Catgut 00</td><td></td></tr><tr><td>Syringe with 30 G needle</td><td>B. Braun</td><td>Omnican 40</td><td>to load micropipette and for anesthesia</td></tr><tr><td>Plasmid isolation kit</td><td>Promega</td><td>Cat. # A2495</td><td>plasmid Midiprep</td></tr><tr><td>Plasmid pEGFP-C1</td><td>Clontech</td><td>Cat. #6084-1</td><td>CMV-promoter + EGFP</td></tr><tr><td>Plasmid pDsRed2-N1</td><td>Clontech</td><td>Cat. #6084-1</td><td>CMV-promoter + DsRed2</td></tr><tr><td>Fast Green dye</td><td>Sigma</td><td>F7258-25G</td><td>for dilution in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>10% Ketamine</td><td>Serum Werk, Bernburg, Germany</td><td>Urotamin</td><td>mix in a rate 1:1 with xylazine</td></tr><tr><td>2% Xylazine</td><td>Serum Werk, Bernburg, Germany</td><td>Xylazin</td><td>mix in a rate 1:1 with ketamine</td></tr><tr><td>Sterilium</td><td>Bode Chemie</td><td>Sterillium</td><td>disinfection</td></tr><tr><td>Vet ointment</td><td>S&amp;amp;K Pharma, GmbH</td><td>Kerato Biciron 5%, Augensalbe</td><td>opthalmic ointment to prevent eye dryness</td></tr><tr><td>To-Pro-3 iodide</td><td>Invitrogen</td><td>T3605</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;10x PBS, pH 7.4&lt;/strong&gt;</td></tr><tr><td>1.37 M NaCl</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>Ibuprofen, Dolormin</td><td>Johnson &amp;amp; Johnson Consumer Health Care Germany</td><td>01094902</td><td>analgesic pediatric solution (NSAID) for postsurgery pain relief</td></tr><tr><td>27 mM KCl</td><td>Carl Roth</td><td>6781.1</td><td></td></tr><tr><td>100 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>T106.2</td><td></td></tr><tr><td>18 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>3904.1</td><td></td></tr></tbody></table>

in vivo microinjection and electroporation of mouse testis

该视频和文章贡献给了显微注射和小鼠睾丸在体内电穿孔的全面描述。睾丸小鼠细胞这种特殊的转染技术使精子发生过程中的独特的研究。 以下协议侧重于睾丸小鼠细胞与质粒构建体转染。具体而言，我们所用的报告载体pEGFP-C1，其表达增强的绿色荧光蛋白（EGFP），也是pDsRed2-N1载体表达红色荧光蛋白（的DsRed2）。两个编码报道基因是人巨细胞病毒立即早期启动子（CMV）的控制下。 对于执行基因转移到小鼠睾丸中，记者质粒构建注入活老鼠的睾丸。为此目的，一个麻醉动物的睾丸被暴露和显微注射的部位制备。我们首选的地方注射是在输出管，与最终连接睾丸网作为睾丸和附睾的精子的解剖运输路线。以这种方式，微注射后的生精小管的填充物被很好地管理和控制由于使用了染色的DNA溶液。其颜色的小管结构观察睾丸足够的填充后，器官被电穿孔。这使该DNA溶液转移入睾丸细胞。在温育后3天，睾丸被除去，在显微镜对绿色或红色荧光下的调查，表示转染的成功。 通常，该协议可以使用用于递送DNA-或RNA-构建体活小鼠睾丸以（过量）表达或敲除基因，促进体内基因功能分析。此外，它适用于研究报道构建或推定的基因边条保守党元素。因此，电穿孔技术的主要优点是快速的性能在低工作量以及在温和的技术设备和技能的组合所需的相比，替代技术。

实验的设置，用于根据该协议在体内进行注射小鼠睾丸和电穿孔，因为它是用在图1中示出，尽管它有可能获得工业化生产的微量，我们优选的，以产生我们自己的移液管通过拉动（ 图1A ）和坡口（ 图1B）的玻璃毛细管，使它们嵌合我们的需要。示出在图1C中的设备用于显微注射和电穿孔。对转染效率的原因，这是重要的，利用方波电穿孔?...

Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

In Vivo Microinjection and Electroporation of Mouse Testis

本文介绍了显微注射和小鼠睾丸中电在体内的睾丸小鼠细胞转染技术，研究精子的独特工艺。所提出的方案涉及以下步骤:玻璃毛细管制备，经由输出管显微注射，并通过电穿孔转染。

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

in-vivo-microinjection-and-electroporation-of-mouse-testis

Research

JoVE Journal

Biology

In Vivo Microinjection and Electroporation of Mouse Testis

27.8K 次观看.  Leibniz Institute for Farm Animal Biology (FBN). 本文介绍了显微注射和小鼠睾丸中电在体内的睾丸小鼠细胞转染技术，研究精子的独特工艺。所提出的方案涉及以下步骤:玻璃毛细管制备，经由输出管显微注射，并通过电穿孔转染。 

视频: In Vivo Microinjection and Electroporation of Mouse Testis

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 ...

科研

生物学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

使用 体内 显微注射和电穿孔的体细胞基因组工程小鼠模型

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

癌症研究

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

生殖细胞的移植和睾丸组织在小鼠异种移植

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

This article reports an in vivo inhibition of CENP-E through abdominal surgery and testicular injection of GSK923295, a valuable model for male meiotic division. Using the immunofluorescence, flow cytometry and transmission electron microscopy assays, we show that CENP-E inhibition results in chromosome misalignment and genome instability in mouse spermatocytes.

使用 体内 抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...

在小鼠睾丸活体显微注射和电穿孔

摘要

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此视频中的章节

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

小鼠视网膜神经节细胞的转染在体内电穿孔

在发展中国家小鼠视网膜体内电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

转基因小鼠内耳的发展

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

使用体内显微注射和电穿孔的体细胞基因组工程小鼠模型

生殖细胞的移植和睾丸组织在小鼠异种移植

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

使用体内抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估

在小鼠睾丸活体显微注射和电穿孔

摘要

探索更多视频

此视频中的章节

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

小鼠视网膜神经节细胞的转染在体内电穿孔

在发展中国家小鼠视网膜体内电穿孔

在子宫内电穿孔：鼠标控制时空基因Transefection

转基因小鼠内耳的发展

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

使用 体内 显微注射和电穿孔的体细胞基因组工程小鼠模型

生殖细胞的移植和睾丸组织在小鼠异种移植

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

使用 体内 抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估

使用体内显微注射和电穿孔的体细胞基因组工程小鼠模型

使用体内抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估