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 border="0" cellpadding="0" cellspacing="3">
	<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 &#216; 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 00</td>
			<td>Catgut 
			Markenkirchen</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, with 30G 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 ddH2O</td>
		</tr>
		<tr>
			<td>10x PBS, pH 7.4</td>
			<td>Carl Roth</td>
			<td>3957.1</td>
			<td>NaCl [1.37 M]</td>
		</tr>
		<tr>
			<td>6781.1</td>
			<td>KCl [27 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T106.2</td>
			<td>Na2HPO4+2H2O [100 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3904.1</td>
			<td>KH2PO4 [18 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Ketamin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Urotamin</td>
			<td>mix in a rate 1:1</td>
		</tr>
		<tr>
			<td>2% Xylazin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Xylazin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilium</td>
			<td>Bode Chemie</td>
			<td>Sterillium</td>
			<td>disinfection</td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>S&#38;K Pharma 
			GmbH</td>
			<td>Kerato Biciron 5% 
			Augensalbe</td>
			<td>or similar to prevent 
			dryness of eyes</td>
		</tr>
		<tr>
			<td>To-Pro-3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</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

27.7K 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

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...