Name: in vivo microinjection and electroporation of mouse testis
Uploaded: 2014-08-23T15:00:00
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）?...

<ol>
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Immunol. 83, 36-39 (2009).</li><li>Yomogida, K., Yagura, Y., Tadokoro, Y., Nishimune, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+expansion+of+germinal+stem+cells+by+ectopically+expressed+human+glial+cell+line-derived+neurotrophic+factor+in+mouse+Sertoli+cells.">Dramatic expansion of germinal stem cells by ectopically expressed human glial cell line-derived neurotrophic factor in mouse Sertoli cells.</a> Biol. 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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 ...

Research

JoVE Journal

Biology

In Vivo Microinjection and Electroporation of Mouse Testis

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

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

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

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

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

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

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

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

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

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

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

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish 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

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

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 ...