我们感谢T.奥的斯，博士，加州大学洛杉矶分校神经生物学系，与我们一起分享TPLSM设施，三Fahlke博士，生理研究所，德国亚琛工业大学，，pEYFP ClC1质粒的捐赠，和先生R.塞拉诺为技术支持。这项工作是由来自美国国立卫生研究院/ NIAMS补助AR047664和AR54816的赠款支持。

实验程序，在体内FDB和IO肌肉电<ol><li>哺乳动物表达质粒体内电协议开始之前，必须扩增产量浓度的范围为2-5微克质粒/μL的TE 注意：我们经常使用的商业扩增试剂盒，并按照制造商的程序 。 商业表达质粒进行体内转染骨骼肌CMV启动子的工作很出色 。 </li><li>分装量的必要（10-20μL）质粒的解决方案，并保存两个0.5 ml Eppendorf管中（每个鼠标脚）。 </li><li>准备一个无菌台氏液中含有2毫克/毫升透明质酸。 </li><li>用一个anesthetizing框，深受麻醉鼠标使用4％异氟醚与经批准的气体麻醉机在O2。放置在一个加热垫（37℃）的动物和维持麻醉，使用灭鼠面罩。监测麻醉深度脚趾捏反射。 </li><li>根据与解剖镜下观察，在一只脚鼠标采用1英寸长的33计消毒针头的脚垫注入10μL的透明质酸酶的解决方案。穿透点靠近脚后跟的皮肤和皮下注射针迈向〜1 / 4“的脚趾基地。 </li><li>如果需要的话，另一只脚重复该过程。 </li><li>断开麻醉和鼠标放置在一个笼子里。允许它从麻醉中完全恢复。 </li><li>一个小时后，麻醉动物和第二次放在加热垫。透明质酸酶的解决方案中描述的相同的步骤，注射20-50微克的质粒DNA（质粒构建的大小而定）。总的注射量应少于20μL/ 英尺注意：当15-20μL是必要的，它是最好，收于皮肤组织胶针切入点。 </li><li>断开麻醉和鼠标放置在一个笼子里。允许它从麻醉中完全恢复，等待10-15分钟。 </li><li>第三次麻醉动物和它放在加热垫。 </li><li>选择一个动物的脚。在足跟部皮肤下放置一个镀金针，并在第二个脚趾的基础。电极相互垂直脚的长轴方向平行。 </li><li>连接头的针（电极），使用微剪辑连接器的电刺激。 Electroporate应用20脉冲，持续时间/每个，20毫秒，以1Hz的肌肉。根据电极的间距，调整脉冲电压幅值（用示波器监测）产生电场〜100 V / cm 时注意：在无宫缩刺激应遵守的水平麻醉是足够的 。 </li><li>如果需要的话，在动物的对侧脚重复上述程序。 </li><li>笼子里的动物，一旦从麻醉中完全恢复下保持观察。注意：如果程序正常，动物应在30分钟内重新获得充分的流动性，事后被送回在动植物公园的动物房。透明质酸和脚垫的DNA注射没有明显的不利影响的动物。一旦从麻醉中恢复，小鼠能够笼子周围正常缓行。作为一个额外的预防措施，添加到饮用水为0.0027毫克/毫升2天止痛药的动物卡洛芬。 </li><li>蛋白的表达可检测转染后2-8天。然而，许多蛋白的持续表达已经观察了好几个月。 </li></ol> 注：所有动物的程序是，由加州大学洛杉矶分校校长的动物研究委员会批准的动物福利法“，对实验动物的人文关怀和使用小灵通的政策规定。 代表性的成果： 体内电上文所述的程序的正确实施，应在有效转染质粒在FDB和IO肌肉。然而，转基因蛋白变异体的表达效率将取决于质粒的规模和复杂性的蛋白质，蛋白质的功能特性，和其他一些变量，我们的控制。正如在图1电穿孔在商业质粒（PMR mCherry）mCherry蛋白编码存在的FDB肌肉所示，大部分的肌纤维转染与我们的协议，其中大多数是显示红色荧光的事实说明。这并不排除个别纤维表现出不同程度的蛋白表达的可能性，或没有在所有3个转，很少有纤维。应Ë指出， 大量 mCherry，EGFP，ECFP和EYFP的，如荧光蛋白的高效表达，不损害肌肉纤维的兴奋性和兴奋收缩偶联物业。事实上，他们是在深水（结果未显示）转肌肉的没有区别。 用来验证本地化表达荧光标记的蛋白在骨骼肌纤维的实际办法。 堤诺表达的转基因蛋白的细胞内定位是同时获得定期评估，使用TPLSM，各自的标签和图像识别标记的细胞结构的荧光图像。其中后者最典型的有：一）二次谐波产生（SHG）的图像，这从一个乐队（居中的M线）9,10的肌节肌球蛋白各向异性出现，B）DI - 8 - ANEPPS荧光图像，可通过标签表面和横向的管状这impermeant电位dye11的肌纤维（T型小管）系统膜获得。在肌纤维染色与DI - 8 - ANEPPS的荧光图像，T型管出现荧光导向纤维长轴约正交的窄带。这些频带间隔不等对方：它们是由很长的距离跨越，跨越的M线和跨越整个的Z - 11行是短暂的分离。 骨骼肌纤维的α-辅肌动蛋白- EGFP的表达及本地化。 α-辅肌动蛋白是标签的变体的结构肌肉蛋白的表达的一个例子如图2所示。这种蛋白质是已知的Z线的重要组成部分，正因为如此，经常使用这structure12标记。我们转染质粒pEGFPN1 -α- actinin1人类（非肌肉）编码FDB和IO肌肉α-辅肌动蛋白在C端与EGFP标记。转染后6天，我们发现，作为评估EGFP荧光分布，α-辅肌动蛋白是主要表达了同样沿纤维轴向间距窄波段。看到每个肌节的单波段（图2A和2D）。这些频段与Z线的共定位表明倍频（图2B）和DI - 8 - ANEPPS（图2E）的图像进行比较EGFP荧光的分布。叠加图像（图2C），α-辅肌动蛋白- EGFP的波段二次谐波频段的替代，说明他们连续两次的M -带位于中间，正好与Z型线的位置。在转的肌肉纤维染色与DI - 8 - ANEPPS，α-辅肌动蛋白- EGFP频段看到每一双T型管（图2F），这是众所周知的侧翼的Z线（即在较短距离分隔）之间为中心，从而表明，转基因α-辅肌动蛋白是有针对性地对Z -线。 在N -末端与EGFP表达标签的DHPRα1s 肌肉转染效率与pEGFPC1.1 DHPRα1S验证TPLSM图像显示（如图3A）大多数纤维表达EGFP的DHPRα1s（一种跨膜蛋白）。转染纤维的最突出的特点是双带的模式EGFP荧光（图3A及3B），将预计，如果这种蛋白质是有针对性地的T管。它也可以在图3中，而显示不同的纤维的荧光强度各级，带状个别纤维的荧光模式似乎保持均匀的沿纤维。在更高的放大倍率（图3B），它可以清楚地看到的乐队之间的不平衡间距是在纤维染色DI - 8 - ANEPPS（例如图2E）观察到类似。说明的倍频带位于EGFP -DHPRα1s乐队之间的间距较大，证实该蛋白在T管的覆盖图像（图3B＆3E）。额外的FRET测量与非荧光的亲脂性阴离子政治部（数据未显示），进一步表明，EGFP的基团是在几纳米的T型管的内层膜单张。 本地化和EYFP的ClC1表达功能的评价纤维转pEYFP - ClC1与EGFP -DHPRα1s观察到类似的模式，它编码一个EYFP的标签构造骨骼肌氯离子通道（ClC1），显示EYFP的荧光条带（图4A）（N -末端） “表达（图3A）和相应的DI - 8 - ANEPPS染色（图2E）所示的T型小管安排。正如预期的那样，EYFP ClC1（图4A）和SHG影像（图4B），覆盖（图4C）所示的叠加，说明，SHG带为中心的大间距ØF EYFP的荧光带之间。 为了评估是否在显着增加的肌肉纤维，如预期般从功能氯离子通道表达的休息电导EYFP ClC1结果的表达，我们从转的FDB肌肉酶分离的肌纤维和研究他们的电生理特性用两个微电极实验装置描述以前6,11,13。图5显示了两种纤维的结果：一是表达大量EYFP - ClC1的全球标准的荧光显微镜的荧光强度测量（未显示）的评估，和其他非转控制。图5A电压的记录（表达EYFP - ClC1从肌肉纤维获得）表明，由于过度的休息电导，纤维几乎是不兴奋的。为了引出一个非常小的积极响应（无超调，图5A），需要使用电流刺激脉冲高达400nA（为0.5ms）。更换一个包含9 - ACA的氯离子通道阻滞剂500微米的外部Tyrode液后，200 nA电流脉冲足以引起动作电位（图5B），虽然慢得多，比那些记录在控制纤维更广泛的（图5C）。正如预期的那样，除了9 - ACA了这种控制纤维的影响不大（图5D）。 <img src="/files/ftp_upload/1520/1520fig1tmb.jpg" alt="图1" border="0" /> 图1 在体内电穿孔法转染效率。明视（A）和荧光（二）转FDB肌肉的图像与PMR - mCherry 。电协议后的天数：12天。请<a href="https://www.jove.com/files/ftp_upload/1520/1520fig1.jpg">点击这里</a>为图1的放大版本。 <img src="/files/ftp_upload/1520/1520figure2.jpg" alt="图2" /> 图2。FDB 纤维的α-辅肌动蛋白- EGFP的表达和定位。小组，A和B是EGFP荧光和二次谐波图像，分别表达α-辅肌动蛋白- EGFP光纤，。 C组是一个图像叠加在A和B。面板D和E是另一种表达α-辅肌动蛋白与DI - 8 - ANEPPS - EGFP和染色，分别纤维的荧光图像。小组F是在D和E日电协议后的图像叠加：6天。 <img src="/files/ftp_upload/1520/1520figure3.jpg" alt="图3" /> 图3。 表达EGFP -DHPRα1s针对FDB纤维 。 A组，一组表达EGFP -DHPRα1s纤维EGFP的荧光图像。 B组是一个正方形的扩大，在面板中为了更好地显示蛋白表达的条带状格局。 C组是倍频影像相应的面板A.小组ð形象，图像的叠加A和C。小组E是面板D.日电协议后表示该地区的扩大：20天。 <img src="/files/ftp_upload/1520/1520figure4.jpg" alt="图4" /> 图4。FDB 纤维EYFP - ClC1的表达和定位。面板A和B是EYFP的荧光和二次谐波图像，分别表达EYFP - ClC1纤维。 C组是A和B小组ð是沿着电协议后面板C.天的形象突出了白线的强度分布在面板的图像叠加：7天。 <img src="/files/ftp_upload/1520/1520figure5.jpg" alt="图5" /> 图5。 电生理评估纤维表达转基因EYFP - ClC1小组 A和B是从响应电流脉冲表达EYFP - 9 - ACA治疗前后ClC1，分别纤维电压记录。前和治疗后分别与9 - ACA，面板C和D是在非转光纤电流脉冲响应（动作电位）引起的电压记录。

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The authors have nothing to disclose.

我们在这里描述应当遵循的，为了达到有效的DNA质粒转染骨骼肌纤维在体内电的详细步骤。我们的方法的主要优点是实现简单，其最小的侵袭，在对动物的健康危害可以忽略不计结果。在现实中，上面介绍的电协议不涉及两个皮下注射每英尺，通过电刺激的协议，所有这一切都是在麻醉状态下的动物的耐受性良好，远远高于。在我们的机构，这些操作是完全确定的非手术的程序，因为它们?...

dna transfection of mammalian skeletal muscles using in vivo electroporation

在细胞生物学中的一个越来越大的兴趣，是为了表达transgenically修改必需的蛋白质（如荧光标记的结构和/或突变的变种）的形式，以调查他们的内源性的分布和功能的相关性。一个有趣的方法，已实施完成这个目标，在完全分化的细胞是<em&gt;在体内</em&gt;通过各种方法进入特定组织，如肝质粒转染<sup&gt; 1</sup&gt;，骨骼肌<sup&gt; 2,3</sup&gt;，甚至大脑<sup&gt; 4</sup&gt;。我们在座的详细描述，以便有效地转染到纤维的遗传物质，必须遵循的步骤<em&gt;趾短屈肌</em&gt;（FDB）<em&gt; interosseus</em（IO）的成年小鼠的肌肉，使用<em&gt;在体内</em&gt;电击方法。实验参数进行了优化，从而最大限度地转，同时尽量减少组织损伤，可能会损害个别纤维表达的蛋白质的质量和数量的肌纤维的数量。我们已经验证了本文中所描述的方法实施中的可溶性蛋白的高收益，即EGFP和ECFP结果<sup&gt; 3</sup&gt;，蛋白酶，FKBP12的，β2aDHPR等;结构蛋白，即minidystrophin和α-辅肌动蛋白和膜蛋白，即α1sDHPR，RYR1，心脏钠/ CA<sup&gt; 2 +</sup&gt;换热器，钠通道，SERCA1 NaV1.4等，当应用FDB，IO和其他肌肉的小鼠和大鼠。一些这些蛋白质的高效表达，得到了验证与生化<sup&gt; 3</sup&gt;和功能的证据<sup&gt; 5</sup&gt;。然而，迄今为止我们所使用的最常见的验证方法是标准的荧光显微镜和双光子激光扫描显微镜（TPLSM），许可证标识不仅是整体的表达，而且还详细的细胞内定位荧光标记的蛋白质结构。该方法同样可以被用于转染的生理相关的蛋白质的表达质粒编码（如下所示），或旨在干扰RNA（siRNA）抑制正常表达的蛋白质的表达（不是由我们尚未测试）。应该指出，转FDB和IO肌纤维，特别是哺乳动物的肌肉生理学调查有关，因为这些肌肉酶分离的纤维是目前最适合的车型之一，调查的兴奋性和兴奋收缩偶联的基本机制下电流或电压钳条件<sup&gt; 2,6-8</sup&gt;。

Watch this Scientific Journal Video about DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation at JoVE.com

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

我们描述了使用电和随后的可视化的表达蛋白，用荧光显微镜活体小鼠的脚部肌肉纤维的高效转染质粒DNA的详细程序。

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 ...

dna-transfection-mammalian-skeletal-muscles-using-vivo

Research

JoVE Journal

Biology

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

17.7K 次观看.  David Geffen School of Medicine, University of California, Los Angeles. 我们描述了使用电和随后的可视化的表达蛋白，用荧光显微镜活体小鼠的脚部肌肉纤维的高效转染质粒DNA的详细程序。

视频: DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

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 ...

科研

生物学

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

游离成年哺乳动物元DNA的细胞核内内微量注射

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

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 ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

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 ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.