Name: dna transfection of mammalian skeletal muscles using in vivo electroporation
Uploaded: 2009-10-19T14:00:00
Description: Watch this Scientific Journal Video about DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation at JoVE.com

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?...

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	<li>Muangmoonchai, R., Wong, S., Smirlis, D., Phillips, I., Shephard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+liver+in+vivo+by+biolistic+particle+delivery.">Transfection of liver in vivo by biolistic particle delivery.</a> Molecular Biotechnology. 20 (2), 145-151 (2002).</li><li>DiFranco, M., Capote, J., Quinonez, M., Vergara, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+dynamic+FRET+signals+from+the+transverse+tubules+in+mammalian+skeletal+muscle+fibers.">Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers.</a> J Gen Physiol. 130 (6), 581-600 (2007).</li><li>DiFranco, M., Neco, P., Capote, J., Meera, P., Vergara, J. 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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 ...

Research

JoVE Journal

Biology

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

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 ...

科研

生物学

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

Analysis of DNA Double-strand Break DSB Repair 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

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 ...

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

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

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, ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

等距和骨骼肌产生偏心力评估隔离肌营养不良症的小鼠模型

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

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

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

In Vivo Microinjection and Electroporation of Mouse Testis

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

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

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

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

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

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 ...