This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology to M.I. (21229004 and 25221304).

研究利用老鼠是按照由东京大学的动物福利委员会制定的准则。 质粒DNA的1.Preparation <ol><li>由亚克隆的cDNA或shRNA序列插入用于哺乳动物细胞12的表达质粒产生用于体内电穿孔的DNA质粒。使用巨细胞病毒立即早期增强和鸡β肌动蛋白启动子融合（CAG）启动子驱动的质粒13，因为它允许强大和稳定的表现。要CAG启动子的控制下的shRNA的表达，使用基于mir30-shRNA的盒系统，用于亚克隆shRNA的14。 </li><li>纯化的质粒DNA使用根据制造商的说明加厚-prep试剂盒，和重悬带的HEPES缓冲盐水（140mM的氯化钠，0.75毫的 Na 2 HPO 4，25mM的HEPES; pH值7.40）的DNA。调节DNA的浓度为≥4微克/微升。 </li><li&gt;准备质粒DNA溶液至4微克/微升的浓度，并添加快绿染料的最小量（0.01％终浓度）来标记注射部位。当需要多个质粒的同时电穿孔，调节质粒DNA溶液至4微克/微升的总浓度。 注意:质粒DNA的最佳组合物应根据各质粒的转染效率来确定。 </li></ol> 2，手术器械和盐水消毒<ol><li>高压釜的手术器械和0.9％NaCl溶液。 </li></ol> 3.玻璃微的制备<ol><li>拉用吸管牵拉玻璃吸管。切移液器的尖端的直径为30-50微米。使用以下参数:热火，600;速度，50;时间，75。 </li></ol> 4.动物外科手术，DNA注射和电注:以上鉴于这一步骤的图1中描述。虽然幼鼠的程序在这里描述的，该方法也适用于用同样的步骤比较成熟的动物。 <ol><li>麻醉用异氟烷大鼠中诱导框直到动物变成通过调节氧气流至0.4升/分和异氟醚浓度为4％（体积/体积）不动。执行脚趾捏，确认正确的麻醉。 </li><li>把大鼠上的预热加热器双目显微镜下，并通过经由面罩连续施用异氟烷维持麻醉。调节氧气流至0.2升/分和异氟醚浓度为2％（体积/体积）。使用眼药水防止眼睛干涩，如果动物的眼睛是睁开的。 </li><li>修复手术胶带腿。 </li><li>清洁与碘伏后大腿的皮肤，使用手术刀切开。 注意:如果手术区域覆盖用H刮手术领域空气。 </li><li>通过创建股四头肌之间的开口了大腿肌肉和股二头肌与缝纫针暴露坐骨神经。 </li><li>湿用0.9％的NaCl溶液中的神经。吸收多余的水用不起毛的纸。 </li><li>插入玻璃微量的基部到柔性管，并且通过轻轻吸填充的DNA溶液（至少一个微升）到微量的足够量。 </li><li>通过轻轻拉动使用针神经的前端侧抬起的露出神经。 注意:不要施加张力的神经，以尽量减少机械应力。 </li><li>将玻璃微插入神经 ​​的末端位置，并注入通过施加压力将DNA溶液（由吹入软管的开口端即 ）。直到神经显示为绿色（1微升最大）注射DNA溶液。因为微量的频繁插入可能损伤神经，不要插入微量的两倍以上。 </li><li>放置TWE察型铂电极约1-2毫米除了神经。填充电极和用0.9％NaCl溶液的神经之间的差距。 注意:不要用电极保持神经，以避免对神经的机械应力。 </li><li>应用电脉冲来使用与所述电极的电穿孔注射部位。第一个脉冲集后，反转电极和应用其他脉冲集。使用下面的参数:电压，50伏;脉冲持续时间，5毫秒;脉冲间隔100毫秒;脉冲数的4倍。 </li><li>清理电穿孔部位用0.9％NaCl溶液。 </li><li>重复步骤4.4-4.11对侧坐骨神经。 </li></ol> 5.后电<ol><li>关闭与氰基丙烯酸酯胶的切口。 </li><li>干燥涂胶后，请用碘伏伤口。 </li><li>松开从面罩的小狗。暖小狗上至少一个小时一个温暖的，以便允许它完全从麻醉中恢复。 ðO不可离开小狗无人看管，直到它已经恢复了足够的意识。 </li><li>从麻醉中苏醒后，返回小狗母亲老鼠。不要返回小狗，直到完全康复。 </li></ol> 6.后手术<ol><li>众议院幼鼠在笼子里，直到进行实验11（ 见图3的例子）。辖卡布洛芬（5mg / kg的; IP），非甾体抗炎药，或叔丁啡（0.1毫克/千克，SC），阿片类镇痛剂，如果需要的话。 注意:如果大鼠小狗不生长良好或炎症围绕手术部位观察到的，排除在实验动物。 </li></ol>

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The authors declare that they have no competing financial interests.

In this paper, we describe a simple and efficient method that allows in vivo gene transfer to myelinating SCs in the rat sciatic nerve using electroporation. This method allows highly selective gene expression in myelinating SCs by simply applying electric pulses to the plasmid DNA-injected sciatic nerve. Because the molecular mechanisms of myelination and demyelination in the peripheral nervous system remain unclear, the present in vivo electroporation method will be a powerful tool to clarify the roles of multiple genes of interest in living animals.
A critical requirement of this method is to keep damage to the nerve during surgery to a minimal level. Should surgical damage cause excessive inflammation, the sciatic nerve may degenerate. To avoid this, one must conduct surgery with extreme care, so as to not damage the blood vessels around the nerve. Mechanical stress to the nerve during the surgery can also be a cause of nerve damage. To minimize mechanical stress, lifting the exposed nerve should be done as gently as possible, and the tweezer-type electrode should be placed close to the nerve without contact. Furthermore, electrical pulses that are too strong can cause undesirable large leg movement, which leads to mechanical stress, or can burn the nerve. If significant damages are observed in the nerves, we recommend reducing the electrical pulse intensities or placing the electrode further away from the nerve.
In our present protocol, CAG promoter-driven plasmids were used as expression vectors. CAG promoter-driven plasmids allow high levels of gene expression in myelinating SCs in vivo. We also have tried a CMV promoter, another widely used universal promoter for mammalian gene expression, but expression of the gene product was very weak. This is consistent with previous results, in which electroporation-mediated transfection was conducted in the embryonic brain20. Therefore, we recommend using CAG promoter-driven plasmids for the in vivo electroporation method.
Because axonal signaling is a key factor in myelination/demyelination21, gene modification in neurons is also important. However, delivery of transgenes using our in vivo electroporation method is limited to SCs. It has been reported that gene delivery into sciatic nerve axons can be achieved when in vivo electroporation is applied to dorsal root ganglion (DRG) neurons in adult rats22. This suggests that delivery of plasmid DNA to the cell body is likely to be critical for in vivo transfection of peripheral axons. Thus, to examine the involvement of axonal molecules in myelination/demyelination, researchers should use neuron-specific genetic methods such as genetically modified animals, neuron-specific viral vectors, or in vivo electroporation to DRG neurons.
Compared with current methods, such as the generation of genetically modified animal lines23 and delivery of transgenes by viral vectors4-6, gene modification of SCs by in vivo electroporation is simpler. This method only requires several days for plasmid DNA construction and one day for electroporation surgery. Plasmid DNA construction does not require a biohazard room that is usually essential for viral vector handling. In addition, one of the advantages of the electroporation method is the capacity for simultaneous expression of multiple gene products using a simple protocol. Our novel technique will be useful for analyzing the interaction of a variety of signaling molecules involved in myelination and demyelination. In particular, by permitting the cotransfection of a number of different intracellular fluorescent probes, our method should be a powerful tool for investigating intracellular signaling dynamics in SCs using live imaging experiments.

The rapid transmission of sensory and motor information in the peripheral nervous system is permitted by the myelin sheath, which is formed by myelinating Schwann cells (SCs)1. Insulation of axons by the myelin sheath enables saltatory conduction, which increases the speed of nerve impulses. In disorders in which the development or maintenance of the myelin sheath is impaired, nerve conduction speed is reduced. This results in neuropathy involving motor and sensory dysfunction. Although there are many studies on the molecular mechanisms of myelination and demyelination in the peripheral nervous system, the roles of the numerous proteins involved in these processes remain unclear.
To study the molecular mechanisms of SC myelination/demyelination in vivo, genetic approaches have been used to modify gene expression in animals. A powerful approach is the use of knockout/knockin or transgenic animals. However, the generation of these animals is expensive and time consuming. For SC-specific gene manipulation, crossing floxed strains with Cre mice or other conditional gene expression methods are necessary. This again is laborious and time intensive. In recent years, a cutting-edge genetic technology, the CRISPR-Cas9 system, has made the generation of genetically modified mice much quicker (about 4 weeks)2,3, but this method is hindered by target sequence limitations, and suffers from off-target effects. As an alternative method, viral vector-mediated gene transfer is a faster and easier method of achieving gene transfer into SCs in vivo4-6. Indeed, the generation of viral vectors is less expensive, and takes a shorter time (within a few weeks), and gene manipulation of SCs can be achieved by simply injecting engineered viral vectors, such as adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors, into the sciatic nerve. Because these viral vectors have different characteristics, users have to choose the one best suited for their purpose. Adenoviral vectors infect axons and SCs in both young and mature sciatic nerves. In particular, adenoviral vectors have higher selectively for non-myelinating SCs than myelinating SCs. Adenoviruses can cause immune responses, and accordingly, immunodeficient strains should be used5. AAV vectors are currently the most widely used viral vectors, and allow in vivo gene transfer with lower toxicity7. AAV can transduce both axons and SCs by direct injection into the nerve fibers8,9. However, AAV-mediated protein expression usually requires 3 weeks or longer to reach maximum levels7,9. Therefore, it is difficult to analyze myelination, which actively progresses during the two week postnatal period. Lentiviral vectors have higher selectively for myelinating SCs than non-myelinating SCs, and do not have toxic effects on sciatic nerves. However, lentiviral vectors do not infect SCs in more mature nerves5, and therefore are unsuitable for analyzing events such as the demyelination process.
Electroporation is another faster and easier approach to achieve in vivo gene transfer. It has been reported that in vivo transfection of SCs can be achieved when electroporation is applied to transected rat sciatic nerves10. However, because this method requires nerve transection for gene delivery, the application is limited to the analysis of the damaged nerves. Here, we describe an alternative method that allows the delivery of transgenes into myelinating SCs in intact rat sciatic nerves using electroporation11. This method requires plasmid construction, which can usually be completed within a week. Then, by simply delivering electric pulses to the site on the sciatic nerve where the plasmid DNA was injected, highly selective transfection of myelinating SCs can be achieved in neonatal as well as in more mature animals. By electroporating multiple plasmids, simultaneous expression of a variety of genes can be easily achieved. The ability to simultaneous express multiple molecules, such as signaling proteins, short-hairpin RNAs (shRNAs) and functional probes, is crucial for investigating complex processes such as myelination and demyelination. The novel in vivo electroporation method described in this paper will be a powerful tool, allowing researchers to analyze the function of a multitude of molecules and their interactions in myelinating SCs.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genopure Plasmid Maxi Kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;">03 143 422 001</td>
			<td style="width:167px;">Plasmid DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green CFC</td>
			<td>WAKO</td>
			<td>069-00032</td>
			<td>Dye for DNA injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC 150T-10</td>
			<td>HARVARD APPARATUS</td>
			<td>30-0062</td>
			<td>Glass capillary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction tubing</td>
			<td>Drummond</td>
			<td>05-2000-00</td>
			<td>Suction tubing for micro injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MODEL P-97</td>
			<td colspan="2">SUTTER INSTRUMENT CO.</td>
			<td>Micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CUY21 Single Cell</td>
			<td>BEX</td>
			<td>Electroporator CUY21 Single Cell</td>
			<td>Pulse generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric warmer</td>
			<td>KODEN</td>
			<td style="width:119px;">CAH-6A</td>
			<td>Warmer during the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isofluolane</td>
			<td style="width:71px;">Mylan</td>
			<td style="width:119px;">1119701G1076</td>
			<td>Anesthetic</td>
		</tr>
	</tbody>
</table>

in vivo gene transfer to schwann cells in the rodent sciatic nerve by electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

用红色荧光蛋白（RFP）转染的坐骨神经-expressing质粒的一个例子示于图2A。细胞呈现两极形态，旺的特征，分别用稀少转RFP。在轴突中没有检测到RFP的荧光。我们通常会发现〜每一根神经100转旺。这种转染效率似乎类似于使用慢病毒载体4 的体内的SC感染效率。 免疫染色实验表明，大多数（〜96％）RFP阳性细胞在P7共标记为S100中，一个SC标记（ 图2B），和标记的共同为MBP在P14 RFP阳性细胞91％，一髓鞘形成SC标记（ 图2C），这表明通过电穿孔该基因转移为脱髓鞘旺高度选择性。 多个基因的导入旺<eM&gt;体内会调查髓鞘形成/脱髓鞘的机制是非常有用的。这里所描述的体内电穿孔方法的主要优点是能够以简单的程序传送的多个基因的能力。图2D示出了具有使用体内电穿孔的GFP和RFP表达质粒的混合物转染的坐骨神经的代表图像。约97％的SC都是GFP和RFP双阳性，提示多基因的高效交付可以简单地通过电穿孔多质粒的混合物来实现。 在啮齿类动物中，髓鞘开始围绕生育，出生后的头两个星期期间急剧增加，然后逐渐减少。因此，通过在这些发育时间窗遗传操纵旺，髓鞘的这些不同阶段所依据的机制可以澄清。慢病毒载体是一个好工具nalyzing髓鞘化，尤其是因为他们有毒性最小，但慢病毒只感染新生儿坐骨神经5,6。相比之下，当转染在P3（ 图2E，顶部）或P14（ 图2E，底部）进行电穿孔介导的基因转移效果很好。 体内电穿孔法新颖的应用说明如下。 图3A显示绿色荧光蛋白表达在不同发育阶段（P7，P14，P21和P31）髓鞘的SC光显微图像。通过光学显微镜分析，形态参数，如长度和直径的变化，可以进行评估。注意，相比于完整的大鼠末梢神经15,16，这些参数具有相似的值，这表明电神经发展而不显著破坏作用。 图3B示出的LacZ-expre的电子显微镜图像ssing脱髓鞘旺。在这种情况下，LacZ基因被用作表达标记。 β半乳糖苷酶使用bluo加仑，不溶性乙醇基板，染色使得能够通过电子显微镜11,17的转染的SC的髓鞘结构的分析。在这些实验中，信号分子的作用，可通过沉默或增加它们的表达，由此允许失功能或获得性功能的影响的分析进行检测。除了 ​​固定的组织的分析， 在体内电介导的基因转移也可以应用于实时成像实验。例如， 图3C示出了髓鞘形成的SC共表达的G-GECO1.1 18，绿色荧光细胞内的Ca 2+指示剂，和R-GECO1mt 19，红色荧光线粒体的 Ca 2+指示剂。通过表达这些指标，我们确定，其控制在髓鞘形成雪旺细胞溶质和线粒体的 Ca 2+浓度信号通路<html> 。因此，本方法可用于研究各种信令机制，特别是当遗传编码荧光探针可用于检测感兴趣的信号。 <img alt="图1" src="/files/ftp_upload/54567/54567fig1.jpg" /> 图 1: 在 I N体内 电穿孔法首先，将麻醉大鼠的坐骨神经示意图露出。第二，质粒DNA注射到坐骨神经。第三，电脉冲通过镊子状电极递送到注射部位。最后，将伤口闭合用胶水。这个程序可以在对侧的神经被重复。 <a href="https://www.jove.com/files/ftp_upload/54567/54567fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "&gt; <img alt="图2" src="/files/ftp_upload/54567/54567fig2.jpg" /> 图 2: 转染坐骨神经代表性的成果 （A）转染坐骨神经的形象代表。神经在P3与RFP表达质粒转染，并固定在P7。 （B）在P7显示与S100，一个SC标志共存一个RFP转染的细胞的形象代表。 （C）的一名代表在P14一个RFP转坐骨神经显示与MBP，髓鞘形成一个标记SC的共存形象。 （D）与GFP和RFP表达质粒转染坐骨神经的形象代表。转旺同时表达GFP和RFP。 （E）的脱髓鞘在P31在P3中，髓鞘形成开始时（顶）转染的SC的图像，并在P31在P14中，当最大量的轴突成为髓鞘（底部）转染的髓鞘的SC的图像，这表明TRANSF髓鞘形成雪旺挠度不仅可以在新生儿神经，而且在更成熟的神经来实现。比例尺= 200微米（A）;为50μm（BE）。这个数字是从我们以前的出版物11修改。 <a href="https://www.jove.com/files/ftp_upload/54567/54567fig2large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54567/54567fig3.jpg" /> 图 3: 在体内 电 （A）髓鞘种姓发展的光学显微分析中的应用 。坐骨神经与P3表达GFP的质粒电穿孔，和分别固定在各个发育阶段（P7，P14，P21和P31）。 GFP阳性的SC的代表图像显示在左侧。平均长度和直径总结为平均±SEM（n = 3的0 - 47从右侧3神经）。髓鞘旺随着开发收益的长度和直径。 （B）用质粒编码的LacZ转染的坐骨神经的电子显微镜图像。转染的SC（白星号，左）微细地与β半乳糖苷酶的反应产物的沉淀物进行标记。 （C）和G-GECO1.1，绿色荧光细胞内的Ca 2+指示剂，和R-GECO1mt，红色荧光线粒体的 Ca 2+指示剂共转染的一个SC的图像。白色虚线矩形内的区域放大显示在右边的面板。比例尺= 50微米（A和C）;为1μm（B）。这个数字是从我们以前的出版物11修改。 <a href="https://www.jove.com/files/ftp_upload/54567/54567fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation at JoVE.com

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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Research

JoVE Journal

Neuroscience

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

8.8K 次观看.  The University of Tokyo. Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

视频: In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

从大鼠坐骨神经轴浆隔离

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

科研

神经科学

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 ...

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 ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...