体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

Summary

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.

Abstract

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.

Introduction

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 vivo^4-6. 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 fibers^8,9. However, AAV-mediated protein expression usually requires 3 weeks or longer to reach maximum levels^7,9. 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.

Protocol

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 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。 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>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。 2，手术器械和盐水消毒高压釜的手术器械和0.9％NaCl溶液。 3.玻璃微的制备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。 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。 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。 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。 修复手术胶带腿。 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。 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。 湿用0.9％的NaCl溶液中的神经。吸收多余的水用不起毛的纸。 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。 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。 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。 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。 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。 清理电穿孔部位用0.9％NaCl溶液。 重复步骤4.4-4.11对侧坐骨神经。 5.后电关闭与氰基丙烯酸酯胶的切口。 干燥涂胶后，请用碘伏伤口。 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。 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。 6.后手术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。

Representative Results

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>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 。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。 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。 请点击此处查看该图的放大版本。 “> 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。 请点击此处查看本图的放大版本。 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。 请点击此处查看本图的放大版本。

Discussion

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 vectors^4-6, 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.

Materials

Genopure Plasmid Maxi Kit	Roche	03 143 422 001	Plasmid DNA purification kit
Fast Green CFC	WAKO	069-00032	Dye for DNA injection
GC 150T-10	HARVARD APPARATUS	30-0062	Glass capillary
Suction tubing	Drummond	05-2000-00	Suction tubing for micro injection
MODEL P-97	SUTTER INSTRUMENT CO.		Micropipette puller
CUY21 Single Cell	BEX	Electroporator CUY21 Single Cell	Pulse generator
Electric warmer	KODEN	CAH-6A	Warmer during the surgery
Isofluolane	Mylan	1119701G1076	Anesthetic