这项工作是从耳聋研究基金会Yc进行S和美国国家科学基金会（内部监督办公室0930096）的拨款，以KFB支持。 KFB：美国国立卫生研究院/ NINDCD 2 RO1 DC04184， <a href="https://commons.era.nih.gov/commons/genericStatus.do?actionRole=nonPI&amp;applID=7854446&amp;uhf-token=Ugf7JvudZTCbTUW9jSAR1snMUgg%3D">3R01DC004184 - 08W1</a> ，NSF DBI 0832862，美国国家科学基金会IOS 0930096

1。为电穿孔电极<ol><li>剪下适当长度的直径为75微米的钨丝（AM系统公司Carlsborg，WA）（约3-5厘米） </li><li>将通过电缆莫仕连接器（针加盖黄铜）的钨丝</li><li>余音钨丝和Molex公司热缩管（SPC技术，芝加哥，IL）和适用于本生灯或热枪热密封萎缩管电线电缆连接器</li><li>为了加强钨丝尖端，电线浸入1.0 1N氢氧化钠（康拉德等，1993），使用文件夹作为另一个电极，电解线与方波Electroporator的。我们使用的条件是5个50伏的脉冲每期100毫秒和50毫秒脉冲之间。电极应加强以一个直径15-20微米</li><li>请在一个托盘内的电线，压成泥，在使用前在电的沟槽</li></ol> 2。设置电穿孔站<ol><li>磁带削尖75μm的钨电极micromanipulators（世界精密仪器）。 </li><li>在解剖显微镜阶段（徕卡）两侧的广场micromanipulators。 </li><li>连接电极保护CUY - 21编辑方波Electroporator（图1）。 </li><li>打开electroporator设立电参数，包括电压，脉冲宽度和脉冲数。被发现三个13伏的脉冲持续时间为5.0ms，100ms的每个脉冲之间，这个实验的最有效的参数。如果电在胚胎创建气泡，降低脉冲的长度。 </li></ol> 3。 Morpholinos显微注射到斑马鱼奥迪囊泡<ol><li>从养殖池的收获斑马鱼的胚胎。在胚胎胚胎孵育中期提高0.3 PPM的亚甲蓝（韦斯特，2005年）在28.5 ° C过夜。 </li><li>与萨特的P - 97电极拉马拉玻璃针</li><li>热身琼脂糖凝胶基地（鱼水的1％琼脂糖凝胶在10毫米培养皿）至37 ° C水浴中</li><li>烧热1％的低熔点琼脂糖（LMPA）鱼水，直到融化，并保持在37℃水浴保温。 </li><li>填写与吗啉溶液（GeneTools，科瓦利斯，或）玻璃针和削减适当直径的尖端。装载到一个显微（库恩）的注射针头。 </li><li>切约10微米的针尖注入矿物油，以确保针尖允许足够吗啉的解决方案交付。 </li><li> Dechorinate胚胎在受精后24小时（HPF）和麻醉MS222（tricaine，Sigma公司），在0.3 PPM的亚甲蓝鱼水。 </li><li>琼脂糖凝胶基地，为方便统一的分析与右侧对齐到3至5麻醉胚胎</li><li>在每个胚胎中放置1至2滴1％LMPA，让固化修复的胚胎</li><li>旋转的培养皿中，使奥迪囊泡右侧</li><li>针放入奥迪囊泡腔注入啉的解决方案与PV820气动PicoPump（世界精密仪器），一个附加的氮气罐（图2） </li></ol> 4。电穿孔程序<ol><li>将安装的电车站后，立即注射胚胎</li><li>放置到组织后的奥迪囊泡的正极，但不穿透;负电极插入大脑前奥迪囊泡</li><li>应用目前使用脚踏开关或按下一个开关。 </li><li>琼脂糖倒入鱼水和删除从琼脂糖纷飞和玻璃吸管的胚胎。使用谨慎不破坏胚胎。对于难以去除的胚胎，用针轻轻地打破任何LMPA周围的胚胎。 </li><li>在一盘与亚甲基蓝鱼水和提高胚胎所需的分析阶段（形态学，免疫组化，原位杂交等和微观anlaysis，）的地点胚胎。 </li></ol> 5。代表性的成果： <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="图1" /> 图1电穿孔站。用削尖的75μm的钨丝电极，通过导线连接到保护CUY - 21编辑方波Electroporator。这些电极贴在micromanipulators。 <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="图2" /> 图2。注水站。所有胚胎注入右耳为达到标准化的目的。 <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="图3" /> 图3。鬼笔环肽染色与3 DPF胚胎微丝。 （一）3 DPF胚胎injecteD与控制莫有强烈的鬼笔环肽染色后的黄斑部（分）。 （二）与对照MO，然后注射电穿孔的胚胎在鬼笔环肽染色时，只注射胚胎相似的水平。 （三）与MIF MOS注入仅奥迪囊泡没有引起减少的鬼笔环肽染色，而随着电MIF MOS注射引起的鬼笔环肽染色时（四）显着减少。上午，前黄斑。 <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="图4" /> 图4。鬼笔环肽染色5 DPF幼虫微丝。没有注射之间的区别只（A）和注射用电击时的标准控制啉（二）。然而，与MIF morpholinos和电穿孔注射5 DPF幼虫呈减少的鬼笔环肽染色后的黄斑部（分）（四），虽然低于3 DPF戏剧性，当与MIF morpholinos（比特彗星注入胚胎相比）。上午，前黄斑。 <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="图5" /> 图5。乙酰化微管蛋白染色胚胎3 DPF。胚胎（一）未收到任何注射或电击。电穿孔（二）的胚胎，但没有收到任何注射。接受注射和电（C，D）与MIF morpholinos的胚胎微管蛋白染色明显减少，与对照组相比。 （时，后黄斑区）。 <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="图6" /> 图6。乙酰化微管蛋白染色胚胎与控制啉DPF 3。 （一）未经任何处理的胚胎表现出较强的乙酰化微管蛋白染色后的黄斑部（分）和广泛的支配。 （二）控制啉注射胚胎。 （三）处理与胚胎电只。 （四）控制与控制啉胚胎注射和电穿孔后黄斑和支配的类似乙酰化微管蛋白的水平相比，没有治疗控制。

<ol>
	<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> J Neurosci Methods. 50, 123-127 (1993).</li><li>Suzuki, M., Takamura, Y., Maeno, M., Tochinai, S., Iyaguchi, D., Tanaka, I., Nishihira, J., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+macrophage+migration+inhibitory+factor+is+essential+for+axis+formation+and+neural+development.">Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.</a> J Biol Chem. 279, 21406-21414 (2004).</li><li>Barald, K. F., Kelley, M. W., W, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+polarization:+new+tunes+in+inner+ear+development.">From placode to polarization: new tunes in inner ear development.</a> Development. 131, 4119-4135 (2004).</li></ol>

基因工具，有限责任公司生产视频和文字中提到的试剂条生产赞助。

我们已成功引入24 HPF斑马鱼胚胎的耳反义寡核苷酸分子轨道。后注射和电提出的胚胎是可行的。如果胚胎存活的电，他们在正常税率的增长相比，收到了没有治疗，以及只有被注射，只有被电穿孔胚胎的胚胎的胚胎。 我们观察到耳后注射和电MIF和MIF - MOS的影响，通过与鬼笔环肽和乙酰化的微管蛋白标签。鬼笔环肽染色后黄斑的感觉毛细胞的微丝表示，造成毛细胞和/或纤毛数字的变化，与MOS只注射的胚胎相比，MIF和MIF - 24 - HPF阶段MO电（图3）。这在毛细胞数目的减少是与Shen 等人的研究结果是一致的。（提交），MOS MIF和MIF导致毛细胞数量减少囊状黄斑。在MOS和电穿孔注射证明，电胚胎毛细胞数量减少，可能有助于在移动马鞍山穿过细胞膜，从而增加相比，奥迪囊泡只有注射的胚胎形态的影响程度。通过乙酰化微管蛋白染色中的statoacoustic神经节的形态变化进行观察。由statoacoustic神经节的神经支配程度受到影响，胚胎被注射，电穿孔显示下降的突起（图5）分支。也有可能减少凝结的神经节细胞和/或神经节细胞的数目下降，因为乙酰化微管蛋白染色的显着下降均注射和电穿孔胚胎。由于MIF已被证明是神经系统发育（Suzuki 等，2004）的关键，电后看到的变化可能增加转染的MIF和 MIF像莫入解决的神经母细胞细胞。 电穿孔直接进入一个发展中的组织的反义寡核苷酸morpholinos是一个有用的工具，为控制基因在胚胎期间该组织的发展，在空间和时间，的表达。 MIF和MIF般进入内耳组织的MO电穿孔后黄斑statoacoustic节的形态异常。有一个在的感觉毛细胞的胚胎被注入了，并已经收到了只注射或只电穿孔的胚胎不治疗或胚胎相比，电穿孔后黄斑的数量减少。还有一个statoacoustic节和凹陷的大小后的感觉补丁的支配程度的下降。电穿孔是一种大分子转成特定的组织与观察所需的组织特定的DNA和RNA，或MO的主要影响，并从其他组织的二次效应这些歧视，包括大脑，神经嵴和利益，有价值的方法内耳的发展periotic间质（Barald和凯利，2004年）。

<ol><li> MOS：0.3毫米MIF，MIF - MO1的组合， 和MIF - MO2是。 米夫莫是 ​​免费的斑马鱼MIF mRNA的起始密码子：5&#39; - acatcggcatgactgcgacagagat - 3&#39;。用于两个MOS MIF - MIF，像 MO1是免费的翻译起始站点：5&#39; - GTTTCTATATTTATGAACGGCATGA - 3“，而MIF，像 MO2从AS序列派生intron1/exon 2边界：5&#39; - GATTCATCCTCTGAAGACGTAAGCC -3&#39;。莫被控制的炒基因工具的核苷酸序列：5&#39; - CCTCTTACCTCAGTTACAATTTATA - 3&#39;。 </li><li>亚甲蓝（0.3 ppm）的鱼水</li><li> MS222（tricaine，Sigma公司;鱼水0.64毫米） </li><li>低熔点琼脂糖（LMPA;罗氏） </li><li>酚红（Sigma公司） </li><li>抗乙酰化微管蛋白（Sigma公司） </li><li>的Alexa Fluor ® 488的羊抗鼠第二抗体（分子探针） </li><li>德克萨斯红鬼笔环肽（分子探针） </li></ol>

direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development

近年来，电已成为一个流行的技术，在体内DNA，RNA和morpholinos的转成各种组织，包括斑马鱼眼，脑，体节。电超过其他的基因操纵方法的优点是，特定的组织，可以有针对性的，在空间和时间的大分子通过引入电流的应用，。在这里，我们描述了使用电穿孔转染发展的斑马鱼的内耳组织中的 MIF和MIF像morpholinos。在过去的研究中，MIF啉注射到胚胎在1 - 8 -细胞期，在广泛的形态学变化，在神经系统和眼睛，以及耳朵。针对内耳组织在开发的后期阶段，我们可以判断在发展中国家内耳MIF的主要作用，而不是次要的影响，可能会导致其他组织的影响。通过使用鬼笔环肽和乙酰化的微管蛋白染色，研究神经元，神经元突起，和毛细胞后黄斑的形态，我们能够在斑马鱼的内耳有针对性的转染的方法，以评估电穿孔的疗效。奥迪囊泡24hpf胚胎注射morpholinos和电穿孔和当时相比，已经收到任何治疗或只已注射或电穿孔胚胎。注射和电穿孔胚胎表明在毛细胞数目减少，降低statoacoustic节（SAG）和更少的凹陷与对照组相比，神经元的神经支配。我们的研究结果表明，在稍后阶段otocysts morpholinos直接传递到避免非特异性的神经系统和神经嵴的影响，在1-8细胞阶段交付morpholinos。它还允许考试定向到内耳和耳朵上的大脑，神经嵴或periotic间质细胞的主要作用不是二次效应的影响。

Watch this Scientific Journal Video about Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development at JoVE.com

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

在24hpf的斑马鱼otocyst morpholinos直接传递到一个方法来已经研制成功。管腔奥迪的囊泡和电的影响渗透到使用morpholinos显微注射，我们能够绕过morpholinos对大脑的影响，并获得具体到内耳的影响。

通过注射和电穿孔的斑马鱼Otocyst的MIF Morpholinos直接运抵影响内耳发展

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

direct-delivery-mif-morpholinos-into-zebrafish-otocyst-injection

Research

JoVE Journal

Biology

14.2K 次观看.  University of Wisconsin, Madison. 在24hpf的斑马鱼otocyst morpholinos直接传递到一个方法来已经研制成功。管腔奥迪的囊泡和电的影响渗透到使用morpholinos显微注射，我们能够绕过morpholinos对大脑的影响，并获得具体到内耳的影响。

视频: Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

科研

生物学

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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 ...

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

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

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

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

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 ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

基因转移到老年人的鸡胚<em&gt;前OVO</em&gt;电穿孔

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

针对鼠标内耳给药方法的比较研究：Bullostomy<em&gt;对战</em&gt;鼓室注射

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...