Name: surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane
Uploaded: 2015-03-16T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

注:所有的程序和动物处理符合NIH道德准则和加州大学的机构动物护理和使用委员会，旧金山批准的协议要求。 1.准备动物外科手术<ol><li>开展外科手术在干净的专用空间。高压灭菌所有的手术器械，消毒与手术前玻璃珠灭菌。 注:在这个协议中，使用日龄10-12（P10-12）FVB小鼠。不同年龄和小鼠的菌株可用于满足特定项目的需要。年纪比P40小鼠是具有挑战性的，因为泡骨头变得更难。 </li><li>麻醉小鼠腹膜内注射盐酸氯胺酮（100毫克/千克），盐酸赛拉嗪（10毫克/千克），以及乙酰丙嗪（2mg / kg的）的混合物。开始外科准备后，才动物不再响应疼痛刺激，如脚趾捏。如果necessarY，施用麻醉剂鸡尾酒的加强剂量（五分之一原剂量），以恢复原来的麻醉平面。 注:请注意在这一步，因为大多数在这个年龄观察到的死亡率是由麻醉剂过量。 </li><li>涵盖了动物的眼睛有保护眼药膏在麻醉过程中，保持眼睛湿润，抑制动物的眨眼反射。 </li><li>将鼠标与颈部伸展在整个手术过程中的加热垫，直到鼠标完全清醒，防止麻醉后体温过低。 </li><li>剃左侧耳后区域与推剪和消毒用70％乙醇和碘伏手术操作之前。 </li></ol> 2.手术过程和载体注射<ol><li>使用耳后的办法，以暴露鼓泡。切开小剪刀皮下组织以露出耳后肌肉。缩回后脂肪组织为th切口电子后侧，分离的肌肉向右侧和左侧垂直于切口以暴露颞骨。确保此切口足够长以提供足够的可视舒适地工作。 </li><li>穿孔鼓室泡与地下25针和通过剥离骨背面以允许接近耳蜗的底转扩大孔根据需要用钳子，然后扩大到足以显现镫骨动脉和圆窗膜（RWM） 。 </li><li>轻轻刺破RWM在中心用硼硅毛细吸管。观察通过RWM在这一点上的流体流出;这是正常的。等到流出稳定（从耳蜗effluxed流体用无菌滤纸干燥）。 注:小心保持和推进玻璃针，使得RWM穿孔是尽可能小时。根据所使用的显微镜​​，持有使用显微操作或手工移液器用吸管持有人。 </li><li>准备使用移液管拉拔器相同的方式膜片钳移液器制备的移液器用于注射。确保该顶端直径较大（大约15微米直径），以便吸取病毒和缩小是很容易做到。 </li><li>制定1〜2微升AAV1-VGLUT3，AAV1-GFP或AAV2-GFP的到注射吸管。后的流出被稳定（5至10分钟），microinject这个固定体积入鼓阶通过同一孔先前在RWM制成。 </li><li>拉出吸管与一个小插件肌肉后迅速密封该RW利基并与一小滴组织粘合剂放置在肌肉，以避免从圆窗泄漏固定。 注意:密封该孔很好针后都与一个小插件肌肉的删除，以防止从耳蜗-此步骤的任何外淋巴泄漏是非常关键的保护听力。未能完全密封的RWM会导致听力下降一段时间。 </li><li>注射后，覆盖孔在听泡与脂肪组织，返回耳后的肌肉和脂肪组织到它们的正常位置，并缝合在层中的卷绕有6-0或更小的可吸收铬酸缝合。 </li><li>消毒用碘伏伤口。 </li></ol> 3.术后护理<ol><li>小鼠放置在一个温暖的笼子里，不随意放置，直到他们完全康复然后再回到与母亲。建议把幼崽回来的母亲之前，请从笼子里的男性。 </li><li>施用皮下卡洛芬（2毫克/千克）对镇痛术后且每隔24小时后3天，以管理炎症和疼痛。每天监测动物遇险，异常消瘦，疼痛，或感染的迹象。通常由手术所有小鼠通常应作用后的第3天。如果遇险或疾病的任何迹象的第3天之后，出现在一个鼠标，考虑安乐死吨他鼠标按机构的指导方针。 </li></ol>耳蜗功能4.评估继病毒输送用听性脑干反应（ABR）录音注:听性脑干反应（ABR）是一个听觉诱发电位经由头皮下放置电极从大脑中的正在进行的电活动中提取和记录。将动物刺激的声音。将所得记录包括五个波反映在听觉通道的连续点中的第一个10毫秒的电活动开始的听觉刺激后。 <ol><li>如在该协议的第1.2节中所述，除了没有乙酰丙嗪麻醉小鼠腹腔注射盐酸氯胺酮和盐酸赛拉嗪的混合物。 </li><li>把鼠标放在整个听力测试一个加热垫，直到鼠标完全清醒，防止麻醉后体温过低。 注:使用下面的声音刺激在这个实验中:点击（5毫秒的持续时间; 31赫兹展示率）。 </li><li>放置皮下针状电极在顶点，左耳（参考）的耳廓的下方，下方的对侧耳朵（地）和如先前在一个隔音室27,28描述开始记录的ABR。记录ABR波形，以5 dB声压级间隔时间从最大幅度下降。 </li><li>确定ABR阈病毒分娩后术后早在4天 注:阈值被定义为最低的刺激水平，在该响应的波峰值的I-V显然并目测反复出现。 </li><li>测量波我来分析活动从耳蜗神经。能产生可检测的ABR波形的最低的刺激电平被定义为阈值。 </li></ol> 5.转基因耳蜗蛋白表达使用免疫<ol><li>通过intrap的过量麻醉小鼠eritoneal注射盐酸氯胺酮和盐酸赛拉嗪的混合物在本协议第1.2描述，除了没有乙酰丙嗪。 </li><li>斩首后只动物不再响应疼痛刺激，如脚趾捏的头部。 </li><li>解剖耳蜗出来，过程整个安装免疫所描述的26。耳蜗全挂载免疫荧光使用抗VGLUT3和抗肌球蛋白抗体7A的详细协议阿基洛夫等人 ，到2013年29说明。 </li><li>的GFP标记，过夜孵育耳蜗全坐骑在4℃下用兔抗GFP抗体以1:250的稀释。 </li><li>冲洗耳蜗两次，每次10分钟，用PBS再孵育2小时在山羊抗兔IgG缀合的Cy2稀释1:4000于PBS中。 </li><li>冲洗用PBS耳蜗两次，每次10分钟，并孵育它们用DAPI 15分钟。 </li><li>安装在载玻片上的耳蜗，观察UND呃用免疫荧光共焦显微镜。 </li></ol>

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H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Functional+and+Structural+Outcome+of+Inner+Ear+Gene+Transfer+via+the+Vestibular+and+Cochlear+Fluids+in+Mice.">The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice.</a> Mol. Ther. 4 (6), 575-585 (2001).</li><li>Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., Mhatre, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+reporter+for+gene+transfer+studies+in+the+cochlea.">Green fluorescent protein as a reporter for gene transfer studies in the cochlea.</a> Hear Res. 114 (1-2), 139-147 (1997).</li><li>Lalwani, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

在这项工作中，我们详细地描述了可用于耳蜗基因疗法，以恢复或抢救是受遗传缺陷损害正常听觉功能的目标的技术。因为它通常是无创伤，这种方法是安全的耳蜗的基因转移或其他潜在的分子疗法30。其他的方法对于耳蜗疗法已被描述，其中包括一个腹侧方法24，在小鼠和豚鼠内耳开窗31,32和内淋巴囊递送33。在我们的经验中，耳后的方法是更快，更简单，并具有...

基因治疗一直被建议作为对遗传性听力损失的潜在的治疗，但在这方面的成功一直难以实现1。迄今为止，病毒介导的方法学都占优势由于相对难以接近耳蜗内靶向特定细胞类型的理论能力。两个腺病毒（AV）和腺相关病毒（AAV）也已用于耳蜗基因递送。自动增值服务是在耳蜗为若干原因是有利的。它们是复制缺陷型病毒，并且可以有效的转基因的分子转移到不同类型的细胞，包括神经元，为一些听力损失的原因的一个重要目标。 AAV进入细胞是通过特异性受体介导2;因此，特定的血清型的选择必须是兼容的细胞类型，以被转导。自动增值服务可以有效地转染毛细胞3和掺入到宿主基因组中，导致稳定的，长期的TRA的表达nsgenic蛋白和该单元4的表型变化。而对于短期应用，例如毛细胞再生未必是有利的，长期表达是遗传缺陷稳定救援非常重要的。因为自动增值服务不与任何人疾病或感染相关的，并证明无耳毒性5,6,7，它们是在基因治疗中的用途为听力损失8的遗传形式的理想候选。 转移外源遗传物质导入用病毒载体的哺乳动物内耳已经研究在过去十年中，并成为一个有前途的技术，用于治疗遗传和获得性形式的听力损失9。耳蜗是潜在的基因治疗有几个原因的理想目标:1）它的体积小，就必须在有限的所需要的病毒量; 2）从它的其他器官系统的限制副作用相对隔离;及3）其流体填充腔促进病毒交付整个迷宫10，11,12,13,14，15。 先天性耳聋的小鼠模型允许使用的研究方法很多监测系统的，可复制的方式内耳的发展。虽然鼠标耳蜗的小尺寸确实存在一些手术难度，鼠标作为遗传性听力损失的研究具有极其重要的模式，有几个实验优于其他品种16。小鼠模型允许范围内通过遗传连锁分析，收集了详细的形态学观察，并模拟情景致病特点的评估;正因为如此，它们是很好的候选病毒介导的基因治疗。在小鼠中结合的技术进步的广泛的遗传研究已经使得有可能产生转基因小鼠在整个实验室17,18，19，20,21的可重现方式。 FurthermorE，有两个获得性和遗传听力损失表型小鼠，使严格的测试，在这个动物模型22，23,24存在众多的车型。因此，校正听力使用病毒介导的基因治疗在小鼠模型是在寻求一种治疗人类疾病的适当的第一步骤。 之前我们已经表明，缺乏的水泡谷氨酸转运体3（VGLUT3）转基因小鼠天生聋哑，由于缺乏谷氨酸释放的IHC带状突触25。由于此突变不导致感觉毛细胞的初级变性，这些突变小鼠有潜在其中测试耳蜗基因治疗先天性听力损失的优良模型。 迄今为止，一些病毒递送技术耳蜗基因治疗已被描述，包括圆窗膜扩散，圆窗膜注射，并且经由内耳开窗递送。有强大的IAL优点和每种方法9的缺点。 这里，我们报告为通过圆窗膜（RWM）病毒介导的基因递送到VGLUT3 KO小鼠内耳的外科方法。在耳后RWM注射方法是微创具有优良的听证会保留，并且是比较快的。如我们先前发表的，在为了恢复听力在该小鼠模型中，源自于AAV2载体携带VGLUT3基因（AAV1-VGLUT3）引入这些耳聋小鼠的耳蜗在出生后第12天（P！@），导致听26的恢复。听力在VGLUT3 KO小鼠经听性脑干反应（ABR）验证，而转基因蛋白的表达用免疫荧光（IF）的验证。这种方法因而表明病毒介导的基因疗法可以纠正一个遗传缺陷否则将导致耳聋。

<table><tbody><tr><td>Ketamine</td><td>Butler Schein</td><td></td><td></td></tr><tr><td>Xylazine</td><td>AnaSed</td><td></td><td></td></tr><tr><td>Acepromazine</td><td>Provided by UCSF LARC</td><td></td><td></td></tr><tr><td>Carprofen analgesia</td><td>Provided by UCSF LARC</td><td></td><td></td></tr><tr><td>Betadine</td><td>Betadine Puredue Pharma</td><td></td><td></td></tr><tr><td>dexamethasone ophthalmic ointment (TobraDex)</td><td>Alcon</td><td></td><td></td></tr><tr><td>Heating pad</td><td>Braintree scientific, inc.</td><td></td><td></td></tr><tr><td>25G needle</td><td>BD</td><td>305127</td><td></td></tr><tr><td>Borosilicate capillary pipette</td><td>World precision instruments, inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Suture PDS*plus Antibacterial</td><td>Ethicon</td><td>PDP149</td><td></td></tr><tr><td>Tissue glue (Vetcode)</td><td>Butler Schein</td><td>31477</td><td></td></tr><tr><td>Rabbit Anti-GFP antibody</td><td>Invitrogen</td><td>A11122</td><td></td></tr><tr><td>Dissecting microscope&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Leica</td><td></td><td>MZ95</td></tr><tr><td>Flaming/ Brown Micropipette&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sutter Instrument Co</td><td></td><td></td></tr><tr><td>Puller Model P-97&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>TDT BioSig III System&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;Tucker-Davis Technologies</td><td></td><td></td></tr></tbody></table>

surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

基因疗法，用于实现由感觉神经性耳聋功能恢复，承诺给予更深入的了解，有助于听力损失潜在的分子和遗传机制。导入载体的进入内耳必须以一种方式在整个耳蜗损伤，同时最大限度地降低到现有结构即广泛分布的试剂来完成。这个手稿描述耳后的手术的方法，可以使用分子，药物，和病毒递送至小鼠10日龄和老年人通过圆窗膜（RWM）用于小鼠耳蜗疗法。这种手术方式，可以快速地直接供货到鼓阶，同时尽量减少失血，避免动物的死亡率。此技术涉及到内和中耳的基本的结构以及颈部肌肉，同时完全保留听力忽略的或没有损害。为了证明这一点的手术技术，囊泡glutam的功效吃转运3敲除（VGLUT3 KO）小鼠将被用作先天性耳聋，它回收输送VGLUT3到内耳后使用腺相关病毒（AAV-1）听力小鼠模型的一个例子。

为了验证该技术的特点和对耳蜗分子治疗耳后的方法实用，AAV1-VGLUT3，AAV1-GFP和AAV2-GFP分别通过RWM送入P10-12小鼠内耳。该方法证明了内毛细胞内成功的转基因表达（IHC）（VGLUT3 图1和GFP 图2和GFP 图3A），外毛细胞（OHC）（GFP 图2）和支持细胞（GFP 图2和图3A）26没有尔蒂伤害显著的器官。表达GFP不同细胞类型的观察看?...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.4K 次观看.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

视频: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

遗传学

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 ...

神经科学

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

医学

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

一种非人类灵长类常见狨中内耳药物的外科手术方法 (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

后半圆管法在新生小鼠内耳基因传递中的研究

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

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. ...

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

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