我们感谢权限发布参考11日第130页上的最初出现的显微注射吸管制造图Humana压; Dlugas拉里和史蒂芬黄，OHSU的教育通信部，录像;拉里Dlugas为视频设计和编辑;亚当M.Ø "奎因，高级设计师，钛师傅/ Envirco公司提供的技术原理设计我们自定义的水平层流罩和Les高士指导维克多Monterroso，MV中，硕士，博士和汤姆Chatkupt的数字电压表，OHSU的比较医学部，与我们的动物保健协议，手术技巧，以及预防性镇痛方案;马塞尔·佩雷让蒂尔，数字电压表，硕士，兽医缝合技术分享他的讲义;爱德华Porsov，MS，设计我们的Adobe Premiere Pro中的视频显微镜的计算机工作站及利亚白乔纳斯LNS的欣克利字幕（波特兰，OR）。这项工作得到了耳聋和行吟从国家研究所的赠款R通讯障碍:以R01直流008595和008595-04S2直流以R01（山）和P30 DC005983（俄勒冈州听力研究中心核心格兰特，彼得·吉莱斯皮，首席研究员）。

1。腹剖腹<ol><li>麻醉的大坝，其胚胎在胚胎每天11.5（E11.5;阴道插件检测当天中午是胚胎发育0.5天），是由腹腔注射戊巴比妥钠麻醉剂溶液（每克体重7.5μL）。工作麻醉剂:50毫克/毫升巴比妥钠溶液180μL，100μL无水乙醇320μL65毫克/毫升的水硫酸镁（调节子宫张力）;丙二醇和400μL（车辆混溶与水和有机组件）。 </li><li>评估麻醉的完整性进行伤害性刺激试验:脸颊和触须触摸爪子挤压尾巴捏和眨眼回应。适用于无菌眼药膏的眼角膜。 </li><li>剃腹部的毛皮从耻骨上区的左肋用剪刀和罚款的刀片（奥斯特＃40刀片）。腹部用70％乙醇，10％的聚维酮碘（优碘），消毒ND 70％的乙醇，按顺序。广场上设置鼠标腹部面朝下，在无菌的悬垂性和加热垫或暖板为2-5分钟（37℃）。 </li><li>在腹侧中线与球头剪刀切开腹部皮肤。延长10-14毫米的切口。确定的白线 ，沿腹中线腹壁缺血，白色的结缔组织带。切开的白线与球放倒剪刀和延长切口10-14毫米。预热（37℃）乳酸林格氏液，立即灌溉腹部。 </li><li>没有植入网站上申请过大的压力，外部的双角子宫环钳。预热，乳酸林格氏液与灌溉外向的子宫角。 </li></ol> 2。 transuterine微量注射<ol><li>编造一个厚壁，具有以下特征的高硼硅玻璃毛细管内注射移液器:12-16μ米外径和20度斜角。 1萨特的P-97小方块长丝的吸管拔，使用下列程序:热量=斜坡测试加3个单位;压力= 200;拉= 0;速度= 46时间= 100）。与"萨特BV-10倒角，使用大口径移液器的104C（金）研磨盘。重悬表达质粒在3-4微克/μL无钙磷酸盐缓冲液（pH值7.2-7.4）。轻轻30秒快速绿色结晶浓缩DNA，涡，在10,000 g旋转10秒。经验的视觉跟踪注射的疗效快绿色的最低金额确定。回填与DNA /快速的绿色解决方案的斜面移液器。加载显微注射吸管连接高压注射器（Picospritzer使用&gt; 99％纯氮气源）移液器支架。 </li><li>透照与强度低，卤素灯可视化胚胎内的着床部位的子宫。确定了BEA婷的心脏，脑泡，肢芽，新生的后脑第 4脑室，和眼睛。冲洗子宫与预热，每2分钟，乳酸林格氏液维持水化。适用于对子宫的温和的压力，重新调整的胚胎，并确定上面提到的解剖标志。 </li><li>东方胚胎，以确定主头静脉，其前部和后部的分支侧面间质领土驻留在otocyst。不能看到otocyst适当的子宫透。 otocyst是位于主头静脉，沿静脉主干，形成美式橄榄球场的立柱或球门柱的形状的前部和后部的分支之间的中间。 otocyst是立柱之间的中间。 </li><li>注射针插入子宫在推定与该otocyst的位置轨迹通过。后通过影视小卡车，通过脉冲微量注射器一次我们想象的示踪染料和近似的枪头位置。推进千分尺控制下的吸管和脉冲再次评估的深度。进一步推进到外侧头间质和脉冲吸管反复。 ，成功otocyst定位将露出背部的淋巴管和扇贝前庭壳形状的锥形形状。释放压力，对子宫，取出胚胎/子宫移液器一项议案，并立即灌溉与子宫预热，乳酸林格氏液。 </li></ol> （三） 在体内电穿孔<ol><li>灌溉用乳酸林格氏液的子宫。新鲜应用乳酸林格氏液电情侣桨式电极的子宫是必要的。乳酸林格氏液的滋润钨电极表面。中心电极的路径注入otocyst。轻轻地压缩与电PA子宫ddles。阴极是在子宫壁的接触外侧注入otocyst和阳极接触子宫壁相邻的未注射otocyst的。与上electroporator的脚踏开关触发方波脉冲列车。电参数为:5，每脉冲43伏和950毫秒脉间延迟50毫秒脉冲。脉冲列车交付后，立即灌溉子宫。记录传递到组织目前的60-100毫安足够染耳上皮细胞祖细胞。注入和electroporate 4-6，E11.5胚胎每大坝。 </li><li>把这些胚胎的第 4脑室，其内耳执行第二个独立transuterine显微注射水溶液的荧光葡聚糖（的Alexa Fluor 488表达质粒表达质粒编码绿色荧光蛋白编码红色荧光蛋白或亚历克斯氟594）准确地注入至少60毫安的电流，每个脉冲是胆小的中电。荧光葡聚糖检测将是出生在后脑，使幼鼠的内耳胚胎发育过程中的操纵（见3.5）的选择。 </li><li>与乳酸林格氏液冲洗子宫角。重新插入腹腔的子宫角。预热，乳酸林格氏液2-4毫升冲洗子宫腔，并允许溢出切口部位排出到无菌的悬垂性。更换悬垂干燥，无菌材料。与非切割针和6-0可吸收缝线缝合腹壁。我们更喜欢运行的针，每针，锁定为腹壁和皮肤。 </li><li>干燥的水坝的毛皮和非甾体类消炎等作为美洛昔康皮下注射管理。返回大坝预热回收笼在无菌的悬垂性。监视和记录水坝的呼吸，切口部位的通畅，阴道血性分泌物。出血是RA重，但如果目前有增无减，安乐死的大坝，而她仍然是麻醉下。请注意她恢复意识，并尝试以走动的时间。返回的大坝鼠标殖民地时，她显示了进食和饮水的迹象，并已开始筑巢建立。通常情况下，这12小时内发生。 </li><li>出生时（出生后第0天），闪光的每个垃圾小狗的用GFP的得克萨斯州或红色滤镜1 stereofluorescence解剖显微镜检测荧光葡聚糖的后脑区域设置适当的。返回到哺乳期大坝只幼仔，显示后脑标签。 </li></ol> 4。代表结果<img src="/files/ftp_upload/3653/3653fig1.jpg" alt="图1" /> 图1。电穿孔介导的基因转移到发展中国家的耳蜗。E11.5 otocyst注射表达质粒编码增强型绿色荧光蛋白（EGFP）和电脉冲（脉冲TRA参数:五，43/50毫秒脉冲和脉间延迟950毫秒）的电压脉冲。 A）从1产后第6天（六）小狗的otocyst注射和示范基地，通过耳蜗中间转EGFP的表达，在E11.5电穿孔的有代表性的内耳。从中间转和顶点只有耳蜗侧壁被删除。 E11.5祖上升到顶点的未转。二）与毛细胞标记（一）所有安装在耳蜗免疫，肌球蛋白7A（MYO7A），表明，EGFP的表达如下Corti器的轨迹，是非常本地化的头发感觉上皮细胞息。 C）从1 E18.5胚胎的otocyst在E11.5注射和电穿孔的有代表性的内耳。激光共聚焦投影演示在MYO7A的积极的感觉毛细胞EGFP的表达。 d）激光共聚焦投影耳蜗感觉上皮从E18.5科尔蒂器官表明绿色荧光蛋白expression的内毛细胞（IHC），外毛细胞（OHC），内（IPC）phalangial的细胞，支柱细胞（PC）和Deiters细胞（DC）。 （二）适用比例尺为（A）。

<ol>
	<li>Gillespie, P. G., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+by+hair+cells:+models,+molecules,+and+mechanisms.">Mechanotransduction by hair cells: models, molecules, and mechanisms.</a> Cell. 139, 33-44 (2009).</li><li>Bok, J., Chang, W., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+and+morphogenesis+of+the+vertebrate+inner+ear.">Patterning and morphogenesis of the vertebrate inner ear.</a> Int. J. Dev. Biol. 51, 521-533 (2007).</li><li>Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell+fate+in+the+sensory+epithelia+of+the+inner+ear.">Regulation of cell fate in the sensory epithelia of the inner ear.</a> Nat. Rev. Neurosci. 7, 837-849 (2006).</li><li>Ohyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+patterning+the+sensory+and+nonsensory+regions+of+the+developing+mammalian+cochlea.">BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea.</a> J. Neurosci. 30, 15044-15051 (2010).</li><li>Pan, W., Jin, Y., Stanger, B., Kiernan, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+is+required+for+the+generation+of+hair+cells+and+supporting+cells+in+the+mammalian+inner+ear.">Notch signaling is required for the generation of hair cells and supporting cells in the mammalian inner ear.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 15798-15803 (2010).</li><li>Gubbels, S. P., Woessner, D. W., Mitchell, J. C., Ricci, A. J., Brigande, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+auditory+hair+cells+produced+in+the+mammalian+cochlea+by+in+utero+gene+transfer.">Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer.</a> Nature. 455, 537-541 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2010).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1027-1032 (2007).</li><li>Chen, C., Smye, S. W., Robinson, M. P., Evans, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electroporation+theories:+a+review.">Membrane electroporation theories: a review.</a> Med. Biol. Eng. Comput. 44, 5-14 (2006).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Brigande, J. V., Gubbels, S. P., Woessner, D. W., Jungwirth, J. J., Bresee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-mediated+gene+transfer+to+the+developing+mouse+inner+ear.">Electroporation-mediated gene transfer to the developing mouse inner ear.</a> Methods Mol. Biol. 493, 125-139 (2009).</li><li>Morsli, H., Choo, D., Ryan, A., Johnson, R., Wu, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+mouse+inner+ear+and+origin+of+its+sensory+organs.">Development of the mouse inner ear and origin of its sensory organs.</a> J. Neurosci. 18, 3327-3335 (1998).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta. Otolaryngol. , 1-77 (1971).</li><li>Sheffield, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector+tropism+for+supporting+cells+in+the+developing+murine+cochlea.">Viral vector tropism for supporting cells in the developing murine cochlea.</a> Hear Res. 277, 28-36 (2011).</li><li>Bedrosian, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery+of+recombinant+viruses+to+the+fetal+murine+cochlea:+transduction+characteristics+and+long-term+effects+on+auditory+function.">In vivo delivery of recombinant viruses to the fetal murine cochlea: transduction characteristics and long-term effects on auditory function.</a> Mol. Ther. 14, 328-335 (2006).</li><li>Reisinger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+functional+equivalence+of+otoferlin+and+synaptotagmin+1+in+exocytosis.">Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.</a> J. Neurosci. 31, 4886-4895 (2011).</li><li>Magnani, E., Bartling, L., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Gateway+to+MultiSite+Gateway+in+one+recombination+event.">From Gateway to MultiSite Gateway in one recombination event.</a> BMC Mol. Biol. 7, 46 (2006).</li><li>Perret-Gentil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Veterinary Suturing. , .</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-1000 &#38; P-97 Pipette Cookbook. , (2011).</li></ol>

没有利益冲突的声明。

发展中国家小鼠内耳的基因转移到小鼠内耳耳基板的发展过程中的发展12,13植入后的第一周。由胚胎9.5天（E9.5），基板已内陷，并演变成一个充满液体的囊泡称为otocyst 2。在泡耳前兆引起成熟内耳内感官和nonsensory的细胞以及神经元支配的前庭和听觉的感觉上皮细胞的机械敏感的毛细胞。年底前胚胎阶段，复杂的，三 ​​维的膜迷路内耳形态成立3,12。通常增益和损失功能的实验方式进行转基因小鼠，以获得进入细胞命运规范和格局的形成，如发育过程的基因调控的洞察力。发展中国家小鼠内耳的动态遗传操作宫内代表免费鼠标转基因的方法，与小鼠实验胚胎学6,11夫妇的基因转移技术。 病毒和电介导的基因转移技术已发展到操纵基因表达在发展中国家的内耳6,11,14,15。腺相关病毒（AAV）载体感染耳祖给的上升感觉和nonsensory的细胞类型，并已用于审问钙依赖小鼠毛细胞的胞吐16的分子基础。 AAV载体产生高滴度，使内耳中的广泛表达不同的病毒血清型显示独特的取向差的细胞表达，可能是有利的。缺点包括一个固定的填充能力，限制大小，可以misexpressed的基因和病毒学专业知识的要求，净化，滴度的病毒。电穿孔介导的基因TRansfer已用于评估一个基本的螺旋-环-螺旋转录因子的作用，在指定6 体内的感官毛细胞的命运。改编为Gateway亚克隆技术表达质粒允许快速和有效的载体建设，推动基因的兴趣，并从IRES序列17的荧光标记蛋白的表达与独特的发起人。感兴趣的基因，可以在24小时的电和表达，能坚持在产后的内在耳取决于所使用的启动表示在耳前体。质粒的构建，纯化，浓缩和定量要求的唯一标准的分子生物学技术。缺点包括在体内质粒〜8KB以上尺寸的增大和胚胎致死率较高，大多数病毒载体相比，转染效率下降。两电和病毒介导的基因转移技术需要掌握的小鼠实验胚胎学高科技niques:生存的手术， 在子宫内的胚胎，操纵和transuterine显微注射。这个手稿的目标是展示每一个关键的数字视频显微镜的方法步骤，以解决后者的问题。 方法论上的视频说明:我们特意选择了不悬垂性与不育领域的切口部位，同时拍摄，使观众可以看到大坝，即头（故意模糊，在大多数的大体解剖位置有关的腹侧中线帧，但辨认），四肢，尾巴。悬垂的切口部位，有效地阻止这种观点，可以混淆欣赏过程中腹的剖腹探查术及注射序列空间有关的解剖关系的努力。但是，我们建议悬垂腹部大坝，使无菌领域外向子宫角上休息，以保持手术无菌。此外，我们故意没有尝试恶魔因为这些方法必须由兽医专家的指导或合格的外科技术员了解到trate在腹壁和皮肤缝合手术切口。马塞尔·佩雷让蒂尔（德克萨斯大学圣安东尼奥），一博士提出了一种优良的讨论，本主题与图案陈述完整，在他的讲义，"兽医缝合的原则。"18 程序建议:发展中国家小鼠内耳的基因转移到在技术上具有挑战性。有一系列的最佳实践，这将消除大量的变异，并确保成功的结果。我们提出具体建议，以促进学习这种实验方法。 请教兽医人员开发的小鼠生存手术动物保健协议:哺乳动物的生存手术的动物保健协议要求生产自麻醉，镇痛，围手术期护理必须定义和协调。作为补充资料（见" <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3653/JoVE_3653_Animal_Care_Protocol_2012.pdf" target="_blank">动物保健议定书"发展</a> "）的动物保健协议的发展，包括语言，可能会形成对申请人的协议的核心，提供详细的讨论。此外，你家机构的动物护理和使用委员会（IACUC）和兽医人员无疑将提供一个合适的协议的制订，要求额外的指导。 在子宫内基因转移建立一个专用的鼠标生存手术部位:腹开腹手术是一个分类作为一个大手术，因为它损害的体腔。大多数IACUCs将不要求无菌的，一流的主要鼠标生存手术100环境。相反，一个专门的区域，包括适当的照明，通风的相关特征研，补充加热，硬，非多孔，disinfectable的表面可能会被要求。我们宁愿进行在一个水平层流罩，保护免受感染的鼠标，降低生理应激我们赖以生存的手术，从而设施术后恢复。我们已经确定了一系列自定义修改振荡阻尼，水平层流罩（Envirco公司低频630），其中包括定向，低功率卤素灯轨道照明;扩大功率基因转移设备的电子电路;和槽的电源插座与台式剪纸线通过。这些修改的一个详细的原理提供的补充资料（见" <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3653/JoVE_3653_Custom_Laminar_Flow_Hood_2012.pdf" target="_blank">层流罩在子宫内基因转移</a> "）。请咨询您的IACUC为您开发您的动物保健协议，以确定一个合适的位置，专门用于鼠标苏rvival手术。 称重管理戊巴比妥钠麻醉前，大坝到最近0.1克的:戊巴比妥钠是一种有效的麻醉剂时，建议的剂量时间表其次，将提供至少105分钟的工作时间。之前确定每个大坝的准确体重计算剂量的麻醉剂的混合物注入。不要估计大坝体重。使用流式细胞Dikison托盘的过敏注射器针头和注射器:人皮伞是理想的无创成孕鼠腹腔注射。此外，这种注射器具有极低的死空间体积和校准范围为水坝，其胚胎在E11.5或E12.5通常需要麻醉卷。 部署预防性镇痛支持胚胎生存:管理上的缝合线如局部麻醉，布比卡因，系统性，非甾体类消炎药，如美洛昔康之前，因此，止痛时，在船上放置回收笼坝坝从麻醉中恢复。在大坝减少不适，有利于术后恢复和维护一个健康的怀孕。 锥显微注射移液器和严格限制的外径:单胚胎致死的最常见的原因是组织和/或血管损伤显微注射移液器构造不良。成E11.5小鼠otocyst的transuterine显微注射法进行较大的中老年胚胎器官（即脑泡，眼，四肢等）比注射不同的约束。 unbeveled，斜面不佳，或不恰当的外直径移液器将导致在子宫内的船只，卵黄囊血管和/或在E11.5的胚胎，这将降低胚胎存活率出血。这是可能的手工小直径打破耐用移液器进行适当斜面，但它是很难经常这样做。在E11.5小鼠胚胎其微小otocyst目标，将是无情的。咨询的P-1000的P-97移液器从萨特仪器食谱2011年为全面论述对移液器的设计和制造19。在这项工作中使用的移液器参数如上所述和他们的制作已充分讨论之前11。在主人的为萨特仪器的BV-10倒角手册的程序说明也是一个极好的资源。 明白为什么误炸发生otocyst transuterine显微注射:视频呈现到E11.5和E12.5 otocyst的的transuterine显微注射的理想结果。误炸otocyst通常导致注入太肤浅，太深，或从制造不足的移液器。图2演示了这些条件。面板A显示的透射成像的otocyst成功后，快速的绿色解决方案transuterine微量注射E12.5胚胎左侧横向视图。作为一个楔形切口背的otocyst后脑（HB）出现。眼（E）有一个沿其边缘和左前肢是突出的色素环。主头静脉（PHV）的主干是下方otocyst。 otocyst是充满了快速的绿色出现黑色，背，淋巴管（ED）和前庭的otocyst（OC）就可以看出端倪。 E11.5-12.5 otocyst的的成功定位将始终呈现出清晰可辨的淋巴管和前庭。 A组，显示相同的图像中没有一个白色的标签，以方便的注入otocyst的严格审查。 <img src="/files/ftp_upload/3653/3653fig2.jpg" alt="图2" /> 图2。一个图案指南误炸所transuterine microinjec的otocystTION。所有面板显示的E12.5胚胎中脑和前脑左左侧来看，除了面板H，这是一个观点的后脑，中脑区域的背。小组在每一行代表图像相同的胚胎。看到了详细的解释otocyst误炸的原因讨论。缩写:E，左眼; ED，淋巴管; FG，绿色快，HB，后脑;磅，肢芽;业主立案法团，otocyst; OT，耳领土; P，吸管; PHV，小学头静脉角，枪头。 肤浅的注射是常见的原因是误炸。板被证明到E12.5胎体的肤浅注射。后脑（HB）缺口和耳领土（OT，圆）是在左，侧视可见一斑。显微注射移液器（P）是在前台。快速绿色（FG）开始枪头（二）从弹出，并与随后的脉冲高压注射器（光盘版），染料扩散成椭圆形。日分布é染料表明，它推出的子宫壁和卵黄囊，胚外膜，笼罩在exocoelomic的腔之间。实验者不应试图重新定位在这个胚胎otocyst，但应该移动到下一个胚胎。多个注射关联与胚胎存活率下降，效果是比E12.5，E11.5急性。 深注射是较为常见的原因是误炸。板跳频表明，通过传递到后脑耳领土注射轨迹。主头静脉（PHV），左眼（E），移液器（P）是显而易见的，在这侧视的E12.5胚胎。快绿（FG）的初始弹射流离失所背面主头静脉/耳领土。与后续脉冲，楔形后脑（HB）是完全充满了快速绿色（小组G）的。一个90度旋转，注射胚胎允许快速绿色的检测范围内的hindbra从背视图（面板H）腔。既浅表和深部注射显微注射吸管的深度与子宫接触后无法感知的结果。行之有效的一种实用的方法是通过子宫前进的吸管，然后立即脉动一次寻找一个快速绿色粉扑粉扑的位置表示枪头的大致位置。基于此范围内发现的脉冲调整的深度。 不足捏造的移液器是误炸的最常见的原因。 IK的面板中显示注射尝试进行手动打破，但没有斜面与枪头。 E12.5胚胎的横向视图显示后脑（HB），左眼（E），和微量注射移液器（P）。请注意，枪头（PT）是大幅鞠躬，并没有穿透子宫（面板）。应用附加力剪吸管的尖端面板J，（箭头），离开点子嵌入子宫ETTE尖。从裂缝吸管少量的快速绿色（FG）被驱逐出场，并发现子宫表面。嵌入式枪头（面板K）必须拆除，罚款，无菌镊子和尖锐废弃物丢弃。如果枪头可以不设或删除成功，安乐死麻醉下坝。 老鼠生存的手术数据表记录程序的意见:学习这些方法的一个关键因素是记录意见，并根据这些意见调整。生产鼠标生存手术数据表，并注意术前，手术和术后的信息。术前的数据包括大坝重量，剂量麻醉药交付，质粒注射等手术数据，包括胚胎注入（即R2是从第二胚胎卵巢在右侧子宫角），注射液的质量（即，没有淋巴管道检测，染料4 <s&gt;日脑室）的当前，中电（即60mAmps，目前的心跳后，电），整体印象（即交付，移液器过于灵活和吸管的轨迹是难以维持）。术后的数据包括下床活动的时间是第一次尝试，切口部位的通畅，在场的阴道出血，外观修饰行为和筑巢等生产生存手术数据表模板中提供的补充资料（见" <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3653/JoVE_3653_Surv_Surg_Data_Sheet_2012.doc">外科鼠标生存资料表</a> "）。质量说明的基础上形成的生产方法的改进，这将导致成功的结果。 使用目前的每脉冲传递到优化转染效率:5组成的方波脉冲串，43volt，50毫秒脉冲与950msec间脉冲延迟。 5mm的铂，桨式电极确保整个耳领土内电场中。类似的转染效率可达到附3mm桨，但他们更难以准确定位。评估转移胚胎在最后的脉冲电流脉冲范式进行了优化，而不是由席卷电压和评估转染效率。理由是，交付每脉冲电流，恒定电压取决于子宫和铂电极之间的电耦合通畅不同。假设子宫新鲜灌溉乳酸林格氏液之前，立即把桨和电荷载体是过剩的，行政的变量是"耦合"桨子宫压力量。充其量电流和零星染&lt;60mAmps 43volts结果温和的压力。适度的压力在43VOLTS结果在60-100mAmps的交付和最有效的转染。 43volts结果，在沉重的压力100mAmps交付和关联坚持改变心脏率和增加胚胎致死。压力过大破裂的卵黄囊（可通过桨感到"砰砰"），并导致胚胎致死。它是可能的变量施加的压力，改变了电极之间的阻抗影响目前交付。脉冲频率为1Hz（50毫秒脉冲每秒）被定义为至少影响胚胎的心脏周期，这是一个积极的胚胎存活相关。我们的结论是最关键的参数，监测建立一个有效的在体内电范式是目前交付的每个脉冲。任何脉冲范式的修改应包括每脉冲当前仔细监测。 采用模块化的方法来学习日é技术:模块1:实践与子宫的外假手术，但不注入或electroporate。水坝容忍腹的剖腹探查术非常好，应该永远不会遇到有关手术方法的术后并发症。老鼠生存的手术技巧无疑是居住在家里机构，所以追求足够的训练。实现掌握手术技巧，然后再进行。模块2:实践到otocyst transuterine显微注射麻醉大坝:一个没有完成的生存手术的期望安乐死麻醉下坝，隔离注入胚胎，并评估的otocyst注射质量。模块3:目标只有2在2个不同的胚胎快速绿色otocysts，不electroporate了，完成了手术，并验证下游胚胎生存。模块4:目标只有2个胚胎的DNA /快速的绿色解决方案，electroporate，验证下游胚胎生存，并确定转EFFICIENCY。模块5:目标只有2个胚胎，DNA /绿色的解决方案快，electroporate，注入4 次的心室标签操纵胚胎的Alexa Fluor，选择在P0标记的胚胎，并确定转染效率。 程序的学习曲线:鼠标生存手术技术的掌握，从一个人的家机构的兽医和手术人员适当的指导，是相当快的，应该只需要3-5水坝新手获得专业知识的实践价值。 transuterine显微注射和体内电穿孔产生有益的转内耳将需要10-50水坝价值的努力，假设有6可行的胚胎和模块化的方法，每次怀孕其次。享受需要精细运动技能的活动（即，演奏乐器的学生们，缝纫，模型制作，或竞技运动），缩短了训练周期比寿本身不。成功掌握这些方法的关键相关细节（即显微注射吸管制造，血管解剖，明确说明，录取）和一个合理的神态自若的一丝不苟。 工作流程:一旦专业知识的不断积累，可以预见将有4-6％坝胚胎; 3-5胚胎生存; 2-4胚胎注射和电穿孔有效地进行分析转染内耳。后脑的Alexa Fluor标记技术，为出生时的排序标签胚胎需要额外的后脑射出，在每一个电穿孔胚胎，但是这是很好的耐受性，并不会产生不利影响程序的效率。因此，有效地从体内电穿孔转染的内耳的回收率相比，毫不逊色的回收率从heterogygous育种范例纯合子突变小鼠。随着经验的积累，每天2-3次，每次实验者水坝是一个合理的工作流量:1.5小时每晚手术大坝和1.5-2小时的手术后监测。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>微消毒箱</td><td> ROBOZ </td><td> RS-9900a </td><td> 8X8.5X1.25英寸</td></tr><tr><td>球尖剪刀</td><td>罚款科学工具</td><td> 14109-09 </td><td></td></tr><tr><td>环钳</td><td>罚款科学工具</td><td> 11106-09 </td><td> 4.8毫米ID/6mm外径</td></tr><tr><td> adson组织钳</td><td>罚款科学工具</td><td> 11027-12 </td><td></td></tr><tr><td>针驱动程序</td><td>罚款科学工具</td><td> 12502-12 </td><td></td></tr><tr><td>过敏注射器托盘</td><td>流式细胞Dickison </td><td> 305536 </td><td></td></tr><tr><td>缝合6-0 </td><td> Syneture </td><td> GL-889 </td><td> 0.7公吨胃肠缝合</td></tr><tr><td>乳酸林格氏注射液USP </td><td>巴克斯特</td><td> 2B2323 </td><td></td></tr><tr><td>快绿</td><td> Sigma Aldrich公司</td><td> F7258 </td><td></td></tr><tr><td>硼硅玻璃毛细管</td><td>哈佛仪器</td><td> 30-0053 </td><td></td></tr><tr><td>戊巴比妥钠溶液</td><td> OVATION制药公司</td><td> NDC的67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td> Fisher Scientific则</td><td> M63-500 </td><td></td></tr><tr><td>丙二醇</td><td> Fisher Scientific则</td><td> P355-1 </td><td></td></tr><tr><td>乙醇</td><td> Sigma Aldrich公司</td><td> E7023-500 </td><td></td></tr><tr><td>美洛昔康</td><td>勃林格Ingeheim </td><td> NADA的141-219 </td><td></td></tr><tr><td>微量普勒</td><td>萨特仪器</td><td>的P-97 </td><td> FB255B框灯丝咨询来自萨特文书的食谱移液器</td></tr><tr><td>微电极倒角</td><td>萨特仪器</td><td> BV-10 </td><td> 104C大型移液器斜边盘;咨询倒角理论的使用说明书</td></tr><tr><td>微量持有人</td><td>华纳仪器</td><td> MP-S15T </td><td>外径为1.5mm移液器和女性Picospritzer油管压力端口。 </td></tr><tr><td>镊子，风格电极</td><td>保得国际股份有限公司</td><td> CUY650P5 </td><td> 5毫米外径</td></tr><tr><td>方波Electroporator </td><td>保得国际股份有限公司</td><td> CUY21EDIT </td><td>脚踏板推荐</td></tr><tr><td> PICOSPRITZER三</td><td>派克汉尼汾</td><td> 051-0500-900 </td><td>脚踏板推荐</td></tr><tr><td>手动控制的微操作机器人</td><td>哈佛仪器</td><td> 640056 </td><td></td></tr><tr><td>水平层流洁净工作台</td><td> Envirco公司</td><td></td><td>低频630-10554自定义修改。见罩原理的补充资料。 </td></tr><tr><td>徕卡stereofluorescence解剖与Lumencor索拉光引擎microcope的</td><td> Bartels和Stout和Lumencor </td><td> MZ10F与Lumencor索拉光引擎</td><td>建议是footpedals，集中MZ10F触发索拉光引擎</td></tr><tr><td>的Alexa Fluor 594葡聚糖</td><td> Invitrogen公司</td><td> D22913 </td><td> 10mg/ml的，水</td></tr><tr><td>的Alexa Fluor 488葡聚糖</td><td> Invitrogen公司</td><td> D22910 </td><td> 10mg/ml的，水</td></tr><tr><td>环保，DRI </td><td>牧羊人专业子宫颈抹片ERS </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

哺乳动物的内耳有6个不同的感觉上皮:3个半规管壶腹嵴，椭圆囊和球囊斑;科尔蒂在连续耳蜗器官。包含前庭毛细胞转导机械刺激到...有特殊意义的平衡，而在Corti器的听觉毛细胞是听觉1主传感器的嵴和斑疹。这些感觉上皮细胞的半规管和耳蜗形态的命运规范期间在小鼠妊娠的第二个星期的地方，主要是在出生前2,3完成。小鼠内耳发育研究的收获转基因胚胎在不同的胚胎或产后阶段，要进入细胞和/或4,5形态表型的分子基础的洞察力，要定期进行。我们推测这种基因转移到发展中国家鼠标在子宫内的内耳&lt;/ EM代表&gt;中的收益和亏损的功能研究方面免费的方法，以传统的鼠标转基因哺乳动物的内耳发育6背后的遗传机制的审讯。 这里展示的实验范式进行基因在小鼠内耳的错误表达研究解决分为三个一般步骤:1）腹开腹手术; 2）transuterine的显微注射; 3） 在体内电穿孔。腹的剖腹手术是老鼠生存的手术技术，允许子宫外化，获得实验获得的植入胚胎7。 transuterine显微注射法是利用斜面，玻璃毛细管微电极引入进入管腔耳泡或otocyst表达质粒在体内的电是方波，直接驶入祖细胞8-10表达质粒的电流脉冲应用。 11日的每一步，包括详细的笔记。小鼠实验胚胎学的技术可能难以学习的散文和静止图像单独。在目前的工作中，我们证明在基因转移过程中的3个步骤。最关键的是，我们部署数字视频显微镜，如何准确显示:1）确定胚胎在子宫内方向; 2）重新调整针对注射的otocyst，胚胎; 3）microinject示踪染料溶液到otocyst混合胚胎11.5天的DNA 12.5; 4）electroporate注入otocyst;和5）产后选择出生在标签电穿孔胚胎。我们提供有代表性的例子，成功转染内耳;图案指南otocyst误炸最常见的原因，讨论如何避免常见的方法错误;写在宫内 Ğ本准则烯转移动物保健协议。

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

在小鼠内耳是一个基板派生的感觉器官，在妊娠期间阐述其发展计划。我们定义一个<em&gt;在子宫内</em&gt;转基因技术包括三个步骤:鼠标腹剖腹，transuterine显微注射，<em&gt;在体内</em&gt;电。我们用数字视频显微镜演示实验胚胎学技术关键。

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

14.0K 次观看.  Oregon Health & Science University. 在小鼠内耳是一个基板派生的感觉器官，在妊娠期间阐述其发展计划。我们定义一个在子宫内转基因技术包括三个步骤:鼠标腹剖腹，transuterine显微注射，在体内电。我们用数字视频显微镜演示实验胚胎学技术关键。

视频: Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

生物学

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

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

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 ...

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

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

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

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 ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

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

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

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 ...

转基因小鼠内耳的发展

摘要

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此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

基因转移到鸡听觉器官通过

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

小鼠视网膜神经节细胞的转染在体内电穿孔

在发展中国家小鼠视网膜体内电穿孔

在子宫内电穿孔:鼠标控制时空基因Transefection

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

在蛋和卵外研究中研究鸟类内耳发育的方法

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

转基因小鼠内耳的发展

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

基因转移到鸡听觉器官通过

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

小鼠视网膜神经节细胞的转染在体内电穿孔

在发展中国家小鼠视网膜体内电穿孔

在子宫内电穿孔:鼠标控制时空基因Transefection

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

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

在蛋和卵外研究中研究鸟类内耳发育的方法