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>

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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?...

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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

Research

JoVE Journal

Neuroscience

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

13.9K 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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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 ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...