转基因小鼠内耳的发展<em>在体内</em>电穿孔
Summary
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>在子宫内</em>转基因技术包括三个步骤：鼠标腹剖腹，transuterine显微注射，<em>在体内</em>电。我们用数字视频显微镜演示实验胚胎学技术关键。
Abstract
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</ EM代表>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。
<p cl屁股=的“jove_content”我们先前所描述的此电为基础的基因转移技术的协议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。Protocol
1。腹剖腹麻醉的大坝，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）。 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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Micro Sterilizing Case | ROBOZ | RS-9900a | 8X8.5X1.25 inches |
| Ball-tipped scissors | Fine Science Tools | 14109-09 | |
| Ring forceps | Fine Science Tools | 11106-09 | 4.8mm ID/6mm OD |
| Adson Tissue Forceps | Fine Science Tools | 11027-12 | |
| Needle driver | Fine Science Tools | 12502-12 | |
| Allergy Syringe Tray | Becton Dickison | 305536 | |
| Suture 6-0 | Syneture | GL-889 | 0.7 metric gastrointestinal suture |
| Lactated Ringer’s Injection USP | Baxter | 2B2323 | |
| Fast green | Sigma Aldrich | F7258 | |
| Borosilicate glass capillary | Harvard Apparatus | 30-0053 | |
| Nembutal Sodium Solution | OVATION Pharmaceuticals Inc. | NDC 67386-501-52 | |
| MgSO4.7H2O | Fisher Scientific | M63-500 | |
| Propylene glycol | Fisher Scientific | P355-1 | |
| Ethanol | Sigma Aldrich | E7023-500 | |
| Meloxicam | Boehringer Ingeheim | NADA 141-219 | |
| Micropipette Puller | Sutter Instruments | P-97 | FB255B box filament; consult Pipette Cookbook from Sutter instruments |
| Microelectrode Beveler | Sutter Instruments | BV-10 | 104C beveling disk for large pipettes; consult owner’s manual for beveling theory |
| Micropipette holder | Warner Instruments | MP-S15T | For 1.5mm outer diameter pipette and female pressure port for Picospritzer tubing. |
| Tweezers-style electrode | Protech International Inc. | CUY650P5 | 5 mm outer diameter |
| Square Wave Electroporator | Protech International Inc. | CUY21EDIT | Footpedal recommended |
| PICOSPRITZER III | Parker Hannifin | 051-0500-900 | Footpedal recommended |
| Manual Control Micromanipulator | Harvard Apparatus | 640056 | |
| Horizontal laminar flow clean bench | Envirco | Custom modifications to LF 630-10554. See supplementary information for hood schematic. | |
| Leica stereofluorescence dissecting microcope with Lumencor SOLA light engine | Bartels and Stout and Lumencor | MZ10F with Lumencor SOLA light engine | Footpedals to focus the MZ10F and to trigger the SOLA light engine are recommended |
| Alexa Fluor 594 Dextran | Invitrogen | D22913 | 10mg/ml, aqueous |
| Alexa Fluor 488 Dextran | Invitrogen | D22910 | 10mg/ml, aqueous |
| Enviro-dri | Shepherd Specialty Papers | www.ssponline.com |
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Tags
Gene TransferDeveloping Mouse Inner EarIn Vivo ElectroporationSensory EpitheliaCristaeMaculaeOrgan Of CortiVestibular Hair CellsAuditory Hair CellsCell Fate SpecificationMorphogenesisGestationTransgenic EmbryosMolecular BasisGain-of-function StudiesLoss-of-function StudiesGene Misexpression StudiesVentral LaparotomyTransuterine Microinjection
Citar este artigo
Wang, L., Jiang, H., Brigande, J. V. Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation. J. Vis. Exp. (64), e3653, doi:10.3791/3653 (2012).