皮质的interneurons的亚型选择性电穿孔
Summary
This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.
Abstract
中枢神经系统（CNS）的成熟的研究依赖于神经元群体的遗传定位。然而，限制所关心的特定的神经元亚型的基因的表达的任务已被证明非常困难，因为特异性的启动子元件的相对稀缺。 GABAergic中间神经元构成的神经元群具有丰富的遗传和形态多样性。事实上，超过11种不同的GABAergic中间神经元亚型已被鉴定在小鼠皮质1。在这里，我们提出了一个适应协议GABA能居的选择性目标。我们通过使用同源的转录增强子元件来实现GABAergic中间神经元亚型选择性靶向因子DLX5和Dlx6，果蝇远侧少（DLL）基因2,3的同系物，通过以驱动特定基因的表达， 在子宫内的电穿孔。
Introduction
从命名的内侧和尾神经节隆起（MGE和专家咨询小组，分别）2瞬态胚胎结构的皮质GABAergic中间神经元的散装起源4。小白蛋白和生长抑素表达的interneurons起源于梅兰日兰，而钙结合蛋白（CR），Vasointestinal肽（VIP）和络丝（RE）表达的interneurons从专家咨询小组发起。这些interneuron亚型可以通过他们的出生日期来区分。梅兰日兰派生亚型胚胎9.5天（E9.5）和E16.5 5,6间出生。相比之下，CGE衍生的interneurons从出生E12.5 E18.5，通过与他们的生产高峰在E15.5 6。这么晚出生人口的遗传定位，但是，仍然遥遥无期。
鼠远侧少（DLX）基因只表达在显影腹侧前脑3。 GABAergic中间神经元及纹状体投射神经元，但不皮质锥体细胞表达Dlx1，2,5，和6个基因在早期发育阶段3。事实上，DLX基因被表达在MGE和CGE脑室下区（SVZ）内所有的GABA能祖细胞。这些基因的表达成为限制在有丝分裂后期阶段7-9选择亚型。先前的实验证据表明，DLX5 / 6增强子元件允许GABA能系在转基因小鼠模型2的选择性靶向。我们测试使用在附加型表达在发育中的小鼠大脑中的上下文这些增强子元件中的一个的。我们的子带最小启动子和在BLUESCRIPT（BS）的骨架质粒的增强型绿色荧光蛋白（EGFP）（ 图1）中克隆的DLX5 / 6增强子元件一起。我们推出的质粒在子宫内电在E15.5的方式选择性地靶向CR-，VIP和重新亚型3,8,10。我们的技术可用于稀疏电穿孔，这有利于对辛格的细胞形态特征的重建。此外，极高的水平在皮层GABA能神经元的基因表达可用于功能研究。我们进行了损失，并使用多种野生型和显性负11的基因功能研究的增益。
Protocol
Representative Results
Discussion
该技术的局限性
尽管该技术允许对细胞过程的细胞中自主分析，它是不适合于人口分析。在电穿孔是非常稀疏的不到一千细胞每大脑电穿孔。因此，该技术不能被用来评估从CGE衍生的interneurons的遗传操作而产生的行为后果。
而电穿孔进行在E13.5-E14.5目标MGE衍生的亚型，效率低10。我们推测，DLX5 / 6增强剂是在有丝分裂后的MGE亚型活性较低，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们非常感谢尹立红提供技术援助。 NVD是一个NARSAD青年研究者奖的获得者，也从美国国立卫生研究院（5 K99 MH095825-02）支持补助。研究在Fishell实验室由卫生心理健康（5 R01 MH095147-02，5 R01 MH071679-09）的神经疾病和中风（5 R01 NS081297-02研究所研究所，研究所，支持，1 P01 NS074972 -01A1）和西蒙斯基金会。
Materials
| Electroporator with pedal | Protech International | CUY21 | |
| 5mm paddle electrodes | Protech International | CUY650P5 | |
| Heating pad | Kent Scientific | DCT-15 | |
| Sutter Instruments P30 Puller | Sutter Instruments | 3282322 | |
| Fluovac Anesthesia Systems | Harvard Apparatus | 726425 | |
| Delicate Operating Scissors 4.75" Straight Sharp/Sharp | Roboz | RS-6702 | |
| 5-0 Silk Black Braid 18" C-1 Box 36 | Roboz | SUT-1073-21 | |
| Micro Clip Applying Forceps 5.5" | Roboz | RS-5410 | |
| 2 Clamp scissors | Roboz | RC-4894 | |
| Holding forceps | Fine Science Tools | 11031-15 | |
| Glass capillary tubing | FHC | 27-30-0 | Borosil 1.0mm OD x 0.75mm ID |
| Fast Green | Sigma-Aldrich | F7258 | |
| Sterile PBS | Life Technologies | 20012-027 |
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