在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集
Summary
在子宫内电转染神经祖细胞在体内的一个有价值的方法。根据电极和电发育时间点的位置，才能有的放矢。皮层细胞的某些子集靶细胞，然后可以在体内或体外基因改变的影响进行分析。
Abstract
在初级神经元文化的体外研究允许突起生长的定量分析。为了研究遗传变异如何影响神经过程的产物，或shRNA的基因结构，可以通过化学转染或病毒转导的主要神经元介绍。然而，初级皮层细胞的细胞类型（从不同层次，抑制性神经元，神经胶质细胞的谷氨酸能神经元）的异构池是转使用这些方法。引进胚胎鼠皮质DNA的结构，允许使用在子宫内电靶细胞的某些子集，而早期胚胎皮层目标的皮质深层电，在后期的胚胎时间点电的目标更多的表面层。此外，差分跨越个别胚胎在背内侧与腹外侧皮层区域的定位结果头上的电极位置。电，转染的细胞可以被解剖，分离，镀在突起生长的体外定量分析。在这里，我们提供了一步一步的方法，定量测量皮层细胞亚群的神经过程的产物。
电在子宫内的基本协议已经Kriegstein 实验室1，2两个其他的Jove文章中详细介绍了。我们将提供我们的协议概述在子宫内电，专注于最重要的细节，说明我们的协议，适用于研究基因功能的神经过程的产物，在子宫内电。
Protocol
在子宫内电的基本协议已经详细描述了在另一个1，2 Kriegstein实验室的朱庇特的文章。这种技术最初是在大隅实验室3和描述我们的协议是基于后在LoTurco 实验室 4发达的地区之一。我们将为您提供概述大鼠胚胎在子宫内电“我们的协议，专注于最重要的细节，说明了我们的协议， 在子宫内电适用于研究基因功能的神经过程的产物。 <…
Discussion
允许初级神经元文化的体外研究的突起生长的定量分析。为了研究遗传变异如何影响神经过程的产物，通过化学转染或病毒转导的主要神经元的shRNA或错误表达结构可以引入。然而，初级皮层细胞的细胞类型（从不同层次，抑制性神经元，神经胶质细胞的谷氨酸能神经元）的异构池是转使用这些方法。引进胚胎鼠皮质DNA的结构，允许使用在子宫内电靶细胞的某些子集，而早期胚胎皮?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者想感谢约瑟夫LoTurco和丹尼斯Selkoe这种技术的有益的讨论。作者感谢美国健康援助基金会的捐助者，为支持这项研究。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Cortical Neuron Preparation | ||||
| Dissection Media: | ||||
| 10X Hanks’ Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free) | Gibco | 14185-052 | ||
| 10X Hanks’ Balanced Salt Solution (HBSS) (with Ca+2 /Mg +2 ) | Gibco | 14065-056 | ||
| 1M HEPES pH 7.4 | Gibco | 15630-080 | ||
| Dishes and Vials: | ||||
| 100 x 15 mm Petri Dishes | Fisherbrand | 08-757-12 | ||
| 60 x 15 mm Petri Dishes | BD Falcon | 351007 | ||
| 15 mL conical vial | Sarstedt | 62-547-205 | ||
| 50 mL conical vial | Sarstedt | 62-554-205 | ||
| Dissection Tools: | ||||
| Scissors | Fine Science Tools | 91402-12 | ||
| Standard Forceps | Fine Science Tools | 11000-12 | ||
| Curved Forceps | Fine Science Tools | 11273-20 | ||
| Fine Forceps | Fine Science Tools | 11255-20 | ||
| Vannas spring scissors | Fine Science Tools | 15000-00 | ||
| Miscellaneous: | ||||
| .25% Trypsin-EDTA | Gibco | 25200 | ||
| Reichert Bright-Line Hemacytometer | Hausser Scientific | 1490 | ||
| Hand-Held Tally Counter | Sigma | Z169021 | ||
| Plating Medium: | ||||
| Dulbecco’s Modified Eagle Medium (D-MEM) | Gibco | 11960-051 | ||
| Fetal Bovine Serum | Sigma | F4135 | ||
| Penicillin-Streptomycin | Gibco | 15140 | ||
| L-glutamine | Gibco | 25030 | ||
| Growth Medium: | ||||
| NEUROBASAL Medium | Gibco | 21103-049 | ||
| B-27 Serum-Free Supplement | Gibco | 17504-044 | ||
| GlutaMAX -I Supplement | Gibco | 35050-061 | ||
| Gentamicin Reagent Solution | Gibco | 15750-060 | ||
| Immunostaining: | ||||
| Fixative, Washes, and Blocking Buffer: | ||||
| Paraformaldehyde | Sigma | P6148 | ||
| Phosphate Buffered Saline | Sigma | P4417 | ||
| Triton X-100 | Sigma | T9284 | ||
| Donkey Serum | Jackson Immuno | 017-000-121 | ||
| Antibodies: | ||||
| beta-III tubulin antibody | Chemicon | MAB1637 | ||
| MAP2 antibody | Chemicon | AB15452 | ||
| Donkey Cy3 anti-mouse | Jackson Immuno | 715-166-151 | ||
| Donkey Cy2 anti-chicken | Jackson Immuno | 703-226-155 | ||
| DAPI | Gibco | D3571 | ||
| Slide Preparation: | ||||
| CC2 Coated Two-Chamber Slides | Lab-Tek | 154852 | ||
| Fluorescent Mounting Media | KPL | 71-00-16 | ||
| 24 x 60 mm Micro Cover Glasses | VWR | 48393-106 | ||
| Clear nail polish | Electron Microscopy Sciences | 72180 | ||
| Electroporation: | ||||
| Ketamine | Henry Schein | 995-2949 | ||
| Xylazine | Henry Schein | 4015809TV | ||
| buprenorphine | Henry Schein | 1118217 | ||
| Picospritzer III | Parker | |||
| BTX square wave electroporator | Fisher | BTXECM830 | ||
| Tweezertrodes, 7 mm, platinum | Harvard Apparatus | 450488 |
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Tags
In Utero ElectroporationPrimary Neuronal CultureGene FunctionSubset Of Cortical NeuronsNeurite OutgrowthGenetic AlterationsShRNACDNA ConstructsChemical TransfectionViral TransductionHeterogeneous Pool Of Cell TypesGlutamatergic NeuronsInhibitory NeuronsGlial CellsEmbryonic Rodent CortexDeep Layers Of The CortexSuperficial Layers Of The CortexDorsal-medial Regions Of The CortexVentral-lateral Regions Of The CortexQuantitative AnalysisStep-by-step Method
Cite This Article
Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons. J. Vis. Exp. (44), e2103, doi:10.3791/2103 (2010).