菱形唇下的Midgestation小鼠胚胎在体外电穿孔
Summary
这项研究描述了一个发展<em>在体外</em>电穿孔技术，允许在较低的菱形唇midgestation胚胎的基因表达操纵。
Abstract
菱形唇是位于后脑的胚胎神经上皮之间的神经管及第四脑室（1检讨）roofplate交界。可分为菱形嘴唇上的菱形唇（URL），其中包括1（R1）和菱产生小脑的神经元和低菱形唇（LRL的），而产生不同的神经脑干谱系2-4。 LRL的衍生工具包括耳蜗核的听觉神经细胞和那些的原子核参与调节平衡和电机控制5-8 precerebellar的。神经再生的LRL的发生在一个大的时间窗口，包括胚胎（五）9.5-16.5 5天，9。不同的神经细胞谱系出现分裂后的细胞在不同发育天，在此期间神经窗口（或出生）LRL的。
基因表达结构的电可以用来操纵LRL的祖细胞的基因表达，并可能改变命运10-12从本地区产生的神经元。通过在子宫内的电LRL的祖细胞在小鼠的基因表达改变已经非常成功地操纵胚胎一天E12.5或以后10 12-14出生的谱系。 在宫内 electroporations前到E12.5已经失败主要是由于穿刺第四脑室roofplate的必要步骤，在提供外源DNA进入LRL的电穿孔相关的杀伤力。然而，许多LRL的派生谱系来自LRL的早于E12.5 9。这些以前出生的谱系，包括外侧网状神经元组成，外部的楔形，下橄榄核的precerebellar系统的功能连接脊髓和大脑皮质的投入小脑5。为了操作的LRL的表达胚胎E12.5年轻，我们开发了在体外系统在胚胎放置到文化电。
这项研究提出了一个在E11.5 LRL的祖细胞的基因表达操纵的高效率和有效的方法。重复性表示培养24小时后的GFP绿色荧光蛋白（GFP）的广泛积极的CAG启动驱动与电穿孔的胚胎。此法的一个重要方面，是基因表达的改变只因为外源基因的表达，并没有因为二次效应，从电和栽培技术的结果。它被确定内源性基因表达模式保持原状电穿孔和培养胚胎。此法可以用来改变命运的细胞从胚胎E12.5年轻的过度表达质粒引进LRL的新兴或击倒（通过RNA干扰）不同亲神经转录因子。
Protocol
1。以电穿孔前的准备由马克西准备（总理或Qiagen公司）的DNA扩增为电。 DNA的浓度应该是最低的1毫克/毫升的高效吸收。 5微升0.01％快速绿色1×PBS（磷酸盐缓冲液）在离心管取出495μLDNA和混合。 2。胚胎丰收 CD-1小鼠（哈伦）建立定时交配。检查阴道塞的存在，并把阴道塞观察胚胎一天（五）0.5的日期。胚胎将可视化插件（E11.5）后11天收获。 …
Discussion
在这项研究中提出的技术在体外电击是一种新颖的方法，可以有效地利用胚胎年龄小于12天的妊娠期，操纵基因表达。胚胎放置到文化允许引入基因的表达和规避杀伤力观察电穿孔胚胎时，被允许留在体内 。这种技术允许基于电的研究，这在以前无法进入的胚胎祖细胞的基因表达操纵。
电技术，导致在引入80％的胚胎基因的高效表达分析。观察图2中…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者想感谢简·约翰逊的MATH1，Ngn1，的Ptf1a抗体和康妮Cepko为“pCAG :: GFP的质粒。这项工作是由NIH的R15的1R15HD059922-01。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Cryostat | Leica | CM-1850 | |
| Biologie tip Dumoxel treated DUMONT forceps | Fine Scientific Tools | 11252-30 | |
| 20 mm MORIA perforated spoon | Fine Scientific Tools | 10370-17 | |
| ECM 830 Square Wave Electroporation Generator | BTX (VWR) | 47745-928 | |
| Harvard Apparatus 7 mm Tweezertrodes* Electrodes | BTX (Fisher) | BTX450165 | |
| Fisher Isotemp CO2 Incubator | Fisher | 1325525 | |
| NAPCO CO2 Gas Regulator | Fisher | 15497020 | |
| 12 Well Tissue Culture Plates | BD Falcon (Fisher) | 877229 | |
| HyClone Liquid Media DMEM/F-12 (1:1); With L-Glutamine and HEPES; 500mL | Thermo Scientific (Fisher) | SH3002301 | |
| HyClone* Donor Equine Serum | Thermo Scientific (Fisher) | SH3007402 | |
| Fetal Bovine Serum, Qualified, Heat Inactivated | Invitrogen | 16140-063 | |
| cellgro* 10,000 IU Penicillin, 10,000μg/mL Streptomycin | Mediatech (Fisher) | MT-30-002-CI | |
| HyClone* L-Glutamine L-Glutamine; 200mM in 0.85% NaCl | Thermo Scientific (Fisher) | SH3003401 | |
| Fast-Green | Fisher | AC41053-0250 | 0.01% |
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Tags
In Vitro ElectroporationLower Rhombic LipMidgestation Mouse EmbryosNeuroepitheliumHindbrainNeural TubeRoofplateFourth VentricleUpper Rhombic LipCerebellumDiverse Neuronal Brainstem LineagesAuditory NeuronsCochlear NucleiPrecerebellar NucleiBalance And Motor ControlNeurogenesisTemporal WindowEmbryonic DaysGene Expression ConstructsLRL ProgenitorsFate Of NeuronsIn Utero Electroporation
Cite This Article
Holland, P. J., George, A. M., Worrell, L. T., Landsberg, R. L. In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos. J. Vis. Exp. (66), e3983, doi:10.3791/3983 (2012).