成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法
Summary
这里介绍的是一种在成年斑马鱼端脑中进行质粒DNA传递和脑细胞标记的电穿孔方法。该协议是一种快速而有效的方法，用于可视化和跟踪单个细胞，并开辟了将电穿孔应用于各种基因操作的新的可能性。
Abstract
电穿孔是一种转染方法，其中电场应用于细胞，在细胞膜中产生临时孔隙，提高渗透性，从而允许将不同的分子引入细胞。本文采用电穿孔技术将质粒引入脑膜细胞，使成年斑马鱼的腹室区域划线。这些细胞的一小部分显示了干细胞的特性，并在斑马鱼大脑中产生新的神经元;因此，研究他们的行为对于确定他们在神经发生和再生中的作用至关重要。通过电穿孔引入质粒，可以长期标记和跟踪单个细胞。此外，Cre重组酶或Cas9等质粒可输送到单个细胞，从而实现基因重组或基因编辑，并为在受控、自然中评估细胞的自主基因功能提供了独特的机会环境。最后，采用这种详细的分步电穿孔方案，成功地将质粒引入大量单细胞。
Introduction
斑马鱼是优秀的动物模型，用于检查刺伤后的大脑再生。与哺乳动物相比，在进化阶梯上，斑马鱼等进化较少的物种通常表现出较高的形成性神经发生率和成人神经干细胞栖息的更广区域，导致在整个过程中不断产生新的神经元成人生活中的大多数大脑区域。与哺乳动物1相比，这一特征似乎与斑马鱼的再生能力显著提升有关，因为斑马鱼在所研究的大多数脑损伤模型中具有显著的潜力，能够有效地产生新的神经元。 3，4，5，6，7，8 。在这里，斑马鱼端脑研究，因为它是一个大脑区域与突出的神经发生在成年。这些成年神经发生区与成年哺乳动物脑中神经原发生区9、10、11同源。
由于存在径向胶质细胞或脑细胞等径向胶质，斑马鱼端脑素中存在丰富的神经原发生区。Ependymoglial细胞作为常驻的成人神经干细胞，负责在完整和再生的大脑3，5中产生新的神经元。系骨追踪实验表明，心室叶膜对损伤有反应，然后增殖并产生新的神经细胞，迁移到病变部位5。由于斑马鱼端脑的电化性质，腹腔细胞排列在心室表面，并建造心室壁。背叶心室壁由背端阴膜细胞层形成（图1A）。重要的是，斑马鱼叶黄体体现了哺乳动物径向胶质和阴叶细胞的特征。长径向过程是径向胶质细胞的典型特征，而细胞延伸和紧密结（及其心室位置）是阴囊细胞12、13、14的典型特征。因此，这些细胞被称为阴叶细胞。
为了在再生过程中遵循单个阴叶细胞的体内行为，需要可靠地标记它们。荧光显微镜的体内细胞标记方法已经描述过，如内源性报告剂或亲脂染料15。与电穿孔相比，这些方法可能需要较长的时间，而且通常无法提供单细胞标记或永久长期跟踪的可能性。然而，电穿孔（除了单细胞标记）提供了将新的DNA引入宿主细胞的可能性。此外，与其他DNA移植到细胞的方法相比，电穿孔已被证明是最有效的方法之一16，17，18，19。
这里介绍的是一个电穿孔协议，已细化，以标记在成年斑马鱼端脑中的单一脑细胞。该协议允许标记单个细胞，以便长期跟踪它们20或操纵特定途径在细胞自主的方式21，22。
Protocol
本议定书中使用的所有动物均按标准饲养条件饲养，实验均按照欧盟和上巴伐利亚州政府的处理准则和条例进行（AZ 55.2-1-54-2532-0916）。 1. 用于电穿孔的质粒混合物的制备 稀释无菌水中感兴趣的质粒，并加入快速的绿色染色剂溶液[1毫克/mL]。确保质粒的最终浓度为±1 μg/μL。以不超过3%的浓度添加污渍，因为它的唯一目的是给溶液着色和可视化心室注射。 制备?…
Representative Results
所述电穿孔方法允许将质粒DNA输送到脑细胞中，这些细胞表面位于斑马鱼端脑素和背端脑细胞层（图1A）。 如果电穿孔的结果为阳性，可以在其他阴极细胞中观察到标记的单阴白细胞（图2A，B中的红细胞）（图2A，B中为白色）。根据电穿孔过程的效率，可以标记更高（<strong class=…
Discussion
这种电穿孔方案是一种可靠的体内方法，用于标记单个细胞。该协议可能需要进一步适应，以标记其他细胞类型，如神经元或寡核苷酸细胞。为了获得成功的标签，可以使用含有不同促进剂的质粒。鸡-β蛋白促进剂，eF1α，CMV和泛素促进剂以前曾被用来驱动不同转基因在ependyglia及其后代23的表达。然而，观察到了不同转基因表达的动力学，这应当加以考虑。例如，CMV启动子驱动?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
特别感谢詹姆斯·科普蒂编辑手稿。我们还感谢德国研究基金会 （DFG） 向 JN 提供的资金，由 SFB 870 和 SPP”嗅觉综合分析”和 SPP 1738″非编码 RNA 在神经系统开发、可塑性和疾病中的新作用”，SPP1757在慕尼黑系统神经学集群（EXC 2145 SyNergy – ID 390857198）框架内的胶质异质性”和卓越战略。
Materials
| Reagent/Material | |||
| Fast Green | Sigma-Aldrich | F7258-25G | For coloring plasmid solution |
| MS222 | Sigma-Aldrich | A5040-25G | MS222 should be stored at RT (up to two weeks) and protected from light |
| Ultrasound gel | SignaGel, Parker laboratories INC. | 15-60 | Electrode Gel |
| Equipment | |||
| Air pump | TetraTec APS 50, 10l-60l | Can be bought in the pet shops | |
| BTX Tweezertrodes Electrodes | Platinum Tweezertrode, BTX Harvard Apparatus | 45-0486 | 1mm diameter |
| Electroporation device | BTX ECM830 Square Wave Electroporation System, BTX Harvard Apparatus | 45-0662 | |
| Injection device | FemtoJet 4i, Eppendorf | 5252000013 | |
| Standard Wall Borosillicate Glass Capillary | Warner Instruments | 64-0766 | Model No: G100-4 |
| Microloader tips | Eppendorf | 5242956003 | |
| Micro-knife | Fine Science Tools | 10056-12 | |
| Joystick micromanipulator | Narishige Japan | MN – 151 | |
| Needle holder | FemtoJet 4i, Eppendorf | 5252000013 | Needle holder comes together with the injection device |
| Needle pulling device | Narishige Japan | Model No: PC-10 | The PC-10 was discontinued by Narishige in 2017 and replaced by the PC-100 |
| Petri dishes | Greiner Bio-One International | 633161 |
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Tags
ElectroporationPlasmid DNAIn Vivo DeliveryZebrafish TelencephalonEpendymoglial CellsCell LabelingCell-autonomous ManipulationCell BiologyCell DelaminationEpithelial HomeostasisCell Division SymmetryGlass CapillaryMicroinjectionAnesthesiaStereomicroscopeMicroknifeTelencephalic VentricleUltrasound GelElectroporation
Cite This Article
Durovic, T., Ninkovic, J. Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon. J. Vis. Exp. (151), e60066, doi:10.3791/60066 (2019).