小鼠海马组织的前子宫内电和器官切片文化
Summary
Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.
Abstract
Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.
Introduction
海马起着记忆和学习中起重要作用，以及情感行为。其中一个主要功能包括整合的短期记忆转化为长期记忆，这需要神经系统的可塑性高的。海马的齿状回充当用于输入信息的主网关，也是两个脑区与正在进行的神经发生1整个成年期1,2。海马结构的发育过程中胚胎发育后期，尤其在最初3〜4周，产后3时。在齿状的早期发展回中需要设一个干细胞库是用于产后以及成年神经4。显影神经元产后以及成年神经发生过程中通过各种阶段，从干细胞通过祖细胞向未成熟和最后的成熟神经元的几个阶段。在神经发生中的表达的不同阶段特定的基因是必需的，以允许成熟和集成新的神经元的进海马电路5,6。
用小鼠遗传学和免疫表型分析，以及允许定义许多这些基因的表达模式和功能分子方法。在另外的微阵列分析，以及染色质免疫沉淀（ChIP）提供关于潜在的直接和间接的靶基因7,8-信息。然而，仍然有关于海马发育的调控机制许多悬而未决的问题，在齿状回尤其是发展。通过向下或向上调节的兴趣和/或它的靶基因和发育过程中按照它们的命运的基因的进一步了解如何特定基因调节的系统是必需的，允许小数量的单元的操作， 在子宫内电的shRNA，利息或Cre重组酶毒素重组基因的cDNA的本身提供了这样一种工具。以确保所希望的DNA或小RNA表达质粒应当用于电穿孔的存在。这种做法是非常成功实施的研究皮质发育9,10，但它是一个更具挑战性的方法检查齿状回中的发展，由于海马结构的更深层次的脑的位置。
前子宫内电后器官切片文化是一种方法来解决这个问题11,12。与此相反，以在子宫内电不是整个胚胎但只有头部被用于允许因此放置电极以更有利的方式直接向海马和齿状回shRNA的/ DNA。我们的团队成功地采用前子宫内电齿状回中的发展过程中8，研究转录因子BCL11B的作用。 BCL11B在齿状回中的发展为r的双重角色egulating祖细胞的增殖和分化的证明了免疫组化。进一步定义了一种机制，BCL11B参与这些过程中，Polleux组11,12的协议进行了调整，以研究在协议部分下面描述的齿状回。在第一种方法的问题是解决BCL11B是否自主调节神经细胞分化的细胞。第二种方法检查桥粒，BCL11B的直接靶基因是否足以拯救BCL11B表型。
Protocol
Representative Results
Discussion
海马在学习和记忆的重要功能。齿状回也是在那里发生神经不仅在发展，而且在整个成年期2脑区之一。产后和成人海马神经发生的收益在涉及许多共同的因素类似的方式。限定这些因素的调节机制将在理解神经变性疾病这又会导致新的治疗和预防措施非常有益的。要获得此信息的一个需要一个系统来操纵单个细胞，并就证明了前子宫内电后器官切片文化观察他们在家乡的环境。
<p class="…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/ Description |
| Flaming/ Brown Micropipette Puller | Sutter Instruments Company (USA) | P-97 | |
| Fine Glass Pipettes | Warner Instruments | G100F-4 | |
| Microgrinder | Narishige, Japan | EG-44 | |
| Anesthetic Bracket unit | Harvard Apparatus | PY2 34-0412 | |
| Halovet Vaporizer | Harvard Apparatus | PY2 34-0398 | |
| Fluovac System | Harvard Apparatus | PY2 34-0387 | |
| IMS Fluosorber | Harvard Apparatus | PY2 34-0415 | |
| Anesthetizing Chamber | Harvard Apparatus | PY2 34-0460 | |
| Electroporator | BEX Company | CUY21 EDIT | |
| Tweezers with disk electrodes | BEX Company | LF650P3 | 3 mm electrodes for E15.5 |
| Tweezers with disk electrodes | BEX Company | LF650P5 | 5 mm electrodes for E18.5 |
| Picospritzer III | Parker Hannifin Corporation | P/N 052-0500-900 | |
| HM 650V Vibrating Blade Microtome, 230V | Thermo Scientific | 920120 | |
| Dissection Microscope | Carl Zeiss Microscopy Gmbh | Stemi SV8 | |
| Inverted Microscope | Leica | Leica DM IL LED | |
| Confocal Microscope | Leica | Sp5II | |
| 6 well dish | BD Falcon | #353502 | |
| 6 well dish | CELLSTAR | #657160 | |
| Tissue culture inserts | BD Falcon | #353090 | |
| Fast Green | Sigma | F7252 | |
| Laminin | Sigma | #L2020 | |
| Poly-L-lysine | Sigma | #P5899 | |
| Spring scissors | Fine Science Tools | 15003-08 | |
| Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | |
| Forceps | Dumont #55 | 11255-20 Inox | |
| HBSS 10X | Life Technology | 14180-046 | |
| BME | Life Technology | 41010-26 |
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