| Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis
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Author:
Date:
2018-03-20
[Abstract] This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system ...
[摘要] 该协议描述了在器官切片制备中靶向胚胎海马祖细胞的体外电穿孔技术。 该技术允许基因摄动检查胚胎海马体的发育过程，同时保持细胞的环境和连接性。 基因扰动可以包括Cre介导的重组，RNAi介导的敲低，基因过表达或这些中任何的组合。 可以在广泛的胚胎阶段进行体外电穿孔，为实验者提供时间控制。 空间控制可以通过在皮氏培养皿中准备大脑来瞄准海马的特定区域来实现。 电穿孔的外植体培养物提供了用于研究包括祖细胞增殖，迁移和细胞命运获取在内的发育过程的高度易处理的系统。
【背景】由于其位于尾端的端脑，海马在可达性方面提出了挑战。胚胎海马甚至更难以接近，需要用子宫外科手术方法进行实验操作。器官切片文化规避了这个问题，同时保留了海马场细胞构筑学的许多方面，包括分子特征和连接性。尽管有描述来自啮齿类动物大脑的海马外植体产后培养的规程（Stoppini等人，1991; ...
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| In utero Electroporation of the Embryonic Mouse Retina
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Author:
Date:
2014-10-05
[Abstract] This protocol is useful to manipulate gene expression in the embryonic retina and compare the result with the contralateral non electroporated retina. In addition, the electroporation of a membrane or cytoplasmic tagged GFP allows to determine the effects of gene manipulation on the outgrowth of retinal ganglion cell axons (Garcia-Frigola et al., 2007) or simply to follow axon outgrowth in mutant embryos. DNA can be directed to different quadrants of the retina (ventral or dorsally) by modifying the position of the electrodes (Petros et al., 2009; Sánchez-Arrones et al., 2013). After the procedure, embryos are left developing to the desired stage, including postnatal stages.
[摘要] 该方案可用于操纵胚胎视网膜中的基因表达，并将结果与对侧非电穿孔视网膜进行比较。 此外，膜或细胞质标记的GFP的电穿孔允许确定基因操作对视网膜神经节细胞轴突的生长的影响（Garcia-Frigola等人，2007）或简单地遵循突变体胚胎中的轴突生长。 通过修改电极的位置，DNA可以被引导到视网膜的不同象限（腹侧或背侧）（Petros等，2009;Sánchez-Arrones等，2013）。 手术后，胚胎发育到期望的阶段，包括产后阶段。
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| Ex utero Electroporation into Mouse Embryonic Neocortex
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Author:
Date:
2013-12-20
[Abstract] This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as increasing the variety of constructs used in each experiment, thereby helping to reduce their overall numbers. Ex utero electroporation followed by short term organotypic slice culture of embryonic brain sections allows immediate access to multiple slices for choosing optimal ones for live-cell imaging experiments, and characterization of various NSC manipulations in the intact stem cell niche. (see also “In utero Electroporation of Mouse ...
[摘要] 这种技术允许高效和可重复地将DNA / RNA转移到多个年龄的啮齿动物的胚胎新皮质中。通过最大化可用于给定实验的胚胎数量，以及增加每个实验中使用的构建体的多样性，从而有助于减少它们的总体数量，子宫内电穿孔在技术上更难以满足子宫电穿孔技术。胚胎电穿孔之后进行短期器官切片培养，可以立即进入多个切片，选择用于活细胞成像实验的最佳切片，以及在完整干细胞生态位中对各种NSC操作的表征。 （参见“子宫内电穿孔小鼠胚胎脑”（Ge，2012）;“胚胎脑切片的器官切片培养”（Calderon de Anda，2013）。此外，子宫内电穿孔新生儿可用于体外原代细胞培养进一步解剖，分离成单个细胞，并根据标准技术在盖玻片或多孔培养皿中铺板：注意，该程序可以在电穿孔后立即进行，在异位基因表达发生之前，或在隔膜培养至仅收集电穿孔细胞的区域。
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