通过体外电穿孔转染对小脑颗粒细胞前体进行标记

Aya Ito-Ishida

doi:10.21769/BioProtoc.778

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Peer-reviewed

Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation

通过体外电穿孔转染对小脑颗粒细胞前体进行标记

AI Aya Ito-Ishida email

发布: 2013年06月20日第3卷第12期 DOI: 10.21769/BioProtoc.778 浏览次数: 15576

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参见作者原研究论文

The authors used this protocol in:

Cover of Neuron, featuring study using the protocol.

Nov 2012

Special Issue on Protocols for Organoid Research

实验方案合集

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Abstract

This protocol will be useful to introduce the genes of interest into the cerebellar granule cells at early stages of development. Since the granule cell precursors are localized in the external granule layer before migration, DNA plasmids can be specifically incorporated into the granule cells by injecting DNA solution into the cerebellar fissures followed by application of electric pulses. This technique can be performed prior to the preparation of either dissociated or organotypic culture, which can be used to study the molecular mechanisms of cell migration, axon elongation and synapstogenesis during development.

Keywords: Electroporation (电穿孔)

Cerebellum (小脑)

Granule cell (颗粒细胞)

Neuron (神经元)

Materials and Reagents

Plasmid Maxi Kit (QIAGEN)
10x HEPES buffered saline (HBS)
Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
1x Phosphate buffered saline (PBS)
10x HBS (see Recipes)

Equipment

Capillary glass (Harvard Apparatus, catalog number: 30-0066 )
Micropipette puller P-97/ IVF (Sutter Instrument)
Aspirator Tube Assembly (Drummond, catalog number: 2-000-000 )
Square pulse electroporator and a foot switch (Nepagene, catalog number: CUY21 )
Platinum plate tweezers-type electrode (Protech International, catalog number: CUY650-P5 )
Dissecting microscope
Dissecting tools: Forceps and some fine scissors
10 cm-petri dish

Procedure

English

中文翻译

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如何引用

Ito-Ishida, A. (2013). Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation. Bio-protocol 3(12): e778. DOI: 10.21769/BioProtoc.778.