An Efficient Electroporation Protocol Supporting In Vitro Studies of Oligodendrocyte Biology.

IF 2.3 Q3 BIOCHEMICAL RESEARCH METHODS
Yugo Ishino, Shoko Shimizu, Shingo Miyata
{"title":"An Efficient Electroporation Protocol Supporting In Vitro Studies of Oligodendrocyte Biology.","authors":"Yugo Ishino, Shoko Shimizu, Shingo Miyata","doi":"10.3390/mps8030064","DOIUrl":null,"url":null,"abstract":"<p><p>Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale studies. Therefore, standardized in vitro systems are needed to facilitate efficient and reliable functional analyses of genes identified in such studies. Here, we describe a practical and efficient method for oligodendrocyte precursor cell (OPC) isolation from mouse brains on postnatal day 6-8 and a gene delivery method for the isolated OPCs. By modifying the magnetic-activated cell sorting (MACS) procedure with reduced processing volumes, we simplified OPC isolation, allowing simultaneous handling of multiple samples and improving workflow efficiency. We also optimized electroporation parameters to achieve robust transfection efficiency with minimal cell death. Transfected OPCs are suitable for both monoculture-based differentiation assays and co-culture with dorsal root ganglion (DRG) explants, in which they reliably differentiate into mature oligodendrocytes and myelinate along the axons. This system enables stable and reproducible in vitro analysis of oligodendrocyte function, supports investigations into both intrinsic differentiation and neuron-glia interactions, and provides a powerful platform for oligodendrocyte research with efficient and timely gene manipulation.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 3","pages":""},"PeriodicalIF":2.3000,"publicationDate":"2025-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12195875/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods and Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/mps8030064","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale studies. Therefore, standardized in vitro systems are needed to facilitate efficient and reliable functional analyses of genes identified in such studies. Here, we describe a practical and efficient method for oligodendrocyte precursor cell (OPC) isolation from mouse brains on postnatal day 6-8 and a gene delivery method for the isolated OPCs. By modifying the magnetic-activated cell sorting (MACS) procedure with reduced processing volumes, we simplified OPC isolation, allowing simultaneous handling of multiple samples and improving workflow efficiency. We also optimized electroporation parameters to achieve robust transfection efficiency with minimal cell death. Transfected OPCs are suitable for both monoculture-based differentiation assays and co-culture with dorsal root ganglion (DRG) explants, in which they reliably differentiate into mature oligodendrocytes and myelinate along the axons. This system enables stable and reproducible in vitro analysis of oligodendrocyte function, supports investigations into both intrinsic differentiation and neuron-glia interactions, and provides a powerful platform for oligodendrocyte research with efficient and timely gene manipulation.

支持少突胶质细胞生物学体外研究的高效电穿孔方案。
少突胶质细胞在中枢神经系统中形成髓磷脂,其功能障碍可引起严重的神经系统症状,因为大规模分析已经强调了病理条件下许多基因表达的改变。虽然体内功能基因分析是可取的,但它们有一些局限性,特别是在大规模研究中。因此,需要标准化的体外系统来促进在此类研究中鉴定的基因的有效和可靠的功能分析。本研究描述了一种从出生后6-8天的小鼠大脑中分离少突胶质前体细胞(OPC)的实用高效方法,以及分离的少突胶质前体细胞的基因传递方法。通过减少处理量修改磁活化细胞分选(MACS)程序,我们简化了OPC分离,允许同时处理多个样品并提高工作流程效率。我们还优化了电穿孔参数,以最小的细胞死亡实现稳健的转染效率。转染后的OPCs既适用于单培养的分化试验,也适用于与背根神经节(DRG)外植体共同培养,在这种情况下,它们可靠地分化成成熟的少突胶质细胞,并沿轴突形成髓鞘。该系统能够稳定、可重复地进行少突胶质细胞功能的体外分析,支持对内在分化和神经元-胶质细胞相互作用的研究,并为少突胶质细胞研究提供了高效、及时的基因操作平台。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Methods and Protocols
Methods and Protocols Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (miscellaneous)
CiteScore
3.60
自引率
0.00%
发文量
85
审稿时长
8 weeks
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信