An Efficient Electroporation Protocol Supporting In Vitro Studies of Oligodendrocyte Biology

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.