Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

Abstract

Source: Lo, J. C. W. et al., Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor. J. Vis. Exp.  (2021)

This video demonstrates the electroporation technique that introduces fluorescent protein-encoding plasmid DNA into mouse primary cerebellar granule cell precursors.

Protocol

1. Electroporation for Hh receptor transgene overexpression:  pEGFP-Smo For electroporation using a 2 mm gap cuvette, prepare the following plasmid-cell electroporation mixture for each reaction of electroporation: 1.2 × 106 mouse primary cerebellar granule cell precursors cells and 10 µg of pEGFP-mSmo (adjust the DNA stock concentration to ~2-5 µg/µL in Tris-EDTA buffer or sterile dH2O) in 100 µL of Opti-MEM.  <stro…

Materials

B27 supplement	Life Technologies LTD	17504044
GlutamMAXTM-I ,100x	Gibco, Life Technologies	35050061	L-glutamine substitute
Matrigel	BD Biosciences	354277	Basement membrane matrix
Neurobasal	Gibco, Life Technologies	21103049
Poly-D-lysine Hydrobromide	Sigma Aldrich	P6407
CU 500 cuvette chamber	Nepagene	CU500
EPA Electroporation cuvette (2 mm gap)	Nepagene	EC-002
Opti-MEM	Life Technologies LTD	31985070	Reduced-serum medium for transfection
pEGFP-mSmo	Addgene	25395
Super Electroporator NEPA21 TYPE II In Vitro and In Vivo Electroporation	Nepagene	NEPA21	Electroporator