Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells
Published: April 30, 2023
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…
Açıklamalar
The authors have nothing to disclose.
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 |
Etiketler
Bu Makaleden Alıntı Yapın
Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells. J. Vis. Exp. (Pending Publication), e20992, doi: (2023).