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Single Cell Electroporation in vivo within the Intact Developing Brain
Published on: July 11, 2008
Single-cell transfection by electroporation using an electrolyte/plasmid-filled capillary.
Manyan Wang1, Owe Orwar, Stephen G Weber
1Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, USA.
Analytical Chemistry
|April 9, 2009
Summary
Single-cell electroporation (SCEP) effectively transfects individual cells using minimal plasmid volumes. Transfection success correlates with cell membrane permeability measured by thiol-ThioGlo 1 fluorescence loss.
Area of Science:
- Cell biology
- Biotechnology
- Molecular biology
Background:
- Single-cell manipulation is crucial for understanding cellular heterogeneity.
- Existing transfection methods often lack precision at the single-cell level.
- Electroporation offers a physical method for membrane permeabilization.
Purpose of the Study:
- To develop and validate a single-cell electroporation (SCEP) technique for adherent cells.
- To establish a method for quantifying electroporation efficiency at the single-cell level.
- To correlate mass transport dynamics with successful gene expression post-transfection.
Main Methods:
- Utilized a pulled capillary for precise delivery of pEGFP plasmid solution to individual cells.
- Applied brief electric pulses to induce electroporation in single adherent cells.
- Quantified electroporation extent by measuring the fluorescence loss of thiol-ThioGlo 1, a marker for reduced glutathione.
- Analyzed fluorescence decay using a mass transport model to determine steady-state fluorescence loss and mass transfer rates.
Main Results:
- SCEP successfully transfected individual cells with low plasmid concentrations (0.16–0.78 µg/µL).
- Electroporation efficiency, indicated by thiol-ThioGlo 1 fluorescence loss (>10%) and mass transfer rate (>0.03 s⁻¹), predicted EGFP expression.
- EGFP fluorescence was detected at 24 hours and increased by 48 hours in successfully transfected cells.
- SCEP demonstrated a higher success rate for single-cell transfection compared to bulk lipid-mediated methods, despite lower EGFP expression levels.
Conclusions:
- Single-cell electroporation (SCEP) is a viable method for efficient, precise transfection of individual adherent cells.
- Thiol-ThioGlo 1 fluorescence loss serves as a reliable indicator of electroporation-induced membrane permeability and transfection success.
- SCEP offers a significant advantage in transfection success rate for single-cell applications over traditional bulk methods.
