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Transient Gene Expression Following Single and Double Exponential Decay Electroporation Pulses

in a 0.2 cm gap electroporation cuvette, then the cell suspension was added. Exponential decay electroporation pulses were administered as specified in Table 1 by a Gene Pulser II system with Capacitance Extender II or an EasyJect Plus electroporator (EquiBio) according to the manufacturers instructions. The cells were then cultured in 4 ml DMEM containing 10% serum in hexwell plates in a humidified 37 C, 5% CO2 environment.

Plasmids and Reporter Enzyme Assays
The expression plasmids pCH1104 and pSVZALAΔ5'5, which contain the SV40 early promoter, were used to produce E. coli β-galactosidase and firefly luciferase respectively. These reporter enzyme activities were assayed in cell extracts prepared 24 hours after electroporation. Adherent cells were washed twice with cold PBS and lysed in 300 l luciferase lysis buffer. Extracts were centrifuged briefly to remove cell debris. Luciferase was quantitated using luciferin (Analytical Luminescence Laboratory, San Diego) employed according to the manufacturers protocol, collecting the light produced by 20l of extract for 10 sec with an MGM Optocomp I luminometer. Galactosidase was determined in the same cell extracts using the chemiluminescent Galacto-Light assay (Tropix, Cambridge, Massachusetts) in the same luminometer.


Conclusion
A single, capacitor discharge electroporation pulse gave better transformation results than a Twin-pulse regime. A single pulse treatment is also much easier to optimize, since there are only two electrical parameters to set.


References
1. EasyJect Electroporator Instruction Manual, Stratagene, La Jolla, Ca
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Prepared by multi-temperature ethanol fractionation with extensive charcoal treatment and dialysis.• Assay: ≥ 95 % (HPCE)• pH ~5.2 (10 mg/mL in 0.15 M NaCl)...
Special grade: for electrophoresis...
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