Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

Abstract

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and β β β β-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

Electroporation at Low Voltages Enables DNA Vaccine to Provide Protection against a Lethal H5N1 Avian Influenza Virus Challenge in Mice

“…Usually electroporation was used for DNA vaccination at a voltage ranging from 100 V/cm to 400-1,200 V/cm [17][18][19][20] . Though the electroporation charge was applied only transiently, current studies have shown that voltages 1 100 V are an unpleasant experience for animals.…”

“…The higher the electroporation voltage used, the greater the pain inflicted. A review of the literature showed that the voltages for electroporation generally range from 100 to 1,200 V/CM [17][18][19][20] . We studied the effect of DNA vaccination when electroporation was performed at lower voltages.…”

Increased levels of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) improve lipid utilisation, insulin signalling and glucose transport in skeletal muscle of lean and insulin-resistant obese Zucker rats

“…Briefly, animals were anaesthetised with isoflurane and the lower hindlimbs were shaved and sterilised (iodine and 70% ethanol [vol./vol.]). To increase transfection efficiency [18][19][20] [20]). …”

Increased ratio of rapsyn to ACh receptor stabilizes postsynaptic receptors at the mouse neuromuscular synapse

“…The tibialis anterior (TA) muscle was surgically exposed. Ten micrograms of plasmid DNA in 10 µl of sterile MilliQ water (Millipore Corp., Billerica, Massachusetts, USA), was layered over the muscle surface (Lee et al 2002). Silver electrodes were placed on either side of the muscle, touching the surface of the DNA solution but not the muscle.…”