Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation.
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 beta-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.
Lee
Min-Jae
MJ
School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea.
Cho
Soon-Shin
SS
Jang
Hyung-Suk
HS
Lim
Young Shin
YS
You
Ji-Ran
JR
Park
Jangwon
J
Suh
Hearan
H
Kim
Jeong-a
JA
Park
Jong-Sang
JS
Kim
Duk-Kyung
DK
D000818:Animals; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D016503:Drug Delivery Systems; D004553:Electric Conductivity; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D007273:Injections, Intramuscular; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009994:Osmolar Concentration; D014677:Pharmaceutical Vehicles / Q000008:administration & dosage*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D014677:Pharmaceutical Vehicles; D012965:Sodium Chloride; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase