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 in-creased as cation concentration of vehicle dec-reased 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.
Animals ; Comparative Study ; DNA/*administration & dosage/metabolism ; Drug Delivery Systems ; Electric Conductivity ; Electroporation/methods ; Escherichia coli/genetics ; Female ; Gene Therapy/*methods ; *Gene Transfer Techniques ; Genes, Reporter ; Injections, Intramuscular ; Luciferase/metabolism ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal/drug effects/*metabolism/pathology ; Osmolar Concentration ; Plasmids/genetics/*metabolism ; Sodium Chloride/*pharmacology ; Transfection ; Vehicles/*administration & dosage ; beta-Galactosidase/metabolism

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 in-creased as cation concentration of vehicle dec-reased 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.
Animals ; Comparative Study ; DNA/*administration & dosage/metabolism ; Drug Delivery Systems ; Electric Conductivity ; Electroporation/methods ; Escherichia coli/genetics ; Female ; Gene Therapy/*methods ; *Gene Transfer Techniques ; Genes, Reporter ; Injections, Intramuscular ; Luciferase/metabolism ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal/drug effects/*metabolism/pathology ; Osmolar Concentration ; Plasmids/genetics/*metabolism ; Sodium Chloride/*pharmacology ; Transfection ; Vehicles/*administration & dosage ; beta-Galactosidase/metabolism

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.
Blotting, Northern ; Bucladesine/pharmacology* ; Bucladesine/antagonists & inhibitors ; Enzyme-Linked Immunosorbent Assay ; Fibronectins/metabolism ; Fibronectins/genetics* ; Fibrosarcoma/genetics* ; Gene Expression Regulation* ; Human ; Luciferase/metabolism ; Precipitin Tests ; Promoter Regions (Genetics) ; RNA, Messenger/metabolism ; Tetradecanoylphorbol Acetate/pharmacology* ; Transfection ; Tumor Cells, Cultured ; beta-Galactosidase/metabolism

A possible role of hepatocyte nuclear factor 1 (HNF1) or HNF3, a predominant trans-acting factors of hepatic or pancreatic beta-cells, was examined on the tissue specific interdependent expression of glucokinase (GK) in liver, H4IIE, HepG2, HIT-T15 and MIN6 cell line. The tissues or cell lines known to express GK showed abundant levels of HNF1 and HNF3 mRNA as observed in liver, H4IIE, HepG2, HIT-T15 and MIN6 cells, whereas they were not detected in brain, heart, NIH 3T3, HeLa cells. The promoter of glucokinase contains several HNF3 consensus sequences and are well conserved in human, mouse and rat. Transfection of the glucokinase promotor linked with luciferase reporter to liver or pancreatic beta cell lines showed high interacting activities with HNF1 and HNF3, whereas minimal activities were detected in the cells expressing very low levels of HNFs. The binding of HNF1 or HNF3 to the GK promoter genes was confirmed by electrophoretic mobility shift assay (EMSA). From these data, we propose that the expression of HNF1 and/or HNF3 may, in part, contribute to the tissue specific expression of GK.
3T3 Cells ; Animal ; Blotting, Northern ; Cell Line ; Cell Nucleus/metabolism ; Cells, Cultured ; DNA-Binding Proteins/genetics/*physiology ; Genes, Reporter ; Glucokinase/*biosynthesis/*genetics ; Hela Cells ; Human ; Liver/metabolism ; Luciferase/metabolism ; Mice ; Models, Genetic ; Nuclear Proteins/genetics/*physiology ; Plasmids/metabolism ; Promoter Regions (Genetics) ; Protein Binding ; Rats ; Support, Non-U.S. Gov't ; Tissue Distribution ; Transcription Factors/genetics/*physiology ; Transcription, Genetic ; Transfection