Fundamental study on gene therapy for diseased myocardium by introduction of myogenetic genes into fibroblasts

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We introduced reporter genes into chicken embryos aiming at their hearts by the electroporation method, and examined various conditions of gene introduction to obtain a good yield of introduced genes. As reporter genes, pmiwZ carrying LacZ gene and pEGFP-C1 containing green fluorescence protein gene were used. Expression of these genes was detected in limb buds and around the heart, but not within the heart. However, expression expression efficiency varied in accordance with conditions tested. Embryos of 48h old were better than those of 72h old. Higher concentration of applied DNA (>10 mg/ml) yielded higher efficiency of expression. Minimal amount of phosphate buffered saline covering both the embryo and the tip of electrodes was necessary to get expression. Linear-shaped electrode (5mm in length and 1mm in diameter) was better than pinpoint or disk-shaped one, and gold was better than platinum, copper or Nichrome as its material. Distance between two electrodes was also important and 8mm was optimal for 48h old chicken embryos. Good results were obtained when pulses of 25V in hight and 50 msec in duration were applied 8 times with 1sec intervals. Application of pulses with higher voltage, shorter duration or less number resulted in decrease of expression efficiency. Thus, we could optimize some of theses conditions which affect the efficiency of gene introduction into animals.