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

采用电穿孔法将报告基因导入鸡胚心脏，并考察了各种基因导入条件，获得了较好的导入基因产量。报告基因采用携带LacZ基因的pmiwZ和含有绿色荧光蛋白基因的pEGFP-C1。在肢体芽和心脏周围检测到这些基因的表达，但在心脏内没有。然而，表达的表达效率随测试条件的不同而不同。48h的胚胎比72h的胚胎好。应用浓度越高（bbb10 mg/ml），表达效率越高。少量的磷酸盐缓冲盐水覆盖胚胎和电极尖端是获得表达所必需的。线形电极（长5mm，直径1mm）优于针状或圆盘状电极，金电极优于铂、铜、镍铬合金电极。两个电极之间的距离也很重要，对于48h龄鸡胚胎，8mm为最佳。高25V、持续时间50msec的脉冲以1sec的间隔施加8次，获得了良好的效果。施加电压较高、持续时间较短或脉冲数量较少的脉冲会导致表达效率下降。因此，我们可以优化一些影响基因导入动物效率的条件。

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.