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

我们通过电穿孔法将报告基因导入鸡胚，并针对鸡胚的心脏，研究了基因导入的各种条件，以获得良好的基因导入率。作为报告基因，使用携带LacZ基因的pmiwZ和包含绿色荧光蛋白基因的pEGFP-C1。这些基因的表达在肢芽和心脏周围被检测到，但在心脏内没有。然而，表达效率根据测试条件而变化。48 h胚龄的胚性较好，72 h胚龄的胚性较好。较高的DNA浓度（>10 mg/ml）产生较高的表达效率。最小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。电极材料以直线形（长5 mm，直径1 mm）优于针形或圆盘形，以金为电极材料优于铂、铜和镍铬合金。电极间距对48 h鸡胚的生长也有重要影响，以8 mm为宜。以25 V高、50 ms宽的脉冲，间隔1 s，重复8次，效果良好。施加较高电压、较短持续时间或较少次数的脉冲导致表达效率降低。因此，我们可以优化这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.