Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs
Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs
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DOI：
10.1089/104454903322625019
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发表时间：
2003-12-01
影响因子：
3.1
通讯作者：
Draghia-Akli, R
中科院分区：
文献类型：
--
作者：
Khan, AS;Smith, LC;Draghia-Akli, R
在许多物种中，利用电穿孔技术可以提高基因转移到骨骼肌后的转基因表达，但最佳条件尚未得到很好的确定。利用含有肌肉特异性分泌型胚胎碱性磷酸酶(SEAP)基因的质粒，我们优化了大型哺乳动物(猪)的电穿孔条件。检测的参数包括电场强度、脉冲次数、注射和电穿孔之间的滞后时间、以及载体的传递量。在总体积为2毫升的0.5毫克SEAP表达载体注射后80秒，在0.4~0.6安培的恒流范围内施加的电脉冲可产生最高水平的表达。进一步的测试表明，未描绘的注射部位的电穿孔降低了SEAP的表达水平。这些结果表明，电穿孔参数如安培、滞后时间和脉冲数量能够调节报告基因在猪中的表达水平。
Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.