作     者:KhanAS SmithLC AbruzzeseRV CummingsKK PopeMA BrownPA Draghia-AkliR 

作者机构:ADViSYSInc.TheWoodlandsTexas77381USA. 

出 版 物:《DNA and Cell Biology》 

年 卷 期:2003年

学科分类:10[医学] 1001[医学-基础医学(可授医学、理学学位)] 1007[医学-药学(可授医学、理学学位)] 1010[医学-医学技术(可授医学、理学学位)] 07[理学] 0710[理学-生物学] 071007[理学-遗传学] 

主  题:碱性磷酸酶/血液 碱性磷酸酶/遗传学 电穿孔/方法 基因表达调控 基因转移技术 肌肉内 骨骼/酶学 质粒/遗传学 

摘      要: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.



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