Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle
Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle
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DOI：
10.1093/hmg/1.6.363
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发表时间：
1992-01-01
影响因子：
3.5
通讯作者：
Jani, Agnes
中科院分区：
文献类型：
--
作者：
Wolff, Jon A.;Ludtke, James J.;Jani, Agnes
质粒pRSVL在小鼠骨骼肌肌内注射后持续表达荧光素酶至少19个月。其他注射的质粒也能长期稳定表达，这表明任何质粒DNA都能在肌肉中稳定存在和表达。质粒DNA在注射后至少19个月的部分肌肉DNA样本中被定量PCR证实。质粒DNA的甲基化模式保持在其细菌形式，表明外源DNA没有在肌肉细胞中复制。从注射肌肉中提取的细胞总DNA电穿孔到细菌中，表明质粒DNA位于染色体外。将注射的肌肉DNA经限制性内切酶酶切和结扎后电穿孔进入细菌中，寻找质粒DNA的染色体整合。在检查的1800多个菌落中未发现含有质粒/染色体连接的质粒。缺乏整合增加了这种基因转移技术理论上的安全性。质粒DNA在肌肉中的长期稳定性表明，肌肉是引入染色体外质粒或病毒DNA进行基因治疗的有吸引力的靶组织。
Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.