Donà Massimo, Sandri Marco, Rossini Katia, Dell'Aica Isabella, Podhorska-Okolow Marzena, Carraro Ugo
Department of Biomedical Sciences, C.N.R. Institute of Neuroscience, Unit for Neuromuscular Biology and Physiopathology, University of Padova, Viale G. Colombo 3, I-35121, Padova, Italy.
Biochem Biophys Res Commun. 2003 Dec 26;312(4):1132-8. doi: 10.1016/j.bbrc.2003.11.032.
The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.
骨骼肌纤维的有丝分裂后特性和长寿性使得任何转染基因都能稳定表达。在成年肌肉和再生肌肉中直接进行体内质粒DNA注射，是一种安全、廉价且简便的方法。在此，我们提出一种优化的电穿孔方案，该方案基于使用刮铲电极将cDNA体内转移至解剖学上明确的肌肉的成年肌纤维中，这些肌纤维可进行功能表征。在我们的操作中，约80%的成年肌纤维通过不同的用于绿色荧光蛋白融合蛋白或β-半乳糖苷酶的质粒在体内被转染。与标准DNA递送相比，荧光素酶活性增加了几个数量级。在解剖学上明确的肌肉中，广泛的基因转移与逆转录病毒递送相当或更好，而最近的人体临床研究表明逆转录病毒递送容易产生严重的副作用。此外，用我们的方法，组织损伤大大减少。因此，本研究通过一种对基因治疗以及基础研究都具有巨大潜力的方案，描述了成年骨骼肌纤维中基因的体内功能电转移。
