Biochemical and Biophysical Research Communications
Functional in vivo gene transfer into the myofibers of adult skeletal muscle
Section snippets
Methods
Plasmids and DNA preparation. The plasmids VR1012 (pCMV-Snap25-GFP, a gift of Prof. T. Pozzan), pCMV-Luc (kindly provided by Dr. M. Cantini), and pCMV-β-gal (pCMV-β-gal, Clontech), containing the cytomegalovirus (CMV) promoter, were grown in DH5α cells and purified using the Plasmid Maxi Kit (Qiagen, Crawley, UK) following the supplier protocol. Identity was confirmed by agarose gel electrophoresis of both uncut and restriction digested plasmids. Contamination with RNA was absent and all the
Influence of electrode shape and electric field on in vivo transfection of mouse muscle fibers
Placed at each side of the tibialis anterior muscle, the spatula-like electrodes shown in Fig. 1 induced much less damage and higher transfection than standard external plates or needle electrodes. These new electrodes delivered low voltage stimulation pulses that induced muscle twitching without lesion of muscle tissue (myofibers and vessels). Three to five days after electroporation, high levels of expression were present (Figs. 2A and B). The entire section of TA showed transfected
Discussion
Viral vectors are highly efficient in gene transfer; on the other hand, serious concerns regarding the possibility of insertional mutagenesis and induction of the host immune response limit their clinical desirability. Several genetically modified viruses, such as retrovirus, herpes simplex virus, Epstein–Barr virus, adenovirus (AdV), and adeno-associated virus (AAV), have been tested for gene delivery into muscle. Of these, AdV and AAV have been found to be most efficient so far for
Acknowledgements
This work was supported in part by institutional funds of the Italian C.N.R. Institute of Neuroscience, Unit for Neuromuscular Biology and Physiopathology, and of the Italian M.U.I.R. (ex 60% to U.C.), Laboratory of Applied Myology, Department of Biomedical Science, University of Padua. We are grateful to Dr. Karen Gustafson for a revision of the English manuscript.
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These authors contributed equally to the present work.