Gwizblank

The vector plasmid gWiz Blank was commercially prepared (Aldevron, Fargo, ND, USA) at concentration of 2 mg/ml in physiological saline. Additionally, concentrations of 1 mg/ml by further dilution and 3.5 mg/ml by concentration (Concentrator plus, Eppendorf, Hamburg, Germany) were prepared.
Plasmid EGFP-N1 (pEGFP-N1, BD Biosciences, San Jose, CA, USA), encoding green fluorescent protein, was used for transfection efficiency experiments. It was isolated after amplification in a competent Escherichia coli (TOP10; Thermo Fisher Scientific) using Maxi-Endo Free Plasmid Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of isolated plasmid was measured with Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA).

All procedures were approved by the University of South Florida Institutional Animal Care and Use Committee (protocol R IS00007249, 2019). Female 7 to 8 week female C57Bl/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were used for this study. For all procedures, the animals were anesthetized in an induction chamber infused with 2.5% isoflurane (Mallinckrodt Veterinary Inc., Mundelein, IL, USA) in O2 and fitted with a standard rodent mask supplied with the same mixture. The empty vector plasmid gWiz-blank was commercially prepared (Aldevron, Fargo, ND, USA) and suspended to 2 mg/mL in physiological saline. Untreated muscle was used as a control group while the experimental groups were intramuscular pDNA injection alone, saline injection followed by pulses, and pDNA injection followed by pulses. Fifty μL of pDNA (gWiz-blank) or saline was injected into the right caudal thigh muscle. A 2-needle electrode with a 5 mm gap (BTX Harvard Apparatus, Holliston, MA, USA) was placed into the muscle surrounding the injection site and eight 20 ms pulses at a voltage to distance ratio of 100 V/cm were immediately applied using an ECM830 pulse generator (BTX). Each mouse was monitored continuously until recovery from anesthesia. After four hours, the animals were euthanized, the muscle samples were collected and snap frozen on dry ice.