DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression u sing time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the nove l Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

　　Key Words:Electroporation-Gene delivery-Whole-body imaging-Katushka-Skeletal muscle

　　Introduction

　　Materials and methods

　　Animals and muscle preparation All animal experiments were conducted in accordance with the recommendations of the European Convention for the Protec tion of Vertebrate Animals used for Experimentation. Experiments were performed on 7–9-week-old female C57Black/ 6 or NMRI mice of own breeding. Animals were maintained in a thermostated environment under a 12-h light/dark cycle and had free access to food (Altromin pellets, Spezialfutter-Werke, Germany) and water. The animals were anesthetized 10 min prior to electrotransfer or scanning by intraperitoneal injections of Hypnorm (0.4 ml/kg, Janssen Saunderton, Buckinghamshire, UK) and Dormicum (2 mg/kg, Roche, Basel, Switzerland). For ex vivo imaging, the animals were killed by cervical dislocation and intact tibialis cranialis (TC) muscles without tendons were excised and immediately scanned.

　　Plasmid constructs The plasmids, pTagFP635, encoding Katushka (Evrogen, Russia), and phGFP-S65T, encoding the green fluorescent protein (GFP) (Clontech, Palo Alto, CA, USA) both under the control of a CMV promoter were used. DNA preparations were performed using Nucleobond AX Maxiprep kits (Machery Nagel), and the concentration and quality of the plasmid preparations were controlled by spectrophotometry. Plasmids were finally dissolved in PBS at a concentration of 0.25 μg/μl unless otherwise specified.

　　In vivo DNA electrotransfer Twenty-microliter plasmid solution was injected i.m. along the fibers into the tibialis cranialis muscle using a 29G insu lin syringe. Plate electrodes with 4-mm gap were fitted around the hind legs. Good contact between electrode and skin was ensured by hair removal and t he use of electrode gel (Eko-gel, Egna, Italy). The electric field was applied using the Cliniporator? (IGEA, Italy) with the following settings: a high voltage (1,000 V/cm (applied voltage?=?400 V), 100 μs) pulse followed by a low voltage (100 V/cm (applied voltage?=?40 V), 400 ms) pulse with a 1-s time lag between the pulses. The Cliniporator? provided online measurement of voltage and current.