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 using 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 novel 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
In vivo gene delivery is gaining increasing momentum for gene therapy as delivery methods become more advanced and safe. This raises the need for precise, sensitive, and easy evaluation of gene transfection efficacy. A method that has become increasingly popular is whole-body imaging of fluorescent proteins (1). Fluorescent proteins provide unique opportunities for non-invasive labeling and tracking of transfected cells in living animals in real time (2). Together with the development of new systems for whole-body imaging, advancements in new fluorescent proteins offer the possibility for direct visualization of gene expression in vivo.
For deep tissue imaging, the optical window for favorable light penetration is in the near-infrared wavelengths (3), which involves proteins with emission spectra in the far-red wavelengths. Most photons in the visual spectrum are absorbed by melanin and hemoglobin in animal tissue, while photons with wavelengths longer than 1,100 nm are absorbed by water. In addition, considerable scattering of the signal occurs at lower wavelengths (3).
Recently, a novel far-red fluorescent, Katushka, was characterized (4). This protein was derived from the sea anemone Entacmaea quadricolor, enhanced to perform with higher brightness and exposed to site-directed mutagenesis to generate proteins with emission spectra in the far-red region. The resulting Katushka was reported to be 7- to 10-fold brighter than other far-red fluorescent proteins, e.g., HcRed and mPlum (4), and is characterized by high pH stability and photostability (4). Few studies have so far used Katushka for in vivo bio-imaging (3;5;6), thus in this study we wanted to characterize the potential of Katushka as a marker for gene expression, including an evaluation of the kinetics and spatial distribution of the Katushka signal.
Until recent years, the preferred method for detecting fluorescence was by use of a charge-coupled device (CCD) camera (1;7). This approach limits the degree of details one can obtain as the camera integrates light from the object, resulting in a planar picture, which is dominated by the light contribution from the first 1–2 mm of tissue. Using new technology, biological samples can be excited with sub-nanosecond laser, while emission of individual photons is detected using a rapid photon multiplier (PMT). In this way, the arrival distribution of photons as a function of time of excitation can be collected from individual points in the tissue; allowing for precise spatial and time distribution of the emitted light. One advantage of determining the fluorescence in real time is a precise determination of the decay time for the molecular quantum mechanical transition, which is responsible for light emission. This lifetime is characteristic for different fluorochromes and by filtering light emission by lifetime, one can obtain a precise measurement, where background luminescence is eliminated. The precision also allows for construction of a 3D volumetric optic tomographic map of the fluorescence.
Our choice of method for gene delivery is DNA electrotransfer. By exposing living cells to short and intense electric pulses, position-dependent changes in the transmembrane potential are induced, rendering cells accessible for cDNA entry (8;9). There are many reports on successful in vivo DNA electrotransfer to muscle tissue as reviewed in Mir et al. (9). We performed the transfer using a combination of one high voltage (HV) and one low voltage (LV) pulses. A pulse combination, which has proven highly efficient for muscle tissue (10;11) and causing minimal adverse effects on the muscle tissue (12;13).
Thus, using time domain optical imaging, we wanted to investigate the novel far-red fluorescent Katushka as a marker for transgenic expression with respect to intensity, lifetime, and spatial distribution, and compare it to the well-known fluorescent marker GFP.
Materials and methods
Animals and muscle preparation All animal experiments were conducted in accordance with the recommendations of the European Convention for the Protection 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 insulin 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 the 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.
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