In Vivo Transfection into Mouse/Rat Muscle, Skin, Joint, Cartilage, Tumor and Others by Electroporation

Electric pulses were delivered using an electric pulse generator (NEPA21 or CUY21; Nepa Gene Co.,Ltd.). Electrodes consisted of a pair of stainless steel needles of 5 mm in length and 0.4 mm in diameter, fixed with a distance (gap) between them of 3 mm or 5 mm (Nepa Gene Co.,Ltd.)

Intramuscular DNA Injection
Anesthetize mice by intraperitoneal injection of 0.01 ml/g body weight of 6 mg/ml pentobarbital sodium solution. Inject the tibialis anterior muscles with 50 µg of purified closed circular DNA of pCAGGS-lacZ plasmid at 1.5 µg/µl in PBS using an insulin syringe with a 27-gauge needle.

Electroporation In Vivo
Insert a pair of electrode needles into the muscle to a depth of 5 mm to encompass the DNA injection sites. Deliver electric pulses using an electric pulse generator. Three 50-msec-long pulses of the indicated voltage (50-100 V) followed by three more pulses of the opposite polarity are administered to each injection site at a rate of one pulse per sec.

β-galactosidase Expression
Five days after DNA transfer, the expression of the lacZ gene is visualized by X-gal staining for -galactosidase activity

The tibialis anterior muscles were fixed in cold 4% paraformaldehyde in PBS for 3 h, then washed in PBS for 1 h, and stained at 37°C for 18 h in the presence of 1mM X-gal to detect E. coli β-galactosidase activity.

For transverse sections, muscles were embedded in O.C.T. compound and frozen in dry ice-acetone.

Serial sections (15μm thick) were sliced with a cryostat and placed on slide glasses coated with 3-amino-propyltriethoxysilane.

The slices were fixed in 1.5% glutaraldehyde for 10 min at room temperature and then washed three times in PBS.

Samples were then incubated at 37°C for 3 h in the presence of 1 mM X-gal.

The muscle sections were counterstained with eosin. The control muscle (without electropulsation) showed only a small number of stained muscle fibers.

Electroporation increased both the number of positively stained muscle fibers and the density of staining.

Jun-ichi Miyazaki, Division of Stem Cell Regulation Research, G6 Osaka University Medical School
*Nature Biotechnology, Volume 16, Number 9, Pages 867-870, September 1998

1: Tweezers w/Variable Gap 3 Needle Fork & Stainless Steel Rectangle Plate Electrode, 5mm x 10mm (CUY663-5X10: NEPA GENE)

2: Pulse generator (CUY21 EDIT Square Wave Electroporator: NEPA GENE).