Gene transfer into muscle by In Vivo electroporation

NEPA21 / CUY21 Applications

[Muscle] Gene transfer into Muscle by In Vivo electroporation

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Electric pulses were delivered using an electric pulse generator (Square Wave Electroporator CUY21EDIT; 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.)

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Injection of plasmid DNA into muscle

In Vivo electroporation

Protocol

1) 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.

2) 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.

3) ß-galactosidase Expression

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

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X-gal Staining

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 ß-galactosidase activity. For transverse sections, muscles were embedded in O.C.T. compound and frozen in dry ice-acetone. Serial sections (15 µm 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 electropul-sation) showed only a small number of stained muscle fibers. Electroporation increased both the number of positively stained muscle fibers and the density of staining..

Electroporation of Whole Muscle

Whole muscle without electroporation

Transverse section with EP

Transverse section without EP

Division of Stem Cell Regulation Research, G6 Osaka University Medical School; Jun-ichi Miyazaki

*nature biotechnology, Volume 16, Number 9, Pages 867-870, September 1998

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