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

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


CRISPR/Cas9 transfection into mouse muscle by in vivo electroporation

Establishment of CRISPR gene repair evaluation system using eGFP KO mice

Figure 1: eGFP gene repair in eGFP KO mouse muscle.

eGFP fluorescence in eGFP KO mouse muscle with attempted eGFP gene repair. Samples No. 1-3 are independent individuals.

To establish an in vivo gene repair evaluation system in muscle, we first investigated transfection conditions using an experimental system in which eGFP expression plasmids were introduced into wild-type mice.

As a result, we found that eGFP fluorescence could be confirmed in muscle with relatively good reproducibility by electroporation with the NEPA21 (Nepa Gene Co, Ltd.), followed by injection of a nucleic acid solution (100 µl) at a depth of 1.5 to 3.5 mm from the epidermis using a CUY568-4-0.5 needle array electrode (Nepa Gene Co., Ltd.), 30 minutes after administration of hyaluronidase.

Next, we examined whether the eGFP gene is repaired in the muscle of eGFP KO mice by direct transfection of CRISPR/Cas9-related nucleic acid.

We found that green fluorescence was observed in the muscles of all individuals, although the extent of fluorescence varied among individuals (Figure 1).


In courtesy of Dr. Hiromi Miura、 Department of Molecular Life Sciences, Basic Medical Science and Molecular Medicine, Tokai University School of Medicine

The Uehara Memorial Foundation Research Report (Volume 31, 2017)

Gene transfer into muscle by In Vivo electroporation

Injection of plasmid DNA into muscle

In Vivo Eletroporation

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

Whole muscle with electroporation

Whole muscle without electroporation

Transverse section with electroporation

Transverse section without electroporation

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 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

Epidermis-Targeted Gene Transfer Using In Vivo Electroporation

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).


beta-Galactosidase expression on d 1 (A) and d 7 (B) after the pCAGGS-lacZ transfer with electroporation at 18V.
beta-Galactosidase was expressed in the upper most cell layers (horny, granular, and prickle cell layers) of the epidermis on d 1 (A), and in the subcutaneous muscle layer on d 7 (B).
Magnification: (A) x 250, (B) x 70

Hiroki Maruyama, Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences

*Epidermal Cells Methods and Protocols, Series: Methods in Molecular Biology, Volume 289, Pages 431-436, October 2004



Drug Delivery and Transfection

Electro Cell Fusion

Fluorescent Staining

Single-Cell/Micro-Particle Transfer

Cell Freezing

Mechanical Vibration