In Vivo/In Utero/In Ovo/Ex Vivo Electroporation Applications

Procedure (In Utero Electroporation)

The actual current is displayed on the screen of the electroporator*. Make sure that the current would become 30-60mA. The current varies according to how the electrode applied or wetness of the uterine horn. Examine the gap between electrodes and the electrode contact areas to fit the current in the appropriate range. If the current is still above the range after the examination, change the voltage setting.
*NEPA21, CUY21 SC or CUY21E (Nepa Gene Co., Ltd.)

GFP Expression

CAG-EGFP was injected into the both lateral ventricles of E14.5 mouse embryos and electronic pulses (33V, 50msec) were charged four times. 3 days later, the embryos (E17.5) were fixed and the brains were removed and examined under a fluorescence stereomicroscope (Fig. A).
Fluorescence was observed in the lateral region of the hemisphere onto which the anode had been
placed and in the medial region of the opposite hemisphere. And brains were frozen and sliced and the fluorescent image was obtained with a confocal laser microscope (Fig. B). GFP positive cells into which DNA was transferred at the ventricular zone (VZ) migrated to the intermediate zone (IZ) and cortical plate (CP). The arrowheads show the border between VZ and IZ and the border between IZ and CP. Dashed line show the border of tissues.
VZ: Ventricular Zone, IZ: Intermediate Zone, CP: Cortical Plate

Hidenori Tabata and Kazunori Nakajima, Department of Anatomy, Keio University School of Medicine

Transfection of a fluorescent protein-expression vector into the developing rat cortex.

Transfection of fluorescent protein-expression vectors into the rat spinal cord.

Masanori Takahashi, Tadashi Nomura, Noriko Osumi, Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine
*Differentiation, Volume 70, Issue 4-5, Pages 155-162, June 2002

Schematic representation of an electroporation set-up.
A fragment of the mouse embryonic hippocampus was placed on a Millipore membrane filter and 5μl EP buffer containing 1mg/ml of plasmid DNA was applied onto the tissue.
A tungsten needle was attached to the surface of a droplet.
After application of square pulses the tissue fragment was returned to a petri dish containing ice-cold HBSS solution.

Electroporation-mediated expression of fluorescent proteins in hippocampal neurons.

Shigeo Okabe, Department of Cellular Neurobiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo
*Neuroreport, Volume 15, Issue 6, Pages 971-975, April 29, 2004

Tetsuo Shirakawa, Center for Advanced Oral Medicine, Hokkaido University Hospital

Takahiko Matsuda, Cepko laboratory, Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School

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.)

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

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

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

Stereomicroscopic views of transfected testes charged with various voltages and observed under visible (A) or excitation (B) light after 5 wk. Voltage is indicated on each testis (A). Loss of testicular weight 5 wk after electric charge in 12-day-old testes (C). All values are means SD (measured number of testes charged with 0, 10, 15, 20, 25, 30, 40, and 50 V are 4, 4, 4, 12, 12, 15, 10, and 12, respectively). Cross-sections of a testis at 5 wk after being charged with 50 V, observed with fluorescence microscope under excitation light (D, left), followed by counterstaining of the same section with hematoxylin (D, right) or another section just stained with hematoxylin (E). In higher voltage charged testes, luminal enlargement of seminiferous tubules (D) or complete degeneration of seminiferous tubules under the capsule (D, left) was observed. In these testes, many fluorescence-positive Sertoli cells were identified easily (E, right), like in 20V charged testes (Day 35).
Bars = 2mm (A, B) or 100μm (D, E).

Kentaro Yomogida, School of Human Environmental Sciences, Mukogawa Women's University
*BIOLOGY of REPRODUCTION, Volume 67, Issue 3, Pages 712-717, September 2002

Harukazu Nakamura and Yuji Watanabe, Graduate School of Life Sciences, and Institute of Development, Aging and Cancer, Tohoku University
*Development Growth & Differentiation, Volume 42, Issue 3, Page 199-201, June 2000

Harukazu Nakamura and Tatsuya Katahira, Graduate School of Life Sciences, and Institute of Development, Aging and Cancer, Tohoku University
*Mechanisms of Development, Volume 121, Issue 9, Pages 1137-1143, September 2004
*Development Growth & Differentiation, Volume 45, Issue 4, Pages 361-367, August 2003

GFP expression becomes detectable by HH6 (about 5 hours after electroporation) in the neural plate, which spreads throughout the central nervous system at HH17. Scale bar: 1mm.

Transgene expression after the electroporation
GFP gene was introduced to the prospective neural plate at HH4, and the embryo was cultured for about 34 hours (HH17 equivalent). GFP expression was occasionally monitored under a fluorescent dissecting microscope. GFP fluorescence was detectable as early as 3 hours after electroporation at the central nervous system and head ectoderm. More than 80% of cells within the transfected area express GFP.

Gene transfection for tissues other than the CNS
Based on the fate map of the chick epiblast, transgenes can be introduced to the somites (A, E), hematopoietic system (B, F), notochord (C, G), or lateral plate mesoderm (D, H).
E-H are the high power views of A-D, respectively.

Kenji Shimamura, Division of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University

(Fig. 1) Two days after the introduction of the vectors. Expression of GFP is observed only in the epithelium (E, left), not in the mesenchyme (M). cSP gene, a luminal epithelium marker, is expressed normally indicating that the electroporation is not harmful for the normal development of the stomach (right). Central figure shows DAPI staining of the explant.
(Fig. 2) Three days after the introduction of the vectors. Small glands are already formed where cSP is not expressed. GFP protein can be found almost all epithelial cells of glandular and luminal epithelia, indicating that the efficiency of electroporation is very high.

Sadao Yasugi, Department of Biological Sciences, Tokyo Metropolitan University

Fertile transgenic plants were regenerated and self-fertilized seeds were obtained in rice and wheat. Transgene integration was confirmed by Southern hybridization. Transmission of the transgene into the next generation (T₁) was indicated by PCR analysis. Transient gene expression was observed in several plant species and silk worm eggs.

Takashi Hagio, Plant Genetic Engineering Unit, National Institute of Agrobiological Sciences
*Improvement research is in progress in his laboratory.

Fig.(1) shows the general setting of the electroporation. Vitelline membrane of the embryo(st.12-13) is usually kept intact. Inject 5-10nl of GFP-mRNA(1 µg/µl, 0.05% Fast Green)into intercellular space of upper-few epithelial layers of the neural plate, and immediately after injection (leaving the needle in the embryo) the electric shock (20-22V, 5msecON, 95msecOFF, 10 shocks) is given by the micro-electrode.
A: Recognition of the target site by Fast Green just after electroporation. This dye fades away 10-15min later.
B: Expression of GFP at the eye 15-20 hours later.
C: Detection of GFP protein in the eye vesicle by FITC-conjugated immuno-staining of thin section.
The local deliveries of mRNAs such as BMP and Shh result in the perturbation of eye development in addition to the up/down regulation of the eye-marker genes.

Kazuhito Takeshima, Radioisotope Research Center, Nagoya University
*Genesis, Volume 33, Issue 2, Pages 81-85, June 2002

Fluorescent images of electroporated honeybee brain at 50 V, targeting the optic lobe (A, B) and mushroom bodies (C-F).
Fluorescent signals were detected around the anode position in the brain electroporated with GFP-expressing plasmid driven by the CMV promoter (A, C, E, F) and not in those electroporated without plasmid (B, D).
(G, H) Magnified views of GFP-expressing areas shown in (A) and (F), respectively.
Positions of electrodes are indicated by circles containing + or -.
Arrowheads indicate the regions in which fluorescence was detected (A, C, E, G) and neural projections from mushroom body cells (F, H).
Frontal view (A, B, F, G, H), rear view (C, D), and top view (E).
Panels (C) and (E) show different views of the same brain.

Takekazu Kunieda and Takeo Kubo, Department of Biological Sciences, Graduate School of Science, University of Tokyo
*University of Tokyo Patent Pending
*Biochemical and Biophysical Research Communications, Volume 318, Issue 1, Pages 25-31, May 21, 2004