Mouse/Rat | ||
Embryos in utero | Embryos cultured | Brain tissues ex vivo |
Brain in vivo | Retina | Muscle |
Skin | Testis |
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![]() ・ In Vivo Electroporator: NEPA21, CUY21 SC or CUY21 EDIT (Nepa Gene Co., Ltd.) ・ In Vivo Electrode: CUY650P3 or CUY650P5 (Tweezers w/3mm or 5mm diameter platinum disk electrode, Nepa Gene Co., Ltd.) ・Micropipette for DNA injection: Fig. B (make the micropipette by a micropipette puller) ・ Aspirator tube assembly (Drummond) ・ Optical fiber light (Technolight, Kenko, #KTS-100RSV) ・ Sterile gauze (K-Pine, 7.5cm x 7.5cm) ・ Surgical instruments: Fine forceps x 2, Surgery scissors x 2, Ring forceps, Needle holder, Surgical tape ・ Nylon suture (Nesco, #HT1605NA75) ・ Silk suture (D&G, #112451) |
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(5)Electroporation Setting | |||||
Age | Electrode (diameter) |
Voltage | Pulse On |
Pulse Off |
Number of Pulses |
E12.5 | 3mm | 33V | 30msec | 970ms | 4 |
E13.5 | 5mm | 30V | 50msec | 950ms | 4 |
E14.5 | 5mm | 33V | 50msec | 950ms | 4 |
E15- | 5mm | 35V | 50msec | 950ms | 4 |
Procedure (In Utero Electroporation)
(1) | A 2 cm midline incision is then made in the abdominal wall along the linea alba using a set of forceps and scissors. A piece of sterile gauze with a hole cut in the center is placed over the incision, and one uterine horn is drawn out through the hole in the gauze. |
(2) | After observing the orientation of the embryos through the wall of the uterine horn, a micropipette is inserted into the lateral ventricle, and 2-5μl of plasmid solution is injected by expiratory pressure using the aspirator tube assembly. When CAG-EGFP is used, a concentration of 1μg/μl is sufficient to visualize the migrating neurons. |
(3) | After the injection, DNA
solution containing 0.01% FastGreen can be seen through the uterine wall
(![]() |
(4) | After soaking the uterine horn with PBS, the head of embryo is pinched with a tweezers-type electrode, and electronic pulses are applied with the electroporator. |
(5) | Electroporation
parameters for ICR mouse. If viability was prioritized over transfection efficiency, the number of pulses can be changed to two. |
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
Procedure | |
1: | Pre-culture embryos in the whole embryo culture system for 1.5-2 hours prior to electroporation. |
2: | Place the embryo in a Petri dish with Tyrode's solution. |
3: | Inject 0.1-0.5 μl of plasmid DNA into the brain ventricle with a fine capillary. |
4: | Apply square pulses using the electroporator (CUY21) and electrodes (chamber-type and forceps-type electrodes are available). 70V, 50 msec at 1-second interval, five pulses are applied for E10.5 mouse embryos. |
5: | Culture the electroporated embryos for 24-48 hours in the whole embryo culture system. |
Transfection of a fluorescent protein-expression vector into the developing rat cortex.
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(A) EGFP-expression vector was electroporated into E11.5 rat telencephalon. The electroporated embryo was cultured in the whole embryo culture system (WEC). (B) 24 hours after electroporation, EGFP-expression was specifically detected at the dorsal part of the telencephalon. Tel: telencephalon; Di: diencephalon |
Transfection of fluorescent protein-expression vectors into the rat spinal cord.
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(A) EGFP-expression vector was transfected into the
spinal cord of E12.5 rat embryos by electroporation. The
electroporated embryos were cultured for 24 hours in the whole
embryo culture system (WEC). (B-E) Time-lapse analysis of neuroepithelial cells in the slice culture system. Histon-EGFP- and DsRed2-expression vectors were co-electroporated into the E12.0 rat spinal cord. The electroporated embryo was cultured for 24 hours in the WEC, then the spinal cord was sliced and time-lapse recording was performed. (B-E) Cell nuclei (B, green) and cytoplasms (C, magenta) of neuroepithelial cells in the slice are simultaneously labeled by the electroporation (E). (D) A DIC image. hb: hindbrain; sc: spinal cord; fl: forelimb; DIC: differential interference contrast |
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.
(a-c) | Organ culture of
hippocampal tissue fragments three days after electroporation with
CAG-eGFP (a), T![]() ![]() |
(d,e) | A mature
hippocampal neuron maintained 14 days in dissociated culture after
electroporation of a ![]() |
(f,g) | A hippocampal neuron 7 days after electroporation of 1:1 mixture of T![]() ![]() |
(h) | Relative fluorescence intensity of hippocampal tissue fragments after electroporation of eGFP-expression plasmids with four different promoter sequences. The tissue fragments were maintained in culture for 4 days, fixed and observed under a confocal microscope. Fluorescence intensities per unit area of the tissue fragments were determined. |
(i) | 2Relative fluorescence intensity of hippocampal tissue fragments isolated at two different developmental stages and electroporated with ![]() |
Bars = 50 µm (a-d, f, g); 10 µm (e). |
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
Figure A: | EGFP expression in the medial preoptic nuclei of a female rat examined 4 days after bilateral electroporation at 10 weeks of age. (An asterisk indicates the trace of the positioning of the electrode) |
Figure B: | EGFP-positive cells (high magnification of Fig. A using a 60x objective lens). EGFP fluorescent signals are observed in the perikarya. |
Figure C: | Estrogen receptor
αimmunoreactivity in the medial preoptic nuclei and the periventricular
nuclei of an adult female rat. 3V: third ventricle |
Figure D: | Estrogen receptor α-positive cells (high magnification of Fig. C using
a 60x objective lens). Estrogen receptor ![]() |
Tetsuo Shirakawa, Center for Advanced Oral Medicine, Hokkaido University Hospital
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Schematic illustration showing the
in vivo electroporation method. (1) DNA injection into subretinal space (2) Electroporation |
Tweezer-type electrodes (3) are placed to hold the head of newborn (P0) rat or mouse (4). |
In vivo electroporated rat retinae harvested at various developmental stages. | |
(A) | Whole-mount preparation of rat retina in vivo electroporated with pCAG-GFP at P0 and harvested at P21. Pictures were taken from the scleral side. |
(B) | Rat retinae in vivo electroporated with pCAG-GFP at P0 were harvested at P2(d-f ), or P5(g-i), and cryosections were prepared. |
Takahiko Matsuda, Cepko laboratory, Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School
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Injection of plasmid DNA into muscle | In Vivo 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.)
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
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Whole muscle with electroporation | Whole muscle without electroporation |
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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 -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
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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
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
(1) | Injection of the plasmid solution colored by Fast Green into the central canal |
(2) | A pair of electrodes are put on the vitelline membrane overlying the embryos, and a 25V, 50 ms rectangular pulse was charged 4 times. |
(3) | We can monitor the transfection efficiency by electroporating GFP expression vector. |
(4) | Strong GFP fluorescence could be detected even 48 hours after electroporation. |
(5) | Right side of the embryo is transfected, hence the left side could be served as the control. (Dorsal view) |
Mechanisms of brain regionalization and neural circuit formation have been studied by misexpression of transcription factors (En1/2, Pax2/5/6, Otx2, Gbx2), secreted factors (Fgf, Shh, semaphoring), signal transduction molecule (Ras, Sprouty2) and receptors (neuropilin) |
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
(A) | shRNA expression vector is electroporated as shown above. |
(B) | Select target DNA sequence of 19 to 21 mer. Sense and antisense sequence were linked to a nucleotide spacer as a loop and put into expression vector that is driven by U6 or H1 promoter. Commercially available expression vector that dreives expression by mouse U6 promoter is effective in chick embryos. |
(C) | After transcription, hairpin is digested to form siRNA, and siRNA forms RISK (RNA-induced silencing complex) to dgrade the target mRNA. |
24 hours after electroporation. | |
(A) | Transfection is monitored by co-electroporated GFP fluorescence. |
(B) | Application of siRNA against En2 by electroporation shows degradation of En2 mRNA. |
(C) | Control side |
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
A: | Schema illustrating the system. |
B: | Hamburger and Hamilton (HH)
stage 4 chick embryo with DNA solution injected to the prospective
neural plate region. DNA solution was colored with a FastGreen
dye. hn: Hensen's node, ps: primitive streak, ao: area opaca, ap: area pellucida |
C: | Schema illustrating the location of DNA solution between the vitelline membrane and the ectoderm. |
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Experimental Procedure |
1: | Dissect an embryo using a paper ring (dried filter paper) out of the egg. |
2: | Rinse gently to remove excess yolk, and place the embryo onto the platform of the cathode chamber. |
3: | Inject DNA solution into the space between the ectoderm and vitelline membrane using a glass pipette (for introduction to the ectoderm derivatives). |
4: | Adjust the target site right onto the cathode, and set the anode above it. Set the electrode distance at 5mm. |
5: | Deliver electric pulses (10V, pulse on 50ms, pulse of 100ms, 5 pulses) using a CUY21 electroporator. |
6: | Place the specimen on an agarose-albumin plate, and incubate at 39![]() |
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
1: | Cut open the proventriculus with small scissors and transfer the tissue to PBS(-) solution. |
2: | Cut the agarose gel with a small well (we use minigel for the separation of DNA) into a piece of appropriate size and put it into the electrode well. |
3: | Fill the well of electrode with PBS(-) and the well of agarose gel with DNA solution (12-15 µl). Put proventriculus tissue into the well with its epithelial side towards the cathode. Two or three samples can be put together. |
4: | Apply pulses of 30V for 15 times with pulse length of 50 ms, and interval time of 75 ms. Remove agarose gel immediately and wash it in Tyrode's solution. Then take out tissues from the gel and wash them well in Tyrode's solution. The tissues can now be cultivated organotypically. |
(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
Direct
gene transfer after the vacuum treatment. For research use, patent royalty is free in Japan. We are ready for patent licensing negotiation. |
1: | Soak mature seeds in water overnight. |
2: | Transfer the seeds into electroporation buffer. | |
3: | Vacuum treatment. | |
4: | Electroporation on ice. | |
5: | Incubate the seeds on ice for a few hours. | |
6: | Grow the plants under the favorable conditions. | |
7: | Select the transformed plants using antibiotics. |
Fig. 1. Electroporation Apparatus and the Chamber (Produced by NEPA GENE Co., Ltd.) | ||
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Apparatus: CUY21 EDIT | Chamber: CUY495P10 | Electroporation on ice |
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.
1: | Starting materials are mature seeds. |
2: | Transformed plants are obtained in relatively short time. |
3: | Plant tissue culture is not necessary. |
4: | Possibly applicable to animal cells and microorganisms. |
Fig. 2. Production of Transgenic Wheat | ||
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GUS Gene Expression | Selection by Geneticin | Transformed Plants |
Fig. 3. Transient GUS Gene Expression | |||
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Rice (japonica) | Rice (indica) | Brassica | Soybean |
Fig. 4. Gene transfer into Silk Worm Eggs |
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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
In vivo electroporation. | |
(A) | The necks of honeybees that had settled in the tube were fixed in place by inserting two U-shaped plastic sheets for surgical ease and later release. |
(B) | A honeybee settled in the tube with her neck fixed prior to the operation. |
(C) | Schematic representation of the micropipette and electrodes placed in the honeybee brain. Purple indicates micropipette filled with DNA solution, blue indicates electrodes. AN, antennae; C, compound eyes; MB, mushroom bodies; Oc, ocelli; OL, optic lobes. |
(D) | DNA injection and electroporation with needle electrodes. |
Arrows and arrowhead indicate electrodes and micropipette for DNA injection, respectively. Scale bar, 300 µm. |
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