Download Zebrafish Electroporation Protocol - Institute of Molecular and Cell

Zebrafish Electroporation Protocol
(developed by Cathleen Teh in Vladimir Korzh’s laboratory in the Institute of
Molecular and Cell Biology, Singapore)
Construction of the Electroporation Chamber
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The top three quarters of a GenePulser® cuvette (Bio-Rad Laboratories, Hercules,
CA, USA) (Figure 1A) with a 0.4-cm electrode gap were removed for convenient
manipulation of the embryo.
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The chamber was half-filled with 1% molten agarose in Hank’s buffer (137 mM
NaCl, 5.4 mM KCl, 0.25mM Na2HPO4, 0.44 mM KH2PO4, 1.3mM CaCl2, 1.0
mM MgSO4, 4.2 mM NaHCO3, pH 7.2). The volume of agarose depends on the
age of the embryo, the older the embryo, the longer its A-P axis and the more
agarose you need. Typically for 24-26 hpf zebrafish embryos the chamber is filled
with 3-4 drops of molten 1% agarose in Hanks buffer from a disposable plastic
Pasteur pipette (app. 120 µl). The chamber top is sealed with Parafilm®.
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To cast a well for the embryo, a blunted microinjection capillary [tip diameter,
0.05 mm; outer diameter (o.d.), 1 mm; inner diameter (i.d.), 0.58 mm; length,
100mm] (Sutter Instruments, Novato, CA, USA) (the lower capillary on Figure
1B), which functions as a mould, was inserted vertically through parafilm into the
center of the chamber containing the molten agarose to the level shown by a line
on Fig.1B and held in place by parafilm until the agarose had set. After that the
capillary and parafilm are removed leaving the well in agarose.
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The size of the well is further adjusted with the blunt capillary by flicking away
excess agarose such that the well can comfortably fit the posterior body of the
embryo including the yolk cell. An uninjected embryo is positioned in the well to
check the quality of the well. Factors that contribute to well quality include the
following:1.) The agarose well should lightly grip the yolk cell without injuring the embryo.
2.) The anterior neural tube should be above agarose level.
3.) The position of the embryo in the electroporation chamber should remain
unchanged after its exposure to the series of electric pulses.
At least 5 electroporation chambers should be prepared for each session as not all
agarose wells would be good enough for electroporation. Besides, the initially
optimal well will deteriorate with repeated use.
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A diagram of the electroporation chamber is shown in Figure 1C. The electrode
plate is shown in black, and the troughs filled with Hank’s buffer are in gray. The
embryo was placed in the agarose well (gray circle) surrounded by Hank’s
buffered agarose (yellow). The orientations of the embryo that resulted in
unilateral transgene expression (blue line) or bilateral expression in the
cerebellum and telencephalon (brown line) are indicated. Troughs that are 1 mm
wide (made by the blunt capillary with an outer diameter of 1mm) were made
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close to the electrode plates, and Hank’s buffer was added to fill the
electroporation chamber to the top.
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The height of the well is important; for efficient electroporation into the brain, the
head must protrude from the well. Embryo could be manipulated using a blunt
capillary under a dissecting microscope which provides large working distance.
The Olympus SZ40 dissecting microscope with transillumination and an
additional source of descending light through the gooseneck is particularly
suitable for this purpose.
Figure 1. Tools for in vivo electroporation into the brain and direction of DNA
microinjection.
Microinjection of DNA into the Neural Tube of Zebrafish Embryos
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Wild-type zebrafish embryos 24–48 hours post-fertilization (hpf) were maintained
in Hank’s buffer containing 0.2 mM 1-phenyl-2-thiourea (PTU), starting from 19
hpf, to inhibit pigment formation.
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Before microinjection, zebrafish embryos were dechorionated, anesthetized using
tricaine and placed dorsal-side up in wedge-shape wells made in 1% agarose and
covered by Hank’s buffer.
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For DNA injection into a cavity of a neural tube, a standard injection protocol
with sharp microcapillary (upper capillary, Fig.1B) was used.
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Using the micromanipulator, the injection capillary was inserted into the yolk cell
of the embryo approximately at the level of first somite (solid line), then through
the floor of the hindbrain near the otic vesicle (broken line) and into the IV
ventricle (arrowhead), Fig.1D.
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DNA was injected using the MPPI-2 pressure injection system (Applied Scientific
Instrumentation, Eugene, OR, USA), Fig.2, until the neural tube was visibly
distended. The injection volume can vary depending on the age of the embryo
(Table 1). Each injected embryo was immediately transferred into the
electroporation chamber filled with Hank’s buffer (to prevent damage to the
injected embryo that may result during transfer).
Electroporation Using Uniform Electric Field
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The position of the microinjected embryo was adjusted such that the tail, posterior
body and yolk cell is inside the well while its anterior neural tube protrudes from
the well. Hank’s buffer was removed to such a level that it barely covers the head.
The electroporation chamber containing the embryo was then clamped tightly
between the metal plates of the safety stand. The electro Square Porator ECM 830
(BTX®; Genetronics, San Diego, CA, USA, (Figure 3) was used to generate
square electric pulses phased 1 s apart.
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The conditions of electroporation depend upon age of the embryo (Table 1). After
electroporation, the embryos were left to recover in small (d=40 mm) glass Petri
dishes containing 2.5 mL of Hank’s buffer with PTU.
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This modification of electroporation is used, firstly, for unilateral labeling of a
relatively limited number of cells in experiments that require analysis of cell
morphology and, secondly, for massive bilateral labeling of cerebellum (Figure
4).
Figure 2. Microinjection was performed using the MPPI-2 pressure injection system
and dissection microscope Olympus SZ40.
Power output
Safety stand
Figure 3. Electro Square Porator ECM 830 with the attached safety stand.
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Fig. 4. Electroporation using uniform electric field. A –electroporation chamber, B –
scheme of unilateral electroporation (M-L axis), C – typical outcome of unilateral
electroporation along M-L axis, GFP mRNA is detected by whole mount in situ
hybridization in the brain of 48 hpf embryo shown in dorsal view, D – scheme of
electroporation along A-P axis, E – typical outcome of electroporation along A-P axis,
GFP mRNA is detected by whole mount in situ hybridization in the cerebellum of 48 hpf
embryo shown in dorsal view.
Electroporation Using Converging Electric Field
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The electric field becomes non-uniform when electrodes of dissimilar size are used.
To generate a converging electric field a plate electrode was used for the cathode and
a 1mm rod electrode for the anode.
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This modification usually results in massive targeted unilateral transgene expression
in the neural tube. The setup is similar to that for electroporation using uniform
electric field but requires an additional pair of electrodes. DNA is injected into the
neural tube of the zebrafish embryo and positioned in the well of the electroporation
chamber as stated above. The difference lies in the delivery of the square electric
pulses. It is no longer delivered through the metal plates of the safety stand but via a
pair of electrodes.
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A pair of electrodes with L-shaped shaft configuration and an electrode length of
1mm (Genetrodes, model 516) were connected to the power output of the Electro
Square Porator ECM 830. The 3mm gap between the electrodes is maintained by the
Genetrode holder.
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In order to generate a converging electric field, the genetrode holder is positioned
such that the negative electrode touches the electrode plate of the electroporation
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chamber while the positive electrode is placed 1mm from the neural tube of the
embryo (Figure 5).
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19-24 hpf zebrafish embryos were optimally electroporated with 5 pulses (spaced
1sec apart) of 17 volts, with a pulse duration of 50msec each. After electroporation,
the embryos recovered in Petri dishes containing Hank’s buffer with PTU.
3mm
Figure 5. Electroporation using converging electric field. The electric field converges
along the anterior-posterior and dorso-ventral axis. The anterior neural tube of
electroporated embryo massively express GFP on the right hand side 1 day after
electroporation as observed under fluorescent microscope (d – diencephalon, m –
midbrain, hb – hindbrain).
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Materials
DNA Isolation
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QIAGEN Plasmid Isolation kit (Qiagen, Hilden, Germany).
Embryo Preparation
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Hank’s buffer
(137 mM NaCl, 5.4 mM KCl, 0.25mM Na2HPO4, 0.44 mM KH2PO4, 1.3mM CaCl2,
1.0 mM MgSO4, 4.2 mM NaHCO3, pH 7.2).
0.2 mM 1-phenyl-2-thiourea (PTU) (Sigma, St. Louis, MO, USA).
Tricaine (Sigma).
Microinjection
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Injection pipette (tip diameter, 0.02 mm) pulled from a glass microcapillary (o.d., 1
mm; i.d., 0.58mm; length, 100 mm) (Sutter Instruments, USA).
Blunt microinjection pipette [tip diameter, 0.05 mm;outer diameter (o.d.), 1 mm;
inner diameter (i.d.), 0.58 mm; length, 100 mm](Sutter Instruments, Novato, CA,
USA) used for embryo positioning.
Micromanipulator.
MPPI-2 pressure injection system (Applied Scientific Instrumentation,Eugene, OR,
USA).
Wedge-shape wells made in 1% agarose and covered by Hank’s buffer.
Petri dishes.
Dissection microscope SZ40 series (Olympus, Tokyo, Japan).
Electroporation
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Several shortened 0.4-cm electrode gap Gene Pulser® cuvettes (Bio-Rad
Laboratories,
Hercules, CA, USA).
1 pair of model 516 Genetrodes (BTX®; Genetronics, San Diego, CA, USA), part
number 10-002509-01.
Electro Square Porator ECM 830 (BTX®; Genetronics, San Diego, CA, USA).
Genetrode holder, gap 1-10mm (BTX®; Genetronics, San Diego, CA, USA).
Hank’s buffer (without PTU).
Screening of Electroporated Zebrafish Embryos
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Any stereomicroscope equipped with the UV lamp and GFP filter.
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Recipes
DNA – circular plasmid prepared using Qiagen midiprep or Qiagen endotoxin free kits or
CsCl grade.
Remember! Qiagen miniprep or phenol-chloroform prep quality DNA is unacceptable.
Hank's Stock Solutions:
Stock #1
8.0 g NaCl
0.4 g KCl
in 100 ml dd H2O
Stock #2
0.358 g Na2HPO4 Anhydrous
0.60 g KH2PO4
in 100 ml ddH2O
Stock #4
0.72 g CaCl2
in 50 ml ddH2O
Stock #5
1.23 g MgSO4x7H2O
in 50 ml dd H2O
Stock #6
0.35 g NaHCO3
10.0 ml dd H2O
Hank’s Buffer / Embryo Medium
1.0 ml Hank's Stock #1
0.1 ml Hank's Stock #2
1.0 ml Hank's Stock #4
95.9 ml dd H2O
1.0 ml Hank's Stock #5
1.0 ml fresh Hank's Stock #6
Use about 10 drops 1 M NaOH to pH 7.2
1% Hank’s Agarose
1% Hank’s agarose was made by adding 0.5g of molecular grade agarose to 50ml of
Hank’s buffer. The mixture was warmed in microwave oven until the agarose melt. The
molten agarose was added to the cut Gene Pulser® cuvette (Bio-Rad
Laboratories,Hercules, CA, USA) to make the electroporation chamber.
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Tricaine
Tricaine (3-amino benzoic acid ethylester, or ethyl m-aminobenzoate) is a powder (Sigma
Cat.# A-5040). It is also available as Finquel (Part No. C-FINQ-UE) from Argent
Chemical Laboratories, Inc. The tricaine solution for anesthetizing zebrafish embryos
was made by combining the following in a glass bottle:
400 mg tricaine powder
97.9 ml DD water
~2.1 ml 1 M Tris (pH 9).
Adjust pH to ~7.
This 20X stock solution is stored in the freezer and diluted 20 times with Hanks’ buffer
when used.
Contacts:
Cathleen Teh ([email protected])
Vladimir Korzh ([email protected])
References :
Teh, C., Chong, S.-W. and Korzh, V. (2003) DNA delivery into neural tube of zebrafish
embryo by electroporation. BioTechniques 35:950-954.
Teh, C., Parinov, S., Korzh, V. (2005) New ways to admire zebrafish: progress in
functional genomics research methodology. BioTechniques, 38, 897-906 (review).
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