Microinjection of DNA, RNA and tracer dyes into early fish embryos
Jochen Wittbrodt, EMBL Heidelberg, Germany
Abstract
Microinjection techniques are widely applied in developmental biology for the analysis of early developmental
processes such as gastrulation, neural induction and patterning or organogenesis. Microinjection experiments into
vertebrate embryos (e.g. mouse, frog, fish) allow to generate transgenic animals by injection of DNA [1, 2]; to interfere
with specific developmental processes by DNA [3], RNA [4] or morpholino oligo [5] injection, or to follow the
fate of individual cells by the injection of fluorescent lineage tracer dyes.
Introduction
Gene transfer technology in fish
has made a great gain in the last
de-cades. Zebrafish (Danio rerio)
and medaka (Oryzias latipes) are
well-establish model organisms used
in developmental biology research
for the analysis of early developmental
processes. Microinjection is
one of the leading methods for the
production of transgenic fish. In
this Application Note, we describe
the sample preparation and the
injection procedure which is performed
with the aid of an Eppendorf
Micromanipulator InjectMan® NI 2
under a standard dissecting microscope.
Experiments
Medaka and zebrafish matings are
set up as described [6, 7]. Embryos
are collected latest 20 minutes after
successful mating or as soon as
eggs are laid and fertilized. Single
embryos are transferred and aligned
into the trenches of an agarose mold
type injection plate [1] (Fig. 2) using
a Pasteur pipette (approximately 20
embryos/trench). Aiming for transient
assays or for the generation of
stable transgenic lines, the embryos
must be at the one-cell stage for
consistent results. Medaka embryos
may be injected in Yamamoto’s
embryo rearing medium, chilled to
4 °C to slow down development. It is
not necessary to remove the chorion
prior to microinjection, as it can be
easily penetrated with the injection
needle. However, embryos tend to
move inside the chorion, so each
embryo has to be oriented properly
just prior to the injection.
Probe preparation and loading
● All probes (DNA, RNA, dyes) are
centrifuged twice at full speed (min.
10,000 x g) for 5 min, 90 % of the
supernatant is transferred to new,
dust-free tubes.
● Probes are loaded from the back
into fire-polished injection needles (e.g.
Eppendorf Femtotips, restricted-use)
with Eppendorf Microloaders (2-5 µl).
1) DNA
For transgenesis, the meganuclease
system should be used [1, 2]. Plasmid
DNA is prepared and purified
using a high-purity plasmid preparation
kit. DNA concentration and purity
can be checked by spectrometry.
The ratio of A260/A280 should be
between 1.8-2.0. For transgenesis
experiments, DNA is co-injected with
the meganuclease (I-SceI). Due to
the low stability of the meganuclease,
aliquots of enzyme solution
should be prepared (e.g. 2 μl) upon
arrival and stored at -80 °C. The
microinjection solution should be
prepared shortly before injection and
kept on ice. Medaka and zebrafish
may be injected using an identical
compositionof injection solution:
DNA 10-20 ng/μl, I-SceI buffer 0.5x
(optional for Medaka: Yamamoto
buffer 0.5x), I-SceI enzyme 0.3 U/μl,
ddH2O ad 30 μl Results in Medaka
are improved by adding Yamamoto
buffer. For consistent results it is
crucial to inject into the cytoplasm
of the embryos and not into the yolk.
● 250.500 pl (approx. 1/6 of the cell
volume) of plasmid DNA at a concentration
of 10 μg/ml (transgenesis
experiments) to 50 μg/ml (mosaic
transient expression) are injected
into the cytoplasm of early
embryos.
2) RNA
● Capped mRNA is synthesized in vitro using an Ambion "mMessage
mMachine" kit.
● RNA is purified through standard
purification columns, precipitated
and resuspended in RNase-free
water.
● The RNA is injected in 1x Ringer´s
solution at concentrations of 50 µg/ml
up to 1 mg/ml (i.e. from 25 pg to 500
pg RNA per cell).
3) Tracer dyes
● Tracer dyes such as FITC-dextran
or rhodamine-dextran are injected at
a concentration of 1.5 % in 1x
Ringer´s solution.
Eppendorf workstation setup
Devices:
InjectMan® NI 2
FemtoJet® express
Universal Stand
Consumables:
Eppendorf Femtotip®
Eppendorf Microloader
Eppendorf Safe-Lock micro test tubes
Injection procedure
● For the Eppendorf FemtoJet® express
microinjector, the starting settings
are 80-100 hPa for compensation
pressure and 500-700 hPa for
injection pressure. The operating
mode is set to “manual“.
● The optimal injection time (to inject
15-20 % of the cell volume) is
established empirically.
● FemtoJet® express and InjectMan®
NI 2 are connected via the interface
cable. The Menu function “Synchron
Pressure“ is active. The needle is
brought down and the injection
pressure is triggered by pressing the
joystick button. Dye or DNA/RNA is
injected as long as the button is
pressed.
● Once the tip of the needle enters
the cytoplasm, up to 500 pl of injection
solution containing 105 to 107
molecules of DNA or RNA are injected.
● Injected embryos are transferred
to hatching solution and kept at
28 °C until hatching.
Solutions
fill up to a final volume of 100 ml
with distilled water; adjust pH to 7.3
Microinjector Femtojet® express
If your research demands injecting
volumes greater than 100 picoliters
and/or longer series at higher pressures
- increasingly seen in functional
genomics and developmental
biology applications - the FemtoJet®
express with its external pressure
supply delivers the precise and continuous
pressure required.
Literature
[1] C. Grabher, J. S. Joly, J. Wittbrodt (2004) Highly efficient zebrafish transgenesis mediated by the meganuclease
I-SceI. Methods Cell Biol 77: 381-401
[2] V. Thermes, C. Grabher, F. Ristoratore, F. Bourrat, A. Choulika, J. Wittbrodt, J. S. Joly (2002)
I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118: 91-8
[3] G. Oliver, F. Loosli, R. Köster, J. Wittbrodt, P. Gruss (1996) Ectopic lens induction in fish in response to the
murine homeobox gene Six3. Mechanisms of Development 60: 233-239
[4] F. Loosli, S. Winkler, J. Wittbrodt (1999) Six3 overexpression initiates the formation of ectopic retina. Genes and
Development 13: 649-654
[5] M. Carl, F. Loosli, J. Wittbrodt (2002) Six3 inactivation reveals its essential role for the formation and patterning
of the vertebrate eye. Development 129: 4057-4063
[6] M. Westerfield (1995) The zebrafish book. The University of Oregon Press, Eugene
[7] T. Yamamoto (1975) Medaka (Killifish), Biology and Strains. Keigaku Publishing Company, Tokyo
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