Microinjection of plasmid DNA or double-stranded RNA into Drosophila embryos
Reinhard Bauer, LIMES Institute, Laboratory of Molecular Developmental Biology, University of Bonn,
Meckenheimer Allee 169, Poppelsdorfer Schloss, D-53115 Bonn, Germany
Abstract
Microinjection of plasmid DNA and double-stranded RNA into Drosophila melanogaster embryos has been used
to manipulate gene expression in many different ways. The best-known application of microinjection techniques
is the generation of transgenic flies by P-element induced germline transformation. This Userguide describes the
set-up for fly embryo injections which we established in our laboratory to generate transgenic fly lines and
to inject double-stranded RNA for knockdown experiments.
Introduction
The introduction of DNA or RNA into embryos by microinjection
techniques is a very common method used
to manipulate gene expression in various model organisms.
In Drosophila melanogaster a P-element, which
is a natural transposon vector jumping in and out of the
fly genome at will, is used as a vehicle to deliver genes
of interest into the genome. The P-element is introduced
by microinjection into the germ line of Drosophila melanogaster where it will be integrated and stably inherited
by progeny. Based on this principle, key techniques have
been developed for disrupting gene expression in only a
subset of cells, and for expressing or silencing [1] genes
ectopically in cells of choice, such as the FLP-FRT system
of mosaic clone analysis [2, 3] and the binary GAL4
system [4].
Materials and methods
Injections into syncytial blastoderm-stage Drosophila embryos are done under an inverted microscope, using
an Eppendorf micromanipulator TransferMan® NK 2 and
an Eppendorf programmable microinjector FemtoJet®
express with external pressure supply (Fig. 1).
Instruments
● Inverted microscope Olympus CKX31 (Olympus, Germany)
● Micromanipulator TransferMan NK 2 (Eppendorf AG, Germany)
● Microinjector FemtoJet express (Eppendorf AG, Germany)
● Pipette puller model P-97 (Sutter Instruments, USA)
● Research pipette 0,5 –10 µl (Eppendorf AG, Germany)
● Centrifuge (e.g., Eppendorf Centrifuge 5424)
Materials
● Micropipette glass tubes; 100 mm long, 1 mm thick (Hilgenberg GmbH, Germany)
● Microloader pipette tip (Eppendorf AG, Germany)
● Cover slips 48 mm x 60 mm, 1 mm thick (Menzel Glaeser, Germany)
● Agar plates with fruit juice
● Small mesh basket for rinsing and dechorionation of embryos
● Double-sided tape (Scotch adhesive transfer tape, VWR, Germany)
● Halocarbon oil 700 (SIGMA)
● Embryo collection chambers
● Sodium hypochloride
● PBT (0.002% Triton X-100 in PBS) and deionized H2O in a squeeze bottle
● Silica gel for desiccation (Kraemer & Martin GmbH, Germany)
● RiboMaxTM Express Large Scale RNA Production system (Promega, USA)
● Plasmid purification kit (Qiagen, USA)
DNA preparation
DNA used for injection is prepared with a Qiagen plasmid
purification kit. For generation of transgenic fly lines an
injection solution with 3:1 ratio of plasmid of interest and
helper plasmid pp25.7D2-3 wc [5] was prepared and ethanol
precipitated. The pellet is dissolved in injection buffer
solution (0.1 mM Na phosphate pH 7.8, 5 mM KCl).
RNA preparation
Double stranded (ds) RNA fragments are generated using
the RiboMaxTM Express Large Scale RNA Production system.
Injections are performed as described by Kennerdell
and Carthew [6] with a final concentration of 0.5 µg/µl
dsRNA. Injections are performed with RNase-free needles
into the posterior domain of the embryo during the syncytial
blastoderm stage.
Preparation of the Drosophila embryos for injection
The flies are transferred into egg-laying chambers 2–3
days before the first injection to acclimatize them to these
conditions. The basic microinjection session follows this
rhythm: Let the females lay eggs for 20–40 minutes on
freshly yeasted apple juice agar plates. Change plates,
transfer all embryos with a paint brush to a mesh basket,
and wash thoroughly with a squeeze bottle of distilled
water. The chorion on the embryos is removed by covering
them for 2 minutes with 6% sodium hypochloride.
At the end of that time rinse very well with PBT, then H2O.
Dry embryos in the mesh basket by putting the basket
on a paper towel. Then pick up 40–60 embryos and
place them on the edge of a thin strip of agar from an
egg laying plate. The eggs should all face in the same
direction, with the anterior end facing out from the agar.
A cover slip with double-stick tape on it is lowered onto
the eggs. Press down firmly and place the cover slip in
the desiccating chamber. The embryos should now have
their posterior end facing the edge of the cover slip. Let
the embryos dry in a container with silica gel for 5–15
minutes. This time span should be adjusted, depending
on relative humidity, until the embryos can be injected
without leaking and without looking wrinkled or bag-like.
After desiccation, put halocarbon oil over the embryos
and place them on the microscope for injection.
Preparation of injection capillaries
For injections a 1 mm borosilicate capillary is pulled to
form an injection capillary, using a horizontal puller. The
settings are different for each machine and need to be
updated each time the heating filament is replaced or
a new type of capillaries is used. If the solution to be
injected does not flow from the tip when beginning the
injections, try to widen the tip of the capillary by touching
an embryo and moving the slide back and forth along the
y axis. Alternatively, break the tip by gently touching the
edge of the slide under the microscope. The break should
be small and subtle. Loading of the injection solution into
the capillary can be easily performed with a Microloader
pipette tip inserted through the back end.
Centrifuge all samples (DNA, RNA) twice at full speed
(12,000 x g) for 5 min. before loading an aliquot into the
capillary.
Injection procedure
Focus the embryos on the cover slip at one end of the
line. Put a filled capillary in the holder and bring the tip
of the needle as close as possible to the first embryo
to receive injection. Do this first manually, then use the
micromanipulator.
As the parameters largely depend on the quality of the
capillary, optimal settings for injection pressure (Pi),
compensation pressure (Pc), and injection time (Ti) of the
FemtoJet express microinjector can´t be specified.
If high-quality capillaries are used the compensation pressure
is usually sufficient for proper injections. In this case
the start settings for the Eppendorf FemtoJet express
microinjector are 1000-2000 hPa for compensation
pressure and 200 hPa for injection pressure, adapted
accordingly.
If medium-quality capillaries are used, the Pi and the
Pc are adjusted and the “manual” injection mode which
allows to determine the injection time individually is used.
The optimal injection time is established empirically.
Now, the capillary should be inserted quickly into the
center of the posterior end (Fig. 2a) of the embryo.
Continue to inject until a droplet can be seen diffusing
into the embryo. Move the embryo off the capillary
quickly (Fig. 2b).
While injecting, it is a good idea to use the “Clean” button
after every 5–6 embryos just to make sure the capillary
does not become clogged. If a clog cannot be removed
with the “Clean” function:
1) Push on the “Clean” button.
2) Move the stage back and forth quickly while pushing
the “clean” button.
3) Put the capillary into the end of an embryo and rip
through the membrane.
4) Carefully re-brake the capillary on the edge of the slide.
Finally, if the capillary continually becomes occluded, it
will have to be changed.
After one injection cycle is finished, place the slide in a
petri dish with apple juice agar and store the dish at 18 OC.
Scoop the larva after two days.
References
[1] Lee, Y., and Carthew, R., 2003. Making a better RNAi vector for Drosophila: use of intron spacers. Methods. 30, 322–329.
[2] Xu, T., Rubin, G., 1993. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 117, 1223-37.
[3] Golic K., Lindquist S., 1989. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.
Cell. 3, 499-509.
[4] Brand, A. H., and Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant
phenotypes. Development 118, 401-415.
[5] Rubin, G. M., Spradling, A. C., 1982. Genetic transformation of Drosophila with transposable element vectors. Science 218,
348-353
[6] Kennerdell J.R., Carthew R.W., 1998. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled
2 act in the wingless pathway. Cell. 23, 1017-26.
I would like to thank Sabine Büttner and Prof. Michael Hoch for their support.
back to top
|