Microinjection Equipment for Zebrafish Embryos
responses to query on bionet.organisms.zebrafish 4/95
QUERY:
I just received money to purchase an injection setup, and I have been
receiving requests for advice on same. I would appreciate some
suggestions on desirable pressure injectors, manipulators and minimal
scope needs (eg. inverted, dissecting, etc.).I know some of this
is described in "The Book" and "Monitor", but I would nevertheless be
interested in some additional comments/attitudes.
Dick Vogt (vogt@biol.sc.edu)
RESPONSE 1:
We do a lot of injections into early cleavage embryos. We use a dissection
scope (wild m3z, has a 10x ocular plus a 6.5-40 mag adjustment, i find this a
convenient mag range). We use a stage, which is a separate unit and was
slightly customized for us by our shop, to hold the injection trays, which
have the embryos in agarose trenches. A stage is not strictly necessary but I
think it significantly improves control. Narashige micromanipulator to hold
the needle. Injection is using a "picospritzer" nitrogen-driven microinjector
from Medical systems corp, Greenvale, N.Y., PLI-100; pretty expensive but
works well. All of the equipment except for the details of the injection
trays is identical to what is used for Xenopus in our lab. The whole system
is pretty easy to use and can be learned in a few days. I think if you have
in mind injections beyond the 16-32 cell stage you will probably need an
inverted scope. If you are further interested in our system, Randy wrote a
methods paper on it, which has a photo of the setup: Moon, R.T, and Christian,
J.L., "Microinjection and expression of synthetic mRNAs in Xenopus embryos"
Technique vol. 1 no. 2 1989, 76-89.
Anne Ungar
Randy Moon's lab
RESPONSE 2:
We use Medical System PLI-100 injectors, and a setup described in
Moon and Christian, Technique vol. 1, 1989. The only thing different is
using the injection trays used in the fish book. The primary advantages
of this injector are a)linear responses when changing settings to vary
doses and b) built in negative pressure to fill needles from a small drop
on parafilm, and c) a clearing feature in case the needle jams. We have
four of them, and have been using them for about 10 years with no major
drawbacks.
Hope all's well--Randy Moon
RESPONSE 3:
You haven't quite given enough info -- the setup depends on what you are
injecting, and in particular what age animal you are working with.
My main setup is a Nikon upright with a stage-mounted hydraulic
micromanipulator. For single-cell injections at late stages (anything
from late gastrula to embryos) we use this rig with a 40x water
immersion objective and DIC. You really need at least that level of
magnification if you want to pop into small, single cells accurately - we also
have a 2x optizoom on the scope to give us a little more mag.
For blastula injections, we can use the same apparatus, but we use a
much lower mag, 4 or 10x. Lately, though, we've found we can be a lot more
crude...we can inject animals on the dissecting scope, just holding the
electrode holder by hand and stabbing away. It's fast, cheap, and fairly
easy, although you can't have too much coffee prior to the experiment.
You haven't said what you'll be injecting. For most fluorescent dyes, we
iontophorese using a WPI current programmer. That box has a lot of nice
knobs and dials, but I think it is overpriced at $1500. Bill Trevarrow
told me he got the same functionality out of a 9V battery, a couple of
resistors, and some duct tape; it may be a matter of style.
We also do pressure injection, using a really nice box from ASI. We just
tap into house air, set a knob to give us a couple of psi for a few msec,
and use a footswitch to spritz away.
Paul Meyers
RESPONSE 4:
We are just going into mass production on a cute inexpensive pressure
injector. I'll send you some information.
Dave Brumbley / dave_asi@rain.com / Tel: (503)485-2284 / Fax: (503)485-0306