Microparticle
Bombardment Protocol
Protocol by Omar Aranout, Daimon Simmons, and Sudhir
Nayak
Prepared by Omar Aranout,
with contributions from Daimon Simmons, and Sudhir Nayak in Tim Schedls lab.
If you have comments, suggestions,
corrections, or improvements please e-mail them to Sudhir Nayak at sudhir2@genetics.wustl.edu. Feel free to cut out all the detail you don't want
and make your own abridged version.
This protocol is based on
the one developed by the Austin Lab. See ÒCreation of Low-Copy Integrated
Transgenic Lines in Caenorhabditis elegansÓ (Genetics. 2001 Mar;157(3):1217-26) for more information about
microparticle bombardement. Aspects
of this protocol use information from the following: Gene. 1999 Mar
18;229(1-2):31-5 and Nucleic Acids Res. 2004 Feb 24;32(4):e40. Suggestions
offered by Mike Nonet and David Pilgrim were also incorporated into the
protocol. We use unc-119(ed3)(ed3) worms, and an UNC-119(+) rescuing vector (MM016, Austin Lab). Transformants
can be recognized easily by initially screening for non-unc (wild-type)
movement.
See Supplement for alternative to using a PDS-1000, egg plate
generation, sucrose floatation, egg plates alternatives and general
troubleshooting information.
o
Worms are initially
grown and maintained on 60mm NGM plates. A pool of approximately 40 plates should be maintained if you want to
shoot 2 times a week. The easiest
way to maintain a large pool of 60mm plates is wash off one nearly starved 60mm
plate to 10 new 60mm plates. If
care is taken to keep this sterile/clean then contamination is minimal and
rarely widespread. We maintain
5-10 unc-119(ed3) plates by
traditional picking just in case.
o
From 60mm plates worms
usually ready to be transferred to 100mm egg plates every 5-7 days at 20C. We
usually use a transfer ratio of between 1:1 and 5:1 60mm plates to 100mm egg
plates depending on the density of the 60mm plates.
o
See Supplement for
protocol on egg plate prepartation.
o
Once transferred, worms
on the 100mm egg plates are ready for bombardment within 3-7 days. Between
10-20 large plates are required per bombardment (0.5-2ml of packed worms). The variability is extremely high and we have not
been able to normalize it. If you
plan on doing a sucrose float then it may be better to use 20 100mm plates, see
Supplement for sucrose floatation protocol.
o
Once worms on 100mm
plates are ready (mostly adults) wash with M9 salts. A picture of an "ideal" egg powder plate where worms have started clearing the bacteria after 4 days is here. Approximately 10-20ml of M9 is enough for 10 plates. Repeat wash procedure with 10-20ml
of M9 to get any remaining worms off the plates. Pool the wash liquid in a 50ml conical and allow to
settle for 5-10 minutes. You can
also spin the worms down at 500-1000g for 1 min. It is important to go back and check the 60mm plates
to make sure you have washed the worms off efficiently.
o
The major barriers to
getting the worms off are bacteria that have grown too dense or there is too
much egg. One solution is to use
less egg when you plate the worms or you can use a higher transfer ratio so the
worms eat most of the extra bacteria. If the egg plates are not producing enough worms you can also use 10x
concentrated OP50 and it works just as well (but it's a little more work), see Supplement.
o
Remove supernatant and
wash with M9 salts 2X, allowing settling for 5-10 minutes each time. If the
worms required extra cleaning (settled worms have chunks floating around with
them), you will need to do sucrose floatation see Supplement.
o
Place one unseeded
large plate in laminar flow hood with the lid off to air-dry for at 30-60
minutes, and put on ice. The
purpose of this is to dry the plate thoroughly so that when you put worms on it
to shoot the transfer liquid is absorbed quickly.
o
We use a hepta-adaptor
with the Biolistic PDS-1000/He equipment. With the hepta adaptor 1-2ml of clean
worms are required. Break the tip of a Pasteur pipette to create a wider bore,
and flame to smooth the edges. Use pipette to transfer worms to the dried/iced
large plate, spreading worms in a monolayer. The best way to spread the worms
is to start at the middle of the plate and proceed in a spiral fashion around
the plate.
o
Once on the plate, the
worms need to stay on ice until the bombardment. If the plate warms up then the worms will form piles
instead of a monolayer, which greatly reduced the number of worms exposed to
the gold beads during shooting.
o
Weigh 15mg of 1 micro
gold beads into a siliconized eppendorf tube. Using non-siliconized tubes also works fine.
o
Add 1 ml of 70% EtOH
o
Vortex for at least 5 minutes.
Vortexing by hand works better than
by machine.
o
Allow to settle for 15
minutes
o
Spin 5 seconds at
maximum speed in bench top microfuge. Aspirate supernatant carefully, some gold
may be lost during aspiration.
|
o
Add 1 mL of dH20;
vortex 1 min o
Allow to settle for 1
min o Spin 5 seconds in microfuge. Aspirate supernatant carefully. Note: pellet will be loose and there will be a streak of gold beads down the side of the tube). |
Repeat 3X |
o
Add 250 microliters of 50% glycerol to bead pellet.
o
Store at room temp.
Once
prepared, the beads will be enough for about 25 bombardements, and can be
stored at room temp. We have used
the same beads up to 1 month with no apparent change in efficiency.
The
following quantities are giving per bombardment, and per 7 bombardments between
parentheses (for the hepta-adaptor, use numbers between parentheses)
o
Vortex beads in 50%
glycerol for at least 5 minutes
o
Aliquot 10 microliters /bombardment (70 microliters ) into a siliconized eppendorf tube. Do this quickly
before the beads have a chance to settle. Start vortexing beads.
Make the following additions
in the order given. You will have to stop vortexing momentarily to make
the additions, do so quickly so that the beads do not settle. Vortex 1 min
between each addition. Using a
vortexer without a tube adapter (i.e. manually) seems to work better.
o
4 microliters /bombardment (28 microliters ) of 0.1M spermidine (stored at -20¡)
o
1 microliters /bombardment (7 microliters ) of DNA construct [concentration should be around
0.5 micrograms /microliters )
o
10 microliters /bombardment (70 microliters ) 2.5M CaCl2
Vortex for 3 minutes after
all additions are completed.
o
Allow beads to settle
for 1 min
o
Spin 5 seconds in
microfuge; remove supernatant using a pipetman to avoid disturbing the pellet
o
Resuspend the pellet, by
racking the eppendorf tube across the length of a rack several times, in 30 ml/bombardment (210 microliters ) of 70% EtOH.
o
Spin 5 seconds in
microfuge, remove supernatant using a pipetman
o
Resuspend the pellet,
in 30 microliters /bombardment (210 microliters ) of 100% EtOH.
o
Spin 5 seconds in
microfuge, remove supernatant using a pipetman
o
Resuspend the pellet in
~10 microliters /bombardment (~77 microliters ) of 100% EtOH.
o
Vortex for at least 3
minutes, continue to resuspend by racking and vortexing until loaded on the
macrocarriers. While loading, continue to resuspend by pipetting up and down.
Load 1ml on a microscope slide to
make sure you have mostly single particles of gold. Some clumps are OK but you don't want too many
because they just kill worms.
o
The hepta-adaptor and the
macrocarriers should always be handled with gloves. The macrocarriers may be
cleaned by dipping into iso-propanol and blotting with Kim-Wipes. Insert one
macrocarrier into a slot in the holder (seven slots total), and secure in place
using a pipetman tip.
o
Make sure that the
beads are fully resuspended, and continue to do so by pipetting up and down as
you load the macrocarriers. Load ~10 microliters of suspended beads in
the center of the macrocarrier. Note: any beads outside the
hole in the holder will not be delivered to the worms.
o
The hepta adaptor must
now be assembled and the worms can be shot. It is not necessary to wait for the
beads to dry completely. Note: We
use 1350 psi rupture disks.
o A (very useful) interactive user guide detailing the
assembly of the hepta-adaptor and using the PDS-1000 system can be found at the
bio-rad website (http://www.biorad.com).
Allow
the worms to recover for 30-60 minutes after bombarding.
o
During recovery dry 10 100mm
NGM plates seeded with OP50 in the laminar flow hood with the lids off. You can also used unseeded plates but worms sometimes
starve too quickly. Usually there
is enough carryover of OP50 and other bacteria that the worms can eat enough to
get the first generation going.
o
Wash off the bombarded
plate with M9 salts (5 ml) and transfer to the 10 100mm plates that should be a
little dry so that the transfer liquid absorbs quickly.
o
Once the liquid is
absorbed grow worms at 20¡
and check in 7-10 days for early transformants. Note: The worms usually will grow very poorly if
incubated at > 23¡.
o
Mark plates with
transformants, and continue to screen/pick worms for one more week. If you have a marker for germ line expression (like
pie-1::GFP) you can pick the early transformants as early as 4 days. If you don't then its best to wait at least 10-14
days before picking anything.
o
The rescue plates are
frequently (~always) contaminated but if contamination from ÒscaryÓ bacteria is
a problem then the hepta adaptor and its associated metal parts can be
autoclaved and inside of the PDS-1000 can be sprayed thoroughly with 70%
ethanol and wiped clean. If the
source of the contamination is the helium tank or its associated hoses, valves,
or regulator then there not much you can do. Fortunately, most contamination seems to be OK and
not much to worry about.
