Schedl Lab

Washington University Genetics

Ballistic Transformation


Prepared by Omar Aranout, with contributions from Daimon Simmons, and Sudhir Nayak in Tim Schedl’s lab.

If you have comments, suggestions, corrections, or improvements please e-mail them to  Siqun Xu at sxu@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 “Creation of Low-Copy Integrated
Transgenic Lines in Caenorhabditis elegans
” (Genetics. 2001 Mar;157(3):1217-26) for more information about micro-particle bombardment. 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) 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.

  • Preparing plates with worms

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Worms are initially grown and maintained on 60mm NGM plates. A pool of approximately 40 plates should be maintained if you want to shoot two 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.

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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.

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See Supplement for protocol on egg plate prepartation.

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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 flotation protocol.

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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. Click here for a picture of unwashed worms collected from 10 egg powder plates.

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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.

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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. Click here for a picture of worms after 1 wash. Click here for a picture of worms after 2 washes.

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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.

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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.

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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.

  • Preparing the gold beads

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Weigh 15mg of 1 micro gold beads into a siliconized eppendorf tube. Using non-siliconized tubes also works fine.

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Add 1 ml of 70% EtOH

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Vortex for at least 5 minutes.
Vortexing by hand works better than by machine.

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Allow to settle for 15 minutes

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Spin 5 seconds at maximum speed in bench top microfuge. Aspirate supernatant carefully, some gold may be lost during aspiration.

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Add 1 mL of dH20;
vortex 1 min

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Allow to settle for 1
min

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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

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Add 250 microliters of 50% glycerol to bead pellet.

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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.

  • Loading the DNA onto the gold beads

The following quantities are giving per bombardment, and per 7 bombardments between parentheses (for the hepta-adaptor, use numbers between parentheses)

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Vortex beads in 50% glycerol for at least 5 minutes

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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.

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4 microliters /bombardment (28 microliters ) of 0.1M spermidine (stored at -20C)

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1 microliters /bombardment (7 microliters ) of DNA construct [concentration should be around 0.5 micrograms /microliters )

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10 microliters /bombardment (70 microliters ) 2.5M CaCl2

Vortex for 3 minutes after all additions are completed.

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Allow beads to settle for 1 min

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Spin 5 seconds in microfuge; remove supernatant using a pipetman to avoid disturbing the pellet

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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.

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Spin 5 seconds in microfuge, remove supernatant using a pipetman

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Resuspend the pellet, in 30 microliters /bombardment (210 microliters ) of 100% EtOH.

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Spin 5 seconds in microfuge, remove supernatant using a pipetman

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Resuspend the pellet in ~10 microliters /bombardment (~77 microliters ) of 100% EtOH.

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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.

  • Loading Macrocarriers

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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.

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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.

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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).

  • Post-bombardment care

Allow the worms to recover for 30-60 minutes after bombarding.

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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.

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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.

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Once the liquid is absorbed grow worms at 20C and check in 7-10 days for early transformants. Note: The worms usually will grow very poorly if incubated at > 23C.

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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.

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The rescue plates are frequently (~always) contaminated but if contamination from ‘scary’ 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.

  • Tim Schedl, Ph.D
    Department of Genetics
    Campus Box #8232
    Washington University School of Medicine
    4566 Scott Ave.
    St. Louis, MO 63110

    Contact

    Email: ts@genetics.wustl.edu
    PHONE: (314)362-6164 [lab]
    FAX: (314)362-7855