Biolistic Transformation in C. Elegans Using Unc-119 Rescue

Biolistic Transformation in C. elegans using unc-119 rescue

Dominique Rasoloson, Cynthia DeRenzo, Dolan Ghosh, Adrian Cuenca, Michael Stitzel, Geraldine Seydoux

Adapted from Shai Shaham, pers. communication, and Pratis et al., 2001

See last page for reagents

This protocol is for bombardment of 3 constructs

Day 1: Master plates

Starting from an almost starved unc-119 (ed3 or 4) plate, take 7 piles of adult unc-119 (worms form piles when starved) and spread on one small NNGM plate with OP50 bacteria spread over the plate. Repeat for another NNGM plate. Two small NNGM plates with worms are needed to bombard 3 constructs. Let the worms grow at 250C.

Day 8: Amplify worms to 12 plates

Resuspend the 2 plates in 8ml M9 buffer. Spin at low speed for 1min. Spread 8ml of supernatant (containing mainly L1’s) to 8 large NEP plates with NA22 bacteria spread evenly over the entire plate (use enriched peptone plates with NA22 bacteria throughout the protocol). Let the worms grow at 250C.

Day 15: Amplify worms to 60 plates

Resuspend 6 of the starved plates in 60ml M9 buffer. Plate 1ml of worm suspension per plate. Incubate the 60 plates in 250C for 2 days, or until they become young adults.

Day 17: DNA preparation

Weigh 35-50mg of 1uM gold beads (BioRad) into siliconized 1.5ml eppendorf tube.

Add 1ml 70% EtOH. Vortex 5mn. Soak for 15mn. Pellet and remove supernatant

Add 1ml sterile water. Vortex 1mn. Soak for 1mn. Pellet and remove supernatant

Resuspend in 500ul sterile 50% glycerol. This bead stock can be used for 2 weeks (or more and should be stored at 40C).

Vortex mix for 5mn. Remove immediately 100ul of bead suspension into each of 3 siliconized eppendorf tubes. Make sure to keep the gold beads in suspension. One tube/construct.

For each of 3 tubes, add in order while vortexing on medium speed:

30ul DNA from Miniprep

100ul 2.5M CaCl2

40ul 0.1M spermidine

Vortex 2mn. Soak for 1mn. Pellet and remove supernatant.

Add 280ul 70%EtOH. Flick tube to mix. Pellet and remove supernatant.

Add 280ul 100%EtOH. Flick tube to mix. Pellet and remove sup. Add 100ul 100%EtOH and resuspend by gently flicking tube. This is your prepped DNA. On the middle of each microcarrier, pipet 11ul of prepped DNA. Do this before prepping worms (below).1 tube (=1construct) will be used to bombard 2 plates.

Day 17: Worm preparation

Autoclave heptaadaptor, microcarrier holder, microcarriers, mesh stop-screens and forceps.

Wash the 60 plates with M9 into 50ml tubes (approx. 400ml/60plates). Spin at low speed for 1 minute; wash worms until M9 solution becomes clear and finally transfer worms to a 15ml tube. Pellet at room temperature. This should give you a pellet of 3-4 mls packed worms. Remove liquid and resuspend worms in M9 to a total volume of 12mls.

Using 5ml-pipette, suck 2ml of worms and add them onto the surface of a dry (1 week post bacterial spreading) enriched peptone plate. Add them drop wise starting at the center and then spiraling around until you reach the edge of the plate. Repeat until all worms are plated (6 plates).

Leave the covers off the plates to evaporate the liquid. This should take no more than 15mn.

While the plates dry, prepare the gene gun. We use the BioRad Biolistic PDS-1000/He particle delivery system with Hepta adaptor. This adaptor saves an enormous amount of time and effort. You can find the illustrations and definitions of the various jargon terms (microcarriers and microcarrier holder) in the BioRad manual. Read this manual to be familiar with the procedures described below.

Wipe down the bombardment chamber and chamber door with 70%EtOH. Unscrew the helium pressure gauge and immerse in isopropanol for 15mn or longer. Screw back the helium pressure gauge.

Open vaccum port, turn on gene gun. Open helium tank valve (check to make sure the He tank pressure is >2200psi). Perform a test bombardment by wetting a rupture disk (1500psi) in isopropanol, placing in retaining cap for hepta adaptor, and tightening onto bombardment chamber.

Close door. Pull vaccum to 27 In. of Hg. Once it reaches 27 In. of Hg press hold. Press Fire button and hold until disk ruptures. Release vacuum (Vent position), open door, unscrew retaining cap and discard the rupture disk.

Day 17: Bombardment

Vortex your DNA preparation at medium speed with cap closed. Stop and quickly transfer 11ul of beads onto the middle of a microcarrier. 1 tube (=1construct) will be used to bombard 2 plates. 14 microcarriers per one construct. Let Ethanol evaporate (this only takes a few minutes).

Place 7 microcarriers (with dried DNA-gold beads) onto the hepta adaptor microcarrier holder using forceps and tighten up with the special tool.

Place a rupture disk soaked in isopropanol in the retaining cap and tighten. Place hepta stopping screen and microcarrier holder in chamber as described in the manual. Place uncovered worm plate (taped to the sample holder using a rolled piece of adhesive tape to create double sided tape) onto second rung in bombardment chamber.

Pull vacuum to 27 In of Hg. Press Fire button until disk ruptures. Release vacuum (Vent position), and remove plate.

Repeat above for all 6 plates. [1 tube (=1construct) is used to bombard 2 plates].

Turn off vacuum. Close helium tank valve. Make sure no pressure is left in the line. Turn the gene gun power OFF.

Day 17: Plating worms

Resuspend bombarded worms off plates with M9 keeping each construct as a separate batch. Each plate will be resuspended with 13ml of M9. Plate 1ml of worms per plate. Should end up with 78 plates.

Once plates have dried, place them into 250C incubator or at RT in a plastic containers to avoid plates drying out.

Let sit for 4 or 5 weeks. Do not pick worms before that.

Day 38: Screening worms

Scan plates for WT worms.

Clone out (to individual plates) 3-5 worms per plate. You can reuse regular NNGM worms plates seeded with OP50 at this step. Place the plates at 250C for 4 days, and then examine the progeny for GFP expression.

Reagents

Biolistic PDS-1000/He particle delivery system Biorad 1652257

Hepta adapter, biolostic Biorad 1652225

Biolistic Microcarriers Biorad 1652335 (500ct)

1uM gold beads Biorad 1652263 (250mg)

Rupture discs Biorad 1652333 (1550psi)

Heptastop screen Biorad 1652226 (50ct)

Spermidine (tissue culture grade) Sigma S-4139 (5g)

Nystatin Sigma N1638 (100mls)

Enriched peptone plates with Nystatin (1L):

1.2 g Nacl2

20 g peptone

25g agar; water to 1L

Autoclave- cool to 550C, then add sterile:

1ml cholesterol (5mg/ml in EtOH)

1ml 1M MgSO4

25ml 1M potassium phosphate (pH6.0)

10mls of Nystatin suspension