Hydra gene gun

Transient Transfection of Hydra by Particle Bombardment -- Protocol from Rob Steele's lab

Materials

• 1.0 M Spermidine

Use free base (Sigma-Aldrich S0266), make up in Milli-Q water, aliquot into 1.5 ml tubes, store at -20°C.

• 2.5 M CaCl2

Make up in Milli-Q water, filter sterilize, store at 4° C.

• Plasmid DNA

Prepare using a Qiagen kit. Do final elution with Milli-Q water. Store at -20°C.

• Gold particles

Prepare 1.0 µM particles according to Bio-Rad instructions (Page 21 in Users’ Manual). After resuspending at 60 mg/ml in 50% glycerol, aliquot in 1.5 ml tubes and store at -20°C.

Preparing DNA-coated gold particles:

1. Place DNA, spermidine, and gold particles on ice. Allow spermidine and DNA to thaw. Keep materials on ice when not in use.
2. Dilute spermidine to a 0.1 M concentration with Milli-Q water.
3. Vortex stock solution of gold particles for 5 minutes at top speed to resuspend and disrupt gold particles.
4. Immediately add 50 µl of gold particles into a 1.5 µl microfuge tube. Be sure to vortex in between transferring particles into more than one tube since continuous agitation of microcarriers is needed.
5. Add 5 µg of DNA into microfuge tube with gold particles.
6. Add Milli-Q water into the tube in an amount that the total volume of the DNA and the water is 5 µl.
7. Set pipettes to 50 µl for the CaCl2 and 20 µl for the spermidine now for prompt use later.
8. Place tube on vortexer with cap open and begin vortexing
9. While vortexing, add 50 µl of 2.5M CaCl2.
10. Immediately add 20 µl of 0.1 M spermidine and cap tube.
11. Continue vortexing at top speed for 3 minutes.
12. Allow microcarriers to settle for 1 minute.
13. Pellet microcariers by spinning in microcentrifuge for 5 seconds (by pressing “Short” button).
14. Remove the liquid and discard.
15. Add 140 µl of 70% ethanol.
16. Spin for 5 seconds in microcentrifuge.
17. Remove the liquid and discard.
18. Add 140 µl of 100% ethanol.
19. Spin for 5 seconds in microcentrifuge.
20. Remove the liquid and discard.
21. Add 48 µl of 100% ethanol.
22. Resuspend pellet by vigorously pipetting up and down with a P200.

Performing a Bombardment:

1. Place a layer of Drierite in a 60 mm dish and put a round piece of filter paper on top of the Drierite. Be sure to keep lid on dish so Drierite does not absorb water.
2. Place macrocarrier into the macrocarrier holder (make sure macrocarrier is smoothly set without wrinkles).
3. Pipette 6 µl of the gold solution onto the center of each of 5 macrocarriers. Spread remaining solution amongst the macrocarriers while being careful not to allow the solution to spread out further than the hole in the macrocarrier holder. Place each holder flatly into a dish with Drierite and replace lid.
4. Let the macrocarrier sit in dish for 10 minutes, allowing all the ethanol to evaporate. The DNA-coated macrocarriers must be used within 2 hours.
5. Turn on the power to the machine by pushing the power button at the upper left of the machine panel. The light should come on.
6. Turn on helium supply (which should go to 900 psi for firing at 650 psi). For a higher psi, set the helium tank to 200-psi above the psi of the rupture disc).
7. Attach hose to the house vacuum stop-cock and turn on the vacuum.
8. Remove the microcarrier launch assembly from the top slot and unscrew the cap.
9. Place a new stopping screen in proper position inside fixed nest of microcarrier launch assembly.
10. Place macrocarrier holder in microcarrier launch assembly (by flipping upside down into the fixed nest) and screw cap on.
11. Unscrew retaining cap and load a 650 psi rupture disc into the cap.
12. Screw back on and tighten the cap with Biolistic Torque Wrench until the metal rod touches the side of the plastic grip.
13. Place microcarrier launch assembly in top slot.
14. Ready the Hydra by placing them in a 35 mm plastic dish and removing the Hydra Medium. Then use an inoculating loop to push the Hydra into a pile with a diameter similar to the rupture disc. Remove the lower rack from the gene gun. Place the dish with the Hydra in the larger dish taped to the rack. Center the Hydra over the hole in the rack
15. Load the rack into the second slot from the top in the gene gun.
16. Close door and push down latch.
17. Pull a vacuum in the gene gun (by switching the middle switch all the way up to “VAC”) until maximum pressure is reached (24+ in. Hg).
18. Switch quickly to “HOLD” (all the way down) to retain the vacuum.
19. Push the “FIRE” button until you hear a pop (at 650 psi).
20. Switch the middle switch to “VENT” (middle position) and wait until pressure reaches 0 psi).
21. Open door and remove plate of Hydra.
22. Unload microcarrier launch assembly and unscrew cap. Discard spent macrocarrier. Place a new macrocarrier into the launch assembly and replace cap. If firing with different DNA, replace stopping screen.
23. Unscrew retaining cap and discard spent rupture disk. Load new rupture disc and replace cap.
24. Repeat process until all the microcarriers have been used.
25. When finished, remove contents from the chamber. Turn off helium supply. Pull a vacuum and fire the helium until the pressure gauge on the helium tank reaches 0. Vent the vacuum, turn it off and remove hose from the nozzle.
26. Turn off the power to the gene gun.