The Helios Gene Gun is the apparatus used in our laboratory for biolistics. It uses pressurized helium gas to accelerate micron sized gold particles which are coated with plasmid DNA. Several factors are important to consider for effective biolistics. These are:
1. The amount of gold used per cartridge (individual bullet) ranges from 0.125 to 1.0 mg. The standard amount used in our laboratory is 0.5 mg/cartridge. The size of gold particles available from Bio-Rad are 0.6, 1.0, and 1.6 μm in diameter. We have found no difference in transfection efficiency between any of these sizes either in cell cultures or in organotypic slice explants. Our laboratory routinely uses 1.0 μm gold for all transfections.
2. The quantity of nucleic acid used for each transfection can vary over a range of 1.0 ng to 5.0 μg/cartridge. In any new experimental situation, we begin with 1.0 μg/cartridge. This 1.0 μg can be made up of single or multiple nucleic acid constructs. DNA transfection efficiencies in our laboratory are similar when using from 1.0μg and 0.1 μg/cartridge. Further dilution to 0.01 μg results in a noticeable decrease in efficiency.
3. The amount of DNA per milligram gold can range from 0.002 to 10.0 μg. A value of 2 is standard in our laboratory. Optimum transfection efficiencies as well as target specificities are greatly influenced by this value and must be determined empirically for each construct–target used. A table in the Helios Gene Gun Manual gives details for varying this parameter.
4. The pressure and distance to shoot at are key parameters, and these values will have to be determined experimentally for each target. For organotypic slice explants, we use between 120 to 180 psi and between 10 to 20 mm distance between the gun barrel and target. For cell lines and primary dissociated cell cultures, we use 120 psi and 17 to 29 mm.
tubes.
water or TE (10 mM Tris,1 mM EDTA, pH 8.0).
Branson 1210).
Biotech, Piscataway, NJ, USA).
Note: An unopened bottle of 100% EtOH must be used each day this procedure is used in order to prevent the absorption of water in EtOH from inhibiting the process. 1. Connect the N2 supply line to the Tubing Prep Station. Cut a 30-inch length of tubing. Connect a 10-mL syringe with adapter tubing to one end of the Gold-Coat tubing and flush the tubing with EtOH. Insert the tubing into the tubing support cylinder. Be sure that the tubing extends past the O-ring seal. Turn on the nitrogen and set the flow to 0.3 LPM. Purge the tubing for at least 15 minutes to dry the inside before loading the gold. 2. Place 25 mg of 1.0 μm gold into a 1.5- mL microfuge tube. 3. Add 100 μL of 0.05 M spermidine to the gold and vortex mix for 5 seconds. 4. Sonicate for 5 to 10 seconds. 5. Add 50 μg plasmid DNA in 100 μL or less total volume and vortex mix immediately. 6. While vortexing, add 100 μL 1 M CaCl2 dropwise. 7. After all CaCl2 is added, turn vortex mixer off and let tube stand for 10 minutes at room temperature. 8. Spin tube for 10 seconds in a microfuge to pellet the gold. 9. Aspirate off most of the liquid but leave approximately 20 μL. 10. Resuspend the pellet in the remaining supernatant by flicking the tube with your finger. Wash the pellet 3 times time, spin approximately 10 seconds between each wash and discard the supernatants, leaving 20 μL of the EtOH supernatant. 11. Add 8.75 μL of 20 mg/mL PVP (in EtOH) to a 15-mL screw cap tube. 12. Add 3.491 mL of 100% EtOH to the 15-mL screw cap tube and mix. 13. Transfer the pellet from step 11 in 200 μL of the PVP/EtOH solution to the 15-mL tube. 14. Wash the microfuge tube with 200 μL PVP/EtOH to suspend any remaining gold and transfer to the 15-mL tube. 15. Vortex mix the gold suspension to break up any clumps and then sonicate briefly to break up any remaining clumps. 16. Invert the tube continuously to keep the gold from settling in the 15-mL tube. 17. Remove the cap of the 15-mL tube. Using a 10-mL syringe with adapter tubing on the end, draw the gold suspension all the way into the Gold-Coat tubing. Remove the tubing from the suspension and continue drawing the suspension into the tubing in order to leave an inch of airspace at the end. 18. Bring the tubing to a horizontal position and slide it into the tubing support cylinder of the Tubing Prep Station. Make sure it goes through O-ring. 19. Let stand for 3 minutes to settle the gold particles. Detach the adapter tubing from the syringe and attach it to the tubing on the peristaltic pump. Remove ethanol at the rate of 0.7 inches/ seconds. 20. Detach the adapter tubing and rotate the Gold-Coat tubing in the support cylinder 180° and let sit for 5 seconds. 21. Turn Tubing Prep Station on to start rotating the tubing. 22. Rotate the tubing for 20 seconds; then purge with N2 at 0.3 LPM. 23. Continue drying the tubing while rotating for 5 minutes. 24. Turn off the motor on the Tubing Prep Station. Close the valve on the flowmeter. Remove the tubing from the tubing support cylinder. 25. Inspect the tubing for blank spots and cut them out with a razor blade. 26. Place a desiccant pellet into a 20-mL scintillation vial and place the vial into the bottom of the tubing cutter. 27. Insert one end of the tubing into the tubing cutter and cut tubing into cartridges. 28. If not using the cartridges immediately, seal the lid of the scintillation vial with parafilm and store at 4°C in a closed chamber with desiccant. Biolistic Transfection of Organotypic Slice Explant Cultures 1. Sterilize the barrel liner and cartridge holder with 70% EtOH. 2. Attach the barrel liner to the Gene Gun. 3. Load cartridges into the cartridge holder (leaving at least one chamber empty) and place in the Gene Gun. 4. Connect the helium hose line to the regulator and Gene Gun. 5. Set the pressure on the regulator. For organotypic cultures we use 120 to 180 psi (exact value is determined empirically). 6. To adjust the pressure in the regulator, keep one cartridge holder position empty. Using this empty position, test the shooting pressure. Adjust if necessary. 7. Cock the cartridge holder to load a cartridge. 8. Position the barrel liner over the target at 10 to 20 mm and shoot the target. 9. After the biolistic run is completed, turn helium supply off and bleed the line. Disconnect the hose line and remove the cartridge holder from the Gene Gun. 10. Place slice cultures into fresh medium. 11. Culture as described above for 4 to 5 days (optimal duration for immunohistochemical assays).
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