Biolistic-Mediated Gene Transfer: A Technique to Deliver Gene of Interest in Target Cells via a Biolistic Gene Gun

プロトコル

1. DNA Preparation

1. Prepare orange macrocarrier biolistic discs by submerging them in 100% ethanol using forceps. Place discs into a large petri dish containing drierite to dry (make sure the drierite does not touch the discs).
2. Once dry, press the macrocarrier discs into the silver disc holders (previously wiped down with 100% ethanol).
3. Vortex gold microcarrier beads suspended in 100% ethanol and aliquot 12 µl into a 1.5 ml microcentrifuge tube, one tube per transformation.
4. Add to each tube in order: 2 µg of DNA (preferably 2 µl of 1 µg/µl of DNA), 10 μl 2.5 M CaCl₂, and 2 μl 1 M spermidine free base.
5. Set up a negative control as in step 1.4 but with no DNA.
6. Vortex each tube and incubate at ambient temperature for 5 min. Gently flick each tube occasionally to resuspend the settled beads during this incubation.
7. Spin tubes at 225 x g for 30 sec to pellet the DNA-coated gold beads. Carefully remove the supernatant (by pipetting or aspiration) and discard.
8. Resuspend beads completely in 600 µl of 100% ethanol by slowly pipetting up and down.
9. Spin tubes at 225 x g for 30 sec to pellet the beads without packing. Carefully remove and discard the supernatant.
10. Resuspend the DNA-coated gold beads in 8 µl of 100% ethanol by slowly pipetting up and down.
11. Pipette the DNA-coated gold beads onto the center of the biolistic disc in a 1 cm diameter and allow to dry.      
    NOTE: A dried gold circle visible on the center of the biolistic disc indicates that a sufficient concentration of gold beads is present.
    NOTE: The macrocarrier discs loaded with DNA-coated gold beads are now ready for use with the gene gun.

2. Operating the Gene Gun

1. Turn on the vacuum pump.
2. Turn on the helium gas by turning the knob counterclockwise until a pressure of approximately 2,200 psi is reached on the pressure gauge.
3. Turn on the gene gun by flipping the red switch on the left.
4. Be sure that the flow rates for the vacuum and the vent are adjusted so the vacuum will reach 28 inches Hg within 15 sec.
5. Be sure the distance between the rupture disc and macrocarrier is approximately 3/8 inch.
6. Clean the entire chamber by wiping down with ethanol.
7. Submerge the rupture discs in 100% ethanol. Allow to dry on a sterile surface (e.g., Petri dish).
8. Use a torque wrench to loosen the rupture disc holder. Insert a clean rupture disc into the holder. Screw the rupture disc holder back into place and tighten with torque wrench by turning it once to the right.
   NOTE: Rupture discs will be replaced following each shoot.
9. Submerge the mesh screens in 100% ethanol. Allow to dry on a sterile surface (e.g., Petri dish).
10. Once dry, place a washed mesh screen on the white plastic mounting plate. Place the macrocarrier disc holder DNA side down into the disc chamber. Screw on the silver cap, and place the mounting plate in the highest slot. 
    NOTE: The mesh screen will be replaced after each shoot.
11. Place a YPD agar plate containing 1 M sorbitol on the bottom plate.
12. Shut chamber door and lock into place.
13. Push and hold the middle red switch up to engage vacuum and allow the vacuum to reach 28 inches Hg. Once proper vacuum level is reached, move this switch to the down position. When ready, hold down the red switch on the right to fire. When the rupture disc pops, immediately release the fire button and push the middle red switch to the middle position to vent the chamber to 0 psi.
14. Clean out rupture disc debris and turn off the gene gun. Then turn off the helium gas by turning the knob clockwise, and finally, turn off the vacuum pump.

資料

Name	Company	Catalog Number	Comments
0.6 μm gold beads	Bio-Rad	165-2262	http://www.bio-rad.com
Spermadine-free base	Sigma- Aldrich	S0266	https://www.sigmaaldrich.com
1350 psi Rupture Discs	Bio-Rad	165-2330	http://www.bio-rad.com
Stopping Screens	Bio-Rad	165-2336	http://www.bio-rad.com
Macrocarriers discs	Bio-Rad	165-2335	http://www.bio-rad.com
PDS-1000/He System	Bio-Rad	165-2257	http://www.bio-rad.com
Microfuge 18 Centrifuge	Beckman Coulter	F241.5P	www.beckmancoulter.com