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

Begin by using forceps to submerge orange macrocarrier biolistic discs in 100% ethanol, then, transfer the discs into a large Petri dish containing drierite, taking care that the discs do not touch the desiccant. Once dry, press the discs into ethanol-wiped silver disc holders, then, vortex a tube of gold beads, and aliquot 12 microliters of the bead solution into one 1.5-milliliter microcentrifuge tube per transformation.

Next, sequentially add 2 micrograms of DNA, 10 microliters of calcium chloride, and two microliters of spermidine free base into each tube of beads. Vortex each sample then, incubate the tubes at ambient temperature for 5 minutes, with occasional gentle flicking to resuspend the settled beads.

At the end of the incubation, pellet the DNA-coated gold beads, and carefully aspirate the supernatant. Slowly resuspend the beads completely in 600 microliters of 100% ethanol, then, pellet the cells again, this time resuspending the beads in 8 microliters of ethanol.

Now dispense each tube of beads onto the center of an individual biolistic disc in 1-centimeter-diameter circles. If a sufficient concentration of beads is present, a gold circle will be visible in the center of each disc once the beads are dry.

To operate the gene gun, first, turn on the vacuum pump, then turn the helium tank knob counterclockwise until a pressure of approximately 2200 PSI is reached on the pressure gauge. Next, flip the left red switch to turn on the gene gun, and adjust the flow rates for the vacuum and the vent so that the vacuum will reach 28 inches of mercury within 15 seconds. Confirm that the distance between the rupture disc and macrocarrier is approximately 3/8ths of an inch at this time as well.

When everything is in position, wipe down the entire chamber with 70% ethanol, and submerge the rupture discs in 100% ethanol. After drying the discs on a sterile surface, use a torque wrench to loosen the rupture disc holder, and insert a clean disc. Screw the rupture disc holder back into place, and turn it once to the right with the torque wrench to tighten it.

Next, submerge the mesh screens in 100% ethanol. Once dry, place a screen onto the white plastic mounting plate, and 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, then, place a YPD agar plate containing 1 molar sorbitol onto the bottom plate.

Now shut the chamber door and lock it into place, then, push and hold the middle red switch in the up position to engage the vacuum, and to allow it to reach 28 inches of mercury. Once the proper vacuum level is reached, and the middle switch has been moved to the down position hold down the right red switch to fire.

When the rupture disc pops, immediately release the fire button and push the middle red switch to the middle position in order to vent the chamber to 0 PSI. If the shot was successful, a gold ring will be visible on the plate. Clean out the rupture disc and macrocarrier disc debris, and turn off the gene gun, then, turn the helium tank knob clockwise to turn off the gas and turn off the vacuum pump.