Biolistic-Mediated Gene Transfer: A Technique to Deliver Gene of Interest in Target Cells via a Biolistic Gene Gun
Transcript
Begin with an optimum volume of gold beads – biologically inert and non-toxic gene delivery vehicles – in a microcentrifuge tube. Then, add cationic agents – spermidine and calcium chloride, followed by the desired DNA fragments to the gold beads. Gently flick the tube and incubate.
Calcium chloride binds to the DNA fragments and facilitates their coating over gold beads via spermidine residues. Next, centrifuge and remove the solvent. Resuspend the DNA-coated beads in ethanol. Gently pipette the beads on a sterile macrocarrier disc. Let it dry.
Set up a biolistic gene gun apparatus comprising a helium gas acceleration tube and a bombardment chamber with a rupture disc and a stopping screen in their respective slots. Next, invert the macrocarrier disc and load it below the rupture disc.
Thereafter, place the target host cells cultured in a suitable solid medium supplemented with sorbitol – an osmotic stabilizer to improve transformation efficiency. Once the setup is ready, fire the gene gun.
As helium travels down the acceleration tube, pressure continues to build until the rupture disc breaks, creating helium shock waves. These shock waves drive the macrocarrier disc carrying gold beads toward the stopping screen, which allows only the gold beads to pass.
The accelerated gold beads penetrate the host cell nucleus, integrating the desired DNA into the host cell genome.
