Botany · Ch 4 — Principles and Processes of Biotechnology
Direct or Vectorless Gene Transfer
Direct or Vectorless Gene Transfer
Direct gene transfer methods share one defining feature: none of them uses a biological vector at all. Instead, the foreign gene is forced into the host plant cell by purely physical or chemical means. Chemical-mediated transfer uses compounds such as polyethylene glycol (PEG) or dextran sulphate to induce plant protoplasts to take up DNA directly from the surrounding solution. Microinjection is exactly what it sounds like: DNA is physically injected straight into a cell's nucleus using an extremely fine glass needle or micropipette, with the protoplast held steady either on an agarose-coated microscope slide or under gentle suction from a holding pipette. Electroporation applies a brief pulse of high voltage across protoplasts, cells or tissue, which transiently opens pores in the plasma membrane large enough for foreign DNA to diffuse through before the membrane reseals. Liposome-mediated transfer (lipofection) encapsulates the DNA inside artificial phospholipid vesicles (liposomes), which is genuinely advantageous compared to injecting bare DNA, because the lipid coat physically shields the DNA from the vacuole's acidic pH and its protease enzymes until the liposome fuses with the vacuole's tonoplast membrane and releases its cargo. Finally, biolistics (the 'gene gun', microprojectile gun, or shotgun method) coats microscopic gold or tungsten particles, only 1-3 micrometres across, with the DNA to be delivered, the …
What this figure shows. A three-stage schematic of a cell membrane (shown as a ring of + and − charges) before, during and after an electric-field pulse: 'Before Pulse' shows the membrane intact with its normal charge distribution; 'During E-field' shows the electric field inducing a voltage across the membrane, opening transient pores through which genes/drugs are introduced into the cell; 'After Pulse' shows the membrane resealed with the gene/dru …
What this figure shows. A stepwise diagram of lipofection: a liposome (containing DNA) approaches and contacts the cell membrane, forming a lipoplex that is taken up into the cell by endocytosis; the resulting endosome undergoes lipid mixing and endosomal maturation; the DNA-carrying lipoplex escapes from the endosome (avoiding degradation in the lysosome), after which the DNA fragments free themselves from the liposome and travel into the nucleus, delivering …
What this figure shows. A cutaway diagram of a biolistic particle gun ('gene gun'): a plastic disc coated with DNA-coated gold particles is loaded into the gun barrel and fired; the disc itself is stopped by a screen partway down the barrel, but the DNA-coated gold particles it was carrying continue travelling through the screen and bombard the target plant cells positioned beyond it, delivering the foreign DNA …