Gene Delivery Systems

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•  Recombinant DNA can be introduced by direct physical or chemical methods or indirectly by vectors

Gene delivery is the process of introducing foreign DNA into host cells for the purpose of genetic modification

  • This delivery may occur directly via physical or chemical methods or indirectly via the use of vectors

Physical Delivery

  • Electroporation – An electric charge is applied to cells, which permeabilises the membrane so that DNA can enter the cell
  • Microinjection – A fine needle is used to directly inject the recombinant DNA into a single host cell
  • Biolistics – Recombinant DNA is coated onto gold particles and fired into tissues by a biolistic device (‘gene gun’)

Chemical Delivery

  • Calcium chloride – Cells are treated in a calcium chloride solution prior to a heat shock (which permeabilises the membrane) 
  • Liposomes – Recombinant DNA is stored within a lipid bilayer vesicle (liposome), which fuses with the cell via endocytosis

Vector Delivery

  • Agrobacterium tumefaciens – A rod-shaped bacterium that can insert a recombinant plasmid into a host cell
  • Tobacco mosaic virus – A retrovirus that can insert genetic material into host cells (primarily tobacco plants)

Methods of Gene Delivery

gene delivery systems

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•  Recombinant DNA can be introduced into whole plants, leaf discs or protoplasts

Recombinant DNA can be introduced into whole plants, leaf discs or protoplasts (plant cells with the cell wall removed)

  • Introducing DNA into whole plants can allow for the targeted recombination of particular plant tissues
  • Recombinant leaf discs can be regenerated under laboratory conditions to form new transgenic plants
  • Protoplasts may take up DNA more easily (due to the absence of a cell wall) and can be regenerated into whole plants

When regenerating whole plants, groups of plant cells form a growing mass (callus) from which plant structures develop

  • Gene delivery systems may be more or less effective depending on the target (whole plant, leaf disc or protoplast)

Plant Transgenesis Techniques

plant transgenesis

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•  Recombinant DNA must be inserted into the plant cell and taken up by its chromosome or chloroplast DNA 

When recombinant DNA is inserted into a plant cell, it is either taken up into a chromosome or into chloroplast DNA

  • Alternatively, the recombinant DNA may remain as an independent construct within the cell (i.e. in an autonomous plasmid)

Integrating the target gene into chloroplast DNA offers certain advantages when developing genetically modified crops

  • Genes found in chloroplast DNA will not be present in pollen produced by transgenic plants (no chloroplasts in pollen grains)
  • This means the target gene cannot be accidentally released into wild plants via cross-pollination