Genetic Transformation Methods

Genetic Transformation Methods

• Genetic transformation involves transferring DNA sequences from one organism to another.
• The transferred DNA needs to be integrated into the recipient organism's DNA (nucleus, mitochondrion, or plastid).
• The transferred DNA sequences can come from the same or different species.
• Crossbreeding is often used when direct transfer is not possible due to sterility or lack of a desired gene.
• Various methods exist for transferring genes, known as transformation or transgenesis, in plants and animals.
• The recipient cell must be competent (able to accept DNA).

Comparison of Genetic Transfer Methods

• Conventional Breeding: Involves successive crosses of related organisms to enhance a desired genetic element.
• Transgenesis: Involves transfer of genes from unrelated organisms.
• Cisgenesis: Involves transfer of genes from a closely related organism.

Techniques in Plant Transformation

• Physical Methods (Direct):

  • Microinjection: Direct injection of DNA into cells.
  • Pressure: Using pressure to deliver DNA.
  • Biolistics (gene gun): Bombarding cells with DNA-coated particles.
  • Electroporation: Using electric pulses to create temporary pores in cell membranes for DNA entry.
  • Microinjection: Injecting DNA using fine needles.
  • Silica/carbon fibers: Using special fibers to deliver DNA.
  • Laser mediated: Using lasers to deliver DNA.
  • SAT (not detailed in the text provided)

• Chemical Methods (Direct):

  • DEAE-dextran: A polymer used to facilitate DNA uptake.
  • Calcium phosphate: A method to introduce DNA into cells.
  • Artificial lipids: Used to deliver DNA into cells.
  • Proteins: Proteins acting as carriers for DNA.
  • Dendrimers: Tree-like molecules used for DNA delivery.

• Biological Methods (Indirect):

  • Agrobacterium-mediated transformation: Uses the soil bacterium Agrobacterium tumefaciens to transfer genes into plant cells via the Ti plasmid.
    • Agrobacterium is a natural genetic engineer.
    • The Ti plasmid is a plasmid of Agrobacterium tumefaciens that plays a crucial role in the transfer process.
  • Rhizogenes (not detailed in the text provided).
  • Virus mediated (not detailed in the text provided).

• In Planta Methods:

  • Meristem transformation: Using plant meristems to produce new plants containing new traits.
  • Floral dip method: A method to transfer DNA genes using plant flowers.
  • Pollen transformation: A method to introduce foreign DNA in plant pollen.

Techniques in Animal Transformation

• Transfer of Genes to Fertilized Eggs/Embryos:
  • Whole nuclei transfer: Transfer of nuclei from a donor cell into an enucleated egg cell.
  • Whole chromosome transfer: Transfer of whole chromosomes into an egg cell.
• Transfer of Genes into Cultured Cells:
  • Coprecipitation: Gene transfer using calcium phosphate.
  • Electroporation: Gene transfer using electric pulses to temporarily open cell membranes.
  • Particle guns (gold or tungsten particles): Gene transfer using the acceleration of particles coated in DNA.
  • Microinjection: Direct injection of DNA into cells using microneedles.
  • Liposomes/sphaeroplasts: Artificial vesicles used to carry DNA into cells.
• Microinjection is commonly used to deliver DNA segments into fertilized eggs
• Agrobacterium is NOT used in animal transformation (direct transfer).

Additional Notes

• Gene gun (biolistic): A device used to deliver DNA, RNA or proteins into cells through high-speed projectiles.
• Electroporation: An electric pulse transiently creating holes in cell membranes to enhance DNA uptake (commonly used for plant protoplasts).
• Whisker-mediated transformation: Whisker crystals used to introduce DNA into plants.
• Chemofusion: Protoplast fusion achieved by treatment with chemicals (like PEG). Fusion and regeneration are common steps involved.