8.4.5 Culture of Transformed Host Cells

Steps in Culturing Transformed Host Cells

1. Inserting the Recombinant DNA into Host Cells:

• The recombinant DNA is inserted into host cells (e.g., bacteria, yeast, or mammalian cells) using a vector, such as a plasmid.
• Transformation techniques include:
• Electroporation: Applying an electrical field to increase the permeability of the cell membrane.
• Calcium ion treatment and heat shock: Facilitating DNA uptake by making the cell membrane more permeable.

2. Identifying Transformed Cells:

• Not all host cells take up the recombinant DNA, so identification is necessary:
• Marker genes: Plasmids often carry marker genes, such as antibiotic resistance genes, to help identify transformed cells.
• Selectable markers: Culturing cells on media containing antibiotics ensures only cells that have taken up the plasmid survive.

3. Culturing Transformed Cells:

• Transformed cells are placed in a nutrient-rich growth medium to allow rapid replication.
• Aerobic conditions are maintained to optimise bacterial or yeast growth.
• Mammalian cells may require a more specialised growth medium with growth factors and controlled temperature.

4. Expression of the Desired Gene:

• The recombinant DNA contains a promoter sequence to initiate transcription of the desired gene.
• Transformed cells produce the target protein or compound encoded by the recombinant DNA.

5. Harvesting the Product:

• The target product (e.g., protein or compound) is extracted and purified from the host cells or their growth medium.
• Purification methods vary based on the product (e.g., chromatography for proteins).

Applications of Transformed Host Cells

• Medical applications: Producing insulin, human growth hormone, or vaccines.
• Agriculture: Developing genetically modified crops with improved traits, such as pest resistance.
• Industry: Producing enzymes for detergents or food processing.

Key Considerations in Culturing Transformed Cells

• Sterility: Maintaining aseptic conditions to prevent contamination.
• Optimisation: Adjusting conditions like temperature, pH, and nutrient levels to maximise yield.
• Ethics: Addressing concerns around genetically modified organisms (GMOs) and their applications.