Applications of Genetic Engineering: Pharmaceuticals, Agriculture, and Biotechnology

Course Introduction and Administrative Details

• The lecturer for this session is Dr. Tibeda.
• This session marks the final class of the module, following the principles and techniques of genetic engineering discussed in the previous session (Monday).
• The focus of today's lecture is the application of genetic engineering in pharmaceutical, agricultural, industrial, and food industries.
• Exam Information:     - The examination is scheduled for August $25$, $2026$.     - The goal for students is to secure a place at Ditto Hall through their performance.     - Dr. Tibeda is available for consultations regarding exam preparation; one student has already requested a session for the day of the lecture.     - Contact details for Dr. Tibeda can be obtained from the class representative.

Pharmaceutical Applications: Insulin Production

• Significance in South Africa: South Africa has a high diabetic burden, making the public sector distribution of insulin a major operation alongside treatment for blood pressure.
• The Role of Insulin:     - It is an anabolic hormone produced by the pancreas, specifically the beta cells ($\beta$-cells) located in the Islands of Langerhans.     - It regulates blood glucose by allowing sugar to be absorbed into body cells from the bloodstream.     - Process of Secretion: When blood glucose levels are high, the Islands of Langerhans sense the increase and signal the secretion of insulin.
• Molecular Structure:     - Insulin is a small protein consisting of a total of $51$ amino acids.     - It is composed of two chains: Chain A ($21$ amino acids) and Chain B ($30$ amino acids).     - These chains are joined by disulfide bonds ($S-S$ bonds).     - An internal disulfide bond within Chain A is critical for making the insulin active; breaking these bonds renders the insulin inactive.
• Physiological Effects:     - It communicates with the liver, muscles, and fat cells to take up glucose.     - Glycogenesis: The process of storing glucose in the form of glycogen in muscles and the liver.     - Lipogenesis: The storage of fat in the body.
• Diabetes Mellitus:     - "Mellitus" means honey, referring to the sweetness of sugar in the urine.     - Hyperglycemia: A condition defined by excessive sugar in the blood, which can hinder wound healing.     - Type 1 Diabetes: An autoimmune condition where the body mistakenly destroys its own beta cells, leading to little or no insulin production. Patients are insulin-dependent and require daily doses.     - Type 2 Diabetes: Characterized by "insulin resistance," where body cells (liver, muscle, fat) do not respond to the insulin produced.     - Hyperinsulinemia: High levels of insulin in the body as the pancreas overworks to compensate for resistance.     - Progression of Type 2: Eventually, beta cells become "worn out" and stop producing insulin. Initial treatment focuses on medication to improve sensitivity, but patients may eventually require direct insulin injections.
• Recombinant DNA Production of Insulin:     - Traditional Method: Historically, insulin was harvested from the pancreases of cows (bovine) or pigs (porcine). This was expensive, required massive amounts of animal tissue, and often caused allergies due to foreign protein structures.     - E. coli Method:         - Initially, separate DNA sequences for Chain A and Chain B were created.         - Because E. coli (a prokaryote) cannot properly fold or glycosylate complex eukaryotic proteins, the chains were produced in two separate batches of E. coli.         - The harvested chains were then mixed in a test tube under oxidizing conditions to form the necessary disulfide bridges.         - Vectors used includes the lacZ gene and $\beta$-galactosidase to prevent the bacterial enzymes from degrading the insulin chains.     - Yeast Method (Saccharomyces cerevisiae):         - Being a eukaryotic host, yeast possesses an endoplasmic reticulum capable of folding, processing, and packaging proteins.         - A synthetic gene linking Chain A and Chain B is inserted into a yeast vector (shuttle vector).         - The yeast secretes a folded "proinsulin" precursor directly into the surrounding liquid, eliminating the need for separate chain production and mixing steps.

Pharmaceutical Applications: Human Growth Hormone (HGH)

• Function: Produced by the pituitary gland (located under the neck), it stimulates the liver to produce an effector called $IGF-1$ (Insulin-like Growth Factor $1$).
• Physiological Role: Essential for bone and cartilage growth, increasing protein synthesis, and mobilizing fatty acids.
• Pathology: A deficiency in HGH leads to dwarfism or stunted growth.
• Historical Production: Previously extracted from the pituitary glands of human cadavers.     - This was inefficient (requiring $50$ to $100$ glands for a one-year supply for one child).     - It was discontinued due to the risk of transmitting fatal neurological/brain diseases (prions).
• Recombinant HGH Production:     - Produced in E. coli using $cDNA$ (complementary DNA).     - $cDNA$ is used because prokaryotes cannot remove introns from eukaryotic mRNA. The $cDNA$ is synthesized from the mRNA of pituitary cells.     - HGH is a large protein containing $191$ amino acids.     - Vector Process: Restriction enzymes like EcoRI and HindIII are used to cut the $cDNA$ and the expression vector. DNA ligase joins them, and the vector is transformed into E. coli.     - Since E. coli does not secrete the hormone, the cells must be destroyed (lysed) to harvest and purify the protein.
• Bovine HGH (Somatotropin): A recombinant version made from bovine genes used to increase growth and milk production in cows. It features two binding receptors: a growth receptor and a prolactin receptor.
• Misuse: Athletes sometimes misuse HGH to increase muscle mass and decrease fat, which can lead to acromegaly (overgrowth of hands, face, and body parts), heart disease, and diabetes.

Recombinant Vaccines and Toxoids

• Vaccine Definition: Biological substances that elicit self-immunity by training the immune system to recognize and fight pathogens without causing the disease.
• Traditional Categories:     - Live-attenuated (weakened): A virulent strain grown in non-natural environments until it loses its virulence but remains alive.     - Inactivated (killed): Pathogens treated with heat or chemicals. They are dead and cannot grow but still trigger an immune response.
• Whole-Cell Vaccines: These use the entire cell, triggering a broad immune response against multiple antigens.
• Genetic Engineering in Vaccines:     - Instead of heat or chemicals, researchers manipulate the genome to remove viral genes, "crippling" the pathogen so it cannot cause disease but can still be used for immunization.
• Bacterial Toxins and Toxoids:     - Toxins: Poisonous proteins produced by bacteria like Clostridium tetani and Corynebacterium diphtheriae.     - Toxoids: Toxins treated (traditionally with formaldehyde) to lose toxicity while retaining the ability to trigger an immune response.     - Mechanism: Toxoids train the body to neutralize the poison (toxin) rather than necessarily killing the bacteria itself.     - Recombinant Production: The toxin gene is identified. It usually has an A subunit (toxic) and a B subunit (non-toxic). The gene for the non-toxic B subunit is inserted into an expression vector and produced in E. coli.
• Subunit Vaccines:     - These contain only a specific piece of the pathogen (an antigen) rather than the whole microbe.     - Example: Hepatitis B Vaccine: The $HBsAg$ gene (surface antigen) is removed from the Hepatitis B virus and inserted into a yeast expression vector.     - Adjuvants: Subunit vaccines often require an adjuvant (e.g., aluminum salts like aluminum hydroxide) to "set off an alarm" in the body so the immune system notices and responds to the antigen.
• DNA Vaccines:     - Fragments of the pathogen's DNA (encoding immunogenic proteins) are inserted directly into a vector and then into the animal.     - The animal's own cells take up the DNA and express the protein, triggering immunity.     - Advantages: Cheap, stable, and the DNA itself is not immunogenic (the body won't attack the DNA, only the protein it produces).

Mammalian Cell Lines in Biotechnology

• Purpose: Used when human proteins are too complex for prokaryotes or yeast to assemble (e.g., specific folding and glycosylation requirements).
• Common Lines:     - CHO Cells: Chinese Hamster Ovary cells; used for monoclonal antibodies and DNase (for cystic fibrosis).     - HEK 293: Human Embryonic Kidney cells; used for viral vector studies.     - Vero Cells: African Green Monkey kidney cells; used for viral vaccine production.
• Pros and Cons:     - Pros: Proteins resemble human versions almost perfectly.     - Cons: Very expensive, slow-growing, and highly susceptible to contamination. Maintaining HEK cells in South Africa can cost over $20,000\,\text{ZAR}$.

Transgenic Animals and "Pharming"

• Definition: Creating animals that carry human genes to produce pharmaceutical products, often in their milk or blood.
• Tissue Specificity: A promoter is attached to the target gene to ensure the protein is only produced in a specific tissue (e.g., mammary glands for milk secretion).
• Process:     - The gene and promoter are injected into a fertilized egg (zygote).     - The zygote is implanted into a surrogate mother treated with hormones to simulate pregnancy.     - The newborn offspring carry the gene and, upon reaching maturity, produce the protein (e.g., blood clotting factors) in their milk.
• Efficiency: One transgenic goat can produce more blood clotting factor in its milk than can be gathered from $1,000$ human blood donors.

Monoclonal Antibodies (MABs)

• Antibodies vs. Antigens: Antibodies are Y-shaped structures that bind to antigens (surface proteins on pathogens).
• Polyclonal vs. Monoclonal: Natural responses are polyclonal (a mixture); monoclonal antibodies recognize only one specific antigen.
• Applications: Used to flag cancer cells for the immune system, block growth receptors in tumors, or deliver toxic drugs directly to a tumor to avoid systemic toxicity.
• Production Technologies:     - Hybridoma Technology: Fusing B cells (which produce antibodies but are unstable) with myeloma cells (immortal cancer cells) to create a "hybridoma" that produces specific antibodies indefinitely.     - Humanization of MABs: Mouse antibodies are often rejected by the human immune system. Recombinant DNA is used to graft the "variable regions" (the specific tips of the Y-shape) from a mouse antibody onto a human "constant region" (the backbone). CHO cells are typically used for this production.

Transgenic Plants in Agriculture

• Agrobacterium tumefaciens: A soil bacterium used as a natural vector. It contains the Ti plasmid (Tumor-inducing plasmid).
• The Ti Plasmid:     - Naturally, it transfers T-DNA into the plant genome, causing crown gall disease (tumors).     - In genetic engineering, the plasmid is "disarmed" by removing the tumor-causing genes and replacing them with beneficial genes.
• Bt Toxin (Insects Resistance):     - The cry gene is taken from Bacillus thuringiensis.     - This gene codes for a protoxin that is toxic to insect larvae (e.g., the cotton bollworm).     - The cry gene is inserted into the Ti plasmid, which then integrates it into the plant's chromosomes, making the plant naturally pest-resistant.
• Herbicide Resistance (Roundup Ready):     - Glyphosate is a broad-spectrum herbicide that kills plants by inhibiting an enzyme necessary for amino acid synthesis.     - The gene for glyphosate resistance is taken from Agrobacterium strain $CP4$.     - This gene is inserted into crops (like soy or corn), allowing farmers to spray entire fields with glyphosate to kill weeds while the crops remain unharmed.

Questions & Discussion

• Question: How does a Roundup Ready crop save a farmer money?
• Answer: By being resistant to the herbicide, it allows for the efficient removal of weeds that would otherwise compete with the crop for nutrients and water, reducing the need for manual weeding or multiple types of treatments.
• Engagement Check: Dr. Tibeda asks, "Are we following?" during the explanation of transformation methods like electroporation and chemical treatment.