Applications of Recombinant DNA Technology

DNA fingerprinting is a technique used to identify an individual by studying the unique patterns of DNA variations inherited in the genome. It is based mainly on VNTRs (variable number tandem repeats), which are highly polymorphic and inherited from parents.

It can be explained through RFLP (Restriction Fragment Length Polymorphism) as follows:

1. DNA is isolated from the sample.
2. The DNA is cut with restriction enzymes at specific sites, producing fragments of different lengths.
3. These fragments are separated by agarose gel electrophoresis.
4. The DNA is transferred to a nylon/nitrocellulose membrane by Southern blotting.
5. A labelled VNTR probe is added and hybridisation occurs with complementary VNTR sequences.
6. The hybridised fragments are detected as band patterns.
7. The band pattern is specific to each individual and is used for forensics and paternity testing.

Thus, in RFLP, the different fragment lengths produced after restriction digestion help reveal the individual-specific DNA profile.

GMOs are genetically modified organisms or transgenic organisms whose genome has been altered by the introduction of one or more foreign genes (transgenes) through genetic engineering.

### Method of development of transgenic plants

1. A gene of interest is selected.
2. A suitable vector is constructed carrying the gene flanked by regulatory sequences such as promoter and terminator.
3. The gene is introduced into plant cells by indirect (vector-mediated) or direct (vector-less) methods.

#### Indirect method: *Agrobacterium tumefaciens*-mediated transfer
- *Agrobacterium tumefaciens* naturally transfers part of its Ti plasmid into plant cells.
- The tumour-inducing region of T-DNA is removed and replaced with the gene of interest; this is called disarming.
- The recombinant T-DNA is transferred into *Agrobacterium*.
- The bacterium infects the plant cells and the T-DNA with the desired gene integrates into the plant nuclear DNA.
- Transformed cells are selected on media containing marker antibiotic and regenerated into shoots and roots.
- Finally, the plantlets are transferred to soil.

This produces a transgenic plant carrying and expressing the foreign gene.

### Direct vs indirect gene transfer

Indirect gene transfer
- The gene is introduced into plant cells using a vector.
- Example: *Agrobacterium tumefaciens* transfers T-DNA into the plant genome.
- It depends on a biological carrier to deliver the gene.

Direct gene transfer
- The gene is introduced without a vector.
- Physical or mechanical methods are used, such as particle bombardment, protoplast transformation, and electroporation.
- The DNA enters the cells directly.

### One indirect method suitable for dicot plants
***Agrobacterium tumefaciens*-mediated gene transfer** is suitable for dicot plants.

Molecular pharming is the production of industrial or therapeutic biomolecules in transgenic plants or animals by using genetic engineering. It involves purposeful addition of genes so that the host produces desired products such as vaccine antigens, therapeutic proteins, diagnostic reagents, nutritional products, bioplastics, and industrial enzymes.

### Applications of transgenic animals in molecular pharming
Transgenic animals are used as bioreactors to produce human proteins in large quantities, especially in milk. Examples given in the chapter include:
- Tracy, a transgenic ewe, producing human $b11-antitrypsin in milk.
- Rosie, the first transgenic cow, producing human **$b1$-lactalbumin-enriched milk.
- Human recombinant proteins produced in transgenic animals include:
- Albumin in cow
- $b1$-fetoprotein in goat
- Growth hormone in goat
- Tissue plasminogen activator in goat
- Coagulation factor IX in mouse
- Coagulation factor VIII** in rabbit

These products are used for therapeutic and medical purposes.

### Gene gun
- A gene gun is a physical method of direct gene transfer.
- DNA is coated on gold or tungsten particles and bombarded into plant tissues at high velocity.
- It is also called biolistics.
- It is used to introduce foreign DNA into plants, especially where *Agrobacterium* methods are less successful.

### Gene therapy
- Gene therapy is a medical technique used to repair faulty genes in humans by introducing correct genetic material into cells.
- It is used for treating diseases such as SCID, cystic fibrosis, hemophilia, muscular dystrophy, etc.
- It may be done ex vivo or in vivo.

### Difference
- Gene gun is a method of gene transfer in plants.
- Gene therapy is a treatment approach in humans to correct defective genes.

So, gene gun is a tool for introducing genes, while gene therapy is the application of gene transfer for treatment.

The development of recombinant subunit vaccines involves the following steps:

1. The antigenic protein of the pathogen is identified.
2. The gene coding for that antigen is isolated.
3. The gene is inserted into a suitable expression vector.
4. The vector is introduced into a host such as E. coli or yeast.
5. The host cells express the recombinant antigen.
6. The antigen is separated and purified.
7. The purified antigen is then used as a vaccine to stimulate immunity.

### Example from the chapter
For Hepatitis B vaccine, the gene coding for HBsAg was inserted into the yeast plasmid pMA56 and expressed in *Saccharomyces cerevisiae*. The produced protein was purified and used for immunisation.

DNA vaccines are vaccines in which the gene for an antigen is cloned into a plasmid and the DNA is directly introduced into the body.

### Main features
- The plasmid contains a promoter, cloning site, origin of replication, selectable marker, and terminator.
- During vaccination, the DNA is injected into muscle, often using a gene gun.
- Some muscle cells take up the DNA and express the antigenic protein.
- This protein triggers the immune system.
- DNA vaccines induce both humoral and cellular immune responses.
- They can also be administered by nasal spray.

Thus, DNA vaccines work by making the body’s own cells produce the antigen and develop immunity.

Monoclonal antibodies produced by rDNA technology have some advantages over those developed only by traditional hybridoma technology:

- They can be made into chimeric or humanised antibodies by adding human segments, so they are more effective and less likely to be rejected.
- They can be engineered for greater specificity and improved activity.
- rDNA methods allow the production of modified antibodies with better therapeutic use.
- They can be designed for targeted therapy and diagnostic applications.

In contrast, ordinary hybridoma-derived antibodies are produced from mouse cells and may be less suitable for use in humans because they can cause immune reactions. Therefore, rDNA technology improves the usefulness of monoclonal antibodies for medical use.

Humulin is the first genetically engineered human insulin produced using rDNA technology.

### Development
1. The insulin gene was isolated from a gene library.
2. It was inserted into an E. coli plasmid.
3. A lac promoter was added next to the gene.
4. The gene was expressed as a fusion protein with **$b2$-galactosidase.
5. Since bacteria cannot process preproinsulin, scientists cloned the A and B chain genes separately.
6. The fusion protein was extracted and treated with cyanogen bromide (CNBr) to separate the insulin-related peptides.
7. The A and B chains were then joined by disulfide bonds to form active human insulin.

The product was marketed in 1982 as Humulinae**, and it was more effective and free from allergic reactions compared to pig and cow insulin.

Humatrope and Protropin are genetically engineered forms of human growth hormone (HGH) produced by rDNA technology.

### About HGH
- HGH is a pituitary hormone containing 191 amino acids.
- It promotes growth by increasing metabolism of amino acids, lipids and carbohydrates.
- Deficiency in childhood causes dwarfism.

### Production
- The HGH gene was prepared using cDNA made from HGH mRNA.
- Since bacteria cannot remove the signal peptide, the signal peptide coding region was removed.
- The complete HGH gene was then inserted into bacteria for production.
- In 1985, genetically engineered HGH was marketed as Humatropeae and Protropinae.

### Importance
These products replaced costly and risky extraction of HGH from cadaver pituitary glands and provided a safer therapeutic hormone for treating dwarfism.

rDNA technology has improved crops by introducing useful genes that give better yield, resistance and quality.

### Applications in crop improvement
- Insect resistance: Example, Bt cotton, which contains the cry gene from *Bacillus thuringiensis* and resists cotton bollworms.
- Resistance to bacteria, viruses, fungi and nematodes.
- Tolerance to herbicides.
- Tolerance to abiotic stresses such as drought, salinity and extreme temperatures.
- Improved nutritional quality: Example, Golden Rice with increased vitamin A.
- Delayed fruit ripening and other quality traits.
- Increased crop yield and better performance in harsh conditions.
- Molecular farming, where plants are used to produce therapeutic products.

Thus, rDNA technology helps develop crops that are more productive, nutritious and resistant to environmental and biological stresses.

The ethical issues related to the use of transgenic animals include:

- Potential risk to human health and environment.
- Transgenic animals or crops may produce proteins that can cause allergic reactions.
- Use of viral vectors and promoters may raise concern about possible viral infection.
- Improved traits may make organisms invasive to native species.
- Random insertion of the transgene may cause unpredictable or non-target effects.
- There are ethical concerns about the creation and use of genetically modified animals.
- In general, the use of GMOs is regulated by bodies such as GEAC to ensure safety.

These points show why biosafety and ethical regulation are necessary.

Vaccinia virus is used as a vector in the development of recombinant vaccines.

### Role of vaccinia virus
- It has a large linear double-stranded DNA genome that can accommodate foreign DNA.
- A small part of vaccinia DNA is cloned into a plasmid and used as an insertion vector.
- Antigen genes from pathogens such as hepatitis virus, herpes simplex virus and influenza virus are inserted into this vector.
- Through homologous recombination, these antigen genes become incorporated into the vaccinia genome.
- The recombinant vaccinia virus then expresses the foreign antigen and induces immunity.

Thus, vaccinia virus acts as a live vector for delivering foreign antigen genes and producing multivalent vaccines.

Recombinant therapeutic agents are useful human proteins and other compounds produced by rDNA technology for treating diseases and improving health.

### Features
- They are safe, pure and efficient.
- They are identical or very similar to natural human proteins, so they usually do not cause allergic reactions.
- They can be produced in large amounts.

### Examples from the chapter
- Monoclonal antibodies
- Insulin
- Human growth hormone (HGH)
- Cytokines
- Blood clotting factors
- Antibiotics

### Importance
- Used in the treatment and diagnosis of many diseases.
- Better than animal-derived products because they are less likely to trigger immune responses.
- Examples include Humulin for diabetes and Humatrope/Protropin for dwarfism.

So, recombinant therapeutic agents have greatly improved modern healthcare.

Humanised antibodies are monoclonal antibodies produced by rDNA technology in which mouse antibodies are modified to carry a few human segments.

### Key points
- They are derived from mouse antibody structures but contain human portions.
- This makes them more effective and less likely to be rejected by the human immune system.
- They are useful in diagnosis and targeted therapy.
- They represent an improvement over ordinary mouse monoclonal antibodies.

Thus, humanised antibodies combine the specificity of mouse antibodies with better compatibility for human use.