Genetic Engineering Technique: Gene Transfer | Science & Technology for UPSC CSE PDF Download

What is Gene Transfer?

Gene transfer is a technique where new DNA is added to the cells of a living organism. This can be done by using carriers like plasmids or modified viruses. The new DNA can be added to the cells outside the organism and then put back into the organism, or it can be directly added to the cells inside the organism.  

Types of Gene Transfer

Steps involved in Gene Transfer

The process of gene transfer can be summarized in four key steps:

(a) Isolation of gene and vector (by PCR)

(b) Digestion of gene and vector (by restriction endonuclease)

(c) Ligation of gene and vector (by DNA ligase)

(d) Selection and expression of transgenic construct

Methods for Gene Transfer

There are two methods of gene transfer:

(a)  Indirect or Vector-Mediated Gene Transfer

Indirect or vector-mediated gene transfer in plants involves the use of a plasmid vector.  One widely employed vector for plant transformation is the Ti-plasmid found in Agrobacterium tumefaciens. 

Agrobacterium-mediated Gene Transfer

• This bacterium carries a relatively large plasmid called the Ti plasmid (which stands for Tumor-inducing plasmid). A specific portion of this plasmid, known as T-DNA (Transfer DNA), is integrated into the plant genome within infected cells, leading to the development of plant tumors, or crown galls. 
• Due to its inherent ability to naturally transfer the T-DNA region from its plasmid into the plant genome upon infecting wounded plant cells, Agrobacterium tumefaciens is often referred to as nature's genetic engineer for plants.
• The foreign gene (e.g. Bt gene for insect resistance) and plant selection marker gene, usually an antibiotic gene like npt II which confers resistance to antibiotic kanamycin are cloned in the T DNA region of Ti-plasmid in place of unwanted DNA sequences.

(b) Direct or vectorless gene transfer method.

In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant without the help of a vector. The following are some of the common methods of direct gene transfer in plants.

The various method of direct gene transfer is: 

(i) Chemical method
Use chemicals like polyethylene glycol and polyvinyl alcohol to help plant cells take in foreign DNA.
Start with plant protoplasts (cells without cell walls) in a magnesium-rich medium.
Add plasmid DNA with the desired gene. Use polyethylene glycol, maintain pH at 8, and briefly heat protoplasts. Cool them on ice to boost DNA uptake.
Allow incubation for DNA integration. Later, reduce polyethylene glycol and increase calcium ions for better transformation efficiency.

(ii) Electroporation
 A pulse of high voltage is applied to protoplasts, cells or tissues which makes transient pores in the plasma membrane through which uptake of foreign DNA occurs.
 

(iii) Biolistic gun
Foreign DNA is attached to tiny gold or tungsten particles, typically measuring 1-3 µm. These particles are then shot onto the target tissue or cells using a device known as a gene gun, micro projectile gun, or shotgun. After bombardment, the treated cells or tissues are grown in a specific medium to encourage the development of plants from the modified cells.
Gene Gun

(iv) Microinjection

The DNA is directly injected into the nucleus using fine tipped glass needle or micro pipette to transform plant cells. The protoplasts are immobilised on a solid support (agarose on a microscopic slide) or held with a holding pipette under suction. 

Microinjection

(v) Lipofection

Artificial phospholipid vesicles called liposomes are valuable for transferring genes. They enable the transfer of genes or DNA from the liposome into the vacuole of a plant.

Achievements of Genetic Engineering

DNA recombinant technology or genetic engineering provides great benefits for the advancement of science and society.

1. Gene Therapy: 

• The first gene therapy used in severe combined immunodeficiency (SCID) patient.
• About 25% of an infant with SCID disorder lack the enzyme adenosine deaminase (ADA) – ADA enzyme involved in purine metabolism.
• These patients have no functioning T & B lymphocytes.
• The affected child of SCID develops recurrent infection early in life because they have no immune response against invading pathogen.
• The ideal approach would be to give the patient a functioning ADA by gene therapy.
• Thus, the genetic disorder can be overcome by introducing a specific gene.

2. Bacteria 

• Human insulin (Humulin) was the first genetically engineered product produced by an American firm Eli Lilly – 5th July 1983.
• Charles Weismann of the University of Zurich obtained interferon through recombinant E.coli (1980).
• Microbes have been engineered to produce Human Growth Hormone (HGH) for curing dwarfism.
• Vaccines are produced by genetic engineering e.g., for Hepatitis-B and Herpes virus).
• Nitrogen fixation genes may be transferred from bacteria to the major food crops to boost food production without using expensive fertilizers.
• Transgenic plant obtained through recombinant DNA technology. The first transgenic plant was tobacco.

Tobacco

• It contains a resistant gene against weedicide (Glycophosate).
• The first transgenic animal was a mouse contain the gene for growth hormone.
• The first introduced transgenic crop in India (2002) is Bt-cotton.
• It is resistant for boll worm (Helicoverpa armigera - Larva of insect). 
• It is formed by the transfer of a pest-resistant gene from Bacillus thuringiensis (bt-2 gene encoding Bt–toxin).
• Bacillus thuringiensis produces a toxic protein called crystal protein (Cry-Protein) this protein is toxic for the larva of a certain insect.

Cry-protein

• This protein kills the insect by inhibiting ion transport in the mid-gut (bt 2 genes is called cry -gene) – In pollution control, microbes have been engineered to break up the crude oil spills.
• Dr Ananda Mohan Chakraborty introduced plasmid from different strains into a single cell of pseudomonas putida. The result was a new genetically engineered bacterium that would clean oil spills called "Superbug" (oil eating bug.)

3. Medical Diagnosis of Disease 

• Recombinant DNA technology is one of the important tools for the diagnosis of several diseases. The diagnostic technique involves the construction of probes consisting of short segments of single-stranded DNA attached to a radioactive or fluorescent marker.
• Such probes are used to identify infections agents such as Salmonella (cause food poisoning), Staphylococcus (pus-forming bacterium), hepatitis virus, HIV; muscular dystrophy, cystic fibrosis and so on. 
• Recombinant DNA technology can also be employed to predict the inheritance of genetic disorders from carrier parents. The chances of birth of an affected child can be predicted by testing the DNA of repetitive prospective genetic disorder carrier parents.

Application

 of Recombinant DNA Products

Application of Genetically Engineered Microbes