Methods of Gene Transfer: Meaning, Applications, Diagrams

Scientists are using various techniques to alter the genome of some organisms. They are doing so to make economically important plant varieties and microorganisms, to treat disease, to monitor the prognosis and therapy treatment of varieties, etc. How are genes transferred to another organism? The Methods of Gene Transfer involve many different methods such as conjugation, transduction, etc.

Gene transfer from one organism to another is a natural process that results in biological trait variation. It is a routine laboratory procedure for bacterial strains like E. coli. Gene transfer in bacteria can be done by three methods, namely, conjugation, transformation and transduction. Read on to explore more about different techniques of natural as well as artificial gene transfer and its applications.

What is Gene Transfer?

Insertion of unrelated genetic information into cells in the form of DNA is known as gene transfer. Gene transfer can be done for a variety of reasons. The treatment of illnesses through gene transfer to provide patients with therapeutic genes is perhaps the most compelling of these reasons. There are also other methods for transferring genes.

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What are the Methods of Gene Transfer?

Different methods of gene transfer used in various applications can be categorised into two broader categories:

The natural method of Gene Transfer: Natural agents such as living cells, bacteria, other DNA molecules, and viruses are utilised to transfer genes in this method.
Artificial method of Gene Transfer: An exogenous gene is transferred to another cell or organism through artificial means such as physical or chemical agents in this method. Some of the main methods of Gene Transfer are as follows:

1. Transformation

i. The process of introducing foreign DNA into bacterial cells is known as transformation (e.g., E. coli). E. coli take up plasmid DNA in ice-cold CaCl₂ (0-5°C) and then undergoes a heat shock (37-45°C for around 90 seconds).
ii. By this technique, the transformation frequency, which refers to the fraction of cell population that can be transferred, is reasonably good, for example, approximately one cell for 1000 (10^-3) cells.

Transformation efficiency

i. The number of transformants per microgram of additional DNA is what this term refers to as transformation efficiency.
ii. The transformation efficiency of E. coli transformation by plasmid is around 107 to 108 cells per microgram of intact plasmid DNA.
iii. Competent bacteria are those that have the ability to take up DNA. Changes in growing circumstances can improve competency.
iv. The transformation process’ mechanism is really not completely known. CaCI₂ is thought to impact the cell wall, causing localised fractures, and be responsible for DNA binding to the cell surface.
v. DNA uptake is stimulated by a short heat shock (a rapid rise in temperature from 5°C to 40°C). Larger DNAs are less effective at transforming in general.

Fig: Transformation

2. Conjugation

i. Conjugation is a microbiological recombination mechanism that occurs naturally in organisms.
ii. Two living bacteria (a donor and a recipient) come together during conjugation, connect through cytoplasmic bridges, and transmit single-stranded DNA (from donor to recipient).
iii. The additional DNA may either integrate with the chromosome (which is uncommon) or stay inside the receiving cell (as is the case with plasmids).
iv. Conjugation can happen between bacterium cells from different genera (e.g., Salmonella and Shigella cells).
v. This is in contrast to the change that occurs within a bacterial genus’ cells. As a result of conjugation, genes from two distinct and unrelated bacteria can be transferred.

3. Transduction

i. The transduction process involves inserting genes into the genome of a host cell utilising viruses, i.e., bacteriophage as carriers. The viruses are used in gene transfer because of the following characteristics:
a. Viruses’ ability to transfer their nucleic acid into cells.
b. Replication and gene expression at a high level.
ii. To enter the host cell, the foreign gene is packed into viral particles.
iii. A receptor-mediated mechanism allows virus particles bearing candidate gene sequences to enter the cell and then into the nuclear genome.
iv. The vector genome passes through a series of complicated steps that result in ds-DNA, which can either remain as an episome or integrate into the host genome and then the candidate gene is expressed.

Fig: Transduction

4. Electroporation

i. The mechanical approach of electroporation is used to introduce polar molecules into a host cell via the cell membrane.
ii. Wong and Neumann used this technique to examine gene transfer in mouse cells for the first time in 1982.
iii. It is currently a commonly used technique for introducing transgenes into the bacterial, fungal, plant, and animal cells, either permanently or transiently.
iv. The main principle behind electroporation is that high voltage electric pulses can cause cell plasma membranes to fuse.
v. Electroporation is a method that involves membrane permeabilisation mediated by an electric field.
vi. Electric shocks can also cause cellular absorption of foreign DNA from the suspending solution (thought to be via pores created by electric pulses).

Fig: Electroporation

5. Liposome-Mediated Gene Transfer

i. Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids in them.
ii. Several methods for encapsulating DNA in liposomes have been developed. The liposome-mediated gene transfer is referred to or known as lipofection.
iii. When DNA fragments are treated with liposomes, the DNA fragments get encapsulated inside liposomes. These liposomes may attach to cell membranes and fuse with them, allowing DNA fragments to be transferred.
iv. As a result, DNA penetrates the cell before reaching the nucleus. The positively charged liposomes attach to cells, combine with DNA, and quickly transfer DNA.
v. Lipofection is a highly effective method for transferring genes to bacterial, animal, and plant cells.

Fig: Liposome-Mediated Gene Transfer

6. Microinjection

i. Dr Marshall A. Barber was the first to suggest DNA microinjection in the early 1800s. In mammals, this technique is frequently employed for gene transfection.
ii. For cultivated cells, DNA transfer via microinjection is commonly employed. This method may also be used to deliver DNA into big cells like oocytes, eggs, and early embryonic cells.
iii. The word “transfection” refers to the physical or chemical transfer of DNA into eukaryotic cells.
iv. A glass micropipette tip with a diameter of 0.5 mm is used to transfer foreign DNA under a powerful microscope.
v. Microinjected cells are put in a container. A holding pipette is placed in the microscope’s field of vision, sucking and holding a target cell at the tip.
vi. The empty needle is removed after the tip of the micropipette is inserted through the cell membrane to transfer the contents of the needle into the cytoplasm.

Fig: Microinjection

7. Particle Bombardment

i. Prof. Sanford and colleagues at Cornell University in the United States created the initial bombardment concept in 1987, coining the name “biolistics” (short for “biological ballistics”) to describe both the procedure and the devices.
ii. Particle bombardment, particle gun, microprojectile bombardment, and particle acceleration are all terms for the same thing. It works by delivering compounds into cells with high-velocity microprojectiles.

Fig: Particle Bombardment

Applications of Gene Transfer Technology

Some of the applications of gene transfer technology are as follows:

i. Gene transfer technology provides the ability to genetically manipulate the cells of higher animals.
ii. Gene therapy is a promising therapeutic option that requires efficient gene delivery into living cells. Since 1990, gene transfer techniques have been employed in human gene therapy trials.
iii. Gene transfer has the potential to be a significant tool for treating a wide range of illnesses. These genes are transferred via a variety of vectors, including retroviral, adenoviral, and adeno-associated virus (AAV) vectors, as well as non-viral methods.
iv. Soyabean, cotton, spruce, papaya, sugarcane, corn, sunflower, rice, maise, wheat, tobacco, and other crops have all been successfully transformed using the Biolistics method.
v. The microinjection technique works well for both primary cells and cells in established cultures.
vi. Electroporation can be used to improve the effectiveness of bacterial cell transformation or transfection.

Summary

Genes contained on the new fragments of DNA could be stably inherited and expressed to give new features to the host bacteria. The scientists then created specific circumstances in the new hosts that enhanced DNA absorption, maintenance, and gene expression. Gene transfer is now a routine laboratory procedure for bacterial strains such as E. coli. Through this article, we understood many different methods of Gene transfer that are used to transfer specific genes into specific organisms.

FAQs on Methods of Gene Transfer

Q.1. What is gene transfer?
Ans: Insertion of unrelated genetic information into cells in the form of DNA is known as gene transfer.

Q.2. What are the 3 methods of gene transfer in bacteria?
Ans: The three methods of gene transfer in bacteria are conjugation, transformation and transduction.

Q.3. What is the electroporation method?
Ans: Electroporation is a type of physical transfection that involves creating temporary pores in cell membranes through which chemicals such as nucleic acids can flow.

Q.4. What is natural gene transfer?
Ans:  The active absorption of DNA by bacterial cells and the heritable incorporation of its genetic information is known as natural gene transfer.

Q.5.  Why is E. coli used in transformation?
Ans: E. coli is used in transformation because of the great efficiency with which DNA molecules are introduced into cells, E. coli is a popular host for gene cloning. Because of its fast growth and capacity to produce proteins in very high quantities, E. coli is a favoured host for protein synthesis.

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