How Does Transfection Work? Step-by-Step Guide for Students
Transfection refers to the introduction of foreign DNA (genetic material other than host genomes) into the cell. The main purpose of transfection is to alter the host genome to express or block the expression of the protein associated with the gene. In this article, the main objective is to develop a keen understanding of the general principle of transfection, it defines the differences between the type of transfections and currently available transfection
methods. It also focuses on the factors that influence the transfection method and, the application of transfection. Transfection can be categorized into transient transfection and stable transfection, it is important to understand that these classifications are based on the stability of the end product of transfection.
There are some important terms that are widely used in transfection technology, the list below describes some of them.
Transformation- It is the term that is used to define and is reserved for the nonviral gene delivery method. The host or recipient of such genetic material can be a bacterial or eukaryotic animal cell.
Transduction- It is also known as the transduction infection. It is used to define the transfer of genetic material into the recipient using a virus as a vector.
Stable Transfection- It is the transfection where the target genetic material gets incorporated into the host genome.
Transient Transfection- It is the condition when the genetic material does not get incorporated into the host cellular genome.
Transfection- It can be defined as the method of nonviral gene delivery method into the recipient cell. Exogenous nucleic acid material includes DNA, RNA, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA).
Principle of Transfection
According to transfection meaning, the exogenous genetic material must enter into the cell passing through the cell membrane. It is important to note that the genetic material whether DNA or RNA is negatively charged due to the protein associated with it. So in an unmanipulated environment, the exogenous DNA can not cross the cell membrane, but during transfection, the genetic material gets conjugated with positively charged compounds or is directly inserted into the nucleus of the host cell. It leads to the removal of the barrier of crossing the charged membrane. Based on the approach used to transfer the exogenous genetic material that is by conjugation with cationic compounds or insertion into the nucleus, transfection can be classified into chemical transfection or physical transfection. Another category of it is known as biological transfection where the transfection is achieved by viral vectors, an example of it includes transduction.
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Difference Between Transient Transfection and Stable Transfection
Transient transfection meaning can be defined as the transfection method in which the exogenous material is not incorporated into the host genome, another key point is that transfection of mRNA can be considered as transient transfection. The reason behind it being mRNA is the coding elements of the cell, which undergo translation to express the protein, when the mRNA is injected into the cell, it expresses the protein that it codes for and gets degraded as the half-life of mRNA is very short. Hence in either case the exogenous material undergoes degradation and is considered as transient transfection.
Stable transfection is the widely used approach; it is the method of incorporation of the exogenous genetic content into the host genome. This method ensures the continuous expression of the protein that the exogenous material codes for. These usually cause permanent alterations in the host genome. The exogenous genetic content is usually transferred directly into the host nucleus.
Comparison of Transient Versus Stable Transfection
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Methods of Transfection
There are three methods of transfection they are physical, chemical, and biological. The biological method of transfection is based on viral vectors and is termed transduction. The physical transfection method is used to incorporate genetic material using sensitive tools and techniques. The chemical transfection uses a transfection reagent to deliver a genetic component; the common example is calcium phosphate transfection.
Overview of Physical and Chemical Transfection
Physical Transfection
Physical transfection includes direct microinjection, biolistic particle delivery, electroporation and laser-mediated transfection.
Direct microinjection- it is the technique in which DNA is inserted with a needle directly into the cell cytoplasm or nucleus. It requires high expertise and skill.
Biolistic particle delivery- In this method, the exogenous material is coated on gold particles and is delivered into the cell using a high-velocity gene gun.
Electroporation- In this method the recipient cell is subjected to electrical pulses in order to temporarily destabilize the cellular membrane. These allow pore formation in the membrane, pores allow the nucleic acid material to pass into cells.
Laser mediated transfection- In this method, a pulse laser is used to transiently permeabilize cells to accept the incoming DNA.
Chemical Transfection
It relies on the use of transfection reagent to allow the genetic material to pass through it, it includes methods like calcium phosphate transfection, PEI transfection. The chemical transfection principle is based on the formation of complexes with negatively charged nucleic acid, which are then subjected to either endocytosis or phagocytosis, and are taken up by the cell. They use cationic polymers, calcium phosphate, or cationic lipid to form a complex with the genetic material.
1. Calcium Phosphate Transfection- This based on the principle of formation of an insoluble precipitate of DNA: calcium phosphate co-precipitate. The flowchart here explains the protocol.
DNA is mixed with calcium chloride (transfection reagent)
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This complex is added to phosphate buffer
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Optimization of the pH works within a narrow window (6.9–7.4)
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Precipitation of the DNA: calcium phosphate complex
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Removal and dispersion of precipitate in cell culture
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Phagocytosis of the co precipitate by the target cell
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2. PEI–DNA Condensation Method- In this method, the genetic material is complexed with a cationic polymer to facilitate the gene delivery, to increase efficiency to target specific cell types. The polymer and the genetic material forms a complex and are then phagocytosed by the cell. In the cellular compartment the host uptakes and integrate the genetic material into the genome.
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Application of Transfection
There are various applications of transfection, some of them are as follows
It is an analytical tool used to study gene or protein expression in various cell types.
Transfection is sometimes used to lower the expression of certain proteins, this achieved by performing siRNA transfection.
It allows large-scale production of recombinant proteins.
Gene therapy is based on the transfection process.
It is used for gene silencing
It is used to produce stable cell lines.
FAQs on Transfection: Principles, Techniques, and Uses
1. What is the basic principle of transfection in cell biology?
The fundamental principle of transfection is the process of introducing foreign nucleic acids, such as DNA or RNA, into eukaryotic cells. This requires the genetic material to cross the cell's outer membrane to enter the cytoplasm or nucleus. The primary goal is to have the host cell express this new genetic information, leading to the production of a specific protein or the silencing of a target gene.
2. What are the main purposes of using transfection in biotechnology?
Transfection is a vital tool in biotechnology with two primary purposes:
Gene Expression: To study the function of a gene by introducing it into a cell and observing the effects of the protein it produces. This is also used to produce large quantities of recombinant proteins for therapeutic or industrial use.
Gene Silencing: To inhibit the expression of a specific gene using molecules like small interfering RNA (siRNA). This helps researchers understand the gene's role by observing what happens when it is "turned off."
3. What are the major categories of transfection methods?
Transfection methods are generally grouped into three major categories based on the technique used to introduce nucleic acids into the cell:
Chemical Methods: These use reagents like calcium phosphate or cationic lipids to form complexes with the nucleic acid, which are then taken up by the cell.
Physical Methods: These use physical force to create temporary pores in the cell membrane. Examples include electroporation (using an electrical pulse) and microinjection (using a fine needle).
Biological Methods: This approach, often called transduction, uses viruses as vectors to deliver the genetic material into the host cell.
4. How is transfection different from transformation and transduction?
These terms describe gene transfer but differ in the target cell and method:
Transfection specifically refers to introducing nucleic acids into eukaryotic cells (e.g., mammalian cells) using non-viral methods.
Transformation is the term for the uptake and expression of foreign genetic material by bacteria, yeast, or plant cells.
Transduction involves the use of a virus as a vector to insert genetic material into a host cell, which can be either eukaryotic or prokaryotic.
5. What is the key difference between transient and stable transfection?
The key difference lies in the long-term fate of the introduced genetic material. In transient transfection, the foreign DNA is expressed for a limited time (e.g., 24-72 hours) but is not integrated into the host cell's genome and is eventually lost. In stable transfection, the foreign DNA successfully integrates into the host cell's chromosomes, becoming a permanent part of the cell's genome and being passed on to all subsequent daughter cells.
6. How does electroporation, a physical transfection method, work?
Electroporation works by applying a controlled, high-voltage electrical pulse to a suspension of cells. This electrical field momentarily destabilises the cell membrane, creating temporary pores or "holes." These pores are large enough for nucleic acids like plasmid DNA to pass through from the surrounding solution into the cell's cytoplasm before the membrane reseals itself.
7. How is transfection applied in the development of gene therapies?
Transfection is a cornerstone of gene therapy. It is used to deliver a functional copy of a gene into the cells of a patient who has a defective or missing gene. For example, in ex vivo gene therapy, a patient's cells are removed, transfected in the lab with the correct gene, and then reintroduced into the patient. The goal is for these genetically modified cells to produce the necessary protein, thereby treating the genetic disorder.
8. What key factors can affect the success and efficiency of a transfection experiment?
The efficiency of a transfection experiment is a critical parameter influenced by several factors:
Cell Health and Type: Healthy, actively dividing cells are generally more receptive. Different cell lines have inherently different levels of transfectability.
Quality of Nucleic Acid: The purity, concentration, and integrity of the DNA or RNA are crucial for good results.
Transfection Method: The chosen method must be optimised for the specific cell type, as some methods are harsher than others.
Reagent-to-DNA Ratio: In chemical methods, the precise ratio of the transfection reagent to the nucleic acid must be carefully optimised to maximise uptake while minimising cell toxicity.
