Introduction
Transfection is an excellent cell culture method used in laboratories to study gene functions and their products in cells. This involves introducing foreign nucleic acids into host cells by using a viral method in which viruses such as retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV) are used; and non-viral method, which includes physical and mechanical methods such as electroporation, sonoporation, magnetofection, gene microinjection, and laser irradiation. These methods are discussed in detail below.
What Is Transfection?
Transfection is a technique of introducing a foreign nucleic acid into a eukaryotic cell (nuclear membrane covers the nucleus and forms complex organisms) to change the genetic makeup. This process helps in studying the genes by altering specific gene expression, normal cellular processes, gene therapeutic effects, and disease molecular mechanisms. It can be used as a gene therapy to treat a noncurable inherited disease. The different types of nucleic acid which can be transfected are DNAs; (deoxyribonucleic acids), RNAs (ribonucleic acids), siRNA (small interfering RNA), shRNA (short hairpin RNA), and miRNA (micro RNA).
How Is Transfection Classified?
Transfection is classified into two types which are:
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Stable Transfection: In this type long-term expression of a transgene (artificially introduced gene) by integrating foreign DNA or with the help of episomal (extrachromosomal DNA of a viral genome or a plasmid) vector as an extra-chromosomal element is kept in the host nuclear genome. This transgene is continuously expressed along with cell replication. This type is used in the case of long-term pharmacology and genetic studies.
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Transient Transfection: The nucleic acid is transfected in the form of oligonucleotides or plasmids. This transgene expression is lost while cell replication. This type is used in the case of short-term studies.
What Are the Different Methods of Transfection?
The transfer of nucleic acid requires a delivery mechanism, such as lipid-based or non-lipid-based delivery vehicles, which increases the contact between vector-vehicle complexes, thereby facilitating the access of the complex into the cell. There are two types of vectors, which are plasmid vector and viral vector which, surrounds the virus and protects it. The different methods of transfections are:
Viral Transfection:
This method uses a viral vector to carry a foreign nucleic acid into a host cell and the viral genetic material is present inside the capsid, which opens after entering the host cell. This type can induce an immune response.
Non-viral Transfection:
These are further divided into chemical methods and physical/mechanical methods. Physical /mechanical method includes:
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Electroporation: This uses electrical voltage to increase cell permeability. It is commonly used in primary cells, B cell lines, and stem cells. But the high voltage can cause apoptosis (programmed cell death), cell necrosis, or damage.
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Sonoporation: This uses the microbubble technique, to create a hole that creates an easy pathway to transfect foreign nucleic acid.
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Magnetofection: This uses magnetic force to transfect foreign nucleic acid. This method causes less destruction to the host cell
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Gene Microinjection: This uses a specialized needle to punch holes in the host cell to transfect foreign nucleic acid.
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Laser Irradiation: This uses a laser beam to create holes in the cell membrane.
Chemical transfection uses chemical compounds to transfect foreign nucleic acid. They are further divided into liposomal-based and non-liposomal-based.
Hybrid Transfection:
This method uses a combination of viral and non-viral transfection. It has higher transfection efficiency.
What Is Viral Transfection?
Viral transfection is called transduction, which uses a viral vector to carry foreign nucleic acid into the host cell. In case of stable transfection retrovirus and lentivirus are used, which have a high risk of triggering mutagenesis, and in case of transient transfection herpes virus, adenovirus and adeno-associated virus (AAV) are used and have less risk of triggering mutagenesis.
Characteristics of specific viruses are:
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Retrovirus: It has RNAs, which get transcribed into double-stranded DNA. It contains an enzyme called integrase, which helps in the integration of foreign nucleic acid into the host cell. It has less potential to trigger inflammation but has a high risk of insertional mutagenesis and gene disruption. It is used to transfect dividing cells, whereas other viruses are used to transfect dividing and non-diving cells. This method of transfection has higher cytotoxicity and can also cause a viral infection.
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Lentivirus: They cantransfect both diving and non-diving cells. It was first developed as gene therapy for acute lymphoblastic leukemia.
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Herpes Virus: It has double-stranded DNAs, they have a great response to neuronal cells which are mostly used in delivering foreign nucleic acid in treating nervous system disease. This is a highly effective method that is used in difficult cells like primary cells (isolated directly from the living cells).
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Adenovirus: It has double-stranded DNAs, has surface proteins on it, which help in communicating with the host cell, and is derived from human adenovirus serotypes 2 and 5. These can transfect dividing and non-dividing cells. These viruses have a huge packaging capacity. The transfection begins with attaching to cell surface receptors and interacting with pontoons (αvβ3 and αvβ5 integrins), through receptor-mediated endocytosis, they reach the nucleus by escaping from the endosome and then the transcription occurs. After the completion of transfection, the cell dies and the virus is released. These viruses cause a strong immune response.
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Adeno Associated Virus: It has single-stranded DNA. These cause less immunogenicity and pathogenicity, therefore these are safe viruses for gene therapy and can be delivered through systemic, CNS, pulmonary, cardiac, or intramuscular routes. They have limited packaging capacity which limits their use in delivering large-sized therapeutic genes. They are used along with other viruses (adenoviruses). These viruses can be alive in a wide range of pH and temperatures
What Are the Advantages of Viral Transfection?
The advantages of viral transfection are:
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It has high efficiency in difficult cells.
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It can be helpful in the formation of stable cell lines in the host genome.
What Are the Disadvantages of Viral Transfection?
The disadvantages of viral transfection are:
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These can cause cytotoxicity (cell damage) and immunogenicity (immune response).
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It is a complex procedure.
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High cost.
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The viruses used have different variations in infectivity.
Conclusion
Therefore the modern cell culture technique can provide a wide range of benefits including studying gene therapeutic effects and disease molecular mechanisms. Each transfection method has its own pros and cons, therefore the selection of a type of transfection depends on the objectives to be achieved.
