There are three common transfection methods: chemical, physical and viral. Physical transfection uses physical means such as biolistics, microinjection and lasers. Chemical transfection uses liposomal and non-liposomal chemicals. And viral transfection urses adenoviruses and retroviruses.
And to just review very briefly, transfection is the method of introducing external DNA or RNA molecules into eukaryotic cells for temporary or long-term expression during laboratory experiments and for clinical gene therapy.
In this article, we will give you a quick review of transfection, and then you will learn all about the concepts behind transfection and the approaches used. We will also take a brief look at the advantages and disadvantages of each of these methods.
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Transfection can be done for both stable and transient expression of the foreign transgene.
The experiment may be designed in a way that the newly introduced foreign nucleic acids remain stable and are therefore expressed for a long time.
Sometimes, even after the transfected cell divides, the transgene is carried over to the daughter progeny where it again expresses itself – for this, the transfected DNA needs to be integrated into the recipient host cell’s genome.
In contrast, for transient transfection, the transfected genetic molecule is stable and expresses itself in the recipient host only for a relatively short amount of time. The transgene is lost when the recipient cell turns over or may be even much earlier.
Methods similar to transfection are transformation and transduction, which are used to transfer foreign nucleic acid molecules to recipient host cells. These methods differ in two aspects – the mode of transfer and the type of host cells used.
For transformation, the recipient host cells are bacterial – in contrast to eukaryotic host cells in the case of transfection. So, the primary difference between transformation and transfection lies in the type of host recipient cells involved.
In the case of transduction versus transfection, the difference is in the way the foreign nucleic acids are transferred to the host cells.
With transduction, foreign nucleic acids are delivered to the recipient host cell by exclusively using a virus or a viral vector as the agent for transmission.
With transfection, though a virus may be used in the experiment to transmit the foreign genetic material, researchers also use non-viral approaches to transfer genetic material to the host cells.
To note, in literature, when researchers use viruses to transfect eukaryotic cells, it is synonymous to transduction.
In the following sections we will take a brief look at the three approaches used for transfection – physical, chemical and biological.
During transfection involving physical methods, the transgene may be introduced into the host cell directly without chemical or biological agents. Common physical transfection approaches are microinjection, electroporation, sonoporation, laserfection, magnetofection and by bioloistics.
Gene microinjection is a technique where the transgene is injected using a sophisticated needle directly into the host cell cytoplasm or nucleus.
Advantage: Microinjection is valuable when the transgene needs to be introduced with clinical precision, such as in gene therapy.
Disadvantage: It requires specialized robots or very well-trained personnel to facilitate the delivery of the transgene into the recipient host cell without damaging it.
Electroporation creates pores in the cell membrane using an electric current. Similar to how it is used for bacterial transformation, in transfection, an electric field is rapidly applied to increase host cells’ permeability, allowing foreign nucleic acid molecules to enter.
Advantage: Electroporation is the preferred method of transfection when working with B cell line cultures, stem cells and cells derived from fresh tissues (primary cells). These cells might not be amenable to other methods of transfection.
Disadvantage: High voltages used in electroporation may cause cell death or significant damage.
Sonoporation and laserfection
Sonoporation and laserfection involves transfection using ultrasound and light waves respectively to create pores in the recipient cell membrane to facilitate entry of the foreign genetic material.
Advantage: Once the cell has been sufficiently permeabilized using these methods, any foreign genetic material in the extra-cellular environment can be introduced into the recipient.
Disadvantage: Both methods, while creating the pores in the recipient cells’ membrane, may cause serious damage to the cells that may even kill them.
Magnetofection is transfection using magnetic force to create pores in the host cells to facilitate entry of the required nucleic acids.
Advantage: Host cell destruction is low, especially compared to other physical methods.
Disadvantages: Transfection efficiency is not high.
Biolistic transfection involves immobilizing the foreign nucleic acid molecules into sub-cellular particles. Then using a special apparatus called gene-gun, they are shot out with a high enough velocity to penetrate the host cell.
Advantages: There are many experimental situations where the biolistic method offers unique advantages.
For one, this method is versatile because it is useful for transfecting cells growing both in vitro, in vivo and deep inside tissues. And it works regardless of the growth rate and other features of the host cells.
In clinical applications, it is useful in gene therapy.
Another advantage of this method is that it saves time during transfection experiments. This is a time-efficient method of transfection because multiple different genetic molecules may be transfected all at once into large populations of recipient cells.
Disadvantages: Gene-guns are expensive.
In chemical transfection, foreign nucleic acids are packed in a chemical that merges with the host cell’s membrane. In this method, the encapsulated transgene enters the cell through endocytosis or phagocytosis. Chemical transfection is categorized as liposomal or non-liposomal.
A schematic overview of chemical transfection is depicted in the figure below.
Here, two approaches to chemical transfection are shown. The first (on the left) shows liposomal transfection in which DNA is encapsulated within positively charged lipid molecules (blue).
The second (on the right) is DNA surrounded by positively charged calcium phosphate molecules (purple).
Let's take a closer look at how these and other approaches to chemical transfection work
In liposomal-based transfection, positively charged aggregated lipid molecules are used to encapsulate the foreign nucleic acid molecules.
These lipid aggregates have the necessary chemical properties that enable smooth merging with the phospholipid layers of recipient cell membrane. This facilitates the encapsulated genetic material’s entrance into the cell.
The other chemical transfection approach is packing or complexing the foreign nucleic acid molecules with a non-liposomal chemical. The basic objective is to mask or neutralize the negative charge on the foreign nucleic acid molecules using a chemical. This overcomes the repulsion between the recipient host cell’s membrane and the transgene so that the latter can enter the cell.
If you see below in the list of chemicals commonly used in non-liposomal chemical transfection, the common feature in most of them is that they either help neutralize the negative charge on the transgene molecules and/ or pack them efficiently to mask the negative charge. Here are some commonly used chemicals used in this method:
A.Calcium phosphate: calcium cations bind to the negatively charged sugar-phosphate backbone of the nucleic acid molecules to be transfected and form a precipitate that is taken up by the recipient cells. While this is a cheap method, the transfection efficiency is low. This is represented on the right-hand side in the picture above where purple calcium cations surround the DNA molecule.
B.Dendrimers: These are three-dimensional organic molecules chemically capable of forming complexes with nucleic acid molecules. These complexes are taken up by the recipient host cells. Though the efficiency is higher than calcium phosphate, it is still lower than viral-based and liposomal-based transfection.
C.Cationic polymers: Because of their positive charge, these molecules can bind to negatively charged transgene molecules. After this, the polymer-transgene complexes are taken up by the transfected cell.
D.Nanoparticles: Due to their small size, nanoparticles can enter the recipient cells easily. Modern methods combine magnetic force along with nanoparticles – for example, the chemical superparamagnetic iron oxide nanoparticle. The magnetic properties facilitate overcoming barriers inside and outside of the recipient cells, so that the transgene is delivered precisely where it is supposed to be.
Adenoviruses, Adenovirus-related viruses and retroviruses are used for transfection work spanning basic research to gene therapy. Virus-mediated transfection is also known as transduction.
This type of transfection is considered highly efficient, even when using cell types that are difficult to transform.
Adenoviruses are double-stranded DNA viruses used to transfect a wide range of host cell types. These viruses transfer the transgene to the recipient, but that DNA does not get integrated with the host cell genome.
Advantages: When DNA is transfected using adenoviruses, it gets inside the recipient cell, but the transfected DNA does not merge with the host chromosomes. So, the transfected DNA expresses itself only for a short time from the cytoplasm, but the genetics of the host cell does not get changed.
This gives rise to a unique advantage – transient expression of the transgene within the host cell, without modifying the genetics of the recipient cell permanently. Further, adenoviruses remain stable during prolonged storage in the lab and can transfect both dividing and non-dividing cells.
Disadvantages: The transfected transgene expression, though initially very high, diminishes fast over a few weeks. So, long-term transgene expression is not possible. Also, when used to transfect in vivo, adenoviruses produce a strong immune response in the host.
Adenovirus-related viruses are single-stranded DNA viruses. They are replication-defective that can transfect both dividing and non-dividing cells. For transfection, the viral genes are replaced by the transgene cassette.
Advantages: If transfected using adenovirus-related viruses, the transgene does not integrate into the host cell genome – therefore, producing transient expression of the transfected gene.
This is capitalized both in the lab for experiments, as well as for certain gene therapies.
Adenovirus-related transfection is very useful in particular types of gene therapy applications where the transgene is intended to be expressed only for a short duration in the hosts’ tissues. This is because the transfected transgene neither integrates with the host chromosomes, nor modifies it permanently. Also, the virus is incapable of replication; so, the infection level can be easily controlled.
This type of transfection is also efficient in different routes of clinical administration for gene therapy like systemic or intramuscular or through the airways.
Disadvantages: The transfection might not be stable and is lost as the cell divides. So, when you want stable and permanent transfection via modification of the host chromosomes, this method might be disadvantageous.
Also, though less immunogenic than adenoviruses, these viruses nevertheless induce immune responses and immunologic memory in the host. So, the viral infection levels and the transfection efficiency may be lower with each round of transfection as the host tissues learn to keep the virus off using its adaptive immune response memory.
When the goal is to obtain stable transfection, a class of RNA viruses called retroviruses are used.
Four types of retroviruses are primarily used for transfection: lentivirus, gammaretrovirus, spumavirus and alpharetrovirus.
Advantages: Retroviruses can be used for stable and long-term expression of the transgene because in this method, the transgene integrates into the host genome. Also, this method of stable expression leads to less immunogenic reactions in the host.
Disadvantages: Sometimes, when using retroviruses for transfection, unwanted mutagenesis in the host’s genome may accidentally happen because the transgenes integrate into the host cell’s chromosomes.
Also, mostly retroviruses can transfect only dividing cells – lentiviruses are exceptions to this though – these viruses can transfect non-dividing cells as well.
Transfection can be used for many purposes – for example, to deliver and integrate a gene permanently in the host cell’s genome, or for introducing a specific RNA molecule to see its effects on the recipient host cell and so on.
Depending on what you want to do, the type of transfection may vary to produce stable or permanent expression of the transfected genetic material.
Chong et al. 2021. Transfection types, methods and strategies: a technical review. PeerJ. doi: 10.7717/peerj.11165
Fus-Kujawa et al. 2021. An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro. Front. Bioeng. Biotechnol. Sec. Preclinical Cell and Gene Therapy
Volume 9 - 2021 | https://doi.org/10.3389/fbioe.2021.701031