perimental applications using transfection include gene editing, modifying host cell gene expression, and for recombinant protein and viral vector production.
Transfection introduces foreign nucleic acids into eukaryotes where they are expressed transiently or stably (long-term).
Gene editing may be done by transfecting the transgene into the recipient host cell, or by using CRISPR-Cas9 – by introducing CRISPR reagents into the target host cell.
For transiently modifying host cell gene expression, an interfering RNA such as siRNA or miRNA may be transfected into the host cells for knocking down expression of target mRNAs.
Transfection is also used to introduce a recombinant DNA construct into expression host cells where this construct expresses a recombinant protein – such as an antibody, for example.
Also, transfection is used in the process of making recombinant vectors – which in turn are used in downstream gene transfer procedures.
Basically, whenever you want to transfer a foreign nucleic acid into a eukaryotic cell, it involves transfection.
In this article, we'll take a closer look at each of these applications of transfection to understand why it is such a helpful and widely used technique.
Specifically, we will take a brief look into how transfection is used in RNAi (RNA interference) experiments, procedures for editing the host genome, and how this technique comes handy in mass producing recombinant proteins using eukaryotic cells as the expression host system.
We will also describe how transfection is used for making recombinant viral vectors, which are used for downstream transfection experiments.
RNAi studies
RNAi, or RNA interference, as the name suggests, is an approach where a foreign RNA is introduced into the host cells to interfere with the gene expression there. This foreign RNA temporarily knocks down expression of one or more genes in the host cells by binding to the corresponding mRNAs of those genes.
RNAi is regularly used for gene expression analyses. In this section, we have two examples describing how RNAi is used for gene expression analyses, and how transfection is used in those experiments.
For RNAi studies, mostly siRNA and miRNAs are transfected. Both have very similar modes of interference. They bind the target mRNA and block its translation and increase the rate of its decay.
While siRNA has a single mRNA as its target in the host cell, miRNAs generally have multiple mRNAs as its target.
Here are two examples of experiments that would need small RNAs to be transfected.
You are studying a specific miRNA – called miRNA A. It is known already that miRNA A has a lot of physiological effects, but its role in gastric cancer is what you want to figure out.
The experiment is designed to examine the effect of miRNA on gastric cancer cells.
Transfection would be a very useful technique because it involves introducing a foreign nucleic acid (miRNA A) into eukaryotic cells (gastric cancer cells). The cell line is transfected with miRNA A. After this, you would perform various assays to find out whether miRNA A has any effect on these cancer cells – for example, causes their apoptosis or increases their growth etc.
For the second example, let’s imagine that it is already known that expressing gene B, we’ll call it, plays a role in lung cancer. You want to explore whether inhibiting its expression may be a therapeutic strategy against lung cancer.
For this, you get a lung cancer cell line that expresses gene B at high levels. You design an siRNA that would bind to the mRNA transcript of gene B and result in its degradation.
In the experiment, you would transfect the lung cancer cell culture with this siRNA to knock down gene B expression. And then you would want to see if this knockdown of gene B indeed has any effect on the cancer cells by causing apoptosis, for example.
Gene editing
For editing genes in host cells, DNA sequences can be transfected and then recombine with the host genome, modifying the genetic makeup of the cell. Another way is to introduce by transfection CRISPR-Cas9 components into the target cells for gene editing.
CRISPR-Cas9 is a genome editing technique. A quick explanation of how this works is that a non-coding RNA known as guide RNA (gRNA), along with a nuclease called Cas9 is transfected into the target host cells.
Using the gRNA and Cas9, the host genome can be cut at a specific desired location to delete DNA sequences or introduce new ones.
Whenever the target cells are eukaryotic, transfection is absolutely essential for gene editing, whether CRISPR-Cas9 is used or any other method.
The figure below illustrates a how a piece of foreign DNA is transfected and recombines with the recipient host cell’s genome, modifying it. Often, the transgene is transfected using a vector, like a viral vector, that promotes integration with the host’s genome.
Transfection can be used to transfect a cell with foreign DNA that integrates with the host cell genome and modifies it.
Recombinant protein production
While microbial cells are often used as expression hosts, there are situations where eukaryotic cells such as insect or mammalian cell lines are preferred as the expression system for mass producing the desired recombinant protein.
For example, when the recombinant protein needs post-translational modifications, such as glycosylation, acylation, acetylation etc., animal cells might just be the best expressions system because some protein modifications can only be possible in cells of higher organisms.
Indeed, for mass producing therapeutic proteins such as antibodies, mammalian cells are still preferred for expression because they are much more closely compatible with the patients’ physiology compared to other expression hosts such as bacteria.
And when you would have to introduce your recombinant construct in the eukaryotic expression host, you will need to use transfection. There lies the importance of transfection in recombinant protein production.
So, in technical terms, for recombinant protein production using eukaryotic cells as the expression host, the corresponding recombinant transgene, cloned in an expression vector, is transfected into the host cell line. Then the transgene expresses itself for high levels of recombinant protein production.
Basically, whenever your expression system comprises mammalian or insect cells, transfection becomes a necessary tool during recombinant protein production because without transfection, you would not be able to introduce the recombinant DNA construct in the target expression cell line.
Recombinant viral vector production
Researchers use viruses as the vehicle, or vector to introduce a transgene into a host cell. However, those viral vectors have to be produced somehow, and they’re often made within eukaryotic cells.
So, in some cases, researchers will be using one vector to introduce a transgene into a eukaryotic cell that will be used to make another vector. And since this whole process involves introducing transgenes into eukaryotic cells, transfection is invariably required.
Transfection is used to synthesize recombinant viral vectors with the cloned transgene. Lentiviral vectors are examples of this. The recombinant viral vector then transduces, or in other words, introduces the desired transgene into the target host cell via viral transduction.
A schematic is presented below of how a recombinant viral vector is produced, and why transfection is necessary in that process.
The viral vector comprises of the following –viral proteins necessary for infection and the proteins necessary for packaging the transgene in the vector. In the first step, these components of the viral vector (viral proteins for infection and packaging) are cloned in 2-3 plasmids. Along with that, the desired transgene is also cloned in another plasmid.
In the next step, these 3-4 plasmids are then transfected together in a cell line – known as packaging cells.
In this packaging cell line, the recombinant viral vector, containing the transgene, is synthesized from these 3-4 plasmids.
And then, this recombinant viral vector is harvested and purified out of this packaging cell line.
We have seen a few examples of experiments where transfection becomes very handy, or even absolutely necessary. Whenever you have a situation where a foreign nucleic acid needs to be introduced into eukaryotic cells, the only way to go about it is transfection. And from gene expression analyses to recombinant protein production, modern bioscience is heavily focused on eukaryotic cells – hand in hand, transfection has also evolved as an absolutely necessary technique.
References
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
Haiyong. 2018. RNA Interference to Knock Down Gene Expression. Methods Mol Biol. 1706: 293-302
