Choosing which type of nucleic acid you’d want to transfect depends on what you want to do in your experiment, and that might have you wondering, what are the different types of nucleic acids that can be transfected, and what is the best option for your experiment?
Nucleic acids commonly transfected into eukaryotic cells are DNA, mRNA and CRISPR-Cas9 molecules. There are also many different types of small RNA molecules that can be transfected into eukaryotic cells that will be discussed.
List of the four types of nucleic acids that are transfected into eukaryotic cells:
- Small non-coding RNAs
- CRISPR Reagents
As a quick reminder, transfection is a method for introducing foreign nucleic acids into eukaryotic cells. For details about this technique and how it is done using physical, chemical or biological (using viruses) means – you may want to review this article.
In modern bioscience, transfection is a cornerstone of multiple types of experimental applications spanning from permanent or transient modification of gene expression to recombinant protein production. Basically, transfection is a consistent part of every experiment that involves transferring foreign nucleic acids into eukaryotic cells.
If you'd like to learn more about some of the different, and really important ways transfection aids vital research applications, you should take a look at this detailed article.
Now that we’ve covered what transfection is, and how and where it is done, in this article, we will discuss what types of molecules are transfected into eukaryotic cells.
Article table of contents:
DNA or CRISPR-Cas9 molecules are transfected for genetic engineering, while mRNAs are transfected to express a transgene for a short term. Small RNA molecules like miRNAs, siRNAs, shRNAs and piRNAs are transfected for transiently knocking down target gene in the host cell.
So now, let’s take a closer look at each of the different types of nucleic acids that are commonly transfected, starting with DNA.
Foreign DNA maybe cloned in a plasmid or in another vector and transfected to express the recombinant construct from the host cell cytoplasm. For example, transfected DNA could be used to produce recombinant proteins.
For gene editing, DNA is transfected in a way that it integrates with the host genome and modifies it.
Figure 1. Fate of transfected DNA. In the top block, the transfected DNA enters the host cell nucleus and recombines with the host genome in the nucleus – modifying it. In the second block, the transgene is transfected in such a way that it remains in the cytoplasm, and maintains and expresses itself there from the vector.
DNA is generally transfected using viral vectors or plasmids.
If the transfected DNA needs to be expressed from a vector, it is cloned downstream of a eukaryotic promoter that is compatible for transcription in the cells being transfected.
Figure 2. Simple illustration of a plasmid used as a vector. The promoter is a eukaryotic promoter for appropriate transgene transcription.
Plasmids used for DNA transfection can be both linear or supercoiled. Supercoiled plasmids, unlike linear plasmids, are less prone to being degraded by the host cells’ nucleases. Because of this, transfection efficiency for supercoiled plasmids is higher compared to linear ones.
However, when you want the foreign DNA to integrate with the host genome, linear plasmids are preferred because of their higher tendency to recombine with host DNA.
When considering whether you should choose viral vectors over plasmids, here are two things to note. One, viral vectors might trigger an immune response in the host cell because viruses are much more immunogenic.
Two, when you are wanting to design your transfection experiment to express the cloned transgene from the vector in the host cell cytoplasm, here is the deal: efficiency of both transfection and recombinant protein production from the transfected DNA is higher for viral vectors compared to plasmids. But viral vectors have high tendency to recombine with the host genome.
mRNAs are transfected to transiently express the encoded polypeptide in the host cell. Small non-coding RNAs like miRNA, siRNAs, shRNAs and piRNAs are transfected in RNA interference (RNAi) assays to transiently knockdown target gene expression.
Here is a brief description of the types of RNAs that are commonly transfected. Details of the experimental applications of RNA transfection are covered here.
Gene expression follows this route – gene expresses mRNA by transcription, which in turn is translated to the corresponding polypeptide.
By transfecting mRNAs, you can get the protein expressed transiently at a high rate within a short span of time because this bypasses the need for the transcription step.
For a transfection experiment where the objective is to express a certain protein in high amounts for a short period of time in the target host cell, transfecting mRNAs may be a better option than transfecting the transgene cloned in a plasmid. This is because the transcription step is not required. When you directly transfect the mRNA into the host cell, translation can readily commence.
However, mRNAs are prone to quick degradation. So, when mRNAs are transfected directly, the expression of the corresponding protein is transient and its translation goes down as the transfected mRNAs are degraded by host cell nucleases.
miRNAs, siRNAs, shRNAs and piRNAs are small non-coding RNAs that are 20-200 nucleotides in size. They do not encode polypeptides themselves but regulate gene expression at a posttranscriptional level, affecting stability and translation of their target mRNAs. They are transfected in RNAi experiments.
The basic idea of how a small RNA works to affect gene expression at a posttranscriptional level is depicted in figure 3.
Figure 3. Mechanism of gene regulation by small non-coding RNA
Small RNAs may be endogenously transcribed – that is, expressed by the cell itself as part of its normal physiological regulation. They have specific mRNAs as their targets. They bind to these target mRNAs and regulate them post-transcriptionally primarily by two mechanisms.
First, promoting or inhibiting the degradation of the target mRNAs. Second, by affecting the rate at which the target mRNAs are translated.
So, by transfecting a host cell with an appropriate small RNA that you would carefully choose, depending on the needs of your experiment – expression of a single gene or many genes may be “knocked down” or boosted up.
There are experimental technicalities that researchers think about when deciding which type of sRNA they want to transfect in a particular experiment. For example, siRNAs are typically specific to a single target mRNA, while miRNAs generally have multiple target mRNAs.
Knock down of a gene means fully/ partially blocking its expression for a relatively short time (transient). This is different from knocking a gene out which involves permanently deleting the gene from the genome of the cell.
The mechanism of action of small RNAs is capitalized in experiments that involve RNAi, as discussed in this article.
CRISPR-Cas9 reagents are transfected into the target cells for gene editing using CRISPR. A non-coding guide RNA (gRNA), along with a Cas9 nuclease is transfected into the target cells so that the host genome can be cut at a specific desired location to delete DNA sequences or add new ones.
CRISPR reagents can be transfected by all three methods of transfection. However, it is important to keep in mind that there are different approaches to physical, chemical and viral transfection that are better suited for transfecting CRISPR reagents.
For instance, when it comes to physical transfection, electroporation is typically used for CRISPR. And when it comes to chemical transfection, lipofection is commonly used. Finally, if a virus is being used to transfect CRISPR reagents, lentiviral transfection is appropriate.
To understand the different transfection approaches and how they work, you might want to look at this article.
We discussed the types of molecules commonly transfected. These molecules span from being DNA or mRNA that encode specific proteins, to various kinds of non-coding RNAs for RNAi or CRISPR applications. This article provides a fundamental perspective when we talk about actual experimental applications of transfection.
The table below summarizes some of the key points of this article, serving as a handy visual.
Table 1. Summary of methods used to transfect nucleic acids
Best Transfection Method
Recombinant protein expression, Gene editing
Viral vectors for cytoplasmic expression, Linear plasmids for genome integration
mRNA, Small non-coding RNA (miRNA, siRNA, shRNA, piRNA)
Transient protein expression, RNA interference (RNAi)
Viral and Chemical methods. Depends on experimental needs.
gRNA with Cas9 nuclease
Gene editing using CRISPR
Physical: Electroporation; Chemical: Lipofection; Viral: Lentiviral transfection
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