Introduction

Transfection is a method of genetic alteration where foreign nucleic acids are introduced into animal cells to induce specific functions. Today, it is one of the most powerful techniques in molecular biology, often used to study the role of genes in health and disease. Transfection is also widely employed in gene therapy development and drug discovery for treating rare genetic disorders and cancer. Furthermore, it enables scalable protein production suitable for biotherapeutics development or industrial applications. Transfection methods can be categorized into two groups: stable transfection and transient transfection. While the former alters the genetic makeup of the host permanently, the latter only induces a short-term gene expression without integrating with the genome. While both stable and transient transfection methods have unique advantages, their utility depends on the specific application. Furthermore, the transfection technique of choice depends on whether a stable or transient transfection is aimed. Therefore, it is crucial to consider the application.

Stable Transfection

Stable transfection involves the integration of the external DNA into the host genome to achieve sustained expression. The transfected cells also gain the ability to pass the DNA to their descendants. Thus, a single transfection process is sufficient to induce long-term changes in the host’s genotype. This makes stable transfection ideal for applications requiring long-term gene expression.

Stable Transfection Methods

Stable transfection efficiency depends on the delivery method and the selection and isolation of transfected cells.

Delivery of the genetic material to the host may not always result in successful integration. Therefore, researchers must enrich the cell culture for the stably transfected cells. To achieve this, researchers co-transfect a marker gene to give selective advantages to transfected cells. For example, a specific drug resistance marker gene confers resistance to the transfected cells such that when the drug is administered to the culture, only the cells with sufficient transfection would survive¹. This selection process allows the isolation of stably transfected cells for further cultivation. Additionally, the transgene can be tagged by a fluorescent protein to confirm the successful integration of the DNA².

Stable Transfection Advantages and Applications

Long-term gene expression is the key benefit of stable transfection. A cell line with sustained transgene expression can reliably synthesize the protein of interest for extended periods. From this perspective, stable transfection lends itself to many life sciences applications.

Stable transfection enables continuous protein production, which is instrumental for the development of therapeutic proteins, vaccines, and industrial applications such as food processing and renewable biofuel production. It also has a central role in disease research and drug discovery, as it can reveal the role of specific genes in cellular processes that contribute to disease progression. In a clinical setting, stable transfection-based compensatory therapies are being investigated to treat rare genetic disorders and autoimmune diseases, where a gene might be introduced to the host genome to compensate for or repair a functional deficit.

Transient Transfection

Whereas stable transfection permanently alters the host genome, transient transfection induces short-term gene expression without genomic perturbations. The corresponding protein synthesis is much more rapid than that for stable transfection. Foreign genes are usually depleted after a few days and are not passed on to the daughter cells. These properties make transient transfection ideal for studying the immediate effects of gene silencing and knockdown or protein expression trials without undesired insertional mutations in the host genome.

Transient Transfection Methods

Transient transfection mainly involves DNA vectors, messenger RNA (mRNA), or RNA-based oligonucleotides, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs)³. RNA-based methods are widely adopted in transient transfection, as they allow researchers to bypass delivery into the nucleus and the transcription steps. Although RNA is less stable than DNA and more prone to rapid degradation before it reaches its target site, transfection reagents can be used to optimize its cellular uptake and target-specific release while preventing degradation.

Transient Transfection Advantages and Applications

Transient transfection can induce desired protein expression without disrupting the host genome. This is particularly advantageous when minimizing the risk of mutagenesis. Transient transfection offers a more rapid protein expression than stable transfection since it does not require nuclear delivery, transcription, and post-transcriptional processing.

The risk-free, rapid, and efficient nature of transient transfection makes it compatible with several biotechnology applications, where researchers can assess the immediate effects of gene overexpression, knockdown, and knockout. Using oligonucleotides, researchers can perform functional studies by targeting an mRNA to inhibit the subsequent protein encoding. Oligonucleotides can also be transfected to interfere with miRNA-mediated inhibition of a target protein to enhance its expression⁴. Finally, transient transfection is compatible with reporter gene analysis. By fusing the gene of interest with a reporter gene that produces a fluorescent or luminescent signal, researchers can quantify and monitor the real-time expression and activity of a gene⁵.

Transient Transfection vs Stable Transfection : A side-by-side Comparison

The two transfection methods vary in many aspects, from the delivery mechanisms to the period of expression and applications. While stable transfection ensures that the foreign DNA is incorporated into the host genome for prolonged and consistent gene expression, transient transfection only promotes short-term genomic and proteomic changes separate from the host nucleus and its genome. Table 1 highlights the differences between the two methods.

Deciding Between Stable Transfection and Transient Transfection

The decision between stable or transient transfection depends on the specific research application, mainly the experiment timeline. Stable transfection is more suitable for cases where researchers intend to generate irreversible changes in the host transcriptome and study these changes longitudinally with only a single transfection. Thus, it can be ideal for establishing disease-associated cell lines and investigating the effects of gene mutations on long-term disease progression. For protein production, stable transfection favors consistency and long-term production over speed and scalability. In contrast, transient selection is more appropriate if altering the genetic makeup poses risks to the host, and only the immediate effects of genomic perturbations are of interest. Accordingly, gene silencing and knockdown studies can benefit from transient expression. Furthermore, it must be preferred over stable expression if rapid and large-scale protein production is a priority over long-term production.

References

1. Kaufman WL, et al. Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation. Nucleic Acids Research 2008;36(17).
2. Peng L, et al. A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye. Bioengineered 2016;8(3):225–231.
3. Chong ZX, Yeap SK, Ho WY. Transfection types, methods and strategies: A technical review. PeerJ 2021;9.
4. Smith CIE, Zain R. Therapeutic oligonucleotides: State of the art. Annual Review of Pharmacology and Toxicology 2019;59(1):605–630.
5. Morales MJ, Gottlieb DI. A polymerase chain reaction-based method for detection and quantification of reporter gene expression in transient transfection assays. Analytical Biochemistry 1993;210(1):188–194.
6. Rybakovsky E, et al. Improving transient transfection efficiency in a differentiated, polar epithelial cell layer. Journal of Biomolecular Techniques 2019;30(2):19–24.

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