Transient Transfection vs Stable Transfection

Transfection methods vary in efficiency, cell toxicity, effects on normal physiology, and level of gene expression, so not all methods can be applied to all cell types and experiments. However, all transfection strategies can be broadly classified into two types: transient transfection and stable transfection. In transient transfection, nucleic acids are introduced to the cell and exist within the cell for a limited period of time. In stable transfection, the nucleic acids persist in the cells long‑term and are passed to the progeny of the transfected cell. The following information compares transient vs stable transfection and the suggested applications for each.

Transient transfection does not integrate nucleic acids into the genome, so transiently transfected genetic material is not passed from generation to generation. Depending on the construct used, the transgene can usually be detected for 1 to 7 days but is typically harvested 24 to 96 hours post-transfection because most of the foreign DNA is degraded by nucleases or diminished during cell division within a few days.

Factors that may influence the optimal harvest time interval include:

• cell type
• research goals
• expression characteristics of the introduced gene
• time it takes for the reporter to reach steady state

Transient transfection is most efficient with supercoiled plasmid DNA due to its enhanced cellular uptake. Other molecules, such as siRNAs, miRNAs, mRNAs, and proteins, can also be used, but they must be high quality and relatively pure for optimal transfection efficiency.

In DNA transfection, the DNA is transported into the nucleus for transcription. In contrast, transfected RNA remains in the cytosol, where mRNA can be expressed within minutes, and siRNA or miRNA can bind to mRNA to silence target genes.

Transiently transfected cells produce high levels of expressed protein due to the large amount of genetic material introduced into the cells. To analyze gene product effectiveness, RNA or protein may need to be isolated for enzymatic activity assays or immunoassays.

Transient transfection is commonly used for temporary cell modifications because it is simpler and easier to execute than stable transfection. It is often used for:

• Plasmid transfection
• mirna, sirna, and RNAi transfection (such as performing RNAi-mediated gene silencing)
• Studying short-term gene effects or gene product expression in gene editing experiments(such as transfecting Cas9 mRNA)
• Protein expression

Recombinant protein expression

Transient transfection of mammalian cells can quickly produce recombinant proteins on a small scale with proper folding and post-translational modifications, which are not available when expressing recombinant proteins in bacterial cells.

Historically, the ability to express milligram-to-gram amounts of recombinant protein has relied on the creation of stable cell lines. Yet, more recently, large-volume transient transfection of HEK293 and CHO cells adapted to suspension culture has met the demand for high amounts of recombinant protein without resorting to the laborious process of stable cell line development.

Recombinant protein expression by transient transfection enables researchers to produce, starting from the vector of interest and suspension-adapted CHO or HEK293 cells, milligram-per-liter quantities of correctly folded and glycosylated recombinant proteins in three to seven days.

Explore mammalian transient protein expression systems

Rapid and ultra high-yield protein production

In many drug discovery applications, transient transfection methods are beneficial for quickly screening protein constructs, enabling the simultaneous evaluation of various candidate molecules in less than one week. Often, transient transfections are carried out in parallel with the more resource-intensive development of stable cell lines, which can take over three months to complete.

Unlike transient transfection, where introduced DNA lasts in cells for several days, stable transfection introduces DNA into host cells, allowing continuous, long-term gene expression without needing repeated transfections. The foreign DNA can either integrate into the cell's genome or remain as an episomal plasmid, supporting extended maintenance, and gene expression over multiple generations.

Stable transfection is useful for producing recombinant proteins and studying long-term DNA effects. However, only a few copies of the DNA usually integrate, leading to lower expression levels compared to transient transfection.

Successful stable transfection requires effective DNA delivery and a way to select cells that have taken up the DNA.

Effective DNA delivery is often determined by cell type and whether the DNA is linear or circular. Only about 1 in 10,000 transfected cells will stably integrate the DNA—linear DNA is taken up less efficiently than supercoiled DNA, but it integrates into the host genome more effectively.

DNA vectors are used most frequently in stable transfection, but siRNA and miRNA can also be stably introduced as short hairpin transcripts from a selectable DNA vector. RNA molecules alone cannot be used for stable transfection.

Selectable markers are included on the DNA construct or on a separate co-transfected vector to select cells that stably express the transfected DNA. After a short recovery period, selective pressure is applied to the cells. When using a co-transfected vector, the ratio of the vector carrying the gene of interest to the vector carrying the selectable marker should be 5:1 to 10:1 to help ensure that cells with the selectable marker also have the gene of interest.

Common selectable markers include genes that confer resistance to selection drugs or compensate for a defective essential gene in the cell line. In selective medium, non-transfected or transiently transfected cells will die, while those expressing the resistance gene or compensating for the essential gene defect will survive. Alternatively, phenotypical or morphological changes can be used as screenable traits.

Selection antibiotics are commonly used to apply selective pressure. The antibiotic chosen will depend on which antibiotic resistance gene or selectable marker is used in the transfection experiment.

Stable cell line generation protocol
View all antibiotics for stable cell transfection

When long-term gene expression is required or when transfected cells need to be used over many experiments, stable transfection is more suitable than transient transfection. Because integration of a DNA vector into the chromosome is a rare event, stable transfection of cells is a more laborious and challenging process, which requires selective screening and clonal isolation.

The ability to establish a stable cell line makes stable transfection a valuable experimental and clinical research application.

Stable transfection is suitable for instances of:

• large-scale protein production in biotechnological and biopharmaceutical settings
• longer-term pharmacology studies
• clinical biotherapeutics
• gene therapy
• research on mechanisms of long-term genetic regulation
• long-term expression in gene therapy

Choose transient or stable transfection based on your experiment's time frame and goals.

Visit Transfection Basics to learn more about performing transfection in your lab.