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How does transient transfection work?
In transient transfection, the introduced nucleic acid exists in the cell only for a limited period of time and is not integrated into the genome. As a result, transiently transfected genetic material is not passed from generation to generation during cell division, and it can be lost by environmental factors or diluted out during cell division. However, the high copy number of the transfected genetic material leads to substantial levels of expressed protein within the temporary period that it exists in the cell.
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Figure 1. Sample transient transfection workflow. Transfected nucleic acids remain in the cell for a limited amount of time.
Depending on the construct used, a transiently expressed gene can generally be detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to 96 hours posttransfection. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The optimal time interval depends on the cell type, research goals, and specific expression characteristics of the introduced gene, as well as the time it takes for the reporter to reach steady state. However, within a few days most of the foreign DNA is degraded by nucleases or diluted by cell division, and after a week, its presence is no longer detected.
Transient transfection is most efficient when supercoiled plasmid DNA is used, presumably due to its more efficient uptake by the cell (see Guidelines for Plasmid Transfection). Many molecules can be transiently transfected into cells, including siRNAs, miRNAs, mRNAs, and even proteins. However, as with plasmid DNA, these macromolecules need to be high quality and relatively pure (see Factors Influencing Transfection Efficiency). While transfected DNA is translocated into the nucleus for transcription, transfected RNA remains in the cytosol, where it is expressed within minutes after transfection (mRNA) or bound to mRNA to silence the expression of a target gene (siRNA and miRNA) (see Guidelines for RNA Transfection).
Due to its simplicity and ease compared to stable transfection, transient transfection is used in a plethora of experimental applications, including any form of RNA transfection (i.e., mRNA or RNAi transfection), many plasmid transfections, as well as for transfection in certain gene editing experiments (e.g., transfection of Cas9 mRNA).
Differences between transient vs stable transfection
Transient transfection
- Nucleic acid does not integrate into the host genome
- Only present and expressed for a limited amount of time
- Simpler and less laborious
- Can be performed using DNA or RNA
- Used for applications that require temporary modifications of cells (e.g., temporary mRNA expression, temporary expression of RNAi molecules)
Stable transfection
- Nucleic acid generally integrates into the host genome
- Supports gene expression over a long period of time
- More laborious and requires the use of selective markers or antibiotics
- Relies on the use of DNA (i.e., from a plasmid or viral vector)
- Used for applications that require the generation of clonal cell lines
Benefits of transiently expressed protein production
- Rapidly go from cloned gene to large quantities of protein in 3–7 days
- Obtain fully post-translationally modified and active mammalian proteins
- Use your available mammalian cell culture facilities with shake flasks and a platform shaker
- Easily purify secreted proteins from serum-free cell culture medium
- Scale up from 50 multi-well plates to to 25 L of culture volume and more
Transient transfection of suspension cells
It is possible to perform large volume transient transfection of HEK293 and CHO cells adapted to suspension culture. This has addressed the need to obtain high amounts of recombinant protein without having to resort to the laborious process of stable cell line development.
Featured 293 and CHO cell transfection products
Gibco Expi293 Expression System
The Gibco Expi293 Expression System includes Expi293 expression medium, Expi293F cells, and the ExpiFectamine 293 transfection kit. These components work together to enable high density transient transfection of Expi293F cells for rapid and ultra high-yield protein production.
Gibco FreeStyle MAX CHO Expression System and FreeStyle MAX 293 Expression System
Gibco FreeStyle MAX Expression System combines FreeStyle media, FreeStyle MAX reagent, and either FreeStyle CHO-S cells or FreeStyle 923 cells. The FreeStyle MAX Systems can replace time-consuming stable cell line generation with large-scale functional protein production in one week.
Gibco FreeStyle MAX Reagent
Gibco FreeStyle MAX Transfection Reagent is a novel transfection reagent to transfect FreeStyle CHO and HEK-293 cells with exceptionally high efficiency, enabling rapid and milligram scale of protein yield per liter.
Find transient transfection reagents
Thermo Fisher Scientific is a trusted supplier for transient transfection materials, including cell culture media, vectors, plasmid purification products, and transfection reagents. Explore transfection reagents based on sample type, including those for:
In addition, our highly recommended Lipofectamine reagents, including the advanced Lipofectamine 3000 reagent, are available for both stable and transient transfection.
Visit Transfection Basics to learn more about performing transfection in your lab.
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