Choosing a Transfection Strategy
Deciding whether you need transient or stable transfection depends on the time frame and ultimate goal of the experiment you wish to conduct. Transiently transfected cells are typically harvested 24–96 hours post-transfection and are often used for studying the effects of short-term expression of genes or gene products, performing RNA interference (RNAi)‑mediated gene silencing, or rapidly producing recombinant proteins on a small scale. Transient transfection with mRNA can deliver even more rapid results; because mRNA is expressed in the cytosol without the need for translocation to the nucleus and the transcription process, it is possible for transfected mRNA to be expressed within minutes after transfection in some systems.
In contrast, stable transfection is more useful when long-term gene expression is required or when transfected cells need to be used over many experiments. 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. As such, it is normally reserved for large-scale protein production, longer-term pharmacology studies, gene therapy, or research on the mechanisms of long-term genetic regulation.
Although transient transfection of mammalian cells has been employed for the production of recombinant proteins with proper folding and post-translational modifications (which are not available when expressing recombinant proteins in bacterial cells) since the invention of transfection reagents, the ability to express milligram-to-gram amounts of recombinant protein has relied mainly on the creation of stable cell lines. More recently, large volume transient transfection of HEK293 and CHO cells adapted to suspension culture has addressed the need to obtain high amounts of recombinant protein without having to resort 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 suspensionadapted CHO or HEK293 cells, milligram-per-liter quantities of correctly folded and glycosylated recombinant proteins in three to seven days.
A major advancement in transient expression technology for rapid and ultra high-yield protein production in mammalian cells is the Expi293™ Expression System, which is based on the high-density culture of Expi293F™ cells in Expi293™ Expression Medium and transfection using the cationic lipid-based ExpiFectamine™ 293 transfection reagent in combination with optimized transfection enhancers. All components work in concert to generate 2- to 10-fold higher protein yields than conventional culture systems such as the FreeStyle™ 293 Expression System, achieving expression levels of greater than 1 g/L for IgG and non-IgG proteins.
Clinical biotherapeutics are frequently generated using stable, high-expression transfectants, because they provide batch-to-batch consistency and low cost at extremely large-scales. However, in many drug discovery applications, it is beneficial to screen protein constructs quickly using transient transfection methods, which allow simultaneous evaluation of various candidate molecules in less than one week. In many instances, transient transfections are performed in parallel while more resource intensive stable cell lines are under development, which can take more than three months to accomplish.
|Transient Transfection||Stable Transfection|
|Transfected DNA is not integrated into the genome, but remains in the nucleus.||Transfected DNA integrates into the genome.|
|Transfected genetic material is not passed onto the progeny; genetic alteration is not permanent.||Transfected genetic material is carried stably from generation to generation; genetic alteration is permanent.|
|Does not require selection.||Requires selective screening for the isolation of stable transfectants.|
|Both DNA vectors and RNA can be used for transient transfection.||Only DNA vectors can be used for stable transfection; RNA by itself cannot be stably introduced into cells.|
|High copy number of transfected genetic material results in high level of protein expression.||Single or low copy number of stably integrated DNA results in lower level of protein expression.|
|Cells are typically harvested within 24–96 hours of transfection.||Requires 2–3 weeks of selection for the isolation of stably transfected colonies.|
|Generally not suitable for studies using vectors with inducible promoters.||Suitable for studies using vectors with inducible promoters.|