There are a number of biological, chemical, and physical methods for introducing nucleic acids into cells. Not all of these methods can be applied to all types of cells and experimental applications, and there is a wide variation amongst them with respect to transfection efficiency, cell toxicity, effects on normal physiology, and level of gene expression. However, all of the transfection strategies can be broadly classified into two general types based on whether the introduced nucleic acid exists in the cell for a limited period of time (transient transfection) or whether it persists in the cells long‑term and is passed to the progeny of the transfected cell (stable transfection).
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 such, 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 high levels of expressed protein within the period that it exists in the cell.
Depending on the construct used, transiently expressed transgene can generally be detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to 96 hours post-transfection. 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; 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. siRNAs, miRNAs, mRNAs, and even proteins can be also used for transient transfection, but as with plasmid DNA, these macromolecules need to be of high quality and also be 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).
In stable transfection, foreign DNA is either integrated into the cellular genome or maintained as an episomal plasmid. Unlike transient transfection, stable transfection allows the long-term maintenance of the exogenous DNA in the transfected cell and its progeny. As such, stable transfection can provide persistent expression of the introduced gene through multiple generations, which can be useful for production of recombinant proteins and analysis of downstream or long-term effects of exogenous DNA expression. However, usually a single or a few copies of the exogenous DNA is integrated into the genome of the stably transfected cell. For this reason, the expression level of stably transfected genes tend to be lower than that of transiently transfected genes.
Because stable integration of foreign DNA into the genome is a relatively rare event, successful stable transfection requires both effective DNA delivery and a way to select cells that have acquired the DNA. One of the most reliable ways to select cells that stably express transfected DNA is to include a selectable marker in the DNA construct used for transfection and then apply the appropriate selective pressure to the cells after a short recovery period.
Frequently used selectable markers are genes that confer resistance to various selection drugs or genes that compensate for an essential gene that is defective in the cell line to be transfected. When cultured in selective medium, cells that were not transfected or were transiently transfected eventually die, and those that express the antibiotic resistance gene at sufficient levels or those that can compensate for the defect in the essential gene survive. Alternatively, phenotypical or morphological changes in the transfected cells can be used as a screenable trait in certain cases. For example, mouse CI127 cells transfected with vectors derived from bovine papilloma virus produce a morphological change (Sarver et al. 1981).
Although linear DNA results in lower DNA uptake by the cells relative to supercoiled DNA, it yields optimal integration of DNA into the host genome (see Factors Influencing Transfection Efficiency). As a rule, stable transfection is limited to DNA vectors, but siRNA and miRNA may be stably introduced into cells when they are delivered as short hairpin transcripts made from a selectable DNA vector (see Vector‑mediated RNAi). However, RNA molecules by themselves cannot be used for stable transfection.