Transient vs Stable Transfection
The method of transfection into a cell can be called either transient or stable. Transfection, one of the basic technologies used in cell and molecular biology, is used by researchers to insert a segment of DNA or RNA into a host cell, usually a mammalian cell in order to produce proteins or alter endogenous gene expression.
Experimental goals and experiment duration determines whether the researcher wants to pursue stable or transient transfection. In a transient transfection, gene expression changes can be studied in a window of 8 to 96 hours post-transfection. This method is useful for short-term expression of genes or gene products, gene knockdown or small-scale protein production. If performed with mRNA, which is only produced outside the nucleus in an unmodified cell, a transient transfection can deliver extremely rapid results. Stable transfection is a longer and more complex process, mainly reserved for protein production on a large scale, research on long-term genetic regulation, extended pharmacology research or gene therapy.
After insertion in a eukaryotic cell, the exogenous nucleic acid may or may not become a part of the cellular genome. If it integrates and becomes an inherent part of the host genome, the introduced DNA is subsequently replicated and expressed, even in daughter cells. Expression of the foreign genetic code is continual and the transfection procedure is termed a stable transfection. If the host cell rejects the inserted DNA segment, the transfection becomes transient because expression of the introduced genetic material is lost; thus, lasting only a short duration of time.
The nature of stable transfection necessitates scientists to use techniques that ensure inclusion of foreign DNA into transfected cell’s genetic composition. Owing to their different natures, stable and transient transfection processes offer varying benefits to scientists. Stable transfection is mainly used for producing proteins on a large scale, studying gene expression and the development of gene therapy treatments for diseases such as cancer. Other factors affecting stable transfection and expression of the incorporated gene include the unpredictable site of integration and the kind of vector used in plasmid construction. At times, the foreign gene may be integrated at a relatively inactive position leading to extremely low expression levels.
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In transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome. The plasmid DNA (or other type of nucleic acid) typically has a reporter gene that allows the scientist to monitor the expression of the reporter gene, usually within 1-2 days post-transfection. Therefore, for a short period of time, the researcher knows there are many copies of the foreign gene in the nucleus of the cell and the gene is being expressed. However, if the gene is not incorporated into the genome, the transfected DNA will be degraded and will not be passed to future generations of the cell. Highly supercoiled DNA appears to be superior for transient transfections.
In stable transfection, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell. This method is a much rarer occurrence and very complex to perform, as sometimes unpredictable regions of the plasmid gets integrated, which may not contain the gene of interest. All stable transfections start out as a transient transfection, but the use of selectable markers expressed on the plasmid DNA enable the researcher to distinguish any cells that have successfully integrated the gene into their genome. A common method used is to design the plasmid DNA to also contain a gene that expresses antibiotic resistance. Continued antibiotic treatment of the cells for long-term results in the expansion of only the stably-transfected cells, while non-stable cells die off due to the lack of antibiotic resistance. Linear DNA appears to be better for stable transfection, although its uptake is still lower than using supercoiled DNA.