Stable Transfection

Stable Transfection

Background:

The possibility to stably integrate genes into the genome of mammalian cells has an important impact on many biomedical research areas as well as for the development of pharmaceutical products. While transient transfection is advantageous for fast analysis of genes and small scale protein production, stable transfection ensures long-term, reproducible as well as defined gene expression.
Major applications for stable transfection are the analysis of gene function and regulation (1), large scale protein production (2), drug discovery and gene therapy (3).

The general mechanism of stable integration
Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the gene of interest has to be introduced into the cell (A), subsequently into the nucleus (B) and finally it has to be integrated into chromosomal DNA (C). 

 

Stable expression can be influenced by two factors: The transfection method used and the vector containing the gene of interest.
The transfection method determines which cell type can be targeted for stable integration. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into notoriously difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and Nucleofection®. Unfortunately, viral methods suffer from several limitations such as time-consuming production of vectors and safety concerns (4).
So far Nucleofection® is the only non-viral method introducing DNA molecules efficiently into the nucleus of virtually any cell type, therefore significantly increasing the chances of chromosomal integration of the transgene.

The type of vector used for stable integration defines the integration mechanism, the regulation of transgene expression and the selection conditions for stably expressing cells. After integration the level and time of expression of the gene of interest depends on the promoter cloned upstream on the expression vector and on the particular integration site. For constitutive expression, promoters such as the CMV promoter are chosen. For a regulated expression inducible promoter systems are available (5).

Furthermore the site of integration can have an effect on the transcription rate of the gene of interest (2). Usually a regular expression plasmid is integrated into the genome of the target cell randomly (6). Integration into inactive heterochromatin results in little or no transgene expression, whereas integration into active euchromatin frequently allows transgene expression. However, random integration often leads to silencing of the transgene. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid (7, 8).

The exact mechanism by which plasmid DNA is integrated is not yet fully understood and remains a matter of research. In viral systems, the foreign DNA is integrated into the host genome via viral integration mechanisms. Plasmid DNA delivered by non-viral methods, on the other hand, is integrated by the cell's machinery itself, possibly via DNA repair and recombination enzymes (9).

Selecting for stable transfected cells
Stably transfected cells can be selected and cultured in various ways: For the selection of stably transfected cells, a selection marker is co-expressed on either the same or on a second, co-transfected vector. A variety of systems for selecting transfected cells exists, including resistance to antibiotics such as neomycin phosphotransferase, conferring resistance to G418, dihydrofolate reductase (DHFR), or glutamine synthetase (2, 10). The culture of the transfected cells can be done either in bulk to obtain a mixed population of resistant cells, or via single cell culture, to obtain cell clones from one single integration event.

Literature

For further information on how to generate stable cell lines please download our respective guideline from this page.

For any additional questions regarding stable transfection with the Nucleofector® Technology please contact our scientific support team.

Please also check our Citation Database for more publications on stable transfection using the Nucleofector® Technology.

to top