Stable Transfection using Nucleofection™
Nucleofector™ Technology and Stable Transfections
The number of stably expression clones resulting from standard transfection experiments is often low, especially for cells which are difficult-to-transfect. In contrast to lipofection reagents the Nucleofector™ Technology is well suited even for the generation of stable expressing clones in difficult-to-transfect cell types. Efficient transfection of difficult-to-transfect cell lines enables the application of stable transfectants to address various scientific questions.
As an example an application note by Gebreselassie et al. demonstrates the use of Nucleofection™ for stably expressing HLA molecules in human lymphoblastoid cells. Furthermore Nucleofection™ was used to establish single-integration clones and clones used for protein production.
Stable Nucleofection™ of your specific cell-type
For the transfection of your specific cell-type, we recommend using the respective cell-type specific Nucleofector™ Kit together with the Optimized Protocol. A special protocol optimization for stable transfection is not necessary. Nevertheless, parameters such as ideal from of DNA, culture conditions, seeding densities, G418 concentration as well as clonal analysis still have to be optimized.
Higher integration rates in difficult-to-transfect cell lines using Nucleofection™. Jurkat cells were transfected using either Nucleofection™ (2 µg DNA) or competitor lipofection reagent L (0.7 µg DNA) according to the respective manufacturers instructions. 24 hours after transfection cells were plated on a 96-well plate containing culture medium supplemented with G418 for selection of stably transfected cells. 30 days after plating cells were analyzed for clonal outgrowth (Integration frequency = number of resistant clones per number of living cells seeded). Due to toxic effects lipofection with 10 µg circular DNA could not be performed.
Background on Stable Tranfection
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
- 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 (see scheme above): 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 following factors:
a) The transfection method used: 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.
b) The vector containing the gene of interest: 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).
c) The site of integration: The integration site 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 on stable transfection
1) Grimm S. (2004). The art and design of genetic screens: mammalian culture cells. Nature Rev Gen, 5: 179-189.
2) Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnol, 22: 1393-1398.
3) Glover, D. J., Lipps, H. J., Jans, D. A. (2005). Towards safe, non-viral therapeutic gene expression in humans. Nature Rev Gen, 6: 299-310.
4) Hacein-Bey-Abina, S. et al. (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 302: 415-419.
5) Worthington, J. et al. (2005) Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer. Gene Ther, 12: 1417-1423.
6) Murnane, J. P., Yezzi, M. J., Young, B. R. (1990) Recombination events during integration of transfected DNA into normal human cells. Nucleic Acids Res, 18: 2733-2738.
7) Ivics, Z. et al. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell, 91: 501-510.
8) Keng, V. W., et al. (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nature Meth, 2: 763-769.
9) Haber, J. E. (1999) DNA repair. Gatekeepers of recombination. Nature, 398: 665-667.
10) Southern, P. J., Berg, P. (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet, 1: 327-341.