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FAQspeak

How do I stably select for my vector in mammalian cells?

Stable transfection involves the maintenance or integration of a plasmid in eukaryotic cells. To accomplish this, first untransfected cells will need to be exposed to the appropriate drug (e.g., G-418) to determine the amount that will kill untransfected cells. Typically, untransfected cells are exposed to a range of drug concentrations to determine the lethal concentration (called a kill curve). Transfect a fresh population of cells with your construct and select using the lethal dose of drug for
2–5 weeks, depending on cell type and drug sensitivity. The antibiotic-resistance gene (e.g., neomycin phosphotransferase) encoded by the construct will protect any transfected mammalian cells from dying when exposed to the drug.

Cells can be transfected with a single construct containing the antibiotic-resistance gene and other desired features, or two vectors can be cotransfected together, one containing the expression construct and other carrying the drug-resistant gene. Often the vector that conveys drug resistance is transfected at a much smaller amount than the expression vector so that if the vector with the antibiotic-resistance gene is integrated into the cell genome, the experimental vector is probably integrated as well. Thus, both plasmids will be maintained by a lethal dose of the appropriate drug.

Under the selective pressure of the antibiotic, resistant clones will form and can be propagated. These clones can then be evaluated for expression levels of the experimental construct. The stably transfected cells will need to be grown in the presence of a lower dose of antibiotic to ensure that the selected plasmid continues to be maintained. At this point, the stably transfected cells can be used for experiments.

For additional information and a protocol for stable transfection, visit the Protocols and Applications Guide chapter on Transfection.