Unlike transient transfection, in which introduced DNA persists in cells for several days, stable transfection introduces DNA into cells long-term. Stably transfected cells pass the introduced DNA to their progeny, typically because the transfected DNA has been incorporated into the genome, but sometimes via stable inheritance of nongenomic DNA.

How does stable transfection work?

Successful stable transfection requires both effective DNA delivery and a way to select cells that have acquired the DNA. Approximately one in 104 transfected cells will stably integrate DNA, although the efficiency varies with cell type and whether linear or circular DNA is used. Integration is most efficient when linear DNA is used.

One of the most reliable ways to select cells that stably express transfected DNA is to include a selectable marker on the DNA construct used for transfection or on a separate vector that is co-transfected into the cell, and then apply the appropriate selective pressure to the cells after a short recovery period. When the selectable marker is expressed from the co-transfected vector, the molar ratio of the vector carrying the gene of interest to the vector carrying the selectable marker should be in the range of 5:1 to 10:1 to ensure that any cell that contains the selectable marker also contains the gene of interest.

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 will die, and those that express the antibiotic resistance gene at sufficient levels or those that can compensate for the defect in the essential gene
will survive.

Recommended transfection reagents

  • Lipofectamine® 3000 reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection performance and reproducible results. It delivers exceptional transfection efficiency into the widest range of difficult-to-transfect and common cell types with improved cell viability.

Selection antibiotics for eukaryotic cells

We offer high-quality selection reagents to complement our wide variety of selectable eukaryotic expression vectors. Geneticin® (G418 sulfate), Zeocin™, hygromycin B, puromycin, and blasticidin antibiotics are the most commonly used selection antibiotics for stable cell transfection. These antibiotics provide unique solutions for your research needs, such as dual selection and rapid, stable cell line establishment.

Geneticin® reagent, also known as G418 sulfate, is commonly used for the selection of mammalian, plant, or yeast cells. The higher purity of our Geneticin® reagent means that 15–30% lower concentrations are required compared to other G418 products; therefore, surviving clonal colonies may arise faster, and cells appear healthier.

Zeocin™ reagent is effective in mammalian cell lines, yeast, insect cells, and bacteria. Resistance to Zeocin™ reagent is conferred by the Sh ble gene, which prevents the binding of Zeocin™ reagent and cleavage of cellular DNA in cells expressing the protein. The concentration required for selection ranges from 50 to 2,000 μg/mL (typically 300 μg/mL), depending on the cell type.

Hygromycin B is an aminoglycosidic antibiotic that inhibits protein synthesis by disrupting translocation and promoting mistranslation of the 80S ribosome. Because its mode of action is different from Geneticin® or Zeocin™ reagents, hygromycin B can be used in dual-selection experiments. Resistance to hygromycin B is conferred by the E. coli hygromycin resistance gene (hygor hph). The concentration for selection ranges from 100 to 1,000 μg/mL (typically 200 μg/mL), and should be optimized for each cell line.

Puromycin, a translation inhibitor in both prokaryotic and eukaryotic cells, is an aminonucleoside antibiotic from Streptomyces alboniger. Resistance is conferred by the puromycin N-acetyltransferase gene (pac) from Streptomyces. Puromycin has a fast mode action, causing rapid cell death even at low antibiotic concentrations, allowing the generation of puromycin-resistant stable cell lines in less than one week. Adherent mammalian cells are sensitive to concentrations of 2–5 μg/mL, while cells in suspension are sensitive to concentrations as low as 0.5–2 μg/mL.

Blasticidin, a potent translational inhibitor in both prokaryotic and eukaryotic cells, is a nucleoside antibiotic from Streptomyces griseochromogenes. Resistance is conferred by the bsd gene product from Aspergillus terreus. E. coli strains are generally sensitive to concentrations of 50 μg/mL, while mammalian cells are sensitive to concentrations as low as 2–10 μg/mL. Cell death occurs rapidly in cells sensitive to blasticidin, and blasticidin-resistant, stable mammalian cell lines can be generated in less than one week at low antibiotic concentrations

Selection of stable transfectants

Selection of stably transfected cells begins with successful transient transfection with a plasmid containing a selectable marker, such as an antibiotic resistance gene. As a negative control, cells should be transfected using DNA that does not contain the selectable marker.

Before starting

  • Ensure that the cell line you are using can produce colonies from isolated cells as some cells require contact with one another to grow. For such cells, adapted or conditioned medium may be beneficial.
  • Choose an appropriate selectable marker.
  • Select a transfection procedure suitable for your cell type.
  • Determine the selective conditions for your cell type by establishing a dose-response curve (kill curve) (Ausubel et al. 1995).

Kill curve

A kill curve should be established for each cell type and each time a new lot of the selective antibiotic is used.

  1. Split a confluent dish of cells at approximately 1:5 to 1:10 (depending on the cell type and cell density post-transfection) into medium containing various concentrations of the antibiotic.
  2. Incubate the cells for 10 days replacing selective medium every 4 days (or as needed).
  3. Examine the dishes for viable cells using the desired method (e.g., Countess® II Automated Cell Counter, hemocytometer with trypan blue staining).
  4. Plot the number of viable cells versus antibiotic concentration to establish a kill curve to determine the most appropriate selective drug concentration required to kill untransfected cells

Selection workflow

Transfect the cells using the desired transfection method. If the selectable marker is on a separate vector, use a 5:1 to 10:1 molar ratio of plasmid containing the gene of interest to plasmid containing the selectable marker.

Note: Perform control transfections with a vector containing the selectable marker but not the gene of interest. If colonies are obtained from cells transfected with the control plasmid but not from cells transfected with plasmid containing the gene of interest, indicating that the gene of interest may be toxic. It is also important to perform replicate transfections in case the transfection fails or the cultures become contaminated.

Forty-eight hours after transfection, passage the cells at several different dilutions (e.g., 1:100, 1:500) in medium containing the appropriate selection drug. For effective selection, cells should be subconfluent, because confluent, non-growing cells are resistant to the effects of antibiotics like Geneticin®. Suspension cells can be selected in soft agar or in 96-well plates for single-cell cloning.

For the next two weeks, replace the drug-containing medium every 3 to 4 days (or as needed).

Note: High cell densities in suspension cultures require frequent medium changes that may deplete critical soluble growth factors, thereby reducing cell viability and the efficiency of the system.

During the second week, monitor cells for distinct “islands” of surviving cells. Depending on the cell type, drug-resistant clones will appear in 2–5 weeks. Cell death should occur after 3–9 days in cultures transfected with the negative control plasmid.

Isolate large (500–1,000 cells), healthy colonies using cloning cylinders or sterile toothpicks, and continue to maintain cultures in medium containing the appropriate drug (for the isolation of clones in suspension culture, see Freshney, 1993).

Transfer single cells from resistant colonies into the wells of 96-well plates to confirm that they can yield antibiotic-resistant colonies. Ensure that only one cell is present per well after the transfer.