How does stable transfection work?

Unlike transient transfection, in which introduced DNA persists in cells for several days, stable transfection introduces DNA into cells long-term, supporting continued gene expression in a cell line without repeated transfections. Typically, stable transfection involves the integration of transfected DNA into the host cell genome, allowing transfected cells to pass this DNA to their progeny. Occasionally, stable transfection can occur via the inheritance of nongenomic DNA.

Figure 1. Sample stable transfection workflow. In stable transfection, transfected DNA is typically incorporated into the host cell genome. Cells can then be selected for and expanded to generate a stable cell line.

Successful stable transfection requires both effective DNA delivery and a way to select cells that have acquired the DNA. Approximately 1 in 104 transfected cells will stably integrate DNA, although the efficiency varies with cell type and whether linear or circular DNA is used (see guidelines for plasmid DNA transfection). 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. When the selectable marker is expressed from a 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. After a short recovery period, an appropriate selective pressure can then be applied to the cells.

Frequently used selectable markers include 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.

The ability to establish a stable cell line makes stable transfection a valuable experimental and clinical research application. Stable cell lines are used for studies of long-term genetic regulation, sustained expression in gene therapy, as well as for large-scale protein production in biotechnological and biopharmaceutical settings.


Selection of antibiotics for stable cell line generation

There are many selection antibiotics to choose from for stable cell line generation. The antibiotic chosen will depend on which antibiotic resistance gene or selectable marker is used in the transfection experiment.

Thermo Fisher Scientific offers 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 common selection antibiotics used for stable cell transfection. These antibiotics provide unique solutions for your research needs, such as dual selection and rapid, stable cell line establishment.

Explore all Gibco selection antibiotics


Protocol for selection of stable cell lines

Selection of stably transfected cells begins with successful transfection of 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)

Kill curve

It is important to note that a kill curve should be established for each cell type and each time a new lot of the selective antibiotic is used. Follow the steps below to generate a kill curve:

  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 media 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 Automated Cell Counter or 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

Basic steps for selecting stable cell lines

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, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Wiley-Blackwell, New York.).

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.

Step 1 : Transfect cells

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.

Step 2 : Passage cells with antibiotic

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.

Step 3 : Monitor for cell "islands"

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.

Step 4 : Isolate colonies

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, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Wiley-Blackwell, New York.).

Step 5 : Transfer single cells

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.


Find stable transfection reagents

Thermo Fisher Scientific offers a variety of stable transfection reagents and products for your stable transfection experiments, including the advanced Lipofectamine 3000 reagent and Neon Electroporation System.

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