Applications: Stable Cell Line Generation

Stable Cell Line Generation

Most cell biology experiments utilize transient transfection protocols that afford peak gene expression between 24-96 hours post transfection. However, if sustained gene expression is required, generation of stable cell lines is a viable option. Furthermore, stable cell lines selected through limiting dilution/colony-picking provide a genetically homogenous and clonal population.

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A. Stable Cell Line Generation GFP B. Stable Cell Line Generation Data

Stable Cell Line Generation and Characterization. HEK 293 cells stably expressing EGFP were generated through transient transfection of EGFP and neomycin plasmid DNA vectors. Monoclonal populations were selected by limiting serial dilution and gene stability was verified for at least ten passages. Cells were assessed by fluorescence and phase microscopy (A) and flow cytometry (B).

Stable cell line generation is made possible by the use of positive selection markers such as G418, hygromycin B, puromycin resistance, etc.. Selection markers can be delivered using the same plasmid that contains the gene of interest (in cis), or on a separate plasmid (in trans) that needs to be co-transfected with the plasmid containing the gene of interest. The cis approach is generally easier and has a higher likelihood of producing drug-resistant stable transfectants that express the gene of interest. The trans method of co-transfection is a good alternative in instances where the target construct does not have the antibiotic-resistance gene in the vector backbone. In such cases, a plasmid mixture containing 5 to 10 parts gene expression plasmid and 1 part antibiotic selection marker plasmid can be introduced into cells. This plasmid ratio helps increase the likelihood that the selected cells will express both the gene of interest and the selection marker.

Stable cell line generation protocol

The protocol for generating stable cell lines requires several steps as shown below:

  1. Antibiotic Kill Curve: Titration of selection antibiotic to determine the concentration required to kill cells that are expressing the transgene (1 week)
  2. Transfection: Introduce the plasmid construct(s) into cells (2 days)
  3. Selection and expansion: Determine polyclonal and monoclonal populations of cells (8-10 weeks)
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Timeline for Stable Cell Line Generation

Timeline for Stable Cell Line Generation. Stable cell line generation can take a total of 9-12 weeks to establish the cell line. It is ideal to first determine the optimal antibiotic concentration for selection for each cell type; this can take up to 1 week. Following this, the target plasmids can be transfected for two days and antibiotic selection can be applied. Once the cells are under adequate selection, clone picking and expansion is the final and most time consuming part of stable cell line generation. After the stable cell line is generated, it is preferable to verify stable expression from a few cell passages before freezing stocks.

For stable cell line generation, use Mirus' high quality cell-culture grade selection antibiotics that are easy-to-use:


All Mirus' broad spectrum plasmid DNA transfection reagents can be used for stable cell line generation. Visit the product pages for more information on each product:

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