How to Make Stable Transfected Cell Line

Stable transfection is the long-term introduction of foreign DNA into cells. Stably transfected cells pass the foreign DNA through progeny. Therefore, to make stably transfected cell lines, foreign DNA has to be integrated into the genome of the cell line. However, stable inheritance can be observed in nongenomic DNA as well. A successful, stable transfection requires effective methods of DNA delivery as well as selection methods for foreign DNA within the cell line. Stably-transfected cell lines are used for a wide range of applications such as the production of recombinant proteins, gene function studies, drug discovery assays, etc.

Key Areas Covered

1. How to Make Stable Transfected Cell Line
      – Stable Cell Line Generation Protocol
2. How to Select Transfected DNA in Cell Line
     – Selection of Stably-Transfected Cell Line

Key Terms: Episomal Maintenance, Integration into the Genome, Plasmid, Selection, Stably Transfected Cell Lines

How to Make Stable Transfected Cell Line- Infographic

How to Make Stable Transfected Cell Line

Transfection is a method of gene transfer in which the genetic material is deliberately introduced into the host cell by chemical or non-chemical methods. There are two types of stably transfected cell lines:

  1. The main type of stable cell lines is generated by the direct integration of transfected DNA into the genome of the host organism. Transfected DNA is integrated into the genome by homologous recombination.
  2. The other method to generate stable cell lines is the episomal maintenance of the transfected DNA. Eukaryotic vectors are used in the transfection of DNA that are not integrated into the genome. However, the stability of episomal DNA is low. Also, episomes are only maintained by certain types of species. Stably transfected cell lines are shown in figure 1.
How to Select Transfected DNA in Cell Lines

Figure 1: Stably Transfected Cell Lines


The steps involved in the generation of stable cell lines are described below.

  1. Generation of a kill curve determining the optimal selection antibiotic concentration – Cells are subjected to increasing amounts of antibiotic concentration to determine the minimum antibiotic concentration required to kill all cells over a week.
  2. Transfection of cells with desired plasmid construct – The method of transfection differs with the type of host cells. Liposome reagents are used in the transfection of adherent cell lines. Electrotransfection or viral methods are used to transfect suspension cell lines.
  3. Select and expand stable polyclonal colonies – At 48-72 hours of transfection, high, optimal, and low concentrations of selective antibiotics are added to the cultures to obtain the stably-transfected cell lines.

How to Select Transfected DNA in Cell Lines

Selection markers are co-expressed with the transfected DNA for the selection of successfully transfected cells. The marker gene could be in the same vector (cis) used to transfect the host cell or on another vector (trans). The resistance to antibiotics such as neomycin, zeocin, hygromycin, puromycin, DHFR, etc. can be used in the selection. In addition, transfected genes can be tagged with GFP.


Stable cell lines can be mainly made by integrating transfected DNA into the genome. Furthermore, the episomal maintenance of transfected DNA can also be used to make stable cell lines for a long period of time in some host organisms. A selection method should be there for the identification of transfected DNA in the cell line. 


1. “Protocol of Stable Cell Line Generation.” Creative BioMart, Available here.

Image Courtesy:

1. “Knockout mouse production 2” By Kjaergaard – Own work (CC BY 3.0) via Commons Wikimedia

About the Author: Lakna

Lakna, a graduate in Molecular Biology and Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things. She has a keen interest in writing articles regarding science.

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