3 Biotech

, 9:357 | Cite as

A novel screening system based on gene targeting to enrich the modified mammalian cells: without leaving selection marker and additional sequence

  • Abtin Behmardi
  • Majid Shahbazi
  • Masoud Golalipour
  • Touraj FarazmandfarEmail author
Protocols and Methods


Gene targeting by homologous recombination (HR) has some disadvantages in screening modified cells that limits their use in targeting gene fragments in long exons. These disadvantages include retention of remaining selection marker after targeting, not removing cells with vector random integration, and leaving loxP sequences after removal of selection markers. Therefore, to overcome these disadvantages, we decided to design a eukaryotic two-step screening system to isolate the favorable, edited cells from undesirable cells in a gene targeting project. This system included two targeting plasmids containing one positive marker and two inducible negative markers. It was designed in such a way that, during the two-step HR and subsequent selection, only the well-edited cells survive and cells with vector random integration, and untargeted and episomal targeting plasmids are eliminated. The percentage of GFP-positive cells in two-step screening method (76.10 ± 3.50) was significantly higher than in the one-step screening method (0.90 ± 0.37) (p < 0.0001). GFP noise caused by the presence of the GFP-episomal expression plasmid had no significant effect on our results. We developed an efficient system to screen and enrich the HR-modified cells from undesired-HR and untargeted cells, without leaving the selection markers in mammalian cells. This method may be a promising method in ex vivo gene therapy approaches, especially when the target is a gene fragment within a large exon.


Gene targeting Homologous recombination Screening Selection marker 



This work was supported by the research department in Golestan University of Medical Sciences, Gorgan, Iran (Grant no. 960226021).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Medical Cellular and Molecular Research Center, Golestan University of Medical SciencesGorganIran

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