Advertisement

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

Abstract

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.

Keywords

Gene targeting Homologous recombination Screening Selection marker 

Notes

Acknowledgements

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.

References

  1. Askew GR, Doetschman T, Lingrel JB (1993) Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol 13:4115–4124CrossRefGoogle Scholar
  2. Cearley JA, Detloff PJ (2001) Efficient repetitive alteration of the mouse Huntington’s disease gene by management of background in the tag and exchange gene targeting strategy. Transgenic Res 10:479–488.  https://doi.org/10.1023/A:1013015616732 CrossRefPubMedGoogle Scholar
  3. Czakó M, Márton L (1994) The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana. Plant Physiol 104:1067–1071CrossRefGoogle Scholar
  4. Dickinson P, Kimber WL, Kilanowski FM et al (2000) Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting. Transgenic Res 9:55–66CrossRefGoogle Scholar
  5. Farazmandfar T, Khanahmad Shahreza H, Haghshenas MR et al (2012) Use of integrase-minus lentiviral vector for transient expression. Cell J 14:76–81PubMedPubMedCentralGoogle Scholar
  6. Farazmandfar T, Haghshenas MR, Shahbazi M (2015) Inhibition of HIV-1 by a lentiviral vector with a novel tat-inducible expression system and a specific tropism to the target cells. Hum Gene Ther 26:680–687.  https://doi.org/10.1089/hum.2015.031 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Iwamoto M, Mori C, Hiraoka Y, Haraguchi T (2014) Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena. Gene 534:249–255.  https://doi.org/10.1016/j.gene.2013.10.049 CrossRefPubMedGoogle Scholar
  8. Kobayashi K, Ohye T, Pastan I, Nagatsu T (1996) A novel strategy for the negative selection in mouse embryonic stem cells operated with immunotoxin-mediated cell targeting. Nucleic Acids Res 24:3653–3655CrossRefGoogle Scholar
  9. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348–352.  https://doi.org/10.1038/336348a0 CrossRefPubMedGoogle Scholar
  10. McCarrick JW, Parnes JR, Seong RH et al (1993) Positive–negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells. Transgenic Res 2:183–190CrossRefGoogle Scholar
  11. Mortensen R (2007) Overview of gene targeting by homologous recombination. Curr Protoc Neurosci Chap 4:4.29.  https://doi.org/10.1002/0471142301.ns0429s40 CrossRefGoogle Scholar
  12. Mullen CA, Kilstrup M, Blaese RM (1992) Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc Natl Acad Sci USA 89:33–37CrossRefGoogle Scholar
  13. Müller U (1999) Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev 82:3–21CrossRefGoogle Scholar
  14. Negroni L, Samson M, Guigonis J-M et al (2007) Treatment of colon cancer cells using the cytosine deaminase/5-fluorocytosine suicide system induces apoptosis, modulation of the proteome, and Hsp90beta phosphorylation. Mol Cancer Ther 6:2747–2756.  https://doi.org/10.1158/1535-7163.MCT-07-0040 CrossRefPubMedGoogle Scholar
  15. Robles-Oteiza C, Taylor S, Yates T et al (2015) Recombinase-based conditional and reversible gene regulation via XTR alleles. Nature Commun 6:8783.  https://doi.org/10.1038/ncomms9783 CrossRefGoogle Scholar
  16. Scahill MD, Pastar I, Cross GAM (2008) CRE recombinase-based positive-negative selection systems for genetic manipulation in Trypanosoma brucei. Mol Biochem Parasitol 157:73–82.  https://doi.org/10.1016/j.molbiopara.2007.10.003 CrossRefPubMedGoogle Scholar
  17. Shimatani Z, Nishizawa-Yokoi A, Endo M et al (2014) Positive-negative-selection-mediated gene targeting in rice. Front Plant Sci 5:748.  https://doi.org/10.3389/fpls.2014.00748 CrossRefPubMedGoogle Scholar
  18. Szeberényi J (2013) Problem-solving test: conditional gene targeting using the Cre/loxP recombination system. Biochem Mol Biol Educ 41:445–449.  https://doi.org/10.1002/bmb.20731 CrossRefPubMedGoogle Scholar
  19. Tzimagiorgis G, Michaelidis TM, Lindholm D, Thoenen H (1996) Introduction of the negative selection marker into replacement vectors by a single ligation step. Nucleic Acids Res 24:3476–3477CrossRefGoogle Scholar
  20. Waldorf AR, Polak A (1983) Mechanisms of action of 5-fluorocytosine. Antimicrob Agents Chemother 23:79–85CrossRefGoogle Scholar
  21. Yanagawa Y, Kobayashi T, Ohnishi M et al (1999) Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker. Transgenic Res 8:215–221.  https://doi.org/10.1023/A:1008914020843 CrossRefPubMedGoogle Scholar

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

Personalised recommendations