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Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9

  • Serena Grossi
  • Gabriele Fenini
  • Paulina Hennig
  • Michela Di Filippo
  • Hans-Dietmar BeerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2109)

Abstract

The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.

Keywords

Human primary keratinocytes CRISPR/Cas9 Knockout Feeder cells 

Notes

Acknowledgments

We thank the Tagesklinik für Kinderchirurgie (Fällanden, Switzerland) for providing skin biopsies. Our work is supported by grants from the Wilhelm Sander-Stiftung, Georg und Bertha Schwyzer-Winiker Stiftung, OPO-Stiftung, Novartis Stiftung für Medizinisch-Biologische Forschung, Theodor und Ida Herzog-Egli-Stiftung, Krebsforschung Schweiz (KFS-3940-08-2016), Vontobel-Stiftung, Bruno Bloch-Stiftung, and the Julius Müller Stiftung für Krebsforschung. P.H. and M.D.F. are members of the Zurich Graduate program in Molecular Life Sciences.

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Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • Serena Grossi
    • 1
    • 2
  • Gabriele Fenini
    • 1
    • 2
  • Paulina Hennig
    • 1
  • Michela Di Filippo
    • 1
  • Hans-Dietmar Beer
    • 1
    • 2
    Email author
  1. 1.Department of DermatologyUniversity Hospital ZurichZurichSwitzerland
  2. 2.Faculty of MedicineUniversity of ZurichZurichSwitzerland

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