Epidermal Cells pp 113-123 | Cite as
Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors
Protocol
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Abstract
Keratinocytes are hard to transfect. Viral vectors are a good alternative to genetically modify primary keratinocytes. A classical method is the use of retroviral vectors by co-culture of keratinocytes with virus-producer cells. This method is efficient in high-calcium conditions with feeder cells. However, sometimes co-culture is not possible and is more laborious as producer cells need to be replaced by feeder cells. Our solution is the use of lentiviral vectors, far more efficient as supernatant on keratinocytes. In this chapter we describe improved detailed protocols for stable genetic modification of human primary keratinocytes of the skin or head and neck, in both low- and high-calcium conditions by lentiviral vectors.
Keywords
Keratinocytes Viral vectors Feeder cells Calcium conditions Lentiviral vectors Genetic modificationNotes
Acknowledgments
This work was funded by Instituto de Salud Carlos III/FEDER (AG; Spain), grants PI14/00900 and PI17/01307. NSG is recipient of a predoctoral scholarship from Universidad de Cantabria/IDIVAL (Spain).
References
- 1.Morgenstern JP, Land H (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18(12):3587–3596CrossRefGoogle Scholar
- 2.Gandarillas A, Davies D, Blanchard JM (2000) Normal and c-Myc-promoted human keratinocyte differentiation both occur via a novel cell cycle involving cellular growth and endoreplication. Oncogene 19(29):3278–3289CrossRefGoogle Scholar
- 3.Roe T, Reynolds TC, Yu G, Brown PO (1993) Integration of murine leukemia virus DNA depends on mitosis. EMBO J 12(5):2099–2108CrossRefGoogle Scholar
- 4.Miller DG, Adam MA, Miller AD (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10(8):4239–4242CrossRefGoogle Scholar
- 5.Higashikawa F, Chang L (2001) Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280(1):124–131. https://doi.org/10.1006/viro.2000.0743 CrossRefPubMedGoogle Scholar
- 6.Rheinwald JG (1989) Methods for clonal growth and serial cultivation of normal human epidermal keratinocytes and mesothelial cells. In: Baserga R (ed) Cell growth and division. IRL Press, Oxford, pp 81–94Google Scholar
- 7.Watt FM, Broad S, Prowse DM (1994) Cultivation and retroviral infection of human epidermal keratinocytes. In: Celis JE (ed) Cell biology: a laboratory handbook. Cambridge University Press, Cambridge, pp 83–89Google Scholar
- 8.Naldini L (1998) Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol 9(5):457–463CrossRefGoogle Scholar
- 9.Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259):263–267CrossRefGoogle Scholar
- 10.Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873–9880CrossRefGoogle Scholar
- 11.Freije A, Ceballos L, Coisy M, Barnes L, Rosa M, De Diego E, Blanchard JM, Gandarillas A (2012) Cyclin E drives human keratinocyte growth into differentiation. Oncogene 31(50):5180–5192CrossRefGoogle Scholar
- 12.Freije A, Molinuevo R, Ceballos L, Cagigas M, Alonso-Lecue P, Rodriguez R, Menendez P, Aberdam D, De Diego E, Gandarillas A (2014) Inactivation of p53 in human keratinocytes leads to squamous differentiation and shedding via replication stress and mitotic slippage. Cell Rep 9(4):1349–1360CrossRefGoogle Scholar
- 13.Nanba D, Matsushita N, Toki F, Higashiyama S (2013) Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector. Stem Cell Res Ther 4(5):127. https://doi.org/10.1186/scrt338 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Chen M, Li W, Fan J, Kasahara N, Woodley D (2003) An efficient gene transduction system for studying gene function in primary human dermal fibroblasts and epidermal keratinocytes. Clin Exp Dermatol 28(2):193–199CrossRefGoogle Scholar
- 15.Laktionov PP, Dazard JE, Vives E, Rykova EY, Piette J, Vlassov VV, Lebleu B (1999) Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes. Nucleic Acids Res 27(11):2315–2324CrossRefGoogle Scholar
- 16.Levy L, Broad S, Zhu AJ, Carroll JM, Khazaal I, Peault B, Watt FM (1998) Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice. Gene Ther 5(7):913–922. https://doi.org/10.1038/sj.gt.3300689 CrossRefPubMedGoogle Scholar
- 17.Graham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36(1):59–74. https://doi.org/10.1099/0022-1317-36-1-59 CrossRefPubMedGoogle Scholar
- 18.DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 7(1):379–387CrossRefGoogle Scholar
- 19.Wiznerowicz M, Trono D (2003) Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 77(16):8957–8961CrossRefGoogle Scholar
- 20.Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P (2006) A versatile tool for conditional gene expression and knockdown. Nat Methods 3(2):109–116CrossRefGoogle Scholar
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