Abstract
The epidermis of the skin and its appendages, such as the hair follicles, are formed and maintained by keratinocyte stem cells. Highly efficient and permanent genetic modifications are valuable tools to examine the multipotency and regenerative capacity of keratinocyte stem cells in skin and hair follicle development, homeostasis, and regeneration. Herein, we describe an ex vivo approach by which primary mouse keratinocytes can be permanently manipulated by lentiviral vectors at the genetic level. This protocol can be used to permanently express a gene-of-interest or selectively silence the expression of an endogenous gene, which can be used in preclinical development of gene-based therapies for skin and systemic disorders.
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References
Yuspa SH, Harris CC (1974) Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp Cell Res 86:95–105
Hager B, Bickenbach JR, Fleckman P (1999) Long-term culture of murine epidermal keratinocytes. J Invest Dermatol 112:971–976
Lichti U, Anders J, Yuspa SH (2008) Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc 3:799–810
Green H, Kehinde O, Thomas J (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci U S A 76:5665–5668
Lichti U, Weinberg WC, Goodman L, Ledbetter S, Dooley T, Morgan D, Yuspa SH (1993) In vivo regulation of murine hair growth: Insights from grafting defined cell populations onto nude mice. J Invest Dermatol 101:124S–129S
Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245–254
Yuspa SH, Kilkenny AE, Steinert PM, Roop DR (1989) Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 109:1207–1217
Dickens S, Van den Berge S, Hendrickx B, Verdonck K, Luttun A, Vranckx JJ (2010) Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds. Tissue Eng Part C Methods 16:1601–1608
Benihoud K, Yeh P, Perricaudet M (1999) Adenovirus vectors for gene delivery. Curr Opin Biotechnol 10:440–447
Khare R, Chen CY, Weaver EA, Barry MA (2011) Advances and future challenges in adenoviral vector pharmacology and targeting. Curr Gene Ther 11:241–258
Eglitis MA, Kantoff P, Gilboa E, Anderson WF (1985) Gene expression in mice after high efficiency retroviral-mediated gene transfer. Science 230:1395–1398
Naldini L, Blömer 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 272:263–267
Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15:871–875
Wiznerowicz M, Trono D (2003) Conditional suppression of cellular genes: Lentivirus vector-mediated drug-inducible RNA interference. J Virol 77:8957–8961
Schroers R, Chen S-Y (2004) Lentiviral transduction of human dendritic cells. Methods Mol Biol 246:451–459
Toyoshima K, Vogt PK (1969) Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 38:414–426
Seitz B, Baktanian E, Gordon EM, Anderson WF, LaBree L, McDonnell PJ (1998) Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate. Graefes Arch Clin Exp Ophthalmol 236:602–612
Acknowledgments
We are grateful to colleagues in our laboratories who have modified and improved the procedure described herein. J.C. is supported by a grant from NIH/NIAMS (AR061485).
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Yasuda, M., Claypool, D.J., Guevara, E., Roop, D.R., Chen, J. (2013). Genetic Manipulation of Keratinocyte Stem Cells with Lentiviral Vectors. In: Turksen, K. (eds) Skin Stem Cells. Methods in Molecular Biology, vol 989. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-330-5_12
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DOI: https://doi.org/10.1007/978-1-62703-330-5_12
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-329-9
Online ISBN: 978-1-62703-330-5
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