Genetic Manipulation of Keratinocyte Stem Cells with Lentiviral Vectors

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.