Efficient retroviral gene transfer to epidermal stem cells.
Skin is an attractive target for gene modification to treat skin diseases, wound healing, or even systemic disorders. Although retroviral transduction results in permanent genetic modification, differentiation and eventually loss of the transduced cells from the epidermis and in temporary transgene expression. Therefore, it is important to develop methods that promote gene transfer to epidermal stem cells, which self-renew and regenerate the epidermis for extended periods of time. Here we describe an efficient protocol that results in high levels of retroviral gene transfer to human epidermal stem cells by immobilizing retrovirus on a recombinant fibronectin (rFN) fragment. In contrast to the traditional method, transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. Notably, transduction on rFN does not require addition of toxic polycations such as polybrene. Overall this method provides a simple, fast, and efficient means to modify human epidermal stem cells for cutaneous gene therapy and for biological studies that require stable genetic modification.
Lei
Pedro
P
Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY, USA.
Andreadis
Stelios T
ST
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D000078404:Epidermal Cells; D005353:Fibronectins / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects; D051379:Mice; D011994:Recombinant Proteins / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*
D005353:Fibronectins; D011994:Recombinant Proteins