Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes

Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and poten tial long life span. Improvement of gene transfer into keratinocytes will b e greatly facilitated by markers that will allow both rapid detection and e fficient selection of transduced cells. For these purposes, a recombinant v ersion of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a repli cation-deficient retroviral vector. High-titer retrovirus was used to trans duce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Bot h cell types stably expressed the EGFP, and this marker allowed rapid purif ication of transduced cells by fluorescence-activated cell sorting. EGFP ex pression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in resp onse to UVB or etoposide. Transduced populations of HK were grafted into SC ID mice, resulting in a functional epidermis. EGFP expression was readily s een by exposing the xenografts to an ultraviolet light source. These studie s demonstrate the feasibility of using EGFP as a convenient and rapid marke r to monitor keratinocyte gene transfer both in vitro and in vivo.