Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice

Abstract

Previous attempts to achieve long-term gene expression in optimised methods for selecting high-titre amphotropic retrovirally transduced human epidermal keratinocytes in packaging cells and for infecting keratinocytes in culture. vivo have been largely unsuccessful. This has been variWhen transduced cells were grafted into mice, graft surously attributed to a failure to target epidermal stem cells, vival was comparable in nude and SCID mice, but it was suboptimal grafting conditions or inactivation of the retroviessential to combine the keratinocytes with a dermal subral vector. In an attempt to overcome these problems we strate. Using these methods the majority of keratinocytes expressed the chick ␤ 1 integrin subunit in primary human expressed the chick ␤ 1 integrin subunit for at least 16 epidermal keratinocytes, which allowed us to monitor retroweeks after grafting. We conclude that epidermal keraviral gene expression on a cell-by-cell basis. We describe tinocytes are attractive recipient cells for gene therapy.

Epithelial stem cells in corneal regeneration and epidermal gene therapy

“…The use of retroviral vectors derived from the MFG prototype, in which the protein of interest is translated from an efficiently spliced genomic RNA under the control of the viral env translation initiation sequences, allowed expression of lacZ or factor IX transgenes for >1 year in human epidermal xenografts [99,103]. Transduction of epidermal stem cells was achieved either by co-culture with packaging cell lines [98,99,104] or by the use of high-titre vector preparations [103].…”

Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis

“…For retroviral infection, keratinocytes were cultured in the presence of AM12 producer lines which had been pre-treated with 4 µg ml -1 mitomycin C (Levy et al, 1998). The producer cells were removed after 3 days and replaced with puromycin-resistant J2-3T3 feeder cells, and 2 µg ml -1 puromycin was added to the medium.…”

Transient activation of FOXN1 in keratinocytes induces a transcriptional programme that promotes terminal differentiation: contrasting roles of FOXN1 and Akt

“…J2-3T3 and NIH 3T3 cells were cultured in DMEM containing 10% donor calf serum. J2-puro are J2-3T3 cells that have been stably transfected with the retroviral vector pBabepuro to render them resistant to puromycin (Levy et al, 1998); they were cultured in DMEM, 10% donor calf serum and 2 µg/ml puromycin.…”