Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice.
Previous attempts to achieve long-term gene expression in retrovirally transduced human epidermal keratinocytes in vivo have been largely unsuccessful. This has been variously attributed to a failure to target epidermal stem cells, suboptimal grafting conditions or inactivation of the retroviral vector. In an attempt to overcome these problems we expressed the chick beta 1 integrin subunit in primary human epidermal keratinocytes, which allowed us to monitor retroviral gene expression on a cell-by-cell basis. We describe optimised methods for selecting high-titre amphotropic packaging cells and for infecting keratinocytes in culture. When transduced cells were grafted into mice, graft survival was comparable in nude and SCID mice, but it was essential to combine the keratinocytes with a dermal substrate. Using these methods the majority of keratinocytes expressed the chick beta 1 integrin subunit for at least 16 weeks after grafting. We conclude that epidermal keratinocytes are attractive recipient cells for gene therapy.
Levy
L
L
Keratinocyte Laboratory, Imperial Cancer Research Fund, London, UK.
Broad
S
S
Zhu
A J
AJ
Carroll
J M
JM
Khazaal
I
I
Péault
B
B
Watt
F M
FM
D016475:3T3 Cells; D000818:Animals; D002645:Chickens; D018920:Coculture Techniques; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics*; D015603:Keratinocytes / Q000637:transplantation / Q000821:virology*; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D012190:Retroviridae; D013997:Time Factors
D019012:Integrin beta1