LONG-TERM COMPLEMENTATION OF DNA-REPAIR DEFICIENT HUMAN PRIMARY FIBROBLASTS BY RETROVIRAL TRANSDUCTION OF THE XPD GENE

Due to their limited life time in culture and their relative resistanc e to DNA transfection, primary fibroblasts derived from UV-hypersensit ive patients could not be used for cloning DNA repair gene and studyin g stable complementation with wild-type DNA repair genes. Primary cell s were only used for complementation analysis after transient expressi on through cell fusion, DNA microinjection and transfection. We report the retroviral-mediated highly efficient transfer and stable expressi on of XPD/ERCC2 gene in fibroblast strains from eight different patien ts using the LXPDSN retroviral vector. Cells derived from skin biopsie s of xeroderma pigmentosum and trichothiodystrophy patients were incub ated with vector-containing suspension and selected with the neomycin- analog G418. LXPDSN vector specifically complemented cells belonging t o the XP-D group. Long-term reversion of repair-deficient phenotype, m onitored by UV survival and UDS analysis, has been achieved in these d iploid fibroblasts. We demonstrate this methodology is a powerful tool to study phenotypic reversion of nucleotide excision repair-deficient cells such as cellular DNA repair properties and we suggest that it m ay be used to study other cellular parameters (cell cycle regulation, p53 stability or immunosurveillance-controlling factors) involved in U V-induced skin cancers and which reliability requires the use of untra nsformed cells.