PubMed1996
Due to their limited life time in culture and their relative resistance to DNA transfection, primary fibroblasts derived from UV-hypersensitive patients could not be used for cloning DNA repair gene and studying stable complementation with wild-type DNA repair genes. Primary cells were only used for complementation analysis after transient expression through cell fusion. DNA microinjection and transfection. We report the retroviral-mediated highly efficient transfer and stable expression of XPD/ERCC2 gene in fibroblast strains from eight different patients using the LXPDSN retroviral vector. Cells derived from skin biopsies of xeroderma pigmentosum and trichothiodystrophy patients were incubated with vector-containing suspension and selected with the neomycin-analog G418. LXPDSN vector specifically complemented cells belonging to the XP-D group. Long-term reversion of repair-deficient phenotype, monitored by UV survival and UDS analysis, has been achieved in these diploid 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 may be used to study other cellular parameters (cell cycle regulation, p53 stability or immunosurveillance-controlling factors) involved in UV-induced skin cancers and which reliability requires the use of untransformed cells.