title: Long-term complementation of DNA repair deficient human primary fibroblasts by retroviral transduction of the XPD gene.
abstract: 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.
authors.affiliation: Laboratory of Molecular Genetics, UPR 42, CNRS, Villejuif, France.
authors.forename: X
authors.initials: X
authors.lastname: Quilliet
authors.forename: O
authors.initials: O
authors.lastname: Chevallier-Lagente
authors.forename: E
authors.initials: E
authors.lastname: Eveno
authors.forename: T
authors.initials: T
authors.lastname: Stojkovic
authors.forename: A
authors.initials: A
authors.lastname: Destée
authors.forename: A
authors.initials: A
authors.lastname: Sarasin
authors.forename: M
authors.initials: M
authors.lastname: Mezzina
mesh_terms: D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000528:radiation effects; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D006201:Hair Diseases / Q000235:genetics; D006801:Humans; D008979:Moloney murine leukemia virus / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D014157:Transcription Factors; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051759:Xeroderma Pigmentosum Group D Protein
chemical_list: D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D004247:DNA; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human