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 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.
Quilliet
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Laboratory of Molecular Genetics, UPR 42, CNRS, Villejuif, France.
Chevallier-Lagente
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Eveno
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Stojkovic
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T
Destée
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Sarasin
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Mezzina
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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
D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D004247:DNA; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human