Long-term complementation of DNA repair deficient human primary fibroblasts by retroviral transduction of the XPD gene

Mutat Res. 1996 Dec 2;364(3):161-9. doi: 10.1016/s0921-8777(96)00024-9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • DNA / biosynthesis
  • DNA Helicases*
  • DNA Repair / genetics*
  • DNA-Binding Proteins*
  • Fibroblasts* / radiation effects
  • Gene Transfer Techniques*
  • Genetic Complementation Test*
  • Genetic Vectors / genetics
  • Hair Diseases / genetics
  • Humans
  • Moloney murine leukemia virus / genetics
  • Proteins / genetics*
  • Transcription Factors*
  • Ultraviolet Rays
  • Xeroderma Pigmentosum / genetics
  • Xeroderma Pigmentosum Group D Protein

Substances

  • DNA-Binding Proteins
  • Proteins
  • Transcription Factors
  • DNA
  • DNA Helicases
  • Xeroderma Pigmentosum Group D Protein
  • ERCC2 protein, human