Author(s): Debenham PG, Webb MB, Stretch A, Thacker J
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Abstract A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV-gpt and HSVtk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSVtk-neo gene. The use of two-gene vectors to assess DNA repair was investigated by cutting the SV-gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types. In such experiments, selection was first applied for the undamaged HSVtk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damaged SV-gpt gene. The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts). Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct. In these experiments, some of the transformed cell colonies showed 'sectoring' on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA. For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated. Mutations of the SV-gpt gene in these cells were isolated while maintaining selection for the HSVtk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events. Spontaneous and X-ray-induced mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence. Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E. coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.
This article was published in Mutat Res
and referenced in Journal of Nephrology & Therapeutics