MUTATIONS INDUCED BY DNA DOUBLE-STRAND BREAKS - THE INFLUENCE OF GENOMIC SITE

The transgenic CHO cell line PL61, carrying a recombinant SV40-gpt gen e, was treated with restriction endonucleases to assess mutagenesis fr om defined DNA double-strand breaks. Mutations in gpt were measured un der two conditions: a stringent condition where selection ensured that the closely-linked neo gene was retained functionally intact or a rel axed condition without the requirement for neo gene function. Despite testing 18 different restriction endonucleases with various numbers of potential break-sites within the transgene, mutations were only found under relaxed selection conditions. These mutations commonly led to c omplete loss of the transgene, suggesting that large deletionspredomin ate when selection is relaxed It is argued, in comparison to mutation data for other genomic sites in CHO cells, that variations in the 'eff ective target size' for mutagenesis may explain the response of the tr ansgene under different conditions.