Abstract
The transgenic CHO cell line PL61, carrying a recombinant SV40-gpt gene, was treated with restriction endonucleases to assess mutagenesis from defined DNA double-strand breaks. Mutations in gpt were measured under two conditions: a stringent condition where selection ensured that the closely-linked neo gene was retained functionally intact, or a relaxed 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 complete loss of the transgene, suggesting that large deletions predominate when selection is relaxed. It is argued, in comparison to mutation data for other genomic sites in CHO cells, that variations in the 'effective target size' for mutagenesis may explain the response of the transgene under different conditions.
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