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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts.

Cultured cells from individuals affected with Fanconi anemia ( FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). We report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental or Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 micrograms/ml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEBr) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEBr colonies independently derived from transfection of human DNA and one of three DEBr colonies independently derived from transfection of Chinese hamster DNA. In addition, Alu-equivalent hamster sequences were detected in three of seven additional independently derived colonies from transfection of Chinese hamster DNA. The DEBr phenotype of these colonies was stably maintained over several subcultures. Our results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA.[1]


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