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© 1988 Oxford University Press

research-article

Transfection of murine multi-potent haemopoietic stem cells with an E.coli DNA alkyltransferase gene confers resistance to the toxic effects of alkylating agents

J. Jelinek 1 2, K. Kleibl 3 4, T.M. Dexter 1 and G.P. Margison 3 5

1Department of Experimental Haematology Manchester M20 9BX, UK
3Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital Manchester M20 9BX, UK

5To whom reprint requests should be sent

O6-alkylguanine-DNA-alkyltransferase (ATase)-deficient murine haemopoietic stem cells were transfected, following electroporation, with a G418-selectable expression vector containing the protein coding region of the Escherichia coli ATase gene ada. Clones of cells that were resistant to G418 or the chloroethylating agent mitozolomide (Mz) were selected and most were shown to express very high levels of bacterial gene-encoded ATase. In comparison with control cells that were transfected with the parent vector, the ATase-expressing clones were considerably more resistant to the toxic effects of the methylating agents N-methyl-N-nitrosourea and methylmethanesulphonate or the chloroethylaling agents Mz or taurine chloroethylnitrosourea, but unchanged in their susceptibility to the bis-chloroethylating agent nitrogen mustard. Thus alkylatlon damage in DNA that can be repaired by the E.coli ATase constitutes the principal lethal lesion produced by alkylating agents in murine haemopoietic stem cells and the ATase deficiency in these cells can be complemented by electroporation-mediated gene transfection.


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