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The human O(6)-methylguanine-DNA methyltransferase (MGMT) gene and its mutants have been used for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O(6)-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing an immune response, we have cloned the canine MGMT drug resistance gene. Comparison of canine and human MGMT-coding regions indicates that there is about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT is also longer, by nine amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT are highly conserved in the canine sequence. To determine whether mutation of the proline residue at position 144 to lysine in the canine MGMT would provide a similar advantage for selection of transduced cells as the human mutant, Moloney murine leukemia virus and human immunodeficiency type 1 vectors encoding the corresponding mutant MGMT were created and used to express separately canine and human MGMTs in cultured cells. Drug resistance assays using BCNU alone or BCNU with BG demonstrated that the wild-type and mutant canine MGMTs provided resistance to the selection agents that was comparable to the human MGMT counterparts.