404. Cloning and Expression of Canine O6-Methylguanine–DNA Methyltransferase Gene in Ex Vivo Cultured Cells

Abstract

The human O6-methylguanine-DNA-methyltransferase (MGMT) gene and its mutants have been used previously for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O6-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing of an immune response, we have cloned the canine MGMT drug resistance gene (GenBank Accession No. AY423553). Comparison of canine and human MGMT coding regions indicated that there was about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT was also longer by 9 amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT were highly conserved in the canine sequence. To determine if mutation of the proline residue at position 144 to lysine (P144K) in the canine MGMT would provide a similar advantage for selection with BG and BCNU of transduced cells as the human P140K mutant, we created the corresponding P144K mutant of canine MGMT. Bicistronic Moloney murine leukemia virus and human immunodeficiency type 1 vectors were used to express separately canine and human MGMTs in cultured canine CTAC cells. Drug-resistance assays demonstrated that the wild-type and the P144K mutant canine MGMTs provided resistance to the BCNU or BG together with BCNU selection, respectively that was comparable to the human MGMT counterparts.