The maps of the DNA constructs used to generate the TMZ-resistant GB cell variants.

The LN229M and U251M cell variants were generated after infection with a lentiviral construct that carries the CDS for human MGMT in tandem with the one for firefly luciferase separated by an IRES sequence and under an EF1α promoter and, in a separate ORF on the same lentivector, the CDS for enhanced GFP under a CMV promoter (Panel A). The GFP reporter facilitated the selection of high MGMT expressing variants of these GB cells which were sorted to purity by FACS. The GL261M cell variant was generated after transfection with a plasmid construct that carries the murine Mgmt CDS under the control of a murine phosphoglycerate kinase (mPGK) promoter (Panel B). The transfected cells were further enriched in murine Mgmt by serial passaging in medium containing increasing concentrations of TMZ.

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