Lentiviral Vectors for EGFR Variants

The pLV1-puro-DEST vector was prepared as previously described [16 (link),34 (link),59 (link)]. Electrophoretic analysis and DNA sequencing were performed to verify the resulting recombinant vectors. The EGFRwt and EGFRvIII cDNA sequences were obtained, and expression plasmids were created [34 (link),59 (link)]. The coding sequences were transferred to pLV1-puro-DEST under the CMV promoter via the LR reaction (Invitrogen, Waltham, MA, USA). Both sequences were confirmed with an Applied Biosystems 3130 Genetic Analyzer. The transfection was performed with Fugene HD (Promega, Madison, WI, USA) and puromycin (InvivoGen, San Diego, CA, USA), which were used to select cells that had successfully incorporated the plasmid.
Site-directed mutagenesis was performed on pENTR/EGFRvIII as previously described [59 (link)]. After the Gateway LR reaction and incorporation of the mutated EGFRvIII cassettes into pLV-puro-DEST vectors, all sequences were confirmed using the Applied Biosystems 3130 Genetic Analyzer. The primers used for the creation of cysteine–serine EGFRvIII mutants are listed in Table S1A.