Hek 293 phoenix ampho packaging cells

A retroviral system was used for transfection of EGFR genes into A549 lung cancer cells. In brief, pBabe-puro vectors (Addgene, Cambridge, MA) containing the cDNA of wild type EGFR and mutant EGFRs (delE746-A750 in exon 19, and L858R in exon 21) were transfected into HEK 293 Phoenix ampho packaging cells (ATCC, Manassas, VA) using Fu-GENE6 transfection reagent (Roche, Lewes, UK). The supernatant was collected for transduction of retrovirus into A549 lung cancer cells 48 hours after transfection. After being selected with puromycin for 3 weeks, the remaining cell colonies were amplified and checked for EGFR expression and used for further analysis.

H1975 lung cancer cells harboring the C797S EGFR mutation were established using retroviral transduction of the EGFR gene with T790M and C797S mutations. Briefly, the C797S mutation was generated by site-directed mutagenesis from pBabe EGFR (L858R/T790M) plasmid (Addgene, Cambridge, MA, USA) using a QuikChange™ Site-Directed Mutagenesis kit (Stratagene California, San Diego, CA, USA) according to the manufacturer’s protocol. The following primers were used for site-directed mutagenesis: forward, 5′-CAGCTCATGCCCTTCGGCAGCCTCCTGGACTATGTCCGGG-3′, and reverse, 5′-CCCGGACATAGTCCAGGAGGCTGCCGAAGGGCATGAGCTG-3′. The resultant pBabe EGFR (L858R/T790M/C797S) plasmid was then transfected into HEK 293 Phoenix ampho packaging cells (ATCC, Manassas, VA) by Fu-GENE6 transfection reagent (Roche, Lewes, UK). Forty-eight hours after transfection, the supernatant was collected for transduction of the retrovirus into the H1975 lung cancer cells. The lung cancer cells were selected with puromycin for 3 weeks, and then the remaining cells were amplified for further analysis.