shAkt macrophage lines

Macrophage lines expressing shAkt or controls were generated by transduction of SMARTvector Inducible Lentiviral shRNA system (Dharmacon, Lafayette CO). Custom made lentiviruses that included one of three sequences: shAkt1: ATCGGAAGTCCATCGTCTC, shAkt2: GGGACTCTCGCTGATCCAC shAkt3: CGTTTGTGCAGCCAGCCCT were developed. A control Lentiviral recombinant containing the sequence TGGTTTACATGTTGTGTGA was also used to generate a control cell line. Lentiviral particles prepared by Dharmacon were used to transduce RAW264.7 macrophages. After 24 hr, transduced macrophages were selected by growth in 6ug/ml puromycin in complete DMEM at 37° C under 5% CO₂ following protocols from Dharmacon. After at least 2 passages under antibiotic selection, each cell line was then assessed for Akt shRNA expression. The expression of Akt shRNA was induced by growth in 1uM doxycycline.