pHUSH vector design and optimization (A) Vector diagrams of the pHUSH vector series: (1) original pHUSH backbone (2) pHUSH-IVS in which the synthetic intron sequence between the TetR ORF and the IRES is removed, and (3) pHUSH-IVSTetROpt with a codon-optimzed TetR (B) Increased TetR expression ensures regulation of GFP-Melk fusion protein in the absence of doxycycline. HEK 293 cells expressing a GFP-MELK fusion were transfected with the pHUSH vector series described above all containing the shB Melk targeting shRNA. After selection with 3 μg/ml puromycin, the resulting stable pools were cultured in the absence (open bars) or presence (filled bars) of 1 μg/ml doxycycline for five days and GFP-Melk expression analyzed by FACS. Data is normalized to the mean fluorescence intensity (10,000 acquired events) for cells containing a pHUSH empty vector control. A representative experiment is shown. (C) Codon optimization of TetR open reading frame (ORF) increases translation. Both the original (WT) and codon-optimized (OPT) TetR ORFs were transiently expressed in 293T cells. Forty-eight hours post-transfection, cell lysates were prepared and Western blotted with an anti-TetR antibody and an anti-tubulin antibody.