Figure 3

Multiple TetO₂ operons within the Pol III promoter enhances regulated knock-down. (A) 293T cells were transfected with 15 ng of pGL3-huMelk in the presence of decreasing molar ratios of TetR and the appropriate H1-shRNA vectors using the indicated molar ratio of TetR:H1-shRNA vector. The maximal level of gene knockdown that can be observed with either promoter is represented by cells transfected with the appropriate H1-shRNA vector and no TetR (0:1). Both H1-shRNA constructs expressed the shB Melk targeting shRNA. Luciferase expression was measured as described in the Methods 48 hours post transfection. (B) Dox regulated Melk knockdown is maintained in differentiated ES cells with the 2 × TetO₂ promoter configuration. Embryoid bodies were generated as described in the Methods from stable pHUSH ES cell clones with shRNA targeting MELK or Luciferase. shMELK ES cell lines were generated with either one TetO₂ (1×-TetO2, colony A6) or two TetO₂ (2×-TetO2, colony 3C11) operons (see also additional file 3). ES cells lines were differentiated into embryoid bodies in the presence or absence of 1 ug/ml doxycycline and the level of Melk expression determined by qRT-PCR and normalized to the housekeeping gene SPF31 (RefSeq NM_014280).