Figure 2.

Divergent promoter arrangement of U6 and UbiC inhibits U6 promoter activity. A. Diagram of U6 and UbiC promoter arrangements. mCIN85 shRNA expression cassette was constructed as described in Materials and Methods, and was inserted into pFRRu to form divergent and tandem promoter arrangements. Minimal interfered U6 promoter activity control was set by replacing the UbiC promoter with a non-promoter cDNA fragment of RFP. B. Residual endogenous mCIN85 protein level to reflect U6 promoter activity. Mouse BMDMs were transduced with retroviruses produced with different vectors as indicated. Four days after viral transduction and drug selection, residual cells were collected and lysed. Western blots were performed using purified rabbit anti-CIN85 as primary antibody. C. Normalized mCIN85 knocking down efficiency reflecting relative U6 promoter activity. Sample sequences are as indicted in Panel B. Values are statistics from three independent experiments.