Fig. 7

Mechanism of gene leakage in the Tet-On 3G system with A2UCOE. A. In the absence of doxycycline and A2UCOE, the Tet-On 3G transactivator does not bind to the tetracycline response element (TRE), and RNA polymerase II (RNA pol II) elongation does not initiate. The integrated gene will be silenced through methylation. B. In the presence of doxycycline, the Tet-On 3G transactivator binds to the TRE, initiating RNA pol II elongation and resulting in transcription. C. In an environment with A2UCOE but without doxycycline, the A2UCOE maintains the sense-strand constitutively expressed genes (rtTA, BSR, mScarlet) in an open chromatin state, preventing methylation and silencing. However, A2UCOE also exerts an unexpected effect on RNA pol II on the anti-sense strand, leading to unexpected elongation through the TRE promoter causing gene transcription even in the absence of doxycycline. D. When the SV40 poly-A sequence is present, it blocks the influence of A2UCOE on RNA pol II, stopping elongation and thereby reducing gene leakage