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. 2013 May 17;41(13):e134. doi: 10.1093/nar/gkt405

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — Two-gene control dynamics of the ST-TA transactivator. (A) Independent control of two different transgenes. HEK-293T cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors at a high transactivator-to-reporter ratio (20:1:1) and grown for 48 h in culture medium containing increasing concentrations of TET or γ-butyrolactone (SCB1) before SEAP and GLuc levels were quantified in the culture supernatant. (B) DPDT relay switch characteristics.

HEK-293T cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors at a low transactivator-to-reporter ratio (1:12:12) and grown for 48 h in culture medium containing increasing concentrations of TET before SEAP and GLuc levels were quantified in the culture supernatant. (C) HEK-293T cells were co-transfected with the tTA- and SCA- expression vectors (pSAM200, pWW122) and corresponding reporter plasmids pDA43 (P_hCMV*−1-GLuc) and pWW124 (P_SPA-SEAP) (ratio 1:1:5:5), grown for 48 h in culture medium containing increasing concentrations of TET before expressed SEAP and GLuc levels were quantified in the culture supernatant. (D) DPDT relay switch characteristics of hMSC-TERT cells. hMSC-TERT cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors in a low transactivator-to-reporter ratio (1:12:12) and grown for 48 h in culture medium containing increasing concentrations of TET before SEAP and GLuc levels were quantified in the culture supernatant. (E) Reversibility of ST-TA-mediated DPDT relay switch characteristics. HEK-293T cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors in a low transactivator-to-reporter ratio (1:12:12) and grown for 24 h in the presence (1 µg/ml) or absence of TET and SEAP, and GLuc levels were profiled in the culture supernatant. The same cells were then washed and incubated with fresh medium and grown for another 24 h in the presence (TET, 1 µg/ml) or absence of TET (TET to no TET), before SEAP and GLuc levels were profiled in the culture supernatant. (F) Schematic of the electric and genetic DPDT relay switch.

Figure 5. — Two-gene control dynamics of the ST-TA transactivator. (A) Independent control of two different transgenes. HEK-293T cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors at a high transactivator-to-reporter ratio (20:1:1) and grown for 48 h in culture medium containing increasing concentrations of TET or γ-butyrolactone (SCB1) before SEAP and GLuc levels were quantified in the culture supernatant. (B) DPDT relay switch characteristics.

HEK-293T cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors at a low transactivator-to-reporter ratio (1:12:12) and grown for 48 h in culture medium containing increasing concentrations of TET before SEAP and GLuc levels were quantified in the culture supernatant. (C) HEK-293T cells were co-transfected with the tTA- and SCA- expression vectors (pSAM200, pWW122) and corresponding reporter plasmids pDA43 (P_hCMV*−1-GLuc) and pWW124 (P_SPA-SEAP) (ratio 1:1:5:5), grown for 48 h in culture medium containing increasing concentrations of TET before expressed SEAP and GLuc levels were quantified in the culture supernatant. (D) DPDT relay switch characteristics of hMSC-TERT cells. hMSC-TERT cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors in a low transactivator-to-reporter ratio (1:12:12) and grown for 48 h in culture medium containing increasing concentrations of TET before SEAP and GLuc levels were quantified in the culture supernatant. (E) Reversibility of ST-TA-mediated DPDT relay switch characteristics. HEK-293T cells were co-transfected with the ST-TA-encoding plasmid pMX1, the P_SPA-driven SEAP- (pWW124) and P_hCMV*−1-driven GLuc- (pDA43) expression vectors in a low transactivator-to-reporter ratio (1:12:12) and grown for 24 h in the presence (1 µg/ml) or absence of TET and SEAP, and GLuc levels were profiled in the culture supernatant. The same cells were then washed and incubated with fresh medium and grown for another 24 h in the presence (TET, 1 µg/ml) or absence of TET (TET to no TET), before SEAP and GLuc levels were profiled in the culture supernatant. (F) Schematic of the electric and genetic DPDT relay switch.