Difference between revisions of "Part:BBa K1431101"
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| − | The | + | Tet-On(Tetracycline-Controlled Transcriptional Activation[http://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation],also known as rtTA2<sup>S</sup>-M2) is a system of inducible gene expression systems for mammalian cells. Tet-On 3G (also known as rtTA-V16) is similar to Tet-On but was derived from rtTA2<sup>S</sup>-S2 rather than rtTA2<sup>S</sup>-M2. The Tet-On 3G protein has 5 amino acid differences compared to Tet-On which appear to increase its sensitivity to doxycycline(Dox) even further. Tet-On 3G is sensitive to 100-fold less Dox and is 7-fold more active than the original Tet-On.<br> |
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| + | <center>https://static.igem.org/mediawiki/parts/8/8b/Tet-On_compare_with_Tet-On_3G.jpg</center> | ||
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| + | <center>Figure 1. Tet-On 3G demonstrates higher sensitivity to doxycycline than Tet-On Advanced. Tet-On 3G and Tet-On Advanced genes<br> were integrated at the same locus in a stable HLF33 cell line expressing luciferase from a TRE promoter.For each of these<br> two double-stable cell lines, induced luciferase expression was measured in response to a range of doxycycline (Dox) <br>concentrations. At 5–10 ng/ml Dox, induced expression was 100–150-fold higher for the Tet-On 3G cell line, and at 50 ng/ml,<br> expression was 4.6 fold higher (data kindly provided by Professor W. Hillen and Dr. C. Berens, University of Erlangen).</center> | ||
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| + | Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (P<sub>TRE3G</sub>,[https://parts.igem.org/Part:BBa_K1431301 BBa_K1431301]) will express high levels of GOI, but only when cultured in the presence of Dox, which is a synthetic tetracycline derivative. In the presence of Dox, Tet-On 3G binds specifically to P<sub>TRE3G</sub> and activates transcription of the downstream GOI. P<sub>TRE3G</sub> lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction.(Source: Clontech)<br> | ||
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| + | The data of our experience shows in [https://parts.igem.org/Part:BBa_K1431301 BBa_K1431301]. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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| + | Note that Tet-On Systems respond well only to doxycycline, and not to tetracycline (Gossen & Bujard, 1995). The half-life of Dox in cell culture medium is 24 hours. To maintain continuous inducible GOI expression in cell culture, the medium should be replenished with Dox every 48 hours. | ||
Latest revision as of 03:09, 18 October 2014
TetOn-3G, an ideal controller of mammalian gene expression with TRE-3G promoter+PolyA
Tet-On(Tetracycline-Controlled Transcriptional Activation[http://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation],also known as rtTA2S-M2) is a system of inducible gene expression systems for mammalian cells. Tet-On 3G (also known as rtTA-V16) is similar to Tet-On but was derived from rtTA2S-S2 rather than rtTA2S-M2. The Tet-On 3G protein has 5 amino acid differences compared to Tet-On which appear to increase its sensitivity to doxycycline(Dox) even further. Tet-On 3G is sensitive to 100-fold less Dox and is 7-fold more active than the original Tet-On.
were integrated at the same locus in a stable HLF33 cell line expressing luciferase from a TRE promoter.For each of these
two double-stable cell lines, induced luciferase expression was measured in response to a range of doxycycline (Dox)
concentrations. At 5–10 ng/ml Dox, induced expression was 100–150-fold higher for the Tet-On 3G cell line, and at 50 ng/ml,
expression was 4.6 fold higher (data kindly provided by Professor W. Hillen and Dr. C. Berens, University of Erlangen).
Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (PTRE3G,BBa_K1431301) will express high levels of GOI, but only when cultured in the presence of Dox, which is a synthetic tetracycline derivative. In the presence of Dox, Tet-On 3G binds specifically to PTRE3G and activates transcription of the downstream GOI. PTRE3G lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction.(Source: Clontech)
The data of our experience shows in BBa_K1431301.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Note that Tet-On Systems respond well only to doxycycline, and not to tetracycline (Gossen & Bujard, 1995). The half-life of Dox in cell culture medium is 24 hours. To maintain continuous inducible GOI expression in cell culture, the medium should be replenished with Dox every 48 hours.