Difference between revisions of "Part:BBa K1431101"

 
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<partinfo>BBa_K1431101 short</partinfo>
 
<partinfo>BBa_K1431101 short</partinfo>
  
Tet-On(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[citation needed]) 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 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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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>
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>) will express high levels of your GOI, but only when cultured in the presence of doxycycline (Dox), which is a synthetic tetracycline derivative(the mechanism of Tet-ON 3G system show below). And the Dox concentrations required for induction of Tet-On Systems are far below cytotoxic levels for either cell culture or transgenic studies.
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[https://static.igem.org/mediawiki/2014/3/37/SUSTC-Shenzhen_Tet_On_3G.png]
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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>
 
<partinfo>BBa_K1431101 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1431101 SequenceAndFeatures</partinfo>
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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.

Tet-On_compare_with_Tet-On_3G.jpg
Figure 1. Tet-On 3G demonstrates higher sensitivity to doxycycline than Tet-On Advanced. Tet-On 3G and Tet-On Advanced genes
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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.