Difference between revisions of "Part:BBa K1431101"

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 rtTA2^S-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^S-S2 rather than rtTA2^S-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.

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 (P_TRE3G,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_TRE3G and activates transcription of the downstream GOI. P_TRE3G 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.

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 <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 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.[http://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation]<br>
+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>,[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(the mechanism of Tet-On 3G system show below). In the presence of Dox, Tet-On 3G binds specifically to PTRE3G 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. <br>
+<center>https://static.igem.org/mediawiki/parts/8/8b/Tet-On_compare_with_Tet-On_3G.jpg</center>
-The Dox concentrations required for induction of Tet-On Systems are far below cytotoxic levels for either cell culture or transgenic studies, and Tet-On 3G responds to even lower concentrations than its predecessors (Zhou et. al,. 2006). 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.(Source: Clontech)<br>
+<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>
+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>
+The data of our experience shows in [https://parts.igem.org/Part:BBa_K1431301 BBa_K1431301].
 <!-- Add more about the biology of this part here
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 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1431101 SequenceAndFeatures</partinfo>
+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.