Part:BBa_K1431101

TetOn-3G, an ideal controller of mammalian gene expression with TRE-3G promoter+PolyA

Tet-On(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.[http://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation]

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(the mechanism of Tet-On 3G system show below). 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)

Figure 1.The Tet-On 3G systems allow inducible gene expression only in the presence of doxycycline. When Dox binds, the transactivator undergoes a conformational change allowing it to bind tet operator (tetO) repeats within the TREG Promoter (PTRE3G). The transactivator activates expression through transcription activation domain repeats.

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.