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本帖最后由 lchanlon 于 2017-2-24 01:54 编辑 8 @. N+ _% U, S' v! K
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回复 富平隐士 的帖子$ D }& d3 c) Q( }5 r. X |' } ^3 B) n
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To advance the study of gene function, scientists were in search of inducible promoters capable of controlling eukaryotic gene expression. Several endogenous promoters had been identified that responded to stimuli, such as hormones or metal ions; however, these systems were confounded by secondary effects. Scientists began to pursue a non-endogenous system for eukaryotes. At the time, bacterial systems from E. coli presented the best candidates for inducible expression.% K2 T6 _; c7 ]/ d* \; l, ?
. J7 e0 x: U" j4 C5 l+ C6 J( ZBacterial systems were tested for functionality in mammalian cells. The lac system with the IPTG inducer was tested first, and IPTG was found to be inefficient, resulting in low levels of induction. In 1992, Manfred Gossen and Hermann Bujard tested the tet system in a mammalian cell system (HeLa) and found that the tet system was functional, and had rapid induction with efficient tetracycline uptake.2 t( U& [ v I! f9 n- T! _
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Tet System and the Tet Response Element (TRE)) O, Z; }0 m/ ?) D3 o9 R f$ ]
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A TRE is 7 repeats of a 19 nucleotide tetracycline operator (tetO) sequence, and is recognized by the tetracycline repressor (tetR). In the endogenous bacterial system, if tetracycline, or one of its analogs like doxycycline, are present, tetR will bind to tetracycline and not to the TRE, permitting transcription. n" q" [* a" a" O& y l0 e
2 L9 F$ [+ t' v2 R+ h+ YTetracycline-dependent promoters are developed by placing a TRE upstream of a minimal promoter.
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Tetracycline On System
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In Gossen et al (1995) , random mutagenesis was used to identify which amino acid residues of tetR were important for tetracycline-dependent repression. Mutating these residues led to the development of a reverse Tet repressor, or rTetR, which reversed the phenotype and created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA ( r everse t etracycline-controlled t rans a ctivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.) e1 S: E9 [) g
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Tetracycline Off System
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The initial system Gossen and Bujard developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. To use tetracycline as a regulator of gene expression, a tetracycline-controlled transactivator (tTA) was developed. tTA was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16), an essential transcriptional activation domain from HSV (herpes simplex virus).& |" x* [( O, ^3 \) J) J3 y- h
- K- p: q P9 v9 W% l. K* ]+ qIn the absence of tetracycline, the tetR portion of tTA will bind these tetO sequences and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR. This precludes tTA binding to the tetO sequences and subsequent increase in expression by the activation domain, resulting in reduced gene expression. This idea of a hybrid transactivator was initially used with the lac system. Tetracycline off is also known as the tTA-dependent system.4 t* U5 e) l* K- S% h) ?
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发表于 2017-2-23 11:22
发表于 2017-2-24 01:54
