VectorBuilder’s piggyBac Tet regulatory protein expression vector can be used for transfection-based, permanent delivery of Tet regulatory protein(s) (tTS, rtTA, etc.) into mammalian cells to help you achieve tetracycline-inducible expression of target genes placed downstream of a tetracycline responsive-element (TRE) promoter.
The Tet-On inducible system is a powerful tool to control the timing of expression of the gene(s) of interest (GOI) in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of a GOI in the absence of tetracycline and its analogs (e.g. doxycycline), and strong, rapid expression in response to the addition of tetracycline or one of its analogs (e.g. doxycycline). This is achieved through a multicomponent system which incorporates active silencing by the tTS protein in the absence of tetracycline and strong activation by the rtTA protein in the presence of tetracycline. In the absence of tetracycline, the tTS protein derived from the fusion of TetR (Tet repressor protein) and KRAB-AB (the transcriptional repressor domain of Kid-1 protein) binds to the TRE promoter, leading to the active suppression of gene transcription. The rtTA protein, on the other hand, derived from the fusion of a mutant Tet repressor and VP16 (the transcription activator domain of virion protein 16 of herpes simplex virus), binds to the TRE promoter to activate gene transcription only in the presence of tetracycline.
While our piggyBac Tet regulatory protein expression vector includes an expression cassette consisting of the Tet regulatory protein(s) driven by a user-selected promoter, the GOI driven by the TRE promoter must be provided using a separate helper vector to achieve tetracycline induced gene expression in the presence of tetracycline, while minimizing leaky expression in the absence of tetracycline.
The piggyBac system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed. The Tet regulatory protein expression cassette is cloned in between the two TRs. When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper would recognize the two TRs on the transposon and insert the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.
PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.
For further information about this vector system, please refer to the papers below.