PiggyBac Inducible Gene Expression Vector (Tet-On, Low Leak)
The PiggyBac inducible gene expression vector combines VectorBuilder’s highly efficient PiggyBac vector system and the Tet-On inducible gene expression system to help you achieve transfection-based permanent integration of tetracycline inducible gene expression cassettes into the host genome.
The Tet-On inducible gene expression system is a powerful tool to control the timing of expression of gene(s) of interest in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of gene(s) of interest 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) with 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 to 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 standard piggyBac inducible gene expression vector expresses tTS and rtTA as a fusion protein, which acts as a gene activation switch, the low leak version of our piggyBac Tet-On vector is designed to allow tissue-specific induction of target transgenes in the presence of tetracycline, while minimizing leaky expression in non-target tissues in the absence of tetracycline. There are three expression cassettes in this vector: 1) the GOI driven by the TRE promoter, 2) the tTS gene driven by a ubiquitous promoter, and 3) the rtTA gene driven by a user-selected tissue-specific promoter. In the absence of tetracycline, the tTS protein, ubiquitously expressed in all tissues, binds to the TRE promoter with high affinity thereby suppressing the GOI expression in all tissues. In the presence of tetracycline, the rtTA protein which is specifically expressed in the target tissue, can bind to the TRE promoter to activate the GOI expression only in the target tissue.
Our piggyBac inducible gene expression vector 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. For the low leak version of this vector, all three expression cassettes consisting of the GOI, the tTS gene and the rtTA gene described above are cloned into this region. 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.