PiggyBac Tet Regulatory Protein Expression Vector
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.
|Mol Cell Biochem. 354:301 (2011)||Review on the PiggyBac system|
|Cell. 122:473 (2005)||Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice|
|Science. 268:1766-9 (1995)||Development of rtTA|
|J Gene Med. 1:4-12 (1999)||Development of tTS|
Our piggyBac Tet regulatory protein expression vector allows highly efficient, transposon-based delivery of Tet regulatory protein(s) into mammalian cells. Using this vector to coexpress the Tet regulatory proteins tTS and rtTA along with the TRE driven GOI can achieve nearly complete silencing of the GOI in the absence of tetracycline, and strong, rapid expression in response to the addition of tetracycline. The piggyBac Tet regulatory protein expression vector along with the helper plasmid are optimized for high copy number replication in E. coli, efficient transfection into a wide range of target cells, and high-level expression of the transgene carried on the vector.
Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.
Technical simplicity: The piggyBac Tet regulatory protein expression vector can be introduced into mammalian cells by conventional transfection. Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.
Very large cargo space: Our transposon vector can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's DNA of interest.
Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of the piggyBac system.
5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.
Promoter: The promoter driving the expression of Tet regulatory protein(s) is placed here.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of Tet regulatory protein(s) is placed here.
rBG pA: Rabbit beta-globin polyadenylation signal. It facilitates transcription termination of the upstream ORF.
CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transfected with the vector to be selected and/or visualized.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
3' ITR: 3' inverted terminal repeat.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.