PiggyBac Inducible Gene Expression Vector (Tet-On)

Overview

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 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.

Our piggyBac inducible gene expression 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 tetracycline inducible gene expression cassette consisting of the tetracycline responsive element (TRE) promoter driving the user-selected GOI and a ubiquitous promoter or tissue-specific promoter driving the regulatory proteins tTS/rtTA is 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. Gene expression can then be turned on in the presence of tetracycline.

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.

References Topic
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.

Highlights

Our Tet-On inducible gene expression vectors are designed to 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 inducible gene 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.

Advantages

Switch-like gene activation: Unlike rtTA only Tet-On systems that usually have significant leaky expression in the absence of induction, our Tet-On gene expression vectors act as true tetracycline-regulated on-and-off switch for controlling gene expression, which can minimize the background expression without induction and result in high sensitivity and high dynamic range of the tetracycline induction.

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 inducible gene 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, transposon-related sequences and the Tet-On components occupy only about 4.6 kb, leaving plenty of room to accommodate the user's sequence of interest.

Disadvantages

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.

Key components

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. 

TRE promoter: Tetracycline-responsive element promoter (2nd generation). This element can be regulated by a class of transcription factors (e.g. tTA, rtTA and tTS) whose activities are dependent on tetracycline or its analogs (e.g. doxycycline).

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 your gene of interest is placed here.

rBG pA: Rabbit beta-globin polyadenylation signal. It facilitates transcription termination of the upstream ORF.

Promoter: The promoter chosen to drive expression of the tTS/rtTA cassette.

tTS: Tetracycline-controlled transcriptional silencer. This protein binds to TRE promoter to actively suppress gene transcription only in the absence of tetracycline and its analogs (e.g. doxycycline).

T2A: Self-cleaving 2A peptide from thosea asigna virus that allows multiple proteins to be made from a polycistronic transcript containing multiple ORFs separated by T2A. The cleavage occurs through a putative “ribosomal skipping” mechanism.

rtTA: Reverse tetracycline responsive transcriptional activator M2 (2nd generation). This protein binds to TRE promoter to activate gene transcription only in the presence of tetracycline or its analogs (e.g. doxycycline). It has higher sensitivity to the inducing drug and lower leaky activity in the absence of the drug compared to its predecessor.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the tTS/rtTA cassette.

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.