Lentivirus Inducible Gene Expression Vector (Tet-On)
VectorBuilder's lentivirus inducible gene expression vector combines the highly efficient third-generation lentiviral vector system with the Tet-On inducible gene expression system to help you achieve 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.
The lentiviral vector system is a highly efficient vehicle for introducing genes permanently into mammalian cells. A lentiviral vector is first constructed as a plasmid in E. coli. For the lentivirus inducible gene expression vector, the tetracycline inducible expression cassette consisting of the tetracycline inducible element (TRE) promoter driving the GOI is placed in-between the two LTRs during vector construction. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two long terminal repeats (LTRs) is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.
When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated into the host genome. The inducible expression cassette placed in-between the two LTRs during vector construction are permanently inserted into host DNA alongside the rest of viral genome.
While our lentivirus inducible gene expression vector includes an inducible gene expression cassette consisting of the TRE promoter driving the user-selected GOI, the TRE binding regulatory proteins rTS and rtTA have to 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. For the lentivirus inducible gene expression vector system, the two-vector system achieves higher levels of transgene induction in the presence of tetracycline compared to an all-in-one vector system. An all-in-one vector consists of two consecutive expression cassettes: the GOI driven by TRE promoter and the tTS/rtTA genes driven by a ubiquitous or tissue-specific promoter. For the lentiviral vectors, internal polyadenylation signal is not suggested to be placed between the LTRs for each individual expression cassette, as this would inhibit virus packaging. Instead, a single polyadenylation signal is placed in the 3’LTR. As a result, transcription from the upstream TRE promoter often continues past the end of the upstream ORF, through the downstream promoters and ORFs (tTS/rtTA genes). This often leads to partial inhibition of expression of the downstream tTS/rtTA, therefore preventing efficient induction of gene expression in the presence of tetracycline. Therefore, we recommend co-transducing target cells with lentivirus carrying the TRE driven GOI and lentivirus expressing the tTS/rtTA cassette to achieve the best induction efficiency.
By design, lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).
For further information about this vector system, please refer to the papers below.
|J Virol. 72:8463 (1998)||The 3rd generation lentivirus vectors|
|Nat Protoc. 1:241 (2006)||Production and purification of lentiviral vectors|
|Science. 268:1766-9 (1995)||Development of rtTA.|
|J Gene Med. 1:4-12 (1999)||Development of tTS.|
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 lentiviral inducible gene expression vector is derived from the third-generation lentiviral vector system. It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level transgene expression.
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.
High-level expression: The TRE promoter can drive very high levels of expression of the GOI in its induced state.
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, lentiviral transduction can deliver genes permanently into host cells due to the integration of the viral vector into the host genome.
High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, titer can reach >109 transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used.
Very broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).
Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.
Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.
Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5’ LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.
Limited cargo space: The wildtype lentiviral genome is ~9.2 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.8 kb, which leaves ~6.4 kb to accommodate the user’s DNA of interest. When the vector goes beyond this size limit, viral titer can be severely reduced. The lentivirus inducible gene expression vector is routinely used for inserting several functional elements besides the ORF of the GOI, such as the TRE promoter and drug resistance cassette. A large ORF plus these additional elements could exceed 6.4 kb, and the result could be compromised viral production.
Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.
RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.
5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of 5'LTR-ΔU3 LTR.
Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.
RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.
cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.
Promoter: The promoter driving your gene of interest is placed here. Users can select between either the 2nd generation (TRE) or the 3rd generation (TRE3G) Tetracycline-responsive element promoter.
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.
WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.
mPGK promoter: Mouse phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression 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 transduced with the vector to be selected and/or visualized.
3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.
SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.
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.