Lentivirus Tet-Inducible shRNA Knockdown Vector

VectorBuilder’s lentivirus tet-inducible shRNA knockdown vector is a highly efficient system for achieving regulated knockdown of target genes in a wide variety of mammalian cell lines and offers an effective tool for studying genes essential for cell development or cell survival. This system utilizes the interaction between repressor (TetR) and operator (TetO) proteins to regulate shRNA expression in the presence or absence of tetracycline or one of its analogs (e.g. doxycycline).

This vector contains a modified human U6 promoter with two repeats of the TetO element (U6/2xTetO) located directly upstream of the shRNA targeting the gene of interest (GOI), which functions to enhance shRNA expression in the presence of tetracycline or one of its analogs (e.g. doxycycline). In the absence of one of these regulators, TetR binds to the TetO elements and suppresses transcription of the shRNA. Conversely, in the presence of tetracycline or one of its analogs, the regulator binds to TetR causing a conformational change such that TetR is no longer able to bind to TetO, allowing transcription of the shRNA driven by the U6 promoter.

VectorBuilder has created shRNA databases that contain optimized shRNAs for common species. For shRNA design we apply rules like those used by the RNAi consortium. When you design shRNA vectors on VectorBuilder’s online platform, you will have the option to search for your target genes in our database. Upon entering your gene name, you will see detailed information on all shRNAs against your GOI available in our database, including a link to UCSC Genome Browser to view these shRNAs in the context of genomic sequence and all the transcript isoforms. Our database ranks all available shRNAs for a target gene in order of their decreasing knockdown scores and recommends testing the top 3 shRNAs with the highest knockdown scores.

By design, our 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 have 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.

Our lentivirus tet-inducible shRNA knockdown vectors are derived from the third-generation lentiviral vector system. This system 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, and efficient vector integration into the host genome. The modified human U6 promoter with two TetO elements drives high-level transcription of the downstream shRNA in mammalian cells only in the presence of tetracycline or one of its analogs (e.g. doxycycline). Our shRNA stem-loop sequences are optimized to mediate efficient shRNA processing and target gene knockdown.

Our lentivirus tet-inducible shRNA knockdown vectors has been validated for efficient target gene knockdown in the presence of doxycycline as shown in Figure 1 below.

Figure 1. EGFP knockdown with the lentivirus tet-inducible shRNA vector system. (A) Lentiviral vectors carrying tet-inducible U6-based scramble or EGFP-targeting shRNA expression cassettes were packaged into the corresponding lentiviral particles and transduced into HEK293T cells stably expressing EGFP. Antibiotic selection with appropriate antibiotics, in this case puromycin, was performed to isolate positively transduced cells followed by treatment with 1 µg/ml doxycycline (dox) to induce shRNA expression. Relative fluorescence intensity (RFI) of EGFP was quantified for all experimental groups using flow cytometry. (B) Cells expressing an inducible EGFP shRNA cassette showed a ~60% reduction in EGFP RFI upon doxycycline induction, while inducible shRNA vectors expressing a non-targeting scramble shRNA had no significant effect on EGFP RFI upon doxycycline induction. (C) Representative fluorescence microscopy images were taken for each group. Exposure: brightfield=10 ms; EGFP=100 ms. Magnification: 100x.

Permanent integration of vector DNA: Lentiviral transduction can deliver genes permanently into host cells due to the integration of the viral vector into the host genome. In this case, this leads to long-term incorporation of a reliable gene expression switch mediated by tetracycline or one of its analogs (e.g. doxycycline).

Tight regulation and efficient induction: The presence of two repeats of the TetO element within the U6 promoter results in enhanced shRNA expression in the presence of tetracycline or one of its analogs (e.g. doxycycline).

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 TU/ml. At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral supernatant 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: Lentiviral vector systems can be used effectively in cultured cells and 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.

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 technical demanding and time consuming relative to conventional plasmid transfection.

Lower efficiency: Lower and less dynamic knockdown is achieved compared to the IPTG-inducible systems.

CMV promoter: Human cytomegalovirus immediate early enhancer/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 has a deletion in 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 CMV 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.

U6/2xTetO: Modified human U6 small nuclear 1 promoter containing two repeats of the tetracycline operator (TetO) sequence to enhance shRNA expression in the presence of tetracycline or one of its analogs (e.g. doxycycline). Pol III promoter which suppresses small RNA expression in the presence of tetracycline repressor protein (TetR) without the small molecule regulator.

shRNA (Sense, Antisense): These sequences are derived from your target sequence and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.

shRNA (Loop): This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

hPGK+intron: Human phosphoglycerate kinase 1 gene promoter (hPGK) fused with the splicing signal from the rabbit beta-globin intron 2. It drives the ubiquitous expression of the downstream ORF.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Regulatory protein: Tetracycline repressor (TetR) and a drug selection gene (such as puromycin resistance gene) linked by an IRES linker. Allows cells to express TetR protein and be resistant to the corresponding drug. In the absence of tetracycline or one of its analogs (e.g. doxycycline), TetR binds to TetO to repress transcription of downstream genes or small RNAs. In the presence of tetracycline or one of its analogs, TetR undergoes a conformational change and is no longer able to bind to TetO, thus allowing downstream genes or small RNAs to be transcribed.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

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 (due to the fact that 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.