Lentivirus IPTG-Inducible shRNA Knockdown Vector

The Lentivirus IPTG-Inducible shRNA Knockdown vector is a highly efficient system for achieving temporal 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 LacI (repressor) and LacO (operator) proteins, derived from the bacterial lactose operon to regulate shRNA expression in the presence and absence of the lactose analogue, isopropyl-ß-D-thiogalactoside (IPTG).

Our Lentivirus IPTG-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 LacO elements drives high-level transcription of the downstream shRNA in mammalian cells in the presence of IPTG, which prevents LacI from binding to LacO. Our shRNA stem-loop sequences are optimized to mediate efficient shRNA processing and target gene knockdown.

Our lentivirus IPTG-inducible shRNA knockdown vector has been validated for highly efficient target gene knockdown in the presence of IPTG as shown in Figure 1 below.

Figure 1. EGFP knockdown with the lentivirus IPTG-inducible shRNA vector system. (A) Lentiviral vectors carrying IPTG-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, puromycin (Puro) or blasticidin (Bsd), was performed to isolate positively transduced cells followed by treatment with 1mM IPTG to induce shRNA expression. Median fluorescence intensity (MFI) of EGFP was quantified for all experimental groups using flow cytometry (FCM); (B) Cells expressing an inducible EGFP shRNA cassette showed a ~42% reduction in EGFP MFI upon IPTG induction. This observation was consistent across inducible shRNA vectors carrying either a puromycin or blasticidin resistance gene. Inducible shRNA vectors expressing a non-targeting scramble shRNA had no effect on EGFP MFI upon IPTG induction. Moreover, in cells transduced with an inducible shRNA vector lacking the LacI repressor, the induction function of the vector was lost and EGFP expression was constitutively inhibited by the EGFP shRNA both with and without IPTG induction.

Tight regulation and maximal induction: The presence of two repeats of the LacO element within the U6 promoter along with an additional LacO element present downstream of the shRNA enables tight regulation of shRNA expression by minimizing background promoter activity in the absence of IPTG and maximizing gene silencing in the presence of IPTG.

Fast response time: IPTG induced gene silencing can be achieved as fast as 48 hours.

Higher efficiency: Can achieve higher and more dynamic knockdown than compared to a Tet-based inducible knockdown system. 

High viral titer: Our vector can be packaged into high-titer virus (>10⁹ TU/ml when virus is obtained through our virus packaging service). At this viral titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral supernatant is used.

Very broad tropism: Our lentiviral 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 vector 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.

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, Δ5' LTR 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 Δ5' 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 importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

U6/2xLacO: Modified human U6 small nuclear 1 promoter containing two repeats of the lac operator sequence. Pol III promoter which suppresses small RNA expression in the presence of LacI.

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.

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

Terminator: Terminates transcription of the shRNA.

LacO: Lac operator. In the absence of IPTG, it is bound by lac repressor (LacI), leading to transcriptional repression of the upstream shRNA. In the presence of IPTG, LacI can no longer bind to LacO, thus allowing upstream shRNA to be transcribed.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream ORF.

Regulatory protein: Lac repressor (LacI) and a drug selection gene (such as puromycin resistance gene) linked by T2A linker. Allows cells to express LacI protein and be resistant to the corresponding drug. In the absence of IPTG, LacI binds to LacO to repress transcription of downstream genes or small RNAs. In the presence of IPTG, LacI undergoes a conformational change and is no longer able to bind to LacO, 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 ΔU3/3' LTR 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.