Lentivirus shRNA Knockdown Vector

The lentivirus shRNA knockdown vector system is a highly efficient method for stably knocking down expression of a target gene in a wide variety of mammalian cells. Once the viral genome is reverse transcribed and permanently integrated into the host cell genome, the shRNA is expressed from the human U6 promoter, leading to degradation of target gene mRNA. The permanent nature of knockdown by lentivirus has several major advantages over transient knockdown by synthetic siRNA (see Advantages section below).

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Our lentivirus U6-based shRNA knockdown vector has been validated for highly efficient gene knockdown as shown in Figure 1 below. The comparison between the U6-based and miR30-based shRNA systems is also presented.

Figure 1. Comparisons of EGFP knockdown through U6-based versus miR30-based shRNA lentiviral systems. (A) Lentiviral vectors carrying the U6-driven shRNA, CMV-driven miR30-based single shRNA, and CMV-driven miR30-based quad shRNA were separately packaged into lentiviral particles. HEK293T cells stably expressing EGFP were transduced with the shRNA lentivirus, and EGFP expression was measured by flow cytometry before and after drug selection using the appropriate antibiotics. (B) Before drug selection, EGFP expression was reduced by ~46% (P<0.001) thru U6-based shRNA, by 13% (P<0.001) thru CMV-driven miR30-based single shRNA, and by 44% (P<0.001) thru CMV-driven miR30-based quad shRNA. (C) After drug selection, EGFP expression was reduced by ~72% (P<0.001) thru U6-based shRNA, by 60% (P<0.001) thru CMV-driven miR30-based single shRNA, and by 67% (P<0.001) thru CMV-driven miR30-based quad shRNA. The relative EGFP expression was calculated by dividing the median fluorescence intensities (MFIs) of the transduced cells by the MFIs of the non-transduced cells. Technical triplicates were performed for the experiment, and SD were presented in the figure. The p-values were calculated based on the Tukey’s test.

Permanent knockdown: Lentiviral integration into the host cell genome is an irreversible process, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the knockdown of the target gene is typically stable and permanent. This can be an important advantage for several experimental goals. It allows long-term analysis of the knockdown phenotype in cell culture or in vivo. It facilitates the isolation of clones having different levels of knockdown and/or different phenotype. When the knockdown vector carries a fluorescence marker such as EGFP, it also allows cells with different amounts of lentiviral integration (and hence potentially different levels of knockdown) to be isolated by flow sorting cells with different fluorescence intensity.

High viral titer: Our vector can be packaged into high-titer virus (>10⁸ 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 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.

Permanent knockdown: Lentiviral integration into the host cell genome is an irreversible process, and the U6 promoter directs constitutive expression of the shRNA. For these reasons, the target gene cannot easily be reactivated once it is knocked down by the Lentivirus shRNA Knockdown vector. This can be an advantage or a disadvantage, depending on experimental goals.