gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
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).
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 has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).
For general information about lentiviral vectors, see our Guide to Vector Systems section on Lentiviral Expression Vectors, and for further information about Lentiviral shRNA Knockdown vectors, please refer to the paper below.
|RNA. 9:493-501 (2003)||Development of lentiviral shRNA vectors|
Our Lentivirus 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 human U6 promoter drives high-level, constitutive transcription of the shRNA in mammalian cells, while our optimized shRNA stem-loop sequences mediate efficient shRNA processing and target gene knockdown.
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.
RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.
Δ5' LTR: 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 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.
Sense, Antisense: These sequences are derived from your target sequences, 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.
hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of 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.
WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.
ΔU3/3' LTR: 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.