gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Lentivirus miR30-Based shRNA Knockdown Vector
The lentivirus miR30-based shRNA knockdown vector system is a highly efficient method for knocking down expression of target gene(s) in a wide variety of mammalian cells. Once the viral genome is reverse transcribed and permanently integrated into the host cell genome, a user-selected promoter drives the expression of a polycistron containing a user-selected ORF and one or more miR30-based shRNAs (shRNAmiR) targeting gene(s) of interest. The shRNAmiR transcript is processed by endogenous, cellular micro-RNA pathways to produce mature shRNAs, which facilitate degradation of target gene mRNAs.
Unlike conventional shRNA vectors, which utilize RNA polymerase III promoters such as U6, miRNA-based shRNA systems are placed under the control of standard RNA polymerase II promoters. This allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications not possible with constitutive U6 promoters.
The ability of RNA polymerase II promoters to efficiently transcribe long transcripts in the miRNA-based shRNA systems also provides additional advantages relative to other knockdown vector systems. Multiple shRNAmiRs can be transcribed as a single polycistron, which is processed to form mature shRNAs within the cell. This allows knockdown of multiple genes or targeting of multiple regions within the same gene using a single transcript. Secondly, in this vector system, a user-selected protein coding gene is also positioned within the same polycistron as the shRNAmiRs. The expression of this ORF can be used to directly monitor shRNA transcription (if a marker ORF is used) or can be used for other purposes requiring co-expression of an ORF and shRNA(s).
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, please refer to our Guide to Vector Systems section on Lentiviral Expression Vectors, and for further information about lentiviral miR30-based shRNA knockdown vectors, please refer to the paper below.
|Cell Rep. 5:1704 (2013)||An Optimized microRNA Backbone for Effective Single-Copy RNAi|
Our Lentivirus miR30-Based shRNA Knockdown vectors incorporate an optimized micro-RNA system and 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 user-selected promoter drives expression of a polycistron containing a user-selected ORF and one or more shRNAmiRs with optimized miR30-based sequences to mediate efficient shRNA processing and target gene knockdown.
Promoter choice: Unlike standard shRNA systems, which utilize RNA polymerase III promoters such as U6, miR30-based shRNAs can be transcribed by diverse RNA polymerase II promoters. This also enables the use of tissue-specific or inducible promoters.
Multiple shRNA co-expression: Because RNA polymerase II efficiently transcribes long RNAs, multiple shRNAmiRs can be expressed as a polycistron from a single promoter.
Co-expression of a reporter ORF: A user-selected gene of interest or reporter gene ORF is co-expressed with the shRNAmiRs, as a polycistron. This facilitates direct monitoring of shRNA transcription.
Permanent knockdown: Lentiviral integration into the host cell genome is an irreversible process. For this reason, 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.
Promoter effects: There is a strong correlation between promoter strength, shRNA expression levels, and knockdown efficacy. Many RNA polymerase II promoters will produce lower shRNA expression than from the strong U6 RNA polymerase III promoter. This can result in reduced target gene knockdown.
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. If a constitutively active promoter is used, the target gene cannot easily be reactivated once it is knocked down. 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.
Promoter: Drives transcription of the downstream ORF and shRNAmiR polycistron. This is an RNA polymerase II promoter, rather than an RNA polymerase III promoter such as U6.
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.
ORF: The open reading frame of your gene of interest or reporter gene is placed here. This can be used to monitor shRNA expression.
5' miR-30E: An optimized version of the human miR30 5’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.
3' miR-30E: An optimized version of the human miR30 3’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.
shRNAs: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNAs.
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 promoter. This 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.
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.