Targeting Human, Mouse and Rat Genomes
- shERWOOD algorithm design for superior knockdown performance
- UltramiR scaffold for increased shRNA processing and potency
- Minimized off-target effects
- All transcripts for a gene are targeted for the most complete knockdown
- Every shRNA is guaranteed to knockdown its intended target*
Superior knockdown with shERWOOD design
shERWOOD-UltramiR shRNA reagents are next generation vector-based RNAi triggers designed using the proprietary shERWOOD algorithm developed and validated in Dr. Gregory Hannon's laboratory at Cold Spring Harbor Laboratory (Knott et al, 2014). An alternate microRNA scaffold "UltramiR" has been optimized for increased shRNA processing and potency based on new information on the key determinants for primary microRNA processing
(Auyeung et al, 2013).
The shERWOOD algorithm is based on the functional testing of over 250,000 shRNA sequences using a high-throughput sensor assay (Knott et al, 2014) and uses key sequence characteristics for predicting shRNA potency to select the rare shRNA designs that are potent at single copy representation in the genome. shERWOOD designs have been applied to the creation of new shERWOOD-UltramiR shRNA collections targeting human, mouse and rat genomes.
Minimized off-target effects
Knockdown specificity of the shERWOOD Ultramir shRNA is highly improved compared to classic stem loop shRNA. This is consistent with publications showing that classic stem loop shRNA can cause significant off-target effects and toxicity (Baek et al, 2014). Several reports have shown that off-target effects can be ameliorated by expressing the same targeting sequence in a primary microRNA scaffold (shRNA-miR).
Molecular Cell (2014)
Simon R.V. Knott, Ashley R. Maceli, Nicolas Erard, Kenneth Chang, Krista Marran, Xin Zhou, Assaf Gordon, Osama El Demerdash, Elvin Wagenblast, Sun Kim, Christof Fellmann and Gregory J. Hannon
*All shRNA constructs in a target gene set are guaranteed to knock down mRNA expression by >70%. Cell line of choice should demonstrate expression of the target gene using the non-targeting controls and should demonstrate gene knockdown using positive control shRNA (targeting PTEN or GAPDH).
Find shERWOOD-UltramiR shRNA
- transEDIT CRISPR/Cas9 Target Sets
- cDNA, ORF & shRNA Clones Special
- Pooled Lentiviral shRNAmir Libraries
[Click to enlarge]
Harnessing the endogenous microRNA pathway to trigger RNAi
Lentiviral, inducible lentiviral and retroviral vector options with choice of promoter
- Deliver to a wide range of cell types including primary and non-dividing
- Fluorescent marker allow direct visual detection of shRNA expression
- Inducible or constitutive promoter options
- Easily create stable cell lines with efficient integration and antibiotic selection
Mammalian promoters may differ in expression level or be silenced over time depending on the target cell line. Variation in expression level can also affect fluorescent marker expression as well as knockdown efficiency. shERWOOD UltramiR shRNA are offered in a choice of promoters for optimal expression, and can be delivered by transfection or transduction.
pZIP Lentiviral Vectors [Vector details]
pZIP Inducible Lentiviral Vector [Vector details]
pLMN Retroviral Vector [Vector details]
The ZIP lentiviral vector is available with many different promoter options (SFFV, human CMV, murine CMV, human EF1a, murine EF1a, TRE-3G) driving expression of shRNA (see schematic on the right). The fluorescent marker and shRNA are on the same transcript allowing the level of fluorescence in the cell to be used as a direct indication of shRNA expression through visual inspection.
The mouse CMV promoter expresses efficiently in a number of human and mouse cell lines and is standard in the ZIP lentiviral vector target gene sets. However, for cell lines where a different promoter may be optimal, the ZIP Promoter Selection Kit is available to quickly test for optimal expression in your target cell line. Simply use the provided pre-packaged viral particles from a panel of vectors expressing ZsGreen from the different promoters to easily detect expression efficiency.
shERWOOD shRNA are expressed with the optimized ultramiR scaffold. The figure below shows the shRNA secondary structure and highlights the sequences that are included in the mature RNAi trigger bound to the targeted mRNA.
|Figure 1. Schematic of shERWOOD-UltramiR shRNA. (A) Passenger (green) and Guide (orange) strand are shown with Dicer and Drosha nuclease cleavage sites are in red. (B) The final step of shRNA processing loads the Guide Strand (orange) into the RISC complex which binds the target mRNA (blue) in a sequence specific manner.|
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- Auyeung et al., 2014. Beyond Secondary Structure: Primary-Sequence Determinants License Pri-miRNA Hairpins for Processing. Cell 152(4):844–858
- Fellmann et al., 2011. Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Mol Cell. 18; 41(6):733-46.
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- Beer et al., 2010. Low-level shRNA Cytotoxicity can contribute to MYC-induced hepatocellular carcinoma in adult mice. Mol Ther 18(1):161-170.
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