Short hairpin RNA(shRNA)
SMARTvector shRNA, GIPZ shRNA, TRIPZ shRNA, TRC shRNA
|제품용도||유전자 발현억제 (knockdown)|
Lentiviral vector-based reagents for RNA interference
Dharmacon lentiviral shRNA products encompass the industry’s broadest, most technologically advanced portfolio of vector-based RNAi reagents for transient, long-term, inducible, and in vivo RNA interference in human, mouse, and rat cells.
Designed using microRNA scaffold-specific attributes for highly efficient processing via the endogenous RNAi pathway while minimizing off-target potential
• SMARTvector Lentiviral shRNA
Advanced rationally-designed microRNA-based shRNA for human, mouse, and rat genes. Choose from seven constitutive promoters and three reporter options to tailor SMARTvector shRNA for specific cells.
• SMARTvector Inducible Lentiviral shRNA
All the benefits of SMARTvector lentiviral shRNAs combined with latest generation of powerful Tet-inducible technology for unprecedented control over gene silencing and reduced off-targeting.
• GIPZ Lentiviral shRNA
Efficient gene silencing with a microRNA-adapted shRNA design. Available as lentiviral vector constructs or high-titer lentiviral particles for human and mouse.
• TRIPZ Inducible Lentiviral shRNA
Inducible shRNA expression allows for tightly regulatable RNAi experiments. Available as lentiviral vector constructs for human.
• SMARTvector Lentiviral shRNA Pooled Libraries
High-titer pooled screening libraries of constitutive shRNAs for pre-defined gene libraries in human, mouse, and rat.
• SMARTvector Inducible Lentiviral shRNA Pooled Libraries
High-titer pooled screening libraries of inducible shRNAs for pre-defined gene libraries in human, mouse, and rat.
• Decode Pooled Lentiviral shRNA Libraries
High-titer pooled screening libraries of constitutive shRNAs for pre-defined gene libraries in human.
• Custom Pooled Lentiviral Screening Libraries
Select from a range of our algorithm-optimized reagents to develop the ideal pooled lentiviral screening resource for CRISPR-Cas9 knockout or RNAi knockdown screens.
Simple hairpin shRNAs
Offers a classic hairpin, rules-based shRNA design utilizing a simple hairpin of 21 base pair sense and antisense stem, and a 6 basepair loop
• TRC Lentiviral shRNA
Lentiviral shRNA collection from The RNAi Consortium (TRC) with coverage in human and mouse.
|Species||Human, Mouse, Rat||Human, Mouse||Human||Human, Mouse|
|Promoter||Choice of 7 constitutive*
and 4 inducible promoters
|Human CMV Pol II||TRE-min-CMV||U6 Pol III|
|Fluorescent reporter gene||GFP/RFP||GFP||RFP|
|In vivo RNAi||●||●||●||●|
|Create stable cell lines||●||●||●||●|
|Recommended for primary and non-dividing cells||●||●||●||●|
|Whole genome library
|Human, Mouse, Rat||Human, Mouse||Human||Human|
Introduction to shRNA
Short hairpin RNA (shRNA) sequences are encoded in a DNA vector that can be introduced into cells via plasmid transfection or viral transduction. Because the shRNA expression cassettes can be incorporated into viral vector systems, including lentivirus, they can integrate into the host genome for the creation of stable cell lines. Additionally, when used in combination with one of several viral delivery systems, they can be delivered into difficult-to-transfect primary cells and used for in vivo applications. Based on the delivery method and vector design, vector-based shRNAs can allow for long-term (or inducible) down-regulation of target genes.
The performance of shRNA is influenced by many factors including the efficiency of transduction or transfection, the promoter driving expression of the shRNA and epigenetic modifications (which can lead to silencing of shRNA expression). Further, the influence that each of these factors have on vector performance can differ depending on the cell line or cell type. When planning an experiment using shRNA the available vector options, including; the shRNA design to be used, the vector features (e.g.,promoter), and the method of delivery should all be taken into consideration based on the requirements of the experiment.
shRNA design is typically divided into two formats, the simple stem-loop shRNA and the microRNA-adapted shRNA.
Simple stem-loop shRNA
Basic shRNAs are modeled on precursor microRNA (pre-miRNA), and are cloned into viral vectors where they are transcribed under the control of RNA Polymerase III (Pol III) promoters. shRNAs are produced as single-strand molecules of 50–70 nucleotides in length, and form stem–loop structures consisting of a 19-29 base-pair region of double-strand RNA (the stem) bridged by a region of single-strand RNA (the loop) and a short 3’ overhang. Once transcribed, shRNAs exit the nucleus, are cleaved at the loop by the nuclease Dicer in the cytoplasm, and enter the RISC to direct cleavage and subsequent degradation of complementary mRNA.
microRNA adapted shRNA
A microRNA-adapted shRNA consists of a shRNA stem structure with microRNA-like mismatches surrounded by the loop and flanking sequence of an endogenous microRNA. microRNA-adapted shRNAs are transcribed from RNA Polymerase ll (Pol ll) promoters, cleaved by the endogenous RNase III Drosha enzyme in the nucleus, and then exported to the cytoplasm where they are processed by Dicer and loaded into the RISC complex. Studies have suggested that the use of a microRNA scaffold, which is processed by both Drosha and Dicer, may promote more efficient processing and reduce toxicity for in vivo RNAi.
RNA interference and manipulation
Figure 1. shRNA approaches include the introduction of genetically engineered viral vectors or plasmid-based vectors expressing silencing sequences embedded in an endogenous microRNA scaffold (1) or simple stem-loop shRNA (2). Expressed sequences (1 and 2, shown in blue) enter the endogenous pathway at an early stage and are efficiently processed into potent silencing molecules using the endogenous microRNA mechanism. All of these approaches lead to target mRNA cleavage (shown in purple) and gene silencing.