shRNA Gene Knockdown Solutions
VectorBuilder offers comprehensive shRNA solutions for your RNAi experiments. You can effortlessly design U6- and miR30-based shRNA vectors with virus packaging, library construction and screening, and stable cell line engineering. Our shRNA services provide a highly efficient one-stop-solution to accelerate your loss-of-function studies.
Highlights
Customizable: Intuitive online design studio with seamless integration of our whole-genome shRNA database.
Comprehensive: Rich collection of vector backbones, components, and downstream options for efficient knockdown.
Streamlined: Full service from experimental design through stable cell line engineering and library screening.
Expert-backed: 100% sequence validated, fast turnaround times, competitive pricing, and powerful technical support.
What We Offer
shRNA Vectors
shRNA Virus
shRNA 3+1 Package
LNP Encapsulation
shRNA Research Discovery Services
Empowered by our free, user-friendly online design platform, you can easily design and order custom and premade shRNA vectors for your gene knockdown experiments.
VectorBuilder offers premium-quality virus packaging services for achieving highly efficient shRNA knockdown in difficult-to-transfect cells. Our proprietary technologies in virus packaging ensure improved titer, purity, viability, and consistency.
Scales ranging from 106 to 109 TU for research-grade packaging; 3rd generation lentivirus with various pseudotyping options available.
Scales ranging from 1010 to 1014 GC for research-grade packaging; more than 18 widely used serotypes available.
Scales ranging from 1010 to 1012 IFU; human Ad5, chimeric Ad5/F35, and gutless adenovirus available.
VectorBuilder offers cloning and packaging of three custom shRNA viruses targeting your GOI and one scramble control virus.
Empowered by our lipid nanoparticle (LNP) encapsulation service, you can easily package your siRNA into homogeneous, high-encapsulation-efficiency LNPs.
VectorBuilder offers custom and premade shRNA libraries and stable cell lines for high-efficiency functional genomics.
We offer design, cloning, and packaging of libraries as well as in vitro and in vivo functional screening and deconvolution for pooled libraries.
Pooled shRNA libraries for human and mouse with whole genome and elite gene scales, with each gene targeted by 5-6 different shRNAs.
Knockdown stable cell lines for applications requiring long-term repression of your GOI with comprehensive QC.
Streamline your research with VectorBuilder’s custom shRNA knockdown solutions.
Contact UsDesign Tips
| Category | Recommendation |
|---|---|
| Number of shRNAs | Test at least three different shRNAs targeting the same GOI to ensure a strong and reliable knockdown. |
| Promoter choice |
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| Target region |
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| General recommendation | Despite the flexibility with miR-based vectors, U6 vectors consistently provide stronger knockdown; U6 is recommended unless specific miR-based features are needed. |
Technical Information
shRNA-mediated gene knockdown
Controlling shRNA expression
shRNA databases
RNA interference (RNAi) is a widely used method of gene modulation where short RNA sequences (~21-23 nucleotides) complementary to the RNA of a gene of interest are introduced into target cells. The introduced RNAs bind the complementary endogenous mRNA, and the resulting double-stranded RNA is degraded by the cell, therefore blocking translation and reducing gene expression. This approach does not completely knock out the gene, as some unbound mRNAs will continue to produce functional protein.
Short hairpin RNA (shRNA), and short interfering RNA (siRNA) are the two commonly used modalities for RNAi. shRNAs are DNA-encoded hairpin structures with higher stability providing long-term knockdown, while siRNAs are short, synthetic RNAs delivered directly to the cells for transient knockdown. Offering several advantages over the conventional siRNA approach, shRNA-mediated gene knockdown is the preferred method for most RNAi applications.
shRNAs can be introduced into target cells using DNA vectors, in both viral and non-viral formats. When cells are transfected or transduced with an shRNA vector, the shRNA is transcribed in the nucleus to form a hairpin structure consisting of a sense strand with the same sequence as the mRNA to be silenced (pink strand in Figure 1), followed by a single-stranded loop and an antisense strand complementary to the sense strand (teal strand in Figure 1). The transcribed shRNA exits the nucleus, is processed by Dicer in the cytoplasm, and is then loaded onto the RNA-induced silencing complex (RISC) for subsequent target mRNA recognition and degradation (Figure 1).
Figure 1. Production and function of forms of RNAi.
There are two widely used approaches to control shRNA expression on vectors: U6-based shRNA expression and miR-based shRNA expression. While U6-based shRNA vectors drive the expression of simple stem-loop shRNAs transcribed by an RNA Polymerase III promoter such as U6, miR-based shRNA vectors are used for expressing shRNAs adapted with a microRNA scaffold under an RNA Polymerase II promoter. shRNAs expressed by both U6- and miR-based vector systems are processed by similar mechanisms within the cytoplasm, ultimately leading to targeted gene silencing. However, after being transcribed within the nucleus, a miR-based shRNA, unlike a U6-based shRNA, is processed in a mechanism similar to that of a primary miRNA due to the presence of endogenous miR-based sequences within its structure (Figure 2).
Figure 2. Mechanisms of U6- and miR-based shRNA mediated gene expression knockdown.
In miR-based shRNA vectors, RNA polymerase II promoters enable the use of tissue-specific, inducible, or variable-strength promoters, allowing for a variety of experimental applications not possible with the constitutive and ubiquitous U6 promoter. Additionally, the ability of Pol II promoters to efficiently produce long transcripts in the miRNA-based shRNA systems provides several advantages over other knockdown vector systems: multiple shRNAmiRs can be transcribed as a single polycistron, which is processed to form multiple mature shRNAs within the cell. This allows knockdown of multiple genes or targeting of different regions within the same gene using a single vector. Additionally, in this vector system, a user-selected protein coding gene can be 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, as illustrated in Figure 4, or can be used for other purposes requiring co-expression of an ORF and shRNA(s). An experimental comparison of the U6- and miR30-based shRNA systems is illustrated in Figure 3: knockdown efficiency before and after drug selection is generally higher with U6-based systems compared to use of one or more shRNAs using the miR30 system.
The table below summarizes the U6- and miR-based shRNA vector systems:
| Category | U6-based shRNA vector | miR-based shRNA vector |
|---|---|---|
| shRNA structure | Simple stem-loop shRNA | shRNA adapted with a microRNA scaffold |
| shRNA length | 50-70 nt | >250 nt |
| Promoter | RNA Pol III promoters such as U6 and H1 | RNA Pol II promoters including ubiquitous, tissue-specific, and inducible promoters |
| shRNA processing mechanism | Processed only by Dicer in the cytoplasm | Processed by Drosha in the nucleus and Dicer in the cytoplasm |
| Number of shRNAs that can be expressed on one vector | Single shRNA (usually) | Single or multiple shRNAs |
| Ability to express other ORFs in the shRNA transcript | No | Yes |
| Gene knockdown efficiency | Often more robust | Often less robust |
| Toxicity | High cellular toxicity | Decreased cellular toxicity |
VectorBuilder’s design studio allows you to create vectors encoding efficient shRNAs utilizing the algorithm found in our online shRNA design tool, which features optimized shRNA databases for common species, enabling you to design shRNAs with high knockdown efficiency for your target genes. For shRNA design, we apply rules similar to those used by the RNAi consortium: for each given RefSeq transcript, we search for all possible 21mers that are considered as candidate target sites. Candidates are excluded if they contain features thought to reduce knockdown efficiency/specificity or clonability, including a run of ≥4 of the same base, a run of ≥7 G or C, GC content <25% or >60%, and AA at the 5’ end. All scores are ≥0, with a mean of ~5, a standard deviation of ~5, and 95% of scores ≤15. An shRNA with a knockdown score of about 15 is predicted to have the best knockdown performance and clonability, while an shRNA with a knockdown score of 0 is predicted to have the worst knockdown performance or is hard to clone.
VectorBuilder’s shRNA design tool provides you with the option to search for target genes in our database. Upon entering your gene name, you will see detailed information on all available shRNAs targeting your gene in our database, including a link to the UCSC Genome Browser to view the shRNA sequences and all transcript isoforms. Our database also ranks all available shRNAs for a target gene in order of their decreasing knockdown scores and recommends the top 3 shRNAs with the highest scores. Please note that knockdown scores are only a theoretical guide. Actual knockdown efficiency could significantly vary from what the scores predict, so target sites with low scores may still work well.
References
Mol Cell. 9:1327 (2002); Characterization of miR30-based gene knockdown.
Nucleic Acids Res. 34:e53 (2006); Development of miR155-based shRNA vectors.
J Gene Med. 9:620 (2007); Development of IPTG-inducible gene knockdown system.
Proc Natl Acad Sci USA. 101:10380 (2004); Development of Cre-lox-regulated gene knockdown system.
Case Studies
U6- and miR30-based shRNA systems
Inducible shRNA systems
Figure 3. Highly efficient EGFP knockdown through U6- and 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) via U6-based shRNA, by 13% (P<0.001) via CMV-driven miR30-based single shRNA, and by 44% (P<0.001) via CMV-driven miR30-based quad shRNA. (C) After drug selection, EGFP expression was reduced by ~72% (P<0.001) via U6-based shRNA, by 60% (P<0.001) via CMV-driven miR30-based single shRNA, and by 67% (P<0.001) via 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 using Tukey’s test.
Figure 4. EGFP knockdown and mCherry expression through miR30-based shRNA lentiviral system. (A) Lentiviral vectors carrying a CMV-driven expression cassette containing mCherry and miR30-based shRNA (scramble control or anti-EGFP) were packaged into lentiviral particles. HEK293T cells stably expressing EGFP were transduced with the miR30-based shRNA lentivirus. After drug selection with the appropriate antibiotics, EGFP and mCherry expression was measured by fluorescence microscopy and flow cytometry. (B) Compared to non-transduced cells and cells transduced with mCherry-shRNA[Scramble] lentivirus, cells transduced with mCherry-shRNA[EGFP] lentivirus showed significant reduction of EGFP expression (P<0.001) in terms of median fluorescence intensities measured by flow cytometry. In addition, while non-transduced cells had undetectable red fluorescence, cells transduced with the miR30-based shRNA lentivirus (both scramble and anti-EGFP) showed strong mCherry expression.
Inducible shRNA systems provide tightly regulated, temporally-controlled knockdown of target genes in a wide variety of mammalian cells. These shRNAs can be controlled by either tetracycline-based mechanisms or IPTG-based mechanisms.
- Tet-inducible shRNA knockdown
- IPTG-inducible shRNA knockdown
This system utilizes the interaction between TetR and TetO proteins to regulate shRNA expression in the presence or absence of tetracycline or one of its analogs.
Figure 5. EGFP knockdown with the lentivirus tet-inducible shRNA vector system. (A) Lentiviral vectors carrying tet-inducible U6-based scramble or EGFP-targeting shRNA expression cassettes were packaged into the corresponding lentiviral particles and transduced into HEK293T cells stably expressing EGFP. Antibiotic selection with appropriate antibiotics, in this case puromycin, was performed to isolate positively transduced cells followed by treatment with 1 µg/ml doxycycline (dox) to induce shRNA expression. Relative fluorescence intensity (RFI) of EGFP was quantified for all experimental groups using flow cytometry. (B) Cells expressing an inducible EGFP shRNA cassette showed a ~60% reduction in EGFP RFI upon doxycycline induction, while inducible shRNA vectors expressing a non-targeting scramble shRNA had no significant effect on EGFP RFI upon doxycycline induction. (C) Representative fluorescence microscopy images were taken for each group. Exposure: brightfield=10 ms; EGFP=100 ms. Magnification: 100x.
This system controls shRNA expression through the bacterial LacI–LacO interaction, turning expression on with IPTG and off when IPTG is absent.
Figure 6. EGFP knockdown with the lentivirus IPTG-inducible shRNA vector system. (A) Lentiviral vectors carrying IPTG-inducible U6-based scramble or EGFP-targeting shRNA expression cassettes were packaged into the corresponding lentiviral particles and transduced into HEK293T cells stably expressing EGFP. Antibiotic selection with appropriate antibiotics, puromycin (Puro) or blasticidin (Bsd), was performed to isolate positively transduced cells followed by treatment with 1mM IPTG to induce shRNA expression. Median fluorescence intensity (MFI) of EGFP was quantified for all experimental groups using flow cytometry (FCM); (B) Cells expressing an inducible EGFP shRNA cassette showed a ~42% reduction in EGFP MFI upon IPTG induction. This observation was consistent across inducible shRNA vectors carrying either a puromycin or blasticidin resistance gene. Inducible shRNA vectors expressing a non-targeting scramble shRNA had no effect on EGFP MFI upon IPTG induction. Moreover, in cells transduced with an inducible shRNA vector lacking the LacI repressor, the induction function of the vector was lost and EGFP expression was constitutively inhibited by the EGFP shRNA both with and without IPTG induction.
Resources
FAQ
A detailed comparison of the two methods is summarized in the table below:
| shRNA-mediated knockdown | siRNA-mediated knockdown | |
|---|---|---|
| Delivery method | Transfection or transduction depending on vector type | Transfection |
| Knockdown duration | Long-term | Transient |
| Episomal or stable integration | Can be either episomal or stable depending on delivery method | Episomal |
| Ability to add selection markers | Yes, fluorescent or drug-selection markers can be added | No |
| Cell-type range | Suitable for a wide range of cell types | Suitable for only cells with high transfection efficiency |
| Off-target effects | Reduced off-target effects | High off-target effects |
| Degradation rate | Low | High |
Either shRNA-mediated knockdown or nuclease-mediated knockout (e.g. CRISPR or TALEN) can be a valuable experimental approach to study the loss-of-function effects of a gene of interest in cell culture. In order to decide which method is optimal for your specific application, there are a few things you should consider.
Mechanisms
- Knockdown vectors: knockdown vectors express short hairpin RNAs (shRNAs) that repress the function of target mRNAs within the cell by inducing their cleavage and repressing their translation. Therefore, shRNA knockdown vectors are not associated with any DNA level sequence change of the gene of interest.
- Knockout vectors: CRISPR and TALEN both function by directing nucleases to cut specific target sites in the genome. These cuts are then inefficiently repaired by the cellular machinery, resulting in permanent mutations, such as small insertions or deletions, at the sites of repair. A subset of these mutations will result in loss of function of the gene of interest due to frame-shifts, premature stop codons, etc. If two closely positioned cut sites in the genome (i.e. within several kb) are targeted simultaneously, this can also result in the deletion of the intervening region.
Effectiveness
shRNA-mediated knockdown will never completely repress the expression of the target gene. Even for the most effective shRNAs, some residual expression of the target gene will remain. In contrast, in a fraction of treated cells, CRISPR and TALEN can generate permanent mutations which may result in complete loss of gene function.
Consistency and uniformity
shRNA vectors generally provide high cell-to-cell uniformity within the pool of treated cells and very consistent results between experiments. In contrast, CRISPR and TALEN produce results that are highly non-uniform from cell to cell due to the stochastic nature of the mutations introduced. To fully knock out the gene of interest in a cell, all copies of the gene in the cell must be knocked out. Given that normal cells have two copies of any gene (except for X- or Y-linked genes) while cancer cells can have more than two copies, such full knockout cells may represent a very small fraction of all the treated cells. For this reason, nuclease-mediated knockout experiments require the screening of clones by sequencing to identify the subset in which all copies of the gene of interest have been knocked out.
Off-target effects
Off-target effects have been reported for both shRNA-mediated knockdown and nuclease-mediated knockout. The off-target phenotype(s) can be estimated by using multiple different shRNAs to target the same gene. If a gene knocked down by multiple different shRNAs results in consistent phenotype(s), then it argues against the phenotype(s) being caused by off-target effects. For CRISPR- or TALEN-mediated knockout, multiple clones containing loss-of-function mutations should be analyzed in order to account for any phenotype(s) that may be due to off-target mutations. Additionally, bioinformatically identified off-target sites could be sequenced in the clones to see if they have been mutated.
Design your homologous recombination donor vector onlineNot all shRNAs will work
Based on our experience and feedback from our customers, we know that generally when 3 or 4 shRNAs are tested for any arbitrary gene, typically 2 or 3 produce reasonable to good knockdown. However, when using shRNAs, it is important to recognize the fact that not all shRNAs will work. Typically, ~50-70% of shRNAs have noticeable knockdown effect, and ~20-30% of them have strong knockdown. If you try a few shRNAs targeting a specific gene, it is possible that by chance, none will produce satisfactory knockdown. When this happens, the best approach is to try more shRNAs, especially the ones that have literature validation. Many researchers also use a “cocktail” of shRNAs (i.e. mixture of different shRNAs) targeting the same gene, which sometimes can improve knockdown efficiency.
The assay for validating the knockdown of your gene is not performed properly
The most common and sensitive assay to evaluate shRNA knockdown efficiency is RT-qPCR. Sometimes, you may need to try several pairs of primers, and then choose the most specific and efficient pair to use. In general, the RT-qPCR primers should span exon-exon junction if possible to avoid amplifying genomic DNA. When using a new pair of primers, we recommend that you run the PCR product on an agarose gel to verify the band, or even validate the PCR product by sequencing. You should always include minus-RT control in RT-qPCR to better estimate the level of genomic DNA contamination. You can our Primer Design Tool to help you better examine the quality of your primers in silico.
Knockdown efficiency can also be assessed by Western blot. However, Western blot is notoriously prone to false positive bands from non-specific antibody binding, which could mistakenly lead to the interpretation that there is no knockdown. Care must therefore be taken to make sure that the antibody used is indeed specific to the gene of interest.
The shRNA might only target a subset of transcript isoforms of your gene
When designing shRNA, we generally recommend those that can target as many transcript isoforms of the gene as possible, unless you are only interested in knocking down a particular isoform. VectorBuilder has created shRNA databases that contain optimized shRNAs for common species. If you design shRNA vectors on VectorBuilder, when you insert the shRNA component into the vector, you will have the option to search the target gene in our database. Then, you will see the detailed information of all the available shRNAs we designed for you, including a link to UCSC Genome Browser to view these shRNAs in the context of genomic sequence and all the transcript isoforms.
Featured Citations
Mechanical stiffness promotes skin fibrosis via Piezo1-Wnt2/Wnt11-CCL24 positive feedback loop
He, et al.
Cell Death & Disease, 2024, doi: 10.1038/s41419-024-06466-3
Products used:
Abstract: Skin fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) caused by fibrotic disorders of the skin. In recent years, ECM stiffness has emerged as a prominent mechanical cue that precedes skin fibrosis and drives its progression by promoting fibroblasts activation. However, how stiffness influences fibroblasts activation for skin fibrosis progression remains unknown. Here, we report a positive feedback loop mediated by the mechanosensitive ion channel Piezo1 and aberrant tissue mechanics in driving skin fibrosis. Piezo1 is upregulated in fibrotic skin in both humans and mice. Piezo1 knockdown dermal fibroblasts lose their fibroproliferative phenotypes despite being grown on a stiffer substrate. We show that Piezo1 acts through the Wnt2/Wnt11 pathway to mechanically induce secretion of C-C motif chemokine ligand 24 (CCL24, also known as eotaxin-2), a potent cytokine associated with fibrotic disorders. Importantly, adeno-associated virus (AAV)-mediated Piezo1 knockdown ameliorated the progression of skin fibrosis and skin stiffness in mice. Overall, increased matrix stiffness promotes skin fibrosis through the inflammatory Piezo1-Wnt2/Wnt11-CCL24 pathway. In turn, a stiffer skin microenvironment increases Piezo1 expression to exacerbate skin fibrosis aggression. Therefore, targeting Piezo1 represents a strategy to break the positive feedback loop between fibroblasts mechanotransduction and aberrant tissue mechanics in skin fibrosis.
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A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma
Pozniak, et al.
Cell, 2024, doi: 10.1016/j.cell.2023.11.037
Products used:
Abstract: To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.
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SGLT2 inhibition eliminates senescent cells and alleviates pathological aging
Katsuumi, et al.
Nature Aging, 2024, doi: 10.1038/s43587-024-00642-y
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Abstract: It has been reported that accumulation of senescent cells in various tissues contributes to pathological aging and that elimination of senescent cells (senolysis) improves age-associated pathologies. Here, we demonstrate that inhibition of sodium–glucose co-transporter 2 (SGLT2) enhances clearance of senescent cells, thereby ameliorating age-associated phenotypic changes. In a mouse model of dietary obesity, short-term treatment with the SGLT2 inhibitor canagliflozin reduced the senescence load in visceral adipose tissue and improved adipose tissue inflammation and metabolic dysfunction, but normalization of plasma glucose by insulin treatment had no effect on senescent cells. Canagliflozin extended the lifespan of mice with premature aging even when treatment was started in middle age. Metabolomic analyses revealed that short-term treatment with canagliflozin upregulated 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, enhancing immune-mediated clearance of senescent cells by downregulating expression of programmed cell death-ligand 1. These findings suggest that inhibition of SGLT2 has an indirect senolytic effect by enhancing endogenous immunosurveillance of senescent cells.
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