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1.  Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy 
Molecular Therapy  2012;21(1):217-227.
Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.
doi:10.1038/mt.2012.160
PMCID: PMC3538299  PMID: 23089734
2.  Therapeutic expression of hairpins targeting apolipoprotein B100 induces phenotypic and transcriptome changes in murine liver 
Gene Therapy  2013;21(1):60-70.
Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therapy.
doi:10.1038/gt.2013.58
PMCID: PMC3881031  PMID: 24152580
AAV; liver; shRNA; miRNA; ApoB; familial hypercholesterolemia
3.  Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules ✯ 
RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.
doi:10.1016/j.ijpara.2011.02.004
PMCID: PMC3094803  PMID: 21447344
Schistosome; Luciferase; RNA interference; U6 gene; Vector-based RNAi; Promoter
4.  Lentiviral miR30-based RNA Interference against Heparanase Suppresses Melanoma Metastasis with Lower Liver and Lung Toxicity 
Aim: To construct short hairpin RNAs (shRNAs) and miR30-based shRNAs against heparanase (HPSE) to compare their safety and their effects on HPSE down-modulation in vitro and in vivo to develop a more ideal therapeutic RNA interference (RNAi) vector targeting HPSE.
Methods: First, we constructed shRNAs and miR30-based shRNAs against HPSE (HPSE-shRNAs and HPSE-miRNAs) and packed them into lentiviral vectors. Next, we observed the effects of the shRNAs on knockdown for HPSE expression, adhesion, migration and invasion abilities in human malignant melanoma A375 cells in vitro. Furthermore, we compared the effects of the shRNAs on melanoma growth, metastasis and safety in xenograft models.
Results: Our data showed that these artificial miRNAs targeting HPSE could be effective RNAi agents mediated by Pol II promoters in vitro and in vivo, although these miRNAs were not more potent than the HPSE-shRNAs. It was noted that obvious lung injuries, rarely revealed previously, as well as hepatotoxicity could be caused by lentivirus-mediated shRNAs (LV shRNAs) rather than lentivirus-mediated miRNAs (LV miRNAs) in vivo. Furthermore, enhanced expression of pro-inflammatory cytokines IL-6 and TGF-β1 and endogenous mmu-miR-21a-5p were detected in lung tissues of shRNAs groups, whereas the expression of mmu-let-7a-5p, mmu-let-7b-5p and mmu-let-7c-5p were down-regulated.
Conclusion: These findings suggest that artificial miRNAs display an improved safety profile of lowered lung injury or hepatotoxicity relative to shRNAs in vivo. The mechanism of lung injuries caused by shRNAs may be correlated with changes of endogenous miRNAs in the lung. Our data here increase the flexibility of a miRNA-based RNAi system for functional genomic and gene therapy applications.
doi:10.7150/ijbs.5425
PMCID: PMC3708037  PMID: 23847439
RNA interference; microRNA(miRNA); heparanase; metastasis; safety
5.  Production of Cloned Pigs with Targeted Attenuation of Gene Expression 
PLoS ONE  2013;8(5):e64613.
The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45–82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.
doi:10.1371/journal.pone.0064613
PMCID: PMC3667777  PMID: 23737990
6.  A direct comparison of strategies for combinatorial RNA interference 
BMC Molecular Biology  2010;11:77.
Background
Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.
Results
Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.
Conclusions
By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.
doi:10.1186/1471-2199-11-77
PMCID: PMC2958852  PMID: 20937117
7.  A miR-21 hairpin structure-based gene knockdown vector 
RNA interference (RNAi) is widely used to study gene functions as a reverse genetic means from first-generation siRNA to second-generation short hairpin RNA (shRNA) or the newly developed microRNA (shRNA-miR). Here we report a gene knockdown vector system based on the mouse miR-21 hairpin structure. In this system, the pre-miRNA hairpin of the miR-21 gene was modified by replacing the 22-nucleotide mature sequence with shRNA sequences that target genes of interest, flanked by 160-bp upstream and 65-bp downstream sequences of the mouse pre-miR-21. We tested this system by knocking down the enhanced green fluorescence protein (EGFP) reporter gene using different vectors, in which shRNA-miR was driven by the polymerase II (pol II) promoter. We found that miR-21 hairpin-based the shRNA-miR can be directly placed under pol II promoter, like UbC or CMV promoter to knockdown the gene of interest. To facilitate the wide application of the miR-21hairpin-based gene knockdown system, we further knocked down the endogenous gene lamin (A/C), which showed that endogenous lamin A/C expression can be efficiently silenced using the miR-21 hairpin -based lentiviral vector. The miR-21hairpin-based gene knockdown vector will provide a new genetic tool for gene functional studies in vitro and in vivo.
doi:10.1016/j.bbrc.2010.03.047
PMCID: PMC2854175  PMID: 20226761
miRNA; miR-21; gene knockdown; lentiviral vector
8.  Criteria for effective design, construction, and gene knockdown by shRNA vectors 
BMC Biotechnology  2006;6:7.
Background
RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.
Results
To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings.
Conclusion
The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.
doi:10.1186/1472-6750-6-7
PMCID: PMC1409772  PMID: 16433925
9.  Increase of the therapeutic effect on non-small-cell lung cancer cells with combination treatment of shRNA against Cyclin D1 and Bcl-xL in vitro 
Overexpression of Cyclin D1 and Bcl-xL proteins has often been found in non-small-cell lung cancer (NSCLC). These two genes may play a significant role in tumorigenesis. However, the combined inhibition of the two genes in vitro is unclear in NSCLC. In this study, the effect of a combined intervention on Cyclin D1 and Bcl-xL in NSCLC is assessed and discussed. Three recombinant plasmids that expressed a cytomegalovirus (CMV) promoter-driven micro30 short hairpin RNA (shRNA) targeting the Cyclin D1 gene (Cyclin D1 shRNA), the Bcl-xL gene (Bcl-xL shRNA) and a combination of the two genes (Cyclin D1-Bcl-xL shRNA), based on the plasmid pcDNA6.2-GW/EmGFP-miR, were constructed. The cell lines A549 and NCI-H441 were divided into four groups; blank control (untreated cells), Cyclin D1 shRNA, Bcl-xL shRNA and Cyclin D1-Bcl-xL shRNA (transfected cells), respectively. The expression of mRNA and protein of Cyclin D1 or Bcl-xL was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The apoptosis and proliferation of the two cell lines were evaluated by dimethylthiazol-diphenyltetrazolium bromide (MTT), cell count and flow cytometry. The recombinant plasmid sufficiently mediated the RNA interference (RNAi) effects in A549 and NCI-H441 cells. The expression levels of mRNA and protein of Cyclin D1 or Bcl-xL in the three intervention groups were significantly reduced compared to the untreated cells (P<0.05). No statistical differences were found among the combined shRNAs and single shRNA regarding Cyclin D1 or Bcl-xL, respectively (P>0.05). In the assessment of proliferation and apoptosis, it was found that in all three intervention groups there was significant inhibition of cell proliferation and promotion of cell apoptosis compared with the untreated cells (P<0.05). Furthermore, the combined interference of the two genes was more effective than either single interference (P<0.05). Our results suggested that the combined targeting of Cyclin D1 and Bcl-xL genes has potential for NSCLC investigation, providing increased efficacy over Cyclin D1 or Bcl-xL inhibition alone.
doi:10.3892/etm.2011.381
PMCID: PMC3438793  PMID: 22969878
non-small-cell lung cancer; RNA interference; Bcl-xL; Cyclin D1; apoptosis
10.  Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways* #  
RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.
doi:10.1631/jzus.B1400001
PMCID: PMC4076603  PMID: 24793764
MicroRNA pathway; shRNA-transgenic pigs; Classical swine fever virus (CSFV); Blastocyst formation; Early lethality
11.  Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs 
BMC Biotechnology  2005;5:13.
Background
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.
Results
To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.
Conclusion
We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.
doi:10.1186/1472-6750-5-13
PMCID: PMC1142307  PMID: 15885150
12.  SUPPRESSION OF KERATOEPITHELIN AND MYOCILIN BY SMALL INTERFERING RNA (AN AMERICAN OPTHALMOLOGICAL SOCIETY THESIS) 
Purpose
Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. In this study, the feasibility of using small interfering RNAs (siRNAs) to suppress the expression of keratoepithelin and myocilin and their capabilities to reduce the related cytotoxic effects caused by mutant myocilins were investigated.
Methods
cDNAs of human KE gene and myocilin gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP by KE-specific shRNAs was evaluated by fluorescence microscopy and Western blotting. Suppression of MYOC-EGFP by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of Western blots. A BiP promoter-driven luciferase reporter assay was used to evaluate the stress response of TM cells induced by misfolded mutant myocilins.
Results
Two KE-specific shRNAs that effectively suppressed the expression of KE-EGFP in HEK293 cells were identified. One shRNA (targeting the coding sequence starting at 1528bp of KE) reduced the expression of KE-EGFP approximately by 50%, whereas the other shRNA (targeting the 3′-UTR region of KE) suppressed greater than 80% of the expression. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of myocilin (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP, ranging from 78% to 90% on average. The activation of BiP gene (as a stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs.
Conclusions
KE- or myocilin-specific shRNAs could effectively suppress the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins. RNA interference may have future therapeutic implications in suppressing these genes.
PMCID: PMC2258103  PMID: 18427622
13.  Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice 
PLoS Genetics  2006;2(1):e10.
RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.
Synopsis
Reverse genetics studies gene functions by altering a gene and observing the consequences. A powerful method of reverse genetics in mammals is gene knockout by homologous recombination, which mutates a gene to prevent its functional expression. Using this method, investigators have revealed the functions of many genes. However, this method is relatively complex, time-consuming, and costly. In addition, this method is limited to studies in mice because it is not well established in other mammalian species. The authors of this study tested an alternative method using RNA interference (RNAi), which is a widely conserved mechanism in eukaryotes and can mediate gene-specific silencing. These investigators used RNA polymerase II (Pol II) to express a short hairpin RNA (shRNA) that triggers destruction of the mRNA-encoding Mn superoxide dismutase (SOD2) in transgenic mice. These mice exhibit phenotypes that were typical in Sod2 knockout mice, including elevated levels of oxidative stress in various tissues, fat deposition in liver and muscles, dilated cardiomyopathy, and premature death. These results open the door of RNAi to a wide array of well-established Pol II transgenic strategies and offer a technically simpler, cheaper, and quicker alternative to gene knockout for reverse genetics in mice and other mammalian species.
doi:10.1371/journal.pgen.0020010
PMCID: PMC1358942  PMID: 16450009
14.  An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi 
Nucleic Acids Research  2005;33(6):e62.
RNA interference (RNAi) mediates gene silencing in many eukaryotes and has been widely used to investigate gene functions. A common method to induce sustained RNAi is introducing plasmids that synthesize short hairpin RNAs (shRNAs) using Pol III promoters. While these promoters synthesize shRNAs and elicit RNAi efficiently, they lack cell specificity. Monitoring shRNA expression levels in individual cells by Pol III promoters is also difficult. An alternative way to deliver RNAi is to use Pol II-directed synthesis of shRNA. Previous efforts in developing a Pol II system have been sparse and the results were conflicting, and the usefulness of those Pol II vectors has been limited due to low efficacy. Here we demonstrate a new Pol II system that directs efficient shRNA synthesis and mediates strong RNAi at levels that are comparable with the commonly used Pol III systems. In addition, this system synthesizes a marker protein under control of the same promoter as the shRNA, thus providing an unequivocal indicator, not only to the cells that express the shRNA, but also to the levels of the shRNA expression. This system may be adapted for in vivo shRNA expression and gene silencing.
doi:10.1093/nar/gni061
PMCID: PMC1074311  PMID: 15805121
15.  Co-expression of Argonaute2 Enhances Short Hairpin RNA-induced RNA Interference in Xenopus CNS Neurons In Vivo 
RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA) into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi) and pReporter (pRe) constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30) enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.
doi:10.3389/neuro.17.001.2009
PMCID: PMC2858607  PMID: 20582287
shRNA; RNAi; knockdown; Pol III promoter; Ago2; AMPA receptor; Xenopus
16.  Organic small hairpin RNAs (OshR): a Do-It-Yourself platform for transgene-based gene silencing 
Methods (San Diego, Calif.)  2013;63(2):101-109.
The RNA interference (RNAi) pathway in animal cells can be harnessed to silence gene expression with artificial small interfering RNAs (siRNAs) or transgenes that express small hairpin RNAs (shRNAs). The transgene-expressing shRNA approach has been adapted into large-scale resources for genome-wide loss-of-function screens, whereas focused studies on a narrow set of genes can be achieved by using individual shRNA constructs from these resources. Although current shRNA repositories generally work, they might fail in certain situations and therefore necessitate other alternatives. We detail here a new highly-accessible and rational design of custom shRNAs that utilizes a refined backbone configuration termed the ‘Organic’ shRNA (OshR) platform. The OshR platform is ‘organic’ because it conforms more naturally to the endogenous vertebrate miRNAs by maintaining specific bulges and incorporating strategic mismatches to insure the desired guide strand is produced while reducing the accumulation of passenger strands that might contribute to off-target effects. We also demonstrate that the reliability of the OshR platform for gene silencing is increased when sequences target the 3' UnTranslated Region (3'UTR) of a gene. We further compare the OshR platform with the current and emerging shRNA designs, and propose that the OshR platform is a novel approach that can allow investigators to generate custom and effective shRNAs for individual gene functional studies.
doi:10.1016/j.ymeth.2013.05.007
PMCID: PMC3966114  PMID: 23707624
RNA interference; shRNA; gene silencing
17.  Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown 
Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery.
doi:10.1007/978-1-60761-657-3_10
PMCID: PMC3679364  PMID: 20387148
RNA interference (RNAi); small interfering RNA (siRNA); short hairpin RNA (shRNA); lentivirus; design; delivery; ASC; Porphyromonas gingivalis; IL-1β; ELISA; THP1
18.  mRNA turnover rate limits siRNA and microRNA efficacy 
Based on a simple model of the mRNA life cycle, we predict that mRNAs with high turnover rates in the cell are more difficult to perturb with RNAi.We test this hypothesis using a luciferase reporter system and obtain additional evidence from a variety of large-scale data sets, including microRNA overexpression experiments and RT–qPCR-based efficacy measurements for thousands of siRNAs.Our results suggest that mRNA half-lives will influence how mRNAs are differentially perturbed whenever small RNA levels change in the cell, not only after transfection but also during differentiation, pathogenesis and normal cell physiology.
What determines how strongly an mRNA responds to a microRNA or an siRNA? We know that properties of the sequence match between the small RNA and the mRNA are crucial. However, large-scale validations of siRNA efficacies have shown that certain transcripts remain recalcitrant to perturbation even after repeated redesign of the siRNA (Krueger et al, 2007). Weak response to RNAi may thus be an inherent property of the mRNA, but the underlying factors have proven difficult to uncover.
siRNAs induce degradation by sequence-specific cleavage of their target mRNAs (Elbashir et al, 2001). MicroRNAs, too, induce mRNA degradation, and ∼80% of their effect on protein levels can be explained by changes in transcript abundance (Hendrickson et al, 2009; Guo et al, 2010). Given that multiple factors act simultaneously to degrade individual mRNAs, we here consider whether variable responses to micro/siRNA regulation may, in part, be explained simply by the basic dynamics of mRNA turnover. If a transcript is already under strong destabilizing regulation, it is theoretically possible that the relative change in abundance after the addition of a novel degrading factor would be less pronounced compared with a stable transcript (Figure 1). mRNA turnover is achieved by a multitude of factors, and the influence of such factors on targetability can be explored. However, their combined action, including yet unknown factors, is summarized into a single property: the mRNA decay rate.
First, we explored the theoretical relationship between the pre-existing turnover rate of an mRNA, and its expected susceptibility to perturbation by a small RNA. We assumed a basic model of the mRNA life cycle, in which the rate of transcription is constant and the rate of degradation is described by first-order kinetics. Under this model, the relative change in steady-state expression level will become smaller as the pre-existing decay rate grows larger, independent of the transcription rate. This relationship persists also if we assume various degrees of synergy and antagonism between the pre-existing factors and the external factor, with increasing synergism leading to transcripts being more equally targetable, regardless of their pre-existing decay rate.
We next generated a series of four luciferase reporter constructs with destabilizing AU-rich elements (AREs) of various strengths incorporated into their 3′ UTRs. To evaluate how the different constructs would respond to perturbation, we performed co-transfections with an siRNA targeted at the coding region of the luciferase gene. This reduced the signal of the non-destabilized construct to 26% compared with a control siRNA. In contrast, the most destabilized construct showed 42% remaining reporter activity, and we could observe a dose–response relationship across the series.
The reporter experiment encouraged an investigation of this effect on real-world mRNAs. We analyzed a set of 2622 siRNAs, for which individual efficacies were determined using RT–qPCR 48 h post-transfection in HeLa cells (www.appliedbiosystems.com). Of these, 1778 could be associated with an experimentally determined decay rate (Figure 4A). Although the overall correlation between the two variables was modest (Spearman's rank correlation rs=0.22, P<1e−20), we found that siRNAs directed at high-turnover (t1/2<200 min) and medium-turnover (2001000 min) transcripts (P<8e−11 and 4e−9, respectively, two-tailed KS-test, Figure 4B). While 41.6% (498/1196) of the siRNAs directed at low-turnover transcripts reached 10% remaining expression or better, only 16.7% (31/186) of the siRNAs that targeted high-turnover mRNAs reached this high degree of silencing (Figure 4B). Reduced targetability (25.2%, 100/396) was also seen for transcripts with medium-turnover rate.
Our results based on siRNA data suggested that turnover rates could also influence microRNA targeting. By assembling genome-wide mRNA expression data from 20 published microRNA transfections in HeLa cells, we found that predicted target mRNAs with short and medium half-life were significantly less repressed after transfection than their long-lived counterparts (P<8e−5 and P<0.03, respectively, two-tailed KS-test). Specifically, 10.2% (293/2874) of long-lived targets versus 4.4% (41/942) of short-lived targets were strongly (z-score <−3) repressed. siRNAs are known to cause off-target effects that are mediated, in part, by microRNA-like seed complementarity (Jackson et al, 2006). We analyzed changes in transcript levels after transfection of seven different siRNAs, each with a unique seed region (Jackson et al, 2006). Putative ‘off-targets' were identified by mapping of non-conserved seed matches in 3′ UTRs. We found that low-turnover mRNAs (t1/2 >1000 min) were more affected by seed-mediated off-target silencing than high-turnover mRNAs (t1/2 <200 min), with twice as many long-lived seed-containing transcripts (3.8 versus 1.9%) being strongly (z-score <−3) repressed.
In summary, mRNA turnover rates have an important influence on the changes exerted by small RNAs on mRNA levels. It can be assumed that mRNA half-lives will influence how mRNAs are differentially perturbed whenever small RNA levels change in the cell, not only after transfection but also during differentiation, pathogenesis and normal cell physiology.
The microRNA pathway participates in basic cellular processes and its discovery has enabled the development of si/shRNAs as powerful investigational tools and potential therapeutics. Based on a simple kinetic model of the mRNA life cycle, we hypothesized that mRNAs with high turnover rates may be more resistant to RNAi-mediated silencing. The results of a simple reporter experiment strongly supported this hypothesis. We followed this with a genome-wide scale analysis of a rich corpus of experiments, including RT–qPCR validation data for thousands of siRNAs, siRNA/microRNA overexpression data and mRNA stability data. We find that short-lived transcripts are less affected by microRNA overexpression, suggesting that microRNA target prediction would be improved if mRNA turnover rates were considered. Similarly, short-lived transcripts are more difficult to silence using siRNAs, and our results may explain why certain transcripts are inherently recalcitrant to perturbation by small RNAs.
doi:10.1038/msb.2010.89
PMCID: PMC3010119  PMID: 21081925
microRNA; mRNA decay; RNAi; siRNA
19.  Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study 
BMC Biotechnology  2007;7:41.
Background
RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.
Results
RNAi-mediated knockdown of gene expression in target cells, controlled by a modified Tet-repressor (TetR) in the presence of doxycycline (Dox) was robust. Expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or an improved second-generation Tet-responsive promoter element (TRE) placed upstream of the promoter was tight and reversible as judged using quantitative protein measurements. We also established and tested a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE. In this system, positive regulation of shRNA production is mediated by novel Tet-dependent transactivators bearing transactivation domains derived from the human Sp1 transcription factor.
Conclusion
Our modified lentiviral vector system resulted in tight and reversible knockdown of target gene expression in unsorted cell populations. Tightly regulated target gene knockdown was observed with vectors containing either a single TO sequence or a second-generation TRE using carefully controlled transduction conditions. We expect these vectors to ultimately find applications for tight and reversible RNAi in mammalian cells in vivo.
doi:10.1186/1472-6750-7-41
PMCID: PMC1959519  PMID: 17634114
20.  Characterization of a potent non-cytotoxic shRNA directed to the HIV-1 co-receptor CCR5 
Background
The use of shRNAs to downregulate the expression of specific genes is now relatively routine in experimentation but still hypothetical for clinical application. A potential therapeutic approach for HIV-1 disease is shRNA mediated downregulation of the HIV-1 co-receptor, CCR5. It is increasingly recognized that siRNAs and shRNAs can have unintended consequences such as cytotoxicities in cells, particularly when used for long term therapeutic purposes. For the clinical use of shRNAs, it is crucial to identify a shRNA that can potently inhibit CCR5 expression without inducing unintended cytotoxicities.
Results
Previous shRNAs to CCR5 identified using conventional commercial algorithms showed cytotoxicity when expressed using the highly active U6 pol III promoter in primary human peripheral blood derived mononuclear cells. Expression using the lower activity H1 promoter significantly reduced toxicity, but all shRNAs also reduced RNAi activity. In an effort to identify shRNAs that were both potent and non-cytotoxic, we created a shRNA library representing all potential CCR5 20 to 22-nucleotide shRNA sequences expressed using an H1 promoter and screened this library for downregulation of CCR5. We identified one potent CCR5 shRNA that was also non-cytotoxic when expressed at a low level with the H1 promoter. We characterized this shRNA in regards to its function and structure. This shRNA was unique that the use of commercial and published algorithms to predict effective siRNA sequences did not result in identification of the same shRNA. We found that this shRNA could induce sequence specific reduction of CCR5 at post transcriptional level, consistent with the RNA interference mechanism. Importantly, this shRNA showed no obvious cytotoxicity and was effective at downregulating CCR5 in primary human peripheral blood derived mononuclear cells.
Conclusion
We report on the characterization of a rare shRNA with atypical structural features having potent RNAi activity specific to CCR5. These results have implications for the application of RNAi technology for therapeutic purposes.
doi:10.1186/1479-0556-7-8
PMCID: PMC2701936  PMID: 19515239
21.  Optimization and characterization of tRNA-shRNA expression constructs 
Nucleic Acids Research  2007;35(8):2620-2628.
Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNALys3-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3′ tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.
doi:10.1093/nar/gkm103
PMCID: PMC1885648  PMID: 17426139
22.  Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo 
BMC Cancer  2010;10:486.
Background
Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV.
Methods
To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting.
Results
Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective.
Conclusions
This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials.
doi:10.1186/1471-2407-10-486
PMCID: PMC2944180  PMID: 20836854
23.  RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences 
Nucleic Acids Research  2009;37(18):6194-6204.
Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3′ untranslated region (3′ UTR). Partially complementary sequences within the 3′ UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.
doi:10.1093/nar/gkp644
PMCID: PMC2764431  PMID: 19656954
24.  Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers 
Journal of Nucleic Acids  2011;2011:131579.
RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.
doi:10.4061/2011/131579
PMCID: PMC3139136  PMID: 21776374
25.  Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors 
Background
RNA interference (RNAi) can potently reduce target gene expression in mammalian cells and is in wide use for loss-of-function studies. Several recent reports have demonstrated that short double-stranded RNAs (dsRNAs), used to mediate RNAi, can also induce an interferon-based response resulting in changes in the expression of many interferon-responsive genes. Off-target gene silencing has also been described, bringing into question the validity of certain RNAi-based approaches for studying gene function. We have targeted the plasminogen activator inhibitor-2 (PAI-2 or SERPINB2) mRNA using lentiviral vectors for delivery of U6 promoter-driven PAI-2-targeted short hairpin RNA (shRNA) expression. PAI-2 is reported to have anti-apoptotic activity, thus reduction of endogenous expression may be expected to make cells more sensitive to programmed cell death.
Results
As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.
Conclusions
Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.
doi:10.1186/1471-2199-5-9
PMCID: PMC514603  PMID: 15291968

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