An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level.
ABSTRACT: RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker ( ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.
Project description:RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.
Project description:RNAi by short hairpin RNA (shRNA) is a powerful tool not only for studying gene functions in various organisms, including mammals, but also for the treatment of severe disorders. However, shRNA-expressing vectors can induce type I interferon (IFN) expression by activation of innate immune responses, leading to off-target effects and unexpected side effects. Several strategies have been developed to prevent type I IFN induction. On the other hand, it has remained unclear whether type I IFNs have effects on shRNA-mediated RNAi. Here, we show that the type I IFNs significantly inhibit shRNA-mediated RNAi. Treatment with recombinant human IFN-? significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFN-?, increased and decreased copy numbers of shRNA and its processed form, respectively, were found in the cells transfected with shRNA-expressing plasmids. Dicer protein levels were not altered by IFN-?. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicer-mediated processing of shRNA to siRNA. Our findings should provide important clues for efficient RNAi-mediated knockdown of target genes in both basic researches and clinical gene therapy.
Project description:Tagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids for Streptococcus mutans UA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters in S. mutans These promoter-sfgfp fusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeled S. mutans UA159, Streptococcus gordonii DL1, and Streptococcus sp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCE Oral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins in Streptococcus mutans and other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactions in situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.
Project description:BACKGROUND: Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. METHODS: We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-? induction were measured by quantitative real-time PCR and ELISA. RESULTS: Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-?, IFN-? and TNF-? in transfected cells. CONCLUSIONS: Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.
Project description:Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant-fungus interactions and tomato root colonization.
Project description:Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.
Project description: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.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.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.
Project description: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.
Project description:RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.
Project description:Whole-genome transgenic RNAi libraries permit systematic genetic screens in individual tissues of Drosophila. However, there is a high incidence of nonspecific phenotypes because of off-target effects. To minimize such effects, it is essential to obtain a deeper understanding of the specificity of action of RNAi. Here, in vivo assays are used to determine the minimum, contiguous nucleotide pairing required between an siRNA and a target mRNA to generate a phenotype. We observe that as few as 16 nucleotides of contiguous homology are sufficient to attenuate gene activity. This finding provides an explanation for the high incidence of off-target effects observed in RNAi-based genetic screens. Toward improving the efficacy of RNAi-induced phenotypes in vivo, we describe siRNA expression vectors that allow coexpression of one or more siRNAs with a fluorescent reporter gene in cultured cells or transgenic flies. This expression system makes use of the small intron from the ftz segmentation gene to provide efficient processing of synthetic siRNAs from a reporter transcript. These studies provide a foundation for the specific and effective use of gene silencing in transgenic Drosophila.