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PubMed2005

RNAi in mice: a promising approach to decipher gene functions in vivo.

RNA interference (RNAi) is a simple and powerful tool widely used to study gene functions in many species. Vector-based systems using RNA polymerase III promoters have been developed to achieve stable expression of small interfering RNA (siRNA) or small hairpin RNA (shRNA) in mammalian cells. Recent investigations demonstrated that when, combined with the Cre-loxP system, the vector-based RNAi can be used to achieve conditional or tissue specific knockdown of endogenous genes with high efficiency in mice. Here, we review these recent progresses and discuss the advantages, limitations and future development of this emerging technology.

Animals↗
PubMed2004

Long-lasting RNAi activity in mammalian neurons.

The effect of RNA interference (RNAi) induced by synthetic small interfering RNAs (siRNAs) on proliferating mammalian cells appears to last for approximately 3-7 days after its induction. Here we show that the RNAi activity induced by a synthetic 21-nucleotide siRNA duplex in postmitotic neurons, mouse primary hippocampal neurons and neurons that differentiated from mouse embryonal carcinoma P19 cells persists for at least 3 weeks, suggesting long-lasting RNAi activity in mammalian neurons. In addition, we also show that an apoptotic (or antiviral) pathway triggered by long dsRNAs is generated during neuronal differentiation of P19 cells, by which the sequence-specific RNAi activity involving long dsRNA appears to be masked.

Animals↗
PubMed2005

Knocking down transport: applications of RNA interference in the study of drug transport proteins.

RNA interference (RNAi) is a gene silencing process mediated by double-stranded RNA (dsRNA). The silencing process is comprised of an initiation step, in which small interfering RNA (siRNA) is introduced to the cell, and an effector step, which involves degrading mRNA molecules of the target gene. RNA interference has been observed in most organisms from plants to vertebrates. As a gene silencing approach, RNAi has proven to be extremely useful in characterizing gene function and developing new tools in cancer therapy and drug delivery. The development of RNAi-related technologies is an emerging area in biomedical research. In this review, recent progress in the application of RNAi to the study of transport proteins is summarized and evaluated; the advantages, disadvantages and future directions of RNAi technology are discussed.

Animals↗
PubMed2005

New genes with roles in the C. elegans embryo revealed using RNAi of ovary-enriched ORFeome clones.

Several RNA interference (RNAi)-based functional genomic projects have been performed in Caenorhabditis elegans to identify genes required during embryogenesis. These studies have demonstrated that the ovary is enriched for transcripts essential for the first cell divisions. However, comparing RNAi results suggests that many genes involved in embryogenesis have yet to be identified, especially those eliciting partially penetrant phenotypes. To discover additional genes required for C. elegans embryonic development, we tested by RNAi 1123 ORFeome clones selected to represent ovary-enriched genes not associated with an embryonic phenotype. We discovered 155 new ovary-enriched genes with roles during embryogenesis, of which 69% show partial penetrance lethality. Time-lapse microscopy revealed specific phenotypes during early embryogenesis for genes giving rise to high penetrance lethality. Together with previous studies, we now have evidence that 1843 C. elegans genes have roles in embryogenesis, and that many more remain to be found. Using all available RNAi phenotypic data for the ovary-enriched genes, we re-examined the distribution of genes by chromosomal location, functional class, ovary enrichment, and conservation and found that trends are driven almost exclusively by genes eliciting high-penetrance phenotypes. Furthermore, we discovered a striking direct relationship between phylogenetic distribution and the penetrance level of embryonic lethality elicited by RNAi.

Animals↗
PubMed2006

Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo.

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.

Animals↗
PubMed2004

Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins.

Post-transcriptional inhibition of HIV-1 replication can be achieved by RNA interference (RNAi). The cellular expression of short interfering RNA (siRNA) or short hairpin RNA (shRNA) homologous to regions of the HIV-1 genome decreases viral replication by the selective degradation of targeted RNA. Here, we demonstrate that another class of noncoding regulatory RNA, termed microRNA (miRNA), can be used to deliver antiviral RNAi. By incorporating sequences encoding siRNA targeting the HIV-1 transactivator protein tat into a human miR-30 pre-microRNA (pre-miRNA) backbone, we were able to express tat siRNA in cells. The tat siRNA delivered as pre-miRNA precursor was 80% more effective in reducing HIV-1 p24 antigen production than tat siRNA expressed as conventional shRNA. Our results confirm the utility of expressing HIV-1 specific siRNA through a miR-30 precursor stem-loop structure and suggest that this strategy can be used to increase the antiviral potency of RNAi.

Base Sequence↗
PubMed2005

Suppression of hLRH-1 mediated by a DNA vector-based RNA interference results in cell cycle arrest and induction of apoptosis in hepatocellular carcinoma cell BEL-7402.

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of mRNA. A DNA vector-based approach has been shown to be able to trigger RNA interference in mammalian cells successfully. LRH-1 is an orphan nuclear receptor predominantly expressed in tissues of endodermal origin, where it controls development and cholesterol homeostasis. In the present study, we demonstrated that the expression of hLRH-1 and cyclin E1 in BEL-7402 cells could be suppressed by up to approximately 80% via DNA vector-based RNA interference. The suppression of hLRH-1 resulted in cell cycle arrest mediated by the down-regulation of cyclin E1. Induction of apoptosis and down-regulation of Gadd45beta were also shown in hLRH-1 knock down BEL-7402 cells. These results, together with the findings that Gadd45beta remained unchanged in cyclin E1 RNAi cells, suggested that the induction of apoptosis by knock down of hLRH-1 was closely related to the down-regulation of Gadd45beta.

Antigens, Differentiation↗
PubMed2002

RNAi functions in cultured mammalian neurons.

In a wide range of organisms, double-stranded RNA triggers posttranscriptional gene silencing or RNA interference (RNAi). Small interfering RNAs, the 21-nt double-stranded RNA intermediates of this natural pathway, have became a powerful tool to knock down specific gene expression in mammalian cell lines and potentially will be useful for the analysis of loss-of-function phenotypes. In mammalian primary neuronal cultures, where genetic manipulations are especially difficult, RNAi might be developed into a highly efficacious tool to study the roles of specific genes in neuron development and functioning. Neurons, however, have been considered the most resistant to RNAi. We report here an application of RNAi to postmitotic primary neuronal cultures. Synthetic siRNA can be readily introduced into neurons and effectively inhibit the expression of endogenous and transfected genes.

Animals↗
PubMed2005

Mammalian RNAi: a practical guide.

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Caenorhabditis elegans and Drosophila melanogaster genomes. Recent advances in the design and delivery of targeting molecules now permit efficient and highly specific gene silencing in mammalian systems as well. RNAi offers a simple, fast, and cost-effective alternative to existing gene targeting technologies both in cell-based and in vivo settings. Synthetic small interfering RNA (siRNA) and retroviral short hairpin RNA (shRNA) libraries targeting thousands of human and mouse genes are publicly available for high-throughput genetic screens, and knockdown animals can be rapidly generated by lentivirus-mediated transgenesis. RNAi also holds great promise as a novel therapeutic approach. This review provides insight into the current gene silencing techniques in mammalian systems.

Animals↗
PubMed2006

RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica.

Ecdysteroids play a major role during developmental growth in insects. The more active form of these hormones, 20-hydroxyecdysone (20E), acts upon binding to its heterodimeric receptor, formed by the two nuclear receptors, EcR and RXR/USP. Functional characterization of USP has been exclusively conducted on the holometabolous insect Drosophila melanogaster. However, it has been impossible to extend such analysis to primitive-hemimetabolous insects since species of this group are not amenable to genetic analysis. The development of methodologies based on gene silencing using RNA interference (RNAi) after treatment with double-stranded RNA (dsRNA) in vivo has resolved such limitations. In this paper, we show that injection of dsRNA into the haemocoel of nymphs and adults of the cockroach Blattella germanica can be used to silence gene function in vivo. In our initial attempt to test RNAi techniques, we halted the expression of the adult-specific vitellogenin gene. We then used the same technique to silence the expression of the B. germanica RXR/USP (BgRXR) gene in vivo during the last nymphal instar. BgRXR knockdown nymphs progressed through the instar correctly but they arrested development at the end of the stage and were unable to molt into adults. The results described herein suggest that RXR/USP function, in relation to molting, is conserved across the insect Class.

Animals↗
PubMed2002

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.

Base Sequence↗
PubMed2006

Vector-based RNAi approaches for stable, inducible and genome-wide screens.

RNA interference (RNAi) has revolutionized the study of biology and offers numerous applications in basic biology as well as in drug discovery research. Since the discovery of RNAi, several tools have been developed to enable loss-of-function studies in mammalian systems. The efficacy of RNAi is dependent on specific and versatile RNAi triggers that have evolved to enable transient, stable and in-vivo applications. Recently developed genome-wide short hairpin RNA (shRNA) and microRNA-adapted short hairpin RNA (shRNAmir) libraries incorporate advances in shRNA design and molecular 'barcodes' to enable more complex RNAi screens and the opportunity to progress to more complex genetics in whole animals.

Animals↗
PubMed2005

Translating RNA interference into therapies for human disease.

RNA interference (RNAi) represents one of the most promising new frontiers in drug discovery. Breakthroughs in understanding RNA's extensive natural role in essential cellular processes have opened up the potential for a whole new class of drugs based on RNAi. Harnessing the natural process of RNAi, short, double-stranded RNA molecules are able to inhibit expression of genes in a sequence-specific manner. By targeting disease-causing genes, RNAi drugs have the potential to be more selective than traditional drugs, and thus more effective as well as less toxic. Over the past few years, important strides have been made in translating the promise of RNAi into therapies for human disease. In contrast to the extensive lead optimization steps typically required in small-molecule and protein drug discovery, RNAi drug candidates can be identified using bioinformatics to select sequences complementary to the target mRNA. The process of selecting an RNAi-based drug candidate may simply involve the synthesis and testing of a relatively small number of short double strands (duplexes) of RNA, incorporating chemical modifications that confer stability and direct these RNA duplexes to the appropriate tissues and cells, and/or formulating these RNA duplexes with appropriate delivery agents to achieve the same goals. As advances in RNAi therapeutics continue, the decades to come should bring a potent new class of drugs based on RNAi.

Animals↗
PubMed2005

Gene silencing using adenoviral RNAi vector in vascular smooth muscle cells and cardiomyocytes.

RNA interference (RNAi) is a new and rapidly progressing technology for facilitating functional gene silencing. To perform highly efficient RNAi in cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells, which are known to have very low transfection efficiency, adenovirus-mediated RNAi was employed. The effects of RNAi on GAPDH transcripts were successfully reduced by nearly 90% in the primary cultured cells, indicating that adenovirus-mediated gene silencing is a promising technique for gene silencing in cardiovascular studies. This chapter describes general guidelines for selecting RNAi target sites, construction of a shuttle vector encoding short hairpin RNAi, and generation of recombinant adenovirus.

Adenoviridae↗
PubMed2005

Inhibition of hepatitis B virus replication by various RNAi constructs and their pharmacodynamic properties.

The strategy of RNA interference (RNAi)-based gene silencing has been suggested to have great potential in treating viral diseases. It provides new hope of being able to complement the limited therapeutic options currently available for chronic hepatitis B virus (HBV) infection. To advance such a strategy towards clinical use, the effects of various parameters on the anti-HBV efficiency of RNAi need to be well-defined. In this study, the efficacy and pharmacodynamic properties of different RNAi target sequences and constructs were examined. Several sequences were found to be effective in cell and animal models, achieving inhibition rates of approximately 80-90 %. Methyl-modified small interfering RNA (siRNA) molecules were found to be more stable inside cells than natural siRNA molecules and offered longer-lasting inhibitory effects. Both were effective at rather low doses (an equimolar ratio with HBV preS2-S protein expression vector). Plasmid DNA vectors were less dose-responsive, but their effectiveness in vivo lasted longer, for approximately 1 month. By analysing these different parameters and their possible mechanisms, some important issues in RNAi therapeutics that should assist the future development of clinical applications have been addressed.

Animals↗
PubMed2005

Assessment of penetrance and expressivity of RNAi-mediated silencing of the Arabidopsis phytoene desaturase gene.

RNA interference (RNAi) is of great value in plant functional genomics. However, the absence of RNAi phenotypes and the lack of uniform level of RNAi silencing has complicated gene identification. Here, the penetrance and expressivity of RNAi-mediated silencing of the phytoene desaturase (PDS) gene in Arabidopsis thaliana were examined quantitatively to provide a reference for the likely severity and distribution of silencing effects. Arabidopsis plants were transformed with an RNAi construct targeting PDS. Transgenic plants were examined for frequency of RNAi-mediated silencing and various silencing phenotypes. mRNA depletion level and RNAi expressivity were assayed by relative reverse transcription polymerase chain reaction (RT-PCR). High penetrance and variable expressivity of RNAi were demonstrated. An inverse correlation between PDS mRNA level and RNAi phenotype was seen. No direct relationship between copy number for the RNAi-generating transgene and phenotype was evident. Decreased RNAi penetrance in T2 plants was observed. It is suggested that variability in RNAi expressivity and postmeiotic decrease in RNAi penetrance constitute barriers for high throughput plant gene characterization.

Arabidopsis↗
PubMed2005

CHR11, a chromatin-remodeling factor essential for nuclear proliferation during female gametogenesis in Arabidopsis thaliana.

Chromatin-remodeling factors regulate the establishment of transcriptional programs during plant development. Although 42 genes encoding members of the SWI2/SNF2 family have been identified in Arabidopsis thaliana, <10 have been assigned a precise function on the basis of a mutant phenotype, and none have been shown to play a specific role during the gametophytic phase of the plant life cycle. A. thaliana chromatin-remodeling protein 11 (CHR11) encodes an imitation of switch (ISWI)-like chromatin-remodeling protein abundantly expressed during female gametogenesis and embryogenesis in Arabidopsis. To determine the function of CHR11 in wild-type plants, we introduced a hairpin construct leading to the production of double-stranded RNA, which specifically degraded the endogenous CHR11 mRNA by RNA interference (RNAi). Transcription of the RNAi-inducing hairpin RNA was driven by either a constitutive cauliflower mosaic virus 35S promoter (CaMV35S) acting at most stages of the sporophytic phase or a newly identified specific promoter acting at the onset of the female gametophytic phase (pFM1). All adult transformants that constitutively lacked sporophytic CHR11 activity showed reduced plant height and small cotyledonary embryos with limited cell expansion. In contrast, RNAi lines in which CHR11 was specifically silenced at the onset of female gametogenesis (megagametogenesis) had normal height and embryo size but had defective female gametophytes arrested before the completion of the mitotic haploid nuclear divisions. These results show that CHR11 is essential for haploid nuclear proliferation during megagametogenesis and cell expansion during the sporophytic phase, demonstrating the functional versatility of SWI2/SNF2 chromatin-remodeling factors during both generations of the plant life cycle.

Adenosine Triphosphatases↗
PubMed2005

Exploiting the RNA interference pathway to counter hepatitis B virus replication.

Chronic infection with hepatitis B virus (HBV) is endemic to sub-Saharan Africa and parts of Asia where persistence of the virus is commonly associated with complicating cirrhosis and hepatocellular carcinoma (HCC). Licensed therapies for HBV are partially effective in selected patients and development of novel treatments remains an important global medical objective. HBV has an unusually compact genome that restricts the ability of the virus to evade potentially therapeutic nucleic acid hybridization. Thus, exploiting the RNA interference (RNAi) pathway, which enables sequence-specific target RNA degradation using small interfering RNA (siRNA), is well suited to developing novel treatment for HBV infection. Several studies, both in vitro and in vivo, have demonstrated that HBV replication can be inhibited in transfected cells by synthetic siRNA duplexes and also Pol III-derived short hairpin RNA (shRNA) sequences. The effectiveness of anti-HBV sequences varies considerably, and is likely to result from differences in activation of the RNAi pathway by individual siRNA species. Exclusion of potentially toxic off-target effects and also development of efficient methods of hepatotropic nucleic acid delivery are important prerequisites before RNAi can be used successfully for anti-HBV treatment.

Gene Expression Regulation, Viral↗