scholarly journals The effect of Dicer knockout on RNA interference using various Dicer substrate interfering RNA structures

2020 ◽  
Author(s):  
Min-Sun Song ◽  
John J Rossi
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Cell Line ◽  
Molecular Mechanism ◽  
Hct116 Cell ◽  
Specific Gene ◽  
Rna Structures ◽  
Guide Strand ◽  
Rnai Silencing ◽  
Interfering Rna

AbstractDicer-substrate siRNA (DsiRNA) was a useful tool for sequence-specific gene silencing. DsiRNA was proposed to have increased efficacy via RNAi gene silencing, but the molecular mechanism underlying the increased efficacy is not precise. We designed the tetra-looped DsiRNA as the tetra-looped RNAs have been reported more stable structure and increased binding efficiency with RNA and protein. To gain a deeper understanding of the Dicer function of DsiRNA, we knocked out Dicer in the HCT116 cell line and analyzed the efficacy of various Dicer substrates on RNAi gene silencing activity. Tetra-looped DsiRNA demonstrated increased efficacy of gene silencing Dicer expressing cells with activity favoring the guide strand. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. Thus, this study allows us to understand the Dicer function of key RNAi silencing and provides valuable resources for RNAi research and applications.

scholarly journals Optimizing RNA interference for application in mammalian cells

2004 ◽  
Vol 380 (3) ◽  
pp. 593-603 ◽  
Author(s):  
René H. MEDEMA
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Mutational Analysis ◽  
Life Sciences ◽  
Novel Technologies ◽  
Genome Wide ◽  
Technological Developments ◽  
Interfering Rna

Over the last 2 years, the scientific community has rapidly embraced novel technologies that allow gene silencing in vertebrates. Ease of application, cost effectiveness and the possibilities for genome-wide reverse genetics have quickly turned this approach into a widely accepted, almost mandatory asset for a self-respecting laboratory in life sciences. This review discusses some of the recent technological developments that allow the application of RNAi (RNA interference) in mammalian cells. In addition, the advantages of applying RNAi to study cell cycle events and the emerging approaches to perform mutational analysis by complementation in mammalian cells are evaluated. In addition, common pitfalls and drawbacks of RNAi will be reviewed, as well as the possible ways to get around these shortcomings of gene silencing by small interfering RNA.

scholarly journals Antiviral Effects of Small Interfering RNA Simultaneously Inducing RNA Interference and Type 1 Interferon in Coxsackievirus Myocarditis

2012 ◽  
Vol 56 (7) ◽  
pp. 3516-3523 ◽  
Author(s):  
Jeonghyun Ahn ◽  
Ara Ko ◽  
Eun Jung Jun ◽  
Minah Won ◽  
Yoo Kyum Kim ◽  
...  
Keyword(s):  
Rna Interference ◽  
Small Interfering Rna ◽  
Antiviral Treatment ◽  
Coxsackievirus B3 ◽  
Hydroxyl Group ◽  
Specific Gene ◽  
Type I ◽  
Antiviral Effects ◽  
Direct Demonstration ◽  
Interfering Rna

ABSTRACTAntiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5′-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5′ end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.

scholarly journals RNA interference as a gene silencing tool to controlTuta absolutain tomato (Solanum lycopersicum)

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2673 ◽  
Author(s):  
Roberto A. Camargo ◽  
Guilherme O. Barbosa ◽  
Isabella Presotto Possignolo ◽  
Lazaro E. P. Peres ◽  
Eric Lam ◽  
...  
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Target Gene ◽  
Target Genes ◽  
Larval Mortality ◽  
Tuta Absoluta ◽  
Specific Gene ◽  
Gene Transcript ◽  
Pest Species ◽  
In Planta

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-AandArginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet forT. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliverin planta-transcribed dsRNA to target insect genes.Tuta absolutalarvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage byT. absolutain these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

scholarly journals RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum)

2016 ◽  
Author(s):  
Roberto A Camargo ◽  
Guilherme O Barbosa ◽  
Isabella Presotto Possignolo ◽  
Lazaro E. P. Peres ◽  
Eric Lam ◽  
...  
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Target Gene ◽  
Target Genes ◽  
Larval Mortality ◽  
Tuta Absoluta ◽  
Specific Gene ◽  
Gene Transcript ◽  
Pest Species ◽  
In Planta

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes [Vacuolar ATPase-A and Arginine kinase] based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ~60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

scholarly journals Branched RNA: A New Architecture for RNA Interference

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Anna Aviñó ◽  
Sandra M. Ocampo ◽  
José Carlos Perales ◽  
Ramon Eritja
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Delivery Systems ◽  
Specific Gene ◽  
Rna Modifications ◽  
Tnf Α ◽  
Innovative Tool ◽  
Branched Rna ◽  
Gene Inhibition ◽  
Novel Structures

Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

scholarly journals Assessing the Functionality of RNA Interference (RNAi) in the Phloem-feeding Maize pest Dalbulus maidis

2021 ◽  
Author(s):  
Tara-Kay L. Jones ◽  
Julio S. Bernal ◽  
Raul F. Medina
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Gene Silencing ◽  
Pest Control ◽  
Messenger Rna ◽  
Specific Gene ◽  
Insect Vector ◽  
Dalbulus Maidis ◽  
Spiroplasma Kunkelii ◽  
Phloem Feeding

Dalbulus maidis [(DeLong & Wolcott), corn leafhopper], a phloem-feeding insect, is the most efficient vector of maize stunting pathogens (Spiroplasma kunkelii, Maize bushy stunt phytoplasma, and Maize rayado fino virus) in the Americas. Studies involving gene editing in insects are rapidly providing information that can potentially be used for insect vector and plant disease control. RNA interference (RNAi), a sequence-specific gene silencing method, is one of the most widely used molecular tools in functional genomics studies. RNAi uses exogenous double-stranded RNA (dsRNA) or small interfering RNA (siRNA) to prevent the production of proteins by inhibiting the expression of their corresponding messenger RNA (mRNA). In this study, we measured the efficacy of gene silencing, and its effects on D. maidis mortality as proof of concept that RNAi is a viable tool for use in genetic pest control of phloem-feeding insects. Oral delivery of dsRNA using an artificial diet was used to silence two key insect genes, vacuolar ATP synthase subunit B, and subunit D (V-ATPase B and V-ATPase D). Our results showed reduced gene expression of V-ATPase B and V-ATPase D after ingestion of dsRNA, and significantly higher mortality, and wing deformation, associated with reduced gene expression, compared to control insects that were not orally fed dsRNA. These results reveal RNAi as a viable tool for use in genetic pest control of phloem-feeding insects, and a way for further functional genomic studies, such as identification of potential target genes for either population suppression or population replacement of this vector of maize diseases.

RNA interference and its therapeutic potential

Open Medicine ◽  
2011 ◽  
Vol 6 (2) ◽  
pp. 137-147 ◽  
Author(s):  
Anubrata Ghosal ◽  
Ahmad Kabir ◽  
Abul Mandal
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Small Interfering Rna ◽  
Therapeutic Potential ◽  
Specific Gene ◽  
History Of ◽  
A Cell ◽  
A New Technique ◽  
Rnai Technique ◽  
Interfering Rna

AbstractRNA interference is a technique that has become popular in the past few years. This is a biological method to detect the activity of a specific gene within a cell. RNAi is the introduction of homologous double stranded RNA to specifically target a gene’s product resulting in null or hypomorphic phenotypes. This technique involves the degradation of specific mRNA by using small interfering RNA. Both microRNA (miRNA) and small interfering RNA (siRNA) are directly related to RNA interference. RNAi mechanism is being explored as a new technique for suppressing gene expression. It is an important issue in the treatment of various diseases. This review considers different aspects of RNAi technique including its history of discovery, molecular mechanism, gene expression study, advantages of this technique against previously used techniques, barrier associated with this technique, and its therapeutic application.

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