RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by long double-stranded RNA (dsRNA). RNAi is an ancient eukaryotic defense mechanism against viruses and mobile elements. In mammals, endogenous RNAi was outstripped during evolution by the current innate and acquired immunity. The RNAi apparatus, which remains essentially intact, serves mostly the microRNA pathway, which regulates endogenous gene expression. Remarkably, several recent publications brought the mammalian endogenous RNAi pathway back into the spotlight. Here, I will provide an up-to-date review of the mammalian endogenous RNAi pathway with a focus on its defensive role and overlaps with miRNA and piRNA pathways.

• Klíčová slova
• Dicer, Oocyte, RNAi, Retrotransposon, Virus,
• MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• RNA interference * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• malá interferující RNA MeSH

The RNA interference (RNAi) and interferons have been an uneasy marriage. Ever since the discovery of RNAi in mammals, the interferon response has been a feared problem. While RNAi became an efficient and widespread method for gene silencing in mammals, numerous studies recognized several obstacles, including undesirable activation of the interferon response, which need to be overcome to achieve a specific and robust RNAi effect. The aim of this text is to provide theoretical and practical information for scientists who want to control interferon response and other adverse effects in their RNAi experiments.

• MeSH
• DNA primery MeSH
• dvouvláknová RNA MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• interferony metabolismus   MeSH
• klonování DNA MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• regulace genové exprese MeSH
• RNA interference * MeSH
• sekvence aminokyselin MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• dvouvláknová RNA MeSH
• interferony MeSH
• malá interferující RNA MeSH

The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and discussion of the benefits and drawbacks of using long double-stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression.

• MeSH
• embryo savčí cytologie   MeSH
• genetické techniky * MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• RNA interference * MeSH
• transgeny MeSH
• umlčování genů MeSH
• vývojová biologie metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.

• MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• RNA interference * MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for many model systems. The protocol presented here describes the cloning of a plasmid construct for use in transgenic RNAi experiments in mouse oocytes. The protocol is intended for production of a transgene by cloning an inverted repeat (IR) into the Zp3 transgenic cassette. The procedure begins with the selection of sequences and formulation of the cloning strategy. Subsequently, the IR is cloned, inserted into the transgenic cassette, and characterized by sequencing. Finally, the transgene is released from the cassette, purified, and provided to the transgenic facility.

• MeSH
• Bacteria metabolismus   MeSH
• klonování DNA * MeSH
• molekulární sekvence - údaje MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty cytologie   MeSH
• plazmidy metabolismus   MeSH
• repetitivní sekvence nukleových kyselin MeSH
• RNA interference * MeSH
• sekvence nukleotidů MeSH
• transgeny * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. Here, we describe selection of the target sequence for efficient RNAi knockdown in mouse.

• MeSH
• 3' nepřekládaná oblast MeSH
• databáze genetické MeSH
• dvouvláknová RNA metabolismus   MeSH
• exprimované sekvenční adresy MeSH
• genetické techniky * MeSH
• komplementární DNA metabolismus   MeSH
• malá interferující RNA metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• myši MeSH
• posttranskripční úpravy RNA MeSH
• RNA interference * MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• dvouvláknová RNA MeSH
• komplementární DNA MeSH
• malá interferující RNA MeSH
• messenger RNA MeSH

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

• Klíčová slova
• Dicer, Mirtron, PKR, dsRNA, siRNA,
• MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• dvouvláknová RNA * metabolismus   genetika   MeSH
• malá interferující RNA * genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• myši MeSH
• protein - isoformy genetika   metabolismus   MeSH
• ribonukleasa III * genetika   metabolismus   MeSH
• RNA interference * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• Dicer1 protein, mouse MeSH Prohlížeč
• dvouvláknová RNA * MeSH
• malá interferující RNA * MeSH
• mikro RNA MeSH
• protein - isoformy MeSH
• ribonukleasa III * MeSH

The RNA interference (RNAi) approach is an efficient and widely used method for silencing plant and animal genes. However, numerous studies have highlighted several obstacles that need to be overcome in order to achieve a specific and efficient silencing effect. Among the major problems of using RNAi in mammalian systems are non-specific effects, such as an undesirable activation of the IFN response, and off-target effects, whereby partial complementarity of an RNAi trigger to non-targeted transcripts causes unwanted silencing effects. The causes of non-specific effects and possible approaches to minimize them are discussed in this review.

• MeSH
• biologické modely MeSH
• interferony genetika   imunologie   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• RNA interference * MeSH
• signální transdukce genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• interferony MeSH
• malá interferující RNA MeSH

RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.

• MeSH
• aktiny genetika   MeSH
• Cnidaria * genetika   MeSH
• fylogeneze MeSH
• Myxozoa * genetika   MeSH
• nemoci ryb * genetika   MeSH
• paraziti * MeSH
• pohyb buněk MeSH
• RNA interference MeSH
• ryby MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny MeSH

The spotted bollworm Earias vittella (Lepidoptera: Nolidae) is a polyphagous pest with enormous economic significance, primarily affecting cotton and okra. However, the lack of gene sequence information on this pest has a significant constraint on molecular investigations and the formulation of superior pest management strategies. An RNA-seq-based transcriptome study was conducted to alleviate such limitations, and de novo assembly was performed to obtain transcript sequences of this pest. Reference gene identification across E. vittella developmental stages and RNAi treatments were conducted using its sequence information, which resulted in identifying transcription elongation factor (TEF), V-type proton ATPase (V-ATPase), and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as the most suitable reference genes for normalization in RT-qPCR-based gene expression studies. The present study also identified important developmental, RNAi pathway, and RNAi target genes and performed life-stage developmental expression analysis using RT-qPCR to select the optimal targets for RNAi. We found that naked dsRNA degradation in the E. vittella hemolymph is the primary reason for poor RNAi. A total of six genes including Juvenile hormone methyl transferase (JHAMT), Chitin synthase (CHS), Aminopeptidase (AMN), Cadherin (CAD), Alpha-amylase (AMY), and V-type proton ATPase (V-ATPase) were selected and knocked down significantly with three different nanoparticles encapsulated dsRNA conjugates, i.e., Chitosan-dsRNA, carbon quantum dots-dsRNA (CQD-dsRNA), and Lipofectamine-dsRNA conjugate. These results demonstrate that feeding nanoparticle-shielded dsRNA silences target genes and suggests that nanoparticle-based RNAi can efficiently manage this pest.

• Klíčová slova
• cotton, dsRNA stability, nanoparticle encapsulated dsRNA, reference genes, spotted bollworm, transcriptome,
• MeSH
• adenosintrifosfatasy MeSH
• dvouvláknová RNA genetika   MeSH
• můry * genetika   MeSH
• nanočástice * MeSH
• protony MeSH
• RNA interference MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• dvouvláknová RNA MeSH
• protony MeSH