We thank Drs. Alison Heffer and Yong Lu for setting up the microinjection apparatus and sharing their invaluable knowledge and experience with insect RNAi. This work was supported by the National Institutes of Health (R01GM113230 to L.P.).

The authors have nothing to disclose.

Mentre un piccolo numero di sistemi modello sofisticati (topi, mosche, vermi) sono stati sviluppati nel corso del 20 ° secolo, il 21 ° secolo ha visto un&#39;ondata di nuovi sistemi di animali in fase di sviluppo nei laboratori di tutto il mondo. Questi nuovi sistemi consentono agli scienziati di affrontare comparativi, domande evolutive che non può essere sondato utilizzando solo gli &#39;standard&#39; sistemi modello. Questa distribuzione di nuovi modelli richiede il rapido sviluppo di metodi ...

Nel 1998, Fuoco e Mello hanno riferito che a doppio filamento di RNA (dsRNA) può indurre inibizione della funzione del gene in Caenorhabditis elegans 1. Questa risposta innescato da dsRNA è stato chiamato interferenza dell&#39;RNA (RNAi), e tale silenziamento genico RNAi-mediata è stato segnalato per essere conservato in animali, piante e funghi 2-7. In piante e alcuni animali, funzioni RNAi sistemica, il che significa che l&#39;effetto può diffondersi ad altre cellule / tessuti dove dsRNA non viene introdotto direttamente (rivisto nel 8-10). Gli scienziati hanno fatto uso di questo endogena risposta RNAi cellulare progettando dsRNAs di indirizzare i geni di interesse, battendo così giù la funzione del gene senza manipolare direttamente il genoma (rivisto nel 11-14). RNAi è un potente strumento per studi funzionali causa dei seguenti vantaggi. In primo luogo, anche con il minimo informazioni sequenza del gene, un gene può essere mirata utilizzando RNAi. Ciò è particolarmente importante per studies di organismi non-modello privi di dati genomici e trascrittomica. In secondo luogo, negli organismi dove la risposta RNAi è robusta sistemica RNAi mediata knockdown gene può essere effettuata in quasi ogni stadio di sviluppo. Questa funzione è molto utile per studiare la funzione dei geni pleiotropici. In terzo luogo, in alcuni casi, effetti RNAi diffuso ai gonadi e discendenti, in modo tale che i fenotipi si osservano nella prole 15,16. Questo fenomeno, noto come genitori RNAi (pRNAi), è particolarmente vantaggioso per i geni che influenzano lo sviluppo embrionale, come numerosa prole prodotta da un solo genitore iniettato può essere esaminato, senza manipolazione diretta di uova. Per queste ragioni, pRNAi è il metodo di scelta. Tuttavia, se pRNAi è inefficace, ad esempio per i geni necessari per la oogenesi, allora embrionale RNAi (eRNAi) deve essere utilizzato. Quarto, RNAi può essere utilizzato per generare l&#39;equivalente di una serie allelica dal fatto che la quantità di dsRNA consegnato può essere variata su un intervallo di produrre debole per difetti forti. Tale gradazione di fenotipi può essere utile per comprendere la funzione del gene quando il gene è coinvolto in un processo complesso e / o completa perdita di funzione è letale. In quinto luogo, la consegna di dsRNA è generalmente facile e fattibile, soprattutto negli animali che mostrano solide risposte RNAi sistemiche. dsRNA può essere introdotto da microiniezione 1,5, alimentazione / ingestione 17,18, ammollo, 19,20 e il virus / batteri-mediata consegna 21,22. Sesto, a differenza di alcuni metodi di targeting / modifica dei geni, non vi è alcuna necessità di screening per gli organismi che trasportano la mutazione o di effettuare incroci genetici per generare omozigoti quando si usa RNAi. Pertanto, rispetto a molte altre tecniche per studiare la funzione del gene, RNAi è veloce, poco costoso, e può essere applicato per schermi di grandi dimensioni 23-25. L&#39;ampio programma di utilità di RNAi fornisce i mezzi per effettuare studi funzionali in una vasta gamma di organismi, ampliando la gamma di specie disponibili per lo studio beyond i sistemi modello tradizionali per i quali sono stati sviluppati strumenti genetici. Ad esempio, gli studi che utilizzano sistemi non-modello sono tenuti a dare intuizioni l&#39;evoluzione dei geni e reti geniche confrontando le funzioni di ortologhi da specie che rappresentano diverse modalità di sviluppo o esporre distinte caratteristiche morfologiche 26-29. Questi tipi di studi forniranno una migliore comprensione della diversità biologica, con conseguenze per la ricerca sia applicata e di base. Essendo il più grande gruppo di animali sul pianeta, gli insetti offrono una grande opportunità per esplorare i meccanismi alla base della diversità. Inoltre, gli insetti sono in genere piccole, hanno cicli di vita brevi, ad alta fecondità, e sono facili da dietro in laboratorio. Negli ultimi due decenni, RNAi è stato applicato con successo negli insetti che abbracciano gli ordini, tra cui Ditteri (vere mosche) 5, lepidotteri (farfalle e falene) 30, Coleotteri (coleotteri) 16,31, Imenotteri (sawfbugie, vespe, formiche e api) 32, Hemiptera (veri insetti), Isotteri (termiti) 34, Blattodea (scarafaggi) 35, ortotteri (grilli, cavallette, locuste e grilli) 36 e Phthiraptera (pidocchi) 37. Applicazione di successo di RNAi ha fornito dati funzionali per gli studi di patterning in embriogenesi precoce (asse anteriore-posteriore 32, dorso-ventrale asse 28, la segmentazione 26,38), determinazione del sesso, 39,40 chitina / cuticola biosintesi 41, ecdysone segnalazione 42, comportamento sociale 43, e altro ancora. Metodi RNAi sviluppati per diverse specie di insetti possono essere di ulteriore vantaggio in quanto sono suscettibili di essere utile per il controllo dei parassiti (rivisto nel 44-46). effetti RNAi saranno nonché specie-specifico gene-specifico, purché regioni non conservate sono scelti per il targeting. Per le specie di insetti utili come le api e bachi da seta, di targeting dei geni vitali per la sopravvivenza divirus o parassiti per controllare l&#39;infezione possono fornire una nuova strategia per proteggere queste specie 47,48. Dermestes maculatus (D. maculatus), comune nome nascondere coleottero, è distribuito in tutto il mondo tranne che per l&#39;Antartide. Come un insetto olometaboli, il ciclo di vita D. maculatus comprende embrionale, larvale, pupa, e stadi adulti (Figura 1). Perché si nutre di carne, D. maculatus è utilizzato nei musei per skeletonize animali morti e entomologi forensi può essere utilizzato per stimare momento della morte 49,50. D. maculatus si nutre di prodotti di origine animale, tra cui carcasse, carne secca, formaggio e le pupe / bozzoli di altri insetti e provoca così danni alle famiglie, alimenti conservati, e la seta, il formaggio, e industrie della carne 51,52. L&#39;applicazione di RNAi in questo coleottero potrebbe fornire un modo efficiente ed ecologico per ridurre al minimo il suo impatto economico. Il nostro laboratorio ha utilizzato D. maculatus come nuovo model insetto per studiare la segmentazione 53. Oltre ad essere suscettibili di laboratorio allevamento, D. maculatus è di interesse per la ricerca di base in quanto è uno sviluppatore intermedio di germi, che lo rende una specie utili per studiare la transizione tra breve e di sviluppo a lungo germe. <img alt="Figura 1" src="/files/ftp_upload/54976/54976fig1.jpg" /> Figura 1: Ciclo di vita di D. maculatus. Fotografie di D. maculatus in diverse fasi della vita, come indicato. Il ciclo di vita da uovo ad adulto dura tre settimane a 30 ° C, ma più a lungo a temperature più basse. (A, F) embrioni appena deposte sono di colore bianco a giallo chiaro e ovali, circa 1,5 mm di lunghezza. Embriogenesi prende ~ 55 ore a 30 ° C. (B, C e G) larve hanno le strisce pigmentate scure e sono ricoperte di setole. Le larve passano attraverso diversi stadi a seconda dell&#39;ambiente e la loro lunghezza può estendersi fino a oltre 1 centimetro. (D, H) </strong&gt; Giovani pupe sono giallo chiaro. Pupation prende ~ 5 - 7 giorni a 30 ° C. (E, I) Poco dopo eclosion, pigmentazione scura appare sopra il corpo adulto coleottero. Gli adulti possono vivere fino a diversi mesi e una femmina può deporre centinaia di embrioni sopra il suo ciclo di vita. <a href="http://ecsource.jove.com/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> In precedenza, abbiamo dimostrato che RNAi è efficace nel abbattendo la funzione del gene in D. maculatus 53. Qui la nostra esperienza di allevamento colonie D. maculatus in laboratorio è condiviso con i protocolli passo-passo sia embrionale e genitori RNAi set-up, iniezione, cura post-iniezione, e l&#39;analisi fenotipica. Il silenziamento genico e analisi metodi dsRNA-mediate introdotti qui non solo di fornire informazioni dettagliate per affrontare questioni in D. maculatus, ma hanno anche un significato potenziale foR applicazione di RNAi in altre specie non-modello scarabeo / insetto.

<table><tbody><tr><td>&lt;em&gt;Dermestes maculatus&lt;/em&gt; live beetles</td><td>Our lab or Carolina Biological Supply</td><td>#144168</td><td>Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out</td></tr><tr><td>Wet cat food</td><td>Fancy Feast</td><td></td><td>Chunks of meat with gravy. Can buy at most pet food and grocery stores</td></tr><tr><td>Dry dog food</td><td>Purina Puppy Chow</td><td></td><td>Can buy at most pet food and grocery stores</td></tr><tr><td>Insect cage (size medium, 30.5 x 19 x 20.3 cm)</td><td>Exo Terra</td><td>PT2260</td><td>For colony maintenance. Can use larger cage if needed</td></tr><tr><td>Insect cage (size mini, 17.8 x 10.2 x 12.7 cm)</td><td>Exo Terra</td><td>PT2250</td><td>For embryo collection</td></tr><tr><td>Petri dish</td><td>VWR</td><td>89038-968</td><td></td></tr><tr><td>Cotton ball</td><td>Fisher</td><td>22-456-883</td><td></td></tr><tr><td>Megascript T7 transcription kit</td><td>Fisher</td><td>AM1334</td><td>For 40 reactions</td></tr><tr><td>Pneumatic pump</td><td>WPI</td><td>PV830</td><td></td></tr><tr><td>Capillary holder</td><td>WPI</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>NARISHIGE</td><td>MN-151</td><td></td></tr><tr><td>Black filter paper (90&amp;nbsp;mm)</td><td>VWR</td><td>28342-010</td><td></td></tr><tr><td>Food coloring (green)</td><td>McCormick</td><td></td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>Hilgenberg</td><td>1406119</td><td></td></tr><tr><td>Needle puller (micropipette puller)</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Microscope glass slide</td><td>WorldWide Life Sciences Division</td><td>41351157</td><td></td></tr><tr><td>Sealing film (Parafilm M)</td><td>Fisher</td><td>13-374-12</td><td></td></tr><tr><td>Model 801 Syringe (10&amp;nbsp;&amp;micro;l)</td><td>Hamilton</td><td>7642-01</td><td></td></tr><tr><td>Needle (32-gauge)&amp;nbsp;</td><td>Hamilton</td><td>7762-05</td><td></td></tr><tr><td>Fixation Solution (Pampel&#039;s)</td><td>BioQuip Products, Inc.</td><td>1184C</td><td>Toxic, needs to be handled in fume hood</td></tr><tr><td>Forcep (DUMONT #5)</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>Cover slip (24 x 50 mm, No. 1.5)</td><td>Globe Scientific</td><td>1415-15</td><td></td></tr><tr><td>Eppendorf Femtotips Microloader pipette tip</td><td>Fisher</td><td>E5242956003</td><td></td></tr><tr><td>Dissecting microscopy for embryo injection</td><td>Leica</td><td>M420</td><td></td></tr><tr><td>Dissecting microscopy for larval phenotypic visualization</td><td>Zeiss</td><td>SteREO Discover. V12</td><td></td></tr><tr><td>DIC microscopy</td><td>Zeiss</td><td>AXIO Imager. M1</td><td></td></tr></tbody></table>

rearing and double-stranded rna-mediated gene knockdown in the hide beetle, dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Laboratorio degli autori ha utilizzato la tecnologia RNAi per studiare l&#39;evoluzione funzionale dei geni che regolano la segmentazione negli insetti 53,55. Mentre tutti gli insetti sono segmentati, i geni che regolano questo processo sembrano aver discostato durante radiazioni insetti 26,38,56-63. Schermi genetiche in Drosophila hanno identificato una serie di nove geni di segmentazione coppia-di regole che sono responsabili per promuovere la formazione ...

Watch this Scientific Journal Video about Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus at JoVE.com

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Qui, vi presentiamo i protocolli per l&#39;allevamento di un coleottero intermedio di germi, Dermestes maculatus (D. maculatus) in laboratorio. Condividiamo anche protocolli per embrionale e genitori RNAi e metodi per l&#39;analisi dei fenotipi embrionali per studiare la funzione dei geni in questa specie.

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

rearing-double-stranded-rna-mediated-gene-knockdown-hide-beetle

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

10.5K Views.  University of Maryland.  Qui, vi presentiamo i protocolli per l'allevamento di un coleottero intermedio di germi, Dermestes maculatus (D. maculatus) in laboratorio. Condividiamo anche protocolli per embrionale e genitori RNAi e metodi per l'analisi dei fenotipi embrionali per studiare la funzione dei geni in questa specie. 

Video: Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA interferenza nelle zecche

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Ricerca

Immunologia e infezioni

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Metodi di consegna orale RNA double-stranded per indurre RNAi nel floema e insetti Pentatomomorfi pianta-sap-alimentazione

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Ambiente

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Riproducibile dsRNA Microinjection e analisi biologica di deposizione delle uova di zanzare e mosche domestiche

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

Interferenza dell'RNA nei Coleotteri acquatici come potente strumento per manipolare l'espressione genica in punti specifici del tempo di sviluppo

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi mediato doppia Gene atterramento e gustative Percezione misura di api mellifere (<em&gt; Apis mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuroscienze

Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera).

Metodo di interferenza dell'RNA mediato dall'elettroporazione in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...

Biologia dello sviluppo

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Applicazioni dell'interferenza dell'RNA nello scarafaggio americano

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

L&#39;interferenza di RNAi per iniezione in dsRNA Drosophila Embrioni

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Biologia

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Su larga scala Knockdown Gene in<em&gt; C. elegans</em&gt; Utilizzo di librerie di alimentazione dsRNA per generare fenotipi Robusti Perdita di funzione

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,

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Applicazioni dell'interferenza dell'RNA nello scarafaggio americano

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