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.

Enquanto um pequeno número de sistemas de modelos sofisticados (ratos, moscas, vermes) foram desenvolvidos durante o século 20, o século 21 tem visto uma onda de novos sistemas animais sendo desenvolvidos em laboratórios de todo o mundo. Estes novos sistemas permitem aos cientistas para abordar questões comparativas, evolutivos que não podem ser sondados utilizando apenas os sistemas de modelo &#39;padrão&#39;. Essa implantação de novos modelos requer o rápido desenvolvimento de métodos ...

Em 1998, Fire and Mello relatado que o RNA de cadeia dupla (ARNcd) pode induzir a inibição da função do gene em Caenorhabditis elegans 1. Esta resposta desencadeada por ARNdc foi denominado ARN de interferência (RNAi), e tal silenciamento de genes mediada por ARNi foi relatado para ser conservado em animais, plantas, fungos e 2-7. Em alguns animais e plantas, as funções de ARNi sistemicamente, o que significa que o efeito pode espalhar a outras células / tecidos onde ARNcd não é introduzida directamente (revisto em 8-10). Os cientistas fizeram uso desta resposta RNAi celular endógeno através da concepção de dsRNAs para alvejar genes de interesse, batendo assim, reduzir a função do gene, sem manipular diretamente o genoma (revisto em 11-14). RNAi é uma poderosa ferramenta para estudos funcionais devido às seguintes vantagens. Em primeiro lugar, mesmo com informação da sequência do gene mínimo, um gene pode ser alvejado usando RNAi. Isto é especialmente importante para rudies de organismos não-modelo que faltam dados genômicos ou transcriptomic. Em segundo lugar, em organismos em que a resposta é robustamente ARNi sistémica, RNAi mediada por silenciamento de genes pode ser realizado em praticamente qualquer estágio de desenvolvimento. Este recurso é muito útil para estudar a função de genes pleiotrópicos. Em terceiro lugar, em alguns casos, os efeitos de ARNi para espalhar as gónadas e descendência, tais fenótipos que são observadas em prole 15,16. Este fenómeno, conhecido como parental RNAi (pRNAi), é especialmente vantajoso para os genes que afetam o desenvolvimento embrionário, tão numerosos descendentes produzidos por um único pai injetada podem ser examinados sem manipulação direta de ovos. Por estas razões, pRNAi é o método de escolha. No entanto, se pRNAi é ineficaz, por exemplo, para os genes necessários para a oogénese, então embrionário RNAi (eRNAi) deve ser usado. Em quarto lugar, o RNAi pode ser utilizado para gerar o equivalente a uma série de alelos em que a quantidade de ARNcd entregues pode ser variada ao longo de uma gama para produzir fraco para defeitos fortes. Tal gradação de fenótipos podem ser úteis para a compreensão da função do gene quando o gene está envolvido num processo complexo e / ou perda completa da função é letal. Em quinto lugar, a entrega de dsRNA é geralmente fácil e viável, especialmente em animais que mostram robustos respostas RNAi sistémicos. dsRNA pode ser introduzido por microinjeção 1,5, alimentação / ingestão 17,18, imersão, 19,20 e vírus / entrega de bactérias mediadas 21,22. Em sexto lugar, ao contrário de alguns genes métodos de segmentação / edição, não há necessidade para triagem de organismos portadores da mutação ou para realizar cruzamentos genéticos para gerar homozigotos ao usar RNAi. Portanto, em comparação com muitas outras técnicas para o estudo da função dos genes, RNAi é rápido, barato, e pode ser aplicado para telas de grande escala 23-25. A ampla utilidade de RNAi fornece meios para realizar estudos funcionais em uma ampla gama de organismos, ampliando a gama de espécies disponíveis para estudo beyond os sistemas de modelos tradicionais para as quais foram desenvolvidas ferramentas genéticas. Por exemplo, estudos usando sistemas não-modelo são obrigados a dar insights sobre a evolução de genes e redes de genes, comparando as funções de ortólogos de espécies que representem os diferentes modos de desenvolvimento ou exibindo características morfológicas distintas 26-29. Estes tipos de estudos irá proporcionar uma melhor compreensão da diversidade biológica, com impactos para a pesquisa aplicada e básica. Sendo o maior grupo de animais no planeta, insetos fornecem uma grande oportunidade para explorar os mecanismos diversidade subjacente. Além disso, os insetos são geralmente pequenos, têm ciclos de vida curtos, alta fecundidade, e são fáceis de criar em laboratório. Nas últimas duas décadas, RNAi tem sido aplicado com sucesso em insetos abrangendo encomendas, incluindo Diptera (moscas verdadeiras) 5, Lepidoptera (borboletas e mariposas) 30, Coleoptera (besouros) 16,31, Hymenoptera (Sawfmentiras, vespas, formigas e abelhas) 32, Hemiptera (percevejos), Isoptera (cupins) 34, Blattodea (baratas) 35, Orthoptera (grilos, gafanhotos, gafanhotos e gafanhotos) 36 e Phthiraptera (piolhos) 37. Aplicação bem sucedida de RNAi forneceu dados funcionais para estudos de padronização na embriogênese precoce (eixo ântero-posterior 32, eixo dorso-ventral 28, segmentação 26,38), a determinação do sexo 39,40, quitina / cutícula biossíntese 41, ecdisona sinalização 42, comportamento social 43, e muito mais. ARNi métodos desenvolvidos para diferentes espécies de insectos pode ser um benefício adicional na medida em que eles são susceptíveis de serem úteis para o controlo de pragas (revisto em 44-46). efeitos de RNAi será tão bem como específicas de espécies específicas de gene, enquanto regiões não conservados são escolhidos para segmentação. Para as espécies de insetos benéficos como abelhas e bichos da seda, tendo como alvo genes vitais para a sobrevivência devírus ou parasitas para controlar a infecção pode proporcionar uma nova estratégia para proteger estas espécies 47,48. Dermestes maculatus (D. maculatus), nome comum hide besouro, é distribuído em todo o mundo, exceto na Antártida. Como um inseto holometabolous, o ciclo de vida D. maculatus inclui embrionário, larval, pupal e estágios adultos (Figura 1). Porque se alimenta de carne, D. maculatus é usado em museus para esqueletizar animais mortos e entomologistas forenses podem usá-lo para estimar o tempo de morte 49,50. D. maculatus alimenta de produtos de origem animal, incluindo carcaças, carne seca, queijo e as pupas / casulos de outros insetos e, assim, provoca danos às famílias, alimentos armazenados, e a seda, queijo e indústrias de carne 51,52. Aplicando RNAi neste besouro poderia fornecer uma maneira eficiente e ambientalmente amigável para minimizar o seu impacto económico. Nosso laboratório tem usado D. maculatus como um novo modelo inseto para estudar segmentação 53. Além de ser passível de criação do laboratório, D. maculatus é de interesse para a investigação fundamental, pois é um desenvolvedor intermediário de germes, tornando-se uma espécie úteis para estudar a transição entre o curto eo desenvolvimento a longo germe. <img alt="figura 1" src="/files/ftp_upload/54976/54976fig1.jpg" /> Figura 1: Ciclo de Vida de D. maculatus. Fotografias de D. maculatus em diferentes fases da vida, como indicado. O ciclo de vida de ovo a adulto demora três semanas a 30 ° C, mas mais tempo em temperaturas mais baixas. (A, F) de embriões recentemente estabelecidas são de cor branca a amarelo claro e oval, aproximadamente 1,5 mm de comprimento. Embriogênese leva ~ 55 h a 30 ° C. (B, C e G) As larvas têm listras escuras pigmentados e são cobertas com cerdas. As larvas passam por vários estádios, dependendo do ambiente e seu comprimento pode estender-se a mais de 1 cm. (D, H) </strong&gt; pupas jovens são a luz amarela. Pupation leva ~ 5 - 7 dias a 30 ° C. (E, I) Logo após a eclosão, pigmentação escura aparece sobre o corpo adulto besouro. Os adultos podem viver até vários meses e uma fêmea pode colocar centenas de embriões sobre sua vida. <a href="http://ecsource.jove.com/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Anteriormente, mostramos que a RNAi é eficaz para derrubar a função do gene em D. maculatus 53. Aqui a nossa experiência criação de colônias de D. maculatus em laboratório é compartilhada junto com o passo-a-passo protocolos para ambos embrionário e parental RNAi set-up, injeção, cuidados pós-injeção, e análise fenotípica. O knockdown e métodos de análise de genes dsRNA mediadas introduzidas aqui não só fornecer informações detalhadas para abordar questões em D. maculatus, mas também têm potencial relevância for aplicação de RNAi em outros não-modelo besouro / espécies de insetos.

<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.

Laboratório dos autores tem utilizado tecnologia de RNAi para estudar a evolução funcional dos genes que regulam a segmentação em insetos 53,55. Enquanto todos os insetos são segmentadas, os genes que regulam este processo parecem ter divergido durante radiações insetos 26,38,56-63. Telas genéticos em Drosophila identificou um conjunto de nove genes de segmentação par de regras que são responsáveis por promover a formação de segmentos corpora...

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

Aqui, apresentamos os protocolos de criação de um besouro intermediário de germes, Dermestes maculatus (D. maculatus) no laboratório. Também compartilhamos protocolos de RNAi embrionário e parental e métodos para análise de fenótipos embrionárias para estudar a função de genes nesta espécie.

Criação e Double-stranded mediada por RNA silenciamento de genes na Esconder 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

Research

JoVE Journal

Genetics

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

10.6K visualizações.  University of Maryland.  Aqui, apresentamos os protocolos de criação de um besouro intermediário de germes, Dermestes maculatus (D. maculatus) no laboratório. Também compartilhamos protocolos de RNAi embrionário e parental e métodos para análise de fenótipos embrionárias para estudar a função de genes nesta espécie. 

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

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.

Interferência de RNA em besouros aquáticos como uma poderosa ferramenta para manipular a expressão genética em pontos de tempo específicos de desenvolvimento

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

Ambiente

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 mediado por Gene Knockdown duplo e Mensuração Percepção gustativa em abelhas do mel (<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 ...

Neurociência

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).

Método de interferência de RNA mediado por eletroporação em Odonata

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

Biologia do Desenvolvimento

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.

Aplicações de interferência de RNA na barata americana

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.

RNAi Interferência por injeção dsRNA em Drosophila Os embriões

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

Biologia

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi Mediated Gene Knockdown e Transgénese por microinjeção no Nematóides Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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.

Em larga escala Knockdown Gene em<em&gt; C. elegans</em&gt; Usando bibliotecas de alimentação dsRNA para gerar fenótipos robustos perda de função

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.

A interferência RNA larval no Red Flour Beetle,<em&gt; Tribolium castaneum</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

A quitosana / RNA de interferência Nanoparticle Mediated silenciamento gênico na Doença Vector Mosquito Larvas

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi gatilho para o interior<em&gt; Anopheles gambiae</em&gt; As pupas

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Criação e Double-stranded mediada por RNA silenciamento de genes na Esconder Beetle,

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