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.

정교한 모델 시스템 (마우스, 날아 벌레)의 소수는 20 세기 동안 개발 동안, 21 세기 새로운 동물 시스템의 물결이 전 세계에 걸쳐 연구실에서 개발중인 보았다. 이 새로운 시스템은 과학자 만 &#39;표준&#39;모델 시스템을 사용하여 탐색 할 수 없습니다 비교, 진화의 문제를 해결 할 수 있습니다. 새로운 모델이 배포 실험실 배양, 유전자 식별 및 새로운 종의 기능적 접근 방법의 급속?...

1998 년 화재와 멜로는 이중 가닥 RNA (dsRNA를이) 예쁜 꼬마 선충 1 유전자 기능의 억제를 유도 할 수 있다고보고했다. dsRNA에 의해 유발이 반응은 RNA 간섭 (RNAi의) 명명하고, 이러한 RNAi를 매개 된 유전자 사일런 싱은 동물, 식물, 균류 및 2-7에서 보존되는 것으로보고되었다. 일부 식물 및 동물에서의 RNAi 기능 전신적 의미 효과 (8-10에서 검토) dsRNA를 직접 도입되지 않은 다른 세포 / 조직으로 확산 될 수있다. 과학자하여 직접 (11-14에서 검토) 게놈을 조작없이 유전자 기능을 쓰러 뜨린, 관심의 유전자를 대상으로 dsRNA에를 설계하여이 내생 세포의 RNAi 응답의 사용을 만들었습니다. RNAi의는 다음과 같은 장점으로 인해 기능 연구를위한 강력한 도구입니다. 먼저도 최소한의 유전자 서열 정보하는 유전자의 RNAi를 사용하여 타겟팅 될 수있다. 이 일에 특히 중요하다게놈 또는 transcriptomic 데이터가없는 비 모델 생물의 udies. 둘째, RNAi의 응답이 견고 전신 인 유기체에서 RNAi를 매개하는 유전자 녹다운 거의 모든 발육 단계에서 수행 될 수있다. 이 기능은다면 발현 유전자의 기능을 연구하는데 매우 유용하다. 셋째, 어떤 경우에는, RNAi의 효과 표현형 자손 15,16 관찰되도록 상기 생식선 및 자손에 퍼졌다. 단일 주입 부모에 의해 생성 된 수많은 자손 계란을 직접 조작하지 않고 검사 할 수있는 부모의 RNAi (pRNAi)로 알려진이 현상은,,, 배아 발달에 영향을 미치는 유전자에 특히 유리하다. 이러한 이유로, pRNAi는 선택의 방법입니다. pRNAi 비효율적 인 경우, oogenesis 필요한 유전자, 예를 들어 다음의 RNAi 배아 (eRNAi)이 사용되어야한다. 넷째, RNAi의 강력한 결함 약한 생산하는 dsRNA의 양은 범위에 걸쳐 변화 될 수있는 전달하는 대립 시리즈의 등가를 생성하는데 사용될 수있다. 표현형 그러한 계조 유전자가 치명적인 복잡한 프로세스 및 / 또는 기능을 완전히 상실 포함될 때 유전자 기능을 이해하는데 도움이 될 수있다. 다섯째, dsRNA를 전달 특히 강력한 조직의 RNAi 반응을 나타내는 동물, 일반적으로 쉽게 가능하다. dsRNA를이 미세 주입 1,5, 공급 / 섭취 17, 18, 침지, 19, 20 및 바이러스 / 박테리아 매개 배달 (21, 22)에 의해 도입 될 수있다. 여섯째, 일부 유전자 타겟팅 / 편집 방법과 달리, 돌연변이를 운반 생물을 선별하거나 RNAi의 사용시 동형 접합체를 생성하기 위해 유전 교배를 실시 할 필요가 없다. 따라서, 유전자 기능을 연구하기위한 다양한 다른 기술에 비해 저렴 RNAi를 빠르게하고, 대형 화면 (23-25)에 적용될 수있다. 의 RNAi의 다양한 유틸리티 beyon 연구 가능한 종의 범위를 확대하고, 생물의 광범위한 기능적 연구를 수행하는 방법을 제공한다유전 적 도구가 개발되어있는 기존의 모델 시스템 거라고. 예를 들어, 비 - 모델 시스템을 사용하여 연구가 다른 개발 모드를 표시하거나 별개의 형태 적 특징 26-29을 나타내는 종 orthologs의 기능을 비교하여 유전자 및 유전자 네트워크의 발전에 대한 통찰력을 제공해야한다. 연구 이러한 유형의 응용 및 기본적인 두 연구의 영향이 생물학적 다양성의 더 나은 이해를 제공 할 것이다. 지구상에서 가장 큰 동물 그룹이기 때문에, 곤충 메커니즘을 기본 다양성을 탐험 할 수있는 좋은 기회를 제공합니다. 또한, 곤충은 일반적으로 작고 짧은 수명주기, 높은 생산력을 가지고 있고, 실험실에서 뒤쪽으로 쉽습니다. 지난 20 년에, RNAi의 성공적 파리목 (사실 파리) 5, 나비목 (나비와 나방) (30)를 포함하여 주문에 걸쳐 곤충 적용되어, 딱정벌레 목 (딱정벌레) 16,31, 벌목 (sawf거짓말, 말벌, 개미와 꿀벌) (32), 매미목 (사실 버그), Isoptera (흰개미) (34), Blattodea (바퀴벌레) (35), 메뚜기 (귀뚜라미, 메뚜기, 메뚜기, 그리고 katydids) 36 Phthiraptera (이가) 37. RNAi를 성공적으로 응용 프로그램, 에크 디손 (42) 신호, 초기 배아에서 패턴의 연구 기능 데이터 (전후방 축 (32), 지느러미 - 복부 축 (28), 세그멘테이션 26,38), 성별 결정 39, 40, 키틴 / 표피의 생합성 (41)를 제공하고있다 사회적 행동 (43), 그리고 더. 다른 곤충 종 개발의 RNAi 방법은 (44-46 검토) 방제에 유용 할 가능성이 있다는 점에서 추가적인 이점이 될 수있다. RNAi의 효과는 비 보존 지역을 타겟팅 선택되는만큼, 유전자 특이뿐만 아니라 종 특이 할 것이다. 의 생존을위한 필수적인 유전자를 대상으로 꿀벌과 누에와 같은 유익한 곤충 종에 대한감염을 제어하는 바이러스 또는 기생충이 종 (47, 48)을 보호하기위한 새로운 전략을 제공 할 수있다. Dermestes maculatus (D. maculatus), 일반 이름 숨기기 딱정벌레, 남극 대륙을 제외하고 전 세계적으로 배포됩니다. holometabolous 곤충으로, D. maculatus 수명주기는 배아, 애벌레, 번데기 및 성인 단계 (그림 1)을 포함한다. 그것은 육체에 공급하기 때문에, D. maculatus는 죽은 동물을 해골로하는 박물관으로 사용되며, 법의학 곤충 학자 죽음 49,50의 시간을 예상하는 데 사용할 수 있습니다. D. maculatus는 시체, 말린 고기, 치즈, 그리고 번데기 / 다른 곤충의 고치를 포함한 동물 제품을 공급함으로써 가정의 손상, 저장 음식, 그리고 실크, 치즈, 육류 산업 51,52됩니다. 이 딱정벌레에서의 RNAi를 적용하면 경제에 미치는 영향을 최소화하기 위해 효율적이고 환경 친화적 인 방법을 제공 할 수있다. 우리 연구소는 새로운 m로 D. maculatus을 사용하고있다ODEL 곤충 세그먼트 (53)를 연구한다. 그것은 단기 및 장기 생식 개발 사이의 전환을 공부하고 그것에게 유용한 종을, 중간 - 생식 개발자로 실험실 양육 의무가있을뿐 아니라, D. maculatus는 기초 연구에 대한 관심이다. <img alt="그림 1" src="/files/ftp_upload/54976/54976fig1.jpg" /> 그림 1 : D. maculatus의 수명주기. 나타낸 바와 같이, 서로 다른 삶의 단계에서 D. maculatus의 사진. 성인에 계란에서 라이프 사이클은 낮은 온도에서 세 30 ° C에서 주하지만 더 오래 걸립니다. (A, F) 갓 배치 배아의 길이는 약 1.5 mm, 밝은 노란색 타원형에 흰색입니다. 배아는 ~ 30 ° C에서 55 시간이 걸립니다. (B, C와 G) 애벌레 어두운 색소 줄무늬가 있고 강모로 덮여있다. 애벌레는 1 이상 cm까지 확장 할 수있는 환경과 길이에 따라 여러 령충을 통해 이동합니다. (D, H) </str옹&gt; 젊은 번데기는 황색이다. 30 ° C에서 칠일 - 번데기는 5 ~합니다. 곧 우화 후 (E, I)는, 어두운 색소 침착은 성인 딱정벌레 몸에 나타납니다. 성인은 몇 개월까지 살 수있는 한 여성은 그녀의 일생을 통해 배아의 수백을 배치 할 수 있습니다. <a href="http://ecsource.jove.com/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 이전에, 우리는 RNAi의는 D. maculatus (53)에 유전자 기능을 쓰러 뜨린에 효과가 있음을 보여 주었다. 다음은 실험실에서 D. maculatus 식민지를 양육 경험은 배아와 부모 모두 RNAi의 설정, 사출, 포스트 분사 관리 및 표현형 분석을위한 단계별 프로토콜과 함께 공유됩니다. 여기에 소개 된 dsRNA를 매개 유전자 최저 및 분석 방법 D. maculatus에 문제를 해결에 대한 자세한 정보를 제공 할뿐만 아니라, FO 잠재적 인 의미가 아니라R 아닌 다른 모델 딱정벌레 / 곤충 종의 RNAi를 적용.

<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.

저자의 실험실은 곤충 53, 55의 분할을 조절하는 유전자의 기능 진화를 연구하는 RNAi의 기술을 사용하고있다. 모든 곤충이 분할되어 있지만, 이러한 과정을 조절하는 유전자를 곤충 방사선 26,38,56-63 동안 갈라 것으로 보인다. 초파리 유전자 스크린 바디 세그먼트 64-70의 형성을 촉진하는 책임 아홉 쌍 룰 분할 유전자 세트를 식별. 여기서, 이?...

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

여기, 우리는 실험실에서 중간 - 세균 딱정벌레, Dermestes maculatus (D. maculatus)를 양육하기위한 프로토콜을 제시한다. 우리는 또한이 종에서 유전자 기능을 연구하기 위해 배아 표현형을 분석 배아 및 부모의 RNAi를위한 프로토콜과 방법을 공유 할 수 있습니다.

양육 및 숨기기 비틀에 RNA 매개 유전자 최저를 두 번 좌초,<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 조회수.  University of Maryland.  여기, 우리는 실험실에서 중간 - 세균 딱정벌레, Dermestes maculatus (D. maculatus)를 양육하기위한 프로토콜을 제시한다. 우리는 또한이 종에서 유전자 기능을 연구하기 위해 배아 표현형을 분석 배아 및 부모의 RNAi를위한 프로토콜과 방법을 공유 할 수 있습니다. 

비디오: 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.

진드기의 RNA 간섭

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

연구

면역학 및 감염병학

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.

체 관 부 식물 수액 공급 Hemipteran 곤충에 RNA 간섭을 유도 하 이중 가닥 RNA 구두 전달 방법

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

환경과학

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.

재현할 수 dsRNA Microinjection 및 산란 검정 집 파리 모기에

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.

특정 발달 시점에서 유전자 발현을 조작하기 위한 강력한 도구로 수생 딱정벌레의 RNA 간섭

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.

꿀벌의 RNAi를 매개로 두 유전자 넉다운과 미각 지각 측정 (<em&gt; 아피스 mellifera</em&gt;)

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

신경과학

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).

오도나타에서 전기 기화 매개 RNA 간섭 방법

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

발생학

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.

미국 바퀴벌레에 있는 RNA 간섭의 신청

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.

dsRNA 주입하여 RNAi 간섭에 Drosophila 태아

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

생물학

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.

대규모 유전자 넉다운에<em&gt; C. 엘레</em&gt; 강력한 기능 상실 표현형을 생성하는 dsRNA를 먹이는 라이브러리 사용

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.

양육 및 숨기기 비틀에 RNA 매개 유전자 최저를 두 번 좌초,

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