이 연구는 대만 (사역의 과학 및 기술, 가장 100-2923-B-002-002-MY3 하 고 H.J.L.를 106-2313-B-002-011-MY3)에서 교부 금에 의해 지원 되었다 체코 공화국 (부여 기관 남쪽 보 헤 미아 대학 GAJU 부여 065/2017/P Y.H.L), 및 스페인 ( 스페인 경제와 경쟁력, CGL2012-36251 및 CGL2015-64727-P X.B., 그리고 카탈루냐어 정부에 부여 부여 2014 SGR 619 X.B.); 그것은 또한 경제 및 지역 개발 (X.B. 페더 자금)에 대 한 유럽 기금에서의 재정 지원을 받았다.

1. 합성 및 dsRNA의 준비
<ol><li>대상 유전자의 3' 되지 않은 지역에 dsRNA 대상 사이트를 식별 합니다. Α-tubulin (욕조) 유전자를 대상으로 사용 되는 dsTub (은행 승인 번호: KX228233), 그리고 dsEGFP의 순서에서 부정적인 dsRNA 제어 설계 향상 된 녹색 형광 단백질 (EGFP; 은행 승인 번호: LC311024).</li>
	<li>T7 발기인 순서를 포함 하는 유전자 특정 뇌관으로 dsRNA 템플릿 합성 표준 PCR 증폭을 수행 (5'-TAATACGACTCACTATAGGG-3'). 린 외PCR 증폭 조건 및 사용 되는 뇌관 정보 그 보고 있습니다. (2017) 12 ( 재료의 표참조).</li>
	<li>DsRNA 준비 T7 전사를 사용 하 여 계속 키트 ( 재료의 표참조), 제조업체의 지침에 따라. 참고: 높은 수량 dsRNA를, (총 40 µ L)에서 1 개의 관에 2 개의 반응을 혼합 하 고 37 ° C에서 밤새 품 어.</li>
	<li>2 µ L DNase의 추가 ( 재료의 표참조) 다이제스트 DNA 템플릿에 37 ° C에서 15 분 동안 dsRNA 샘플으로.</li>
	<li>추가 200 µ L 추출 시 약 ( 재료의 표참조) 40 µ L 클로 프롬, 그리고 소용돌이.</li>
	<li>4 ° C에서 13000 x g에서 10 분 동안 centrifuge 다음 새로운 microcentrifuge 튜브에 상쾌한 (150 µ L)를 수집 합니다.</li>
	<li>150 µ L 소 프로 파 놀을 추가 하 고 얼음에 15 분 동안 잠복기 여 dsRNA를 침전.</li>
	<li>4 ° C에서 13000 x g에서 15 분 동안 centrifuge 다음 상쾌한을 제거 합니다.</li>
	<li>DsRNA 펠 릿을 세척을 70% 에탄올의 200 µ L를 추가 합니다. 4 ° c.에 13000 x g에서 10 분 원심 분리기</li>
	<li>에탄올을 제거 하 고 에탄올 세척 단계를 반복 합니다.</li>
	<li>에탄올과 3 분 동안 원심 진공 집중 하 여 펠 렛 건조 dsRNA 다음 RNase 무료 물 dsRNA를 resuspend.</li>
	<li>마이크로 볼륨 대 일 분 광 광도 계를 사용 하 여 순화 dsRNA의 수량을 결정 ( 재료의 표참조) 제조업체의 지침에 따라 고 원하는 농도 (예를 들면,의 준비를 위해 µ 0.25 µ g/L dsRNA lipoplexes)입니다.</li>
	<li>최대 3 개월 동안-20 ° C에서 dsRNA 샘플을 저장 합니다.</li>
</ol>2입니다. dsRNA Lipoplexes의 준비
<ol><li>Transfection 시 약의 4 µ L를 희석 ( 재료의 표참조) 5% 포도 당 솔루션의 4 µ L을 추가 하 여. 5% 포도 당 솔루션의 4 µ L을 추가 하 여 (1 µ g) dsRNA의 4 µ L를 희석.</li>
	<li>추가 희석된 liposome 시 약 해결책 즉시 희석된 dsRNA 솔루션으로 한 번에. 짧게 그들을 혼합 하 25 ° c.에 15 분 동안 품 어 소용돌이 참고: 역순으로 솔루션을 혼합 하지 마십시오.</li>
	<li>DsRNA lipoplexes 사용 하기 위해 준비가 되었습니다. 준비, 1 시간 이내 사용 하 고 1 시간 후 삭제. 참고: 제조업체의 지침에 따라 lipoplexes는 가능한 빨리 사용 되어야 한다.</li>
</ol>3. Hemolymph Midgut 주스에서 세포 외 효소의 수집
<ol><li>
곤충 염 분 버퍼 (10 x 주식)
	<ol>
<li>두 배 증류수 109.3 g NaCl, KCl 15.7 g, 6.3 g CaCl2와 8.3 g MgCl2·6H2o. 900 mL를 혼합</li>
		<li>PH 6.8를 조정 하 고 최대 1000 mL를 채우기.</li>
	</ol>
</li>
	<li>
Hemolymph 컬렉션
	<ol>
<li>그들은 3 분 동안 이동 하지 않습니다 때까지 얼음에 바퀴벌레를 anesthetize.</li>
		<li>바퀴벌레 (엄지와 검지 손가락), 두 손가락으로 누른 바퀴벌레의 복 부 측을 올려.</li>
		<li>해가 위를 사용 하 여 뒷 다리의 고관절의 끝을 잘라. 참고: 고관절 및 trochanter 사이의 연결된 교차로 잘라. 고관절의 다른 부분의 절단 hemolymph를 수집 하는 비효율적 이었다.</li>
		<li>부드럽게 가운데 손가락으로 복 부를 짜 내 고 10 µ L micropipette 절 개에서 출혈 하는 hemolymph를 수집 합니다.</li>
		<li>Microcentrifuge 튜브에 여러 바퀴벌레 (5 개인)에서 hemolymph를 풀. 참고: Microcentrifuge 튜브 melanization를 방지 하기 위해 얼음에 계속. 남성 바퀴벌레에서 얻은 hemolymph의 평균 볼륨 2-3 µ L 이다.</li>
		<li>10 대 벤치탑 분리기와 짧게는 hemolymph 아래로 회전 s.</li>
		<li>원하는 볼륨 (즉, 10 µ L), hemolymph의 전송 및 1 x 곤충 염 분 버퍼의 50 µ L을 혼합.</li>
		<li>Hemocytes 아래로 회전 하 4 ° C에서 1000 x g에서 10 분 원심 분리기. 깨끗 한 microcentrifuge 튜브는 상쾌한 전송.</li>
		<li>A28015를 측정 하 여 마이크로 볼륨 대 일 분 광 광도 계를 사용 하 여 총 단백질 농도 계량.</li>
		<li>동일한 단백질 농도 (6 mg / µ L의 총 단백질)을 각 샘플을 조정 합니다.</li>
	</ol>
</li>
	<li>
Midgut 주스 컬렉션
	<ol>
<li>얼음에 바퀴벌레 anesthetize 바퀴벌레 복 부 측을 수정 해 부 접시에 찬 1 x 곤충 염 분 버퍼 곤충 핀을 사용 하 여 합니다.</li>
		<li>고급 핀셋으로 복 부를 해 부. 전체 소화 관 (앞, 중간, 그리고 뒷 창 자 포함) 곤충 염 분 버퍼 x 1 신선한 요리를 제거 합니다.</li>
		<li>전면 및 뒷 창 자 제거 하 고 신속 하 게 100 µ L 곤충 염 분 버퍼를 포함 하는 microcentrifuge 튜브는 midgut 전송.</li>
		<li>수영장 6 바퀴벌레 같은 튜브 및 10에 대 한 소용돌이에서 midguts s.</li>
		<li>Hemocytes 및 창 자 조직 회전 하 4 ° C에서 1000 x g에서 10 분 원심 분리기. 깨끗 한 microcentrifuge 튜브는 상쾌한 전송.</li>
		<li>A28015를 측정 하 여 마이크로 볼륨 대 일 분 광 광도 계를 사용 하 여 총 단백질 농도 계량.</li>
		<li>동일한 단백질 농도 (6 mg / µ L의 총 단백질)을 각 샘플을 조정 합니다.</li>
	</ol>
</li>
</ol>4입니다. dsRNA 저하 분석 결과
<ol><li>갓 벗은 dsRNA의 dsRNA lipoplexes (1 µ g) 4 µ L를 준비 합니다.</li>
	<li>믹스 hemolymph, 또는 midgut dsRNA 솔루션 곤충 염 분 버퍼 (제어)의 10 µ L 추출 효소 주스.</li>
	<li>EGTA (20mm) 또는 RNase 무료 물 효소 활동의 저해에 대 한 컨트롤 샘플을 2 µ L를 추가 합니다.</li>
	<li>1 시간 또는 더 많은 (6, 12, 또는 24 시간) 25 ° c.에 품 어</li>
	<li>200 µ L 추출 시 약 및 40 µ L 클로 프롬, 소용돌이 다음을 추가 합니다.</li>
	<li>4 ° C에서 13000 x g에서 10 분 동안 centrifuge 다음 새로운 microcentrifuge 튜브에 상쾌한 (150 µ L)를 수집 합니다.</li>
	<li>150 µ L 소 프로 파 놀을 추가 하 고 얼음에 15 분 동안 잠복기 여 dsRNA를 침전.</li>
	<li>4 ° C에서 13000 x g에서 15 분 동안 centrifuge 다음 상쾌한을 제거 합니다.</li>
	<li>DsRNA 펠 릿을 세척을 70% 에탄올의 200 µ L를 추가 합니다. 4 ° c.에 13000 x g에서 10 분 원심 분리기</li>
	<li>에탄올을 제거 하 고 에탄올 세척 단계를 반복 합니다.</li>
	<li>에탄올과 10 µ L RNase 무료 물으로 3 분 Resuspend dsRNA에 대 한 원심 진공 집중 하 여 펠 렛 건조 dsRNA 제거 합니다.</li>
	<li>1.5 %agarose 젤에 젤 전기 이동 법을 통해 치료 dsRNA의 무결성을 확인 하십시오.</li>
</ol>5. 구강 관리 dsRNA의
<ol><li>건조 식품 (차 개)의 광고 libitum 다이어트 바퀴벌레를 유지 합니다. 바퀴벌레에서 물 공급을 하루 dsRNA 섭취 실험 전에 박탈.</li>
	<li>유연한 집게 (그림 1, 5 단계)와 함께 그것의 날개를 잡아 하 여 바퀴벌레를 잡아. 참고: 바퀴벌레의 신체 부위를 잡아 하지 마십시오.</li>
	<li>준비는 dsRNA lipoplexes, 뿐만 아니라 벌 거 벗은 dsRNA, 먹이, 단원 2에서에서 설명한 대로. 참고: DsRNA lipoplexes 먹이 기 전에 갓 준비 되어야 한다.</li>
	<li>DsRNA lipoplexes 또는 벌 거 벗은 dsRNA의 4 µ L를 피드 (250 ng)는 micropipette와.</li>
	<li>천천히 피펫으로 dsRNA 솔루션의 방울, 바퀴벌레의 mouthparts에 가까운 고 바퀴벌레는 물방울을 완전히 섭취.</li>
	<li>8 일 또는 16 일 지속적으로 섭취 실험을 하루에 두 번 (1 시간 후에 빛 고을 조명 하기 전에 1 시간)을 수행 합니다. 참고: 모든 바퀴벌레는 물만 섭취 실험 동안 dsRNA 해결책 또는 제어 시 약 (예를 들어, nuclease 무료 물, 포도 당 솔루션 및 liposome 시 약)와 수동 먹이 통해 인수.</li>
	<li>DsRNA 실험의 섭취의 16 일 후 바퀴벌레에 물병을 제공 합니다.</li>
</ol>6. 최저 평가
<ol><li>선택한 시간에 포인트 (2, 9, 17 일 dsRNA 섭취 분석 결과 후), dsEGFP 및 dsTub 처리 곤충을 수집 하 고 (예를 들어, midgut) 선택한 조직 해 부.</li>
	<li>실시간 양이 많은 PCR (qRT-PCR)을 수행 합니다. QRT-PCR 증폭 조건 및 뇌관 정보 린 외에서 보고. (2017) 12.</li>
	<li>평가 dsRNA 치료 곤충 매일 사망률을 확인 하 고는 더 이상 바퀴벌레를 제거 하 고 제어 합니다.</li>
</ol>

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<li>Lin, Y. -H., Huang, J. -H., Liu, Y., Belles, X., Lee, H. -J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+delivery+of+dsRNA+lipoplexes+to+German+cockroach+protects+dsRNA+from+degradation+and+induces+RNAi+response%5BTitle%5D)+AND+%22Pest+Manag.+Sci%22%5BJournal%5D)">Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response.</a> Pest Manag. Sci. 73, 960-966 (2017).</li>
<li>Gharavi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chiral+cationic+polyamines+for+chiral+microcapsules+and+siRNA+delivery%5BTitle%5D)+AND+%22Bioorg.+Med.+Chem.+Lett%22%5BJournal%5D)">Chiral cationic polyamines for chiral microcapsules and siRNA delivery.</a> Bioorg. Med. Chem. Lett. 23, 5919-5922 (2013).</li>
<li>Whyard, S., Singh, A. D., Wong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ingested+double-stranded+RNAs+can+act+as+species-specific+insecticides%5BTitle%5D)+AND+%22Insect+Biochem.+Mol.+Biol%22%5BJournal%5D)">Ingested double-stranded RNAs can act as species-specific insecticides.</a> Insect Biochem. Mol. Biol. 39, 824-832 (2009).</li>
<li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+responses+of+migratory+locusts+to+systemic+RNA+interference+via+double-stranded+RNA+injection+and+feeding%5BTitle%5D)+AND+%22Insect+Mol.+Biol%22%5BJournal%5D)">Differential responses of migratory locusts to systemic RNA interference via double-stranded RNA injection and feeding.</a> Insect Mol. Biol. 22, 574-583 (2013).</li>
<li>Liu, J., Smagghe, G., Swevers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+response+of+BmToll9-1+and+RNAi+machinery+genes+to+exogenous+dsRNA+in+the+midgut+of+Bombyx+mori%5BTitle%5D)+AND+%22J.+Insect+Physiol%22%5BJournal%5D)">Transcriptional response of BmToll9-1 and RNAi machinery genes to exogenous dsRNA in the midgut of Bombyx mori.</a> J. Insect Physiol. 59, 646-654 (2013).</li>
<li>Airs, P. M., Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+interference+for+mosquito+and+mosquito-borne+disease+control%5BTitle%5D)+AND+%22Insects%22%5BJournal%5D)">RNA interference for mosquito and mosquito-borne disease control.</a> Insects. 8, 4(2017).</li>
<li>Taning, C. N. T., et al. <a target="_blank" href="https://link.springer.com/article/10.1007/s10340-016-0736-9">Oral RNAi to control Drosophila suzukii: Laboratory testing against larval and adult stages.</a> J. Pest Sci. 89, 803-814 (2016).</li>
<li>Wu, S. Y., McMillan, N. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipidic+systems+for+in+vivo+siRNA+delivery%5BTitle%5D)+AND+%22AAPS+J%22%5BJournal%5D)">Lipidic systems for in vivo siRNA delivery.</a> AAPS J. 11, 639-652 (2009).</li>
<li>Tam, Y. Y. C., Chen, S., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+lipid+nanoparticles+for+siRNA+delivery%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Advances in lipid nanoparticles for siRNA delivery.</a> Pharmaceutics. 5, 498-507 (2013).</li>
<li>Xia, Y., Tian, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+surface+properties+on+liposomal+siRNA+delivery%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Effect of surface properties on liposomal siRNA delivery.</a> Biomaterials. 79, 56-68 (2016).</li>
</ol>

저자는 공개 없다.

이 프로토콜은 독일 바퀴벌레의 midgut 주스에서 ribonuclease 소화에 대 한 보호를 포함 하는 dsRNA lipoplexes의 구두 배달을 통해 효과적인 RNAi에는 메서드를 제공 합니다. 다양 한 곤충 종에 다른 연구에서와 같이, 구두 전달의 dsRNA 통해 가난한 RNAi 효과 대부분 차지 dsRNA8,,910의 저하에 의해. 이 프로토콜은 구두 전달의 dsRNA에에서 저?...

RNAi 많은 진핵생물1dsRNA 분자에 의해 트리거되는 post-transcriptional 입을 통로의 메커니즘을 통해 최저의 유전자 발현을 효과적인 방법으로 입증 되었습니다. 연구의 지난 10 년간, RNAi 주사를 통해 특정 유전자의 표현이 없애고 또는 곤충2,3의 다양 한 taxa에 dsRNA의 먹이로 동작을 개발에서 유전자의 기능 연구를 유용한 도구가 되고있다. 특이성 및 고갈 시키는 효과의 견고성, RNAi의 응용 프로그램은 현재 해충 제어 관리4,5에 대 한 잠재적인 전략으로 간주 되고있다. 그러나, RNAi의 효율 대상 다른 유전자 전달 방법에 따라 곤충 종 사이 널리 다릅니다. 증거의 성장 몸은 ribonucleases에 의해 타락은 dsRNA의 불안정 RNAi5,6의 제한 된 효능에 중요 한 요소는 나왔다. 예를 들어, Manduca sexta 에 낮은 RNAi 감도 사실 hemolymph 섞인 dsRNA 신속 하 게 저하 1 시간7에 의해 설명 되어 있다. 유사 하 게, 효율적으로 섭취 dsRNA 저하, midgut에서 알칼리 nucleases의 존재 다른 곤충 순서8,,910낮은 RNAi 효율성과 강하게 상관 된다.
DsRNA의 구두 전달 특히 해충 제어 전략에 RNAi의 응용 프로그램에 대 한 흥미 롭 지만 midgut에 nucleases에 의해 dsRNA의 저하를 지체 하는 방법을 하지 아직 개발 되었습니다, 어떤 효과적인 보장을 했합니다 먹이 통해 RNAi입니다. 그러나, 구두 전달의 dsRNA에 RNAi의 응답 다량 dsRNA, 누에나방에 예를 들어 50 µ g의 먹이 또는 지속적으로 8 일 (총에서 8 µ g dsRNA) 메뚜기 종에서 먹이 보고 되었습니다. 독일 바퀴벌레, Blattella germinica, dsRNA11,12,,1314의 주입에 의해 RNAi에 매우 민감 하지만 먹이 통해 dsRNA에 응답 하지 않습니다. 최근, 린 외. 그는 dsRNA liposome 사업자 결과 최저에 성공적인 RNAi에 α-tubulin 유전자 발현에에서 캡슐화 독일 바퀴벌레15midgut 및 트리거 상당한 사망률 (2017) 증명. DsRNA는 midgut에의 저하는 구두 RNAi에 대 한 제한 요소, liposome 사업자 저하, 쉽게 다른 곤충에 적용 가능한 용기에 강한 nuclease 활동에서 dsRNA를 보호 하기 위해 차량으로 서브. 메모, 특정 transfection 시 약을 선택 하기 위한 이유의 현재 프로토콜에서 liposome 캐리어는 그것 테스트 되었습니다 곤충 세포 선 transfection에 대 한 더 적은 독성으로 사용 ( 재료의 표참조)에 따라에 제조 업체의 지침입니다. Gharavi 외 에 다른 liposome transfection 시스템의 비교에 따르면 (2013) 16, transfecting 작은 간섭 RNA (siRNA)의 효율은 약이 고 다른 곤충17,18 dsRNA 전달 시스템에 사용 된 다른 상용 시스템 사이의 동일 . 또한, 우리의 먹이 방법은 충분히 신중 하 게 각 바퀴벌레에 의해 dsRNA의 적절 한 양을 섭취 하 고 결과 강력 하 고 확인 합니다. 요약 하자면, 현재 프로토콜 및 결과 dsRNA 안정성 향상 dsRNA lipoplexes를 사용 하 여 하는 해충에 대 한 유망한 접근 미래에 RNAi의 전략 구두 납품의 디자인에 문을 열어 보여줍니다.

<table><tbody><tr><td>GenJe Plus DNA in vitro Transfection reagent</td><td>SignaGen</td><td>SL100499</td><td>for lipoplexes preparation</td></tr><tr><td>Blend Taq plus</td><td>TOYOBO&amp;nbsp;</td><td>BTQ-201</td><td>for PCR</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>ABI&amp;nbsp;</td><td>4385612</td><td>for qPCR</td></tr><tr><td>FirstChoice RLM-RACE Kit</td><td>Invitrogen</td><td>AM1700</td><td>for 3&#039; UTR identification&amp;nbsp;</td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Invitrogen</td><td>AMB13345</td><td>for dsRNA synthesis</td></tr><tr><td>TURBO DNase</td><td>Invitrogen</td><td>AM2239</td><td>remove DNA template from dsRNA</td></tr><tr><td>TRIzol</td><td>Invitrogen</td><td>15596018</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>RQ1 RNase-Free Dnase</td><td>Promega</td><td>M6101</td><td>remove DNA template from total RNA&amp;nbsp;</td></tr><tr><td>chloroform&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C2432</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>I9516</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>ethanol</td><td>Sigma-Aldrich</td><td>24102</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Diethyl pyrocarbonate, DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>for RNase free water preparation</td></tr><tr><td>glucose solution</td><td>Sigma-Aldrich</td><td>G3285</td><td>for lipoplexes preparation</td></tr><tr><td>Sodium chloride, NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>insect saline buffer formula</td></tr><tr><td>Potassium chloride, KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>insect saline buffer formula</td></tr><tr><td>Calcium chloride, CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1016</td><td>insect saline buffer formula</td></tr><tr><td>Magnesium chloride hexahydrate, MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670</td><td>insect saline buffer formula</td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E3889</td><td>enzyme inhibitor&amp;nbsp;</td></tr><tr><td>dissecting scissor</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>fine tweezers</td><td>F.S.T.</td><td></td><td>cockroach dissection</td></tr><tr><td>flexible tweezer</td><td>F.S.T.</td><td></td><td>cockroach holding&amp;nbsp;</td></tr><tr><td>pipetman</td><td>RAININ</td><td>P10</td><td>sample preparation</td></tr><tr><td>microcentrifuge tube</td><td>Axygen</td><td>MCT175C, PCR02C</td><td>sample preparation</td></tr><tr><td>pipette tip&amp;nbsp;</td><td>Axygen</td><td></td><td>sample preparation</td></tr><tr><td>vortexter</td><td>Digisystem</td><td>vm1000</td><td>sample preparation</td></tr><tr><td>Minispin centrifuge</td><td>The Gruffin Group</td><td>GMC 206</td><td>for liquid spin down&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>ALC</td><td>PK121R</td><td>sample preparation</td></tr><tr><td>pH meter&amp;nbsp;</td><td>JENCO</td><td>6071</td><td>for pH adjust</td></tr><tr><td>micro-volume spectrophotometer</td><td>Quawell</td><td>Q3000</td><td>nucleic acid quantitative</td></tr><tr><td>PCR Thermal cycler</td><td>ABI&amp;nbsp;</td><td>2720</td><td>for template PCR or&amp;nbsp; dsRNA synthesis incubation&amp;nbsp;</td></tr><tr><td>quantitative real-time PCR</td><td>ABI&amp;nbsp;</td><td>StepOne plus</td><td>gene expression quantitative</td></tr><tr><td>Centrifugal Vacuum Concentrators</td><td>eppendorf</td><td>5301</td><td>for dsRNA or total RNA extraction</td></tr><tr><td>Multipette&amp;nbsp;</td><td>eppendorf</td><td>xstream</td><td>for real-time PCR sample loading&amp;nbsp;</td></tr><tr><td>Agarose I</td><td>amresco</td><td>0710</td><td>for nucleic acid electrophoresis</td></tr><tr><td>tub gene specfifc forward preimer</td><td>tri-I biotech</td><td></td><td>GGG ACA AGC CGG AGT GCA GA</td></tr><tr><td>tub gene specfifc reverse preimer</td><td>tri-I biotech</td><td></td><td>TCC TGC TCC TGT CTC GCT GA</td></tr><tr><td>dsTub template forward primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&amp;nbsp;</td></tr><tr><td>dsTub template reverse primer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&amp;nbsp;</td></tr><tr><td>dsEGFP template forward preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td></tr><tr><td>dsEGFP template reverse preimer</td><td>tri-I biotech</td><td></td><td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td></tr><tr><td>tub qPCR forward primer</td><td>tri-I biotech</td><td></td><td>GGA CCG CAT CAG GAA ACT GGC</td></tr><tr><td>tub qPCR reverse preimer</td><td>tri-I biotech</td><td></td><td>CCA CAG ACA GCC TCT CCA TGA GC</td></tr><tr><td>ef1 qPCR forward primer</td><td>tri-I biotech</td><td></td><td>CGC TTG AGG AAA TCA AGA AGG A</td></tr><tr><td>ef1 qPCRreverse preimer</td><td>tri-I biotech</td><td></td><td>CCT GCA GAG GAA GAC GAA G</td></tr></tbody></table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

RNA 간섭 (RNAi) 수많은 유전자의 생물 학적 기능을 잠복 근무에 대 한 광범위 하 게 적용 하 고 필수 유전자 발현의 중단에 의해 해충 제어 도구 운영으로 envisaged 되었습니다. 다른 메서드를 주입, 먹이, 및 몸을 담글, 이중 가닥 RNA (dsRNA)의 배달에 대 한 보고 되었습니다, 비록 구두 전달의 dsRNA 통해 RNAi의 효율은 매우 다른 곤충 집단 변수. 이전에 게시 하는 많은 연구에 의해 표시 된 독일 바퀴벌레, Blattella germanica, dsRNA 주입에 매우 민감한입니다. 현재 연구는 dsRNA liposome 사업자와 캡슐화 midgut 주스 dsRNA의 저하를 지체 시키는 충분 한지 설명 하는 방법을 설명 합니다. 특히, dsRNA 리 크게 캡슐화의 지속적인 먹이 midgut tubulin 식 감소 하 고 바퀴벌레의 죽음에 지도. 결론적으로, 수립 및 활용 dsRNA nucleases에 대 한 보호, dsRNA lipoplexes 수 곤충 해충에 대 한 RNAi의 실제적인 사용을 미래에 있습니다.

DsRNA의 구두 전달 프로토콜의 단순화 된 구조는 그림 1, dsRNA lipoplexes의 준비에 대 한 주요 단계 표시 됩니다에 표시 됩니다.
B. germanica, 비보 전 분석 결과 dsTub lipoplexes midgut 주스 incubated 했다 실시 됐다는 dsRNA의 무결성의 midgut 주스에서 dsRNA 저하 시 liposome 사업자에 의해 주어진 보호를 조사 ?...

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA 방해의 실용적인 사용: Liposome 캐리어 바퀴벌레에 대 한 이중 가닥 RNA의 구두 납품

이 원고는의 리에 이중 가닥 RNA의 구강 섭취를 통해 독일 바퀴벌레는 midgut에 유전자 발현의 고갈 보여 줍니다.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

practical-use-rna-interference-oral-delivery-double-stranded-rna

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Biology

9.3K 조회수.  National Taiwan University. 이 원고는의 리에 이중 가닥 RNA의 구강 섭취를 통해 독일 바퀴벌레는 midgut에 유전자 발현의 고갈 보여 줍니다.

비디오: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

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 ...

연구

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 ...

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 ...

발생학

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

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.

질병 벡터 모기 유충의 키토산 / 간섭 RNA 나노 입자 중재 유전자 음소거

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

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

성인 Anopheles 감비아 모기에 효과적인 경구 RNA 간섭 (RNAi) 투여

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

파인우드 선충류에서의 RNA 간섭의 적용, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

유전자 기능 분석을 위한 Trichogramma dendrolimi 의 RNAi 기술 발전

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

저자 스포트라이트: RNA 간섭을 통한 Wolbachia-Induced Thelytokous partthenogenesis 및 Genetic Toolkit Development 조사

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.