Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

1. 모기 종과 양육을 <ol><li> A. 유지 gambiae의 G3와 A. aegypti 리버풀 IB12의 (아래의 대표 연구에 사용) 균주 또는 표준 실험실 연습에 또는 이전에 23, 24 설명한대로 따라 관심의 다른 균주. </li></ol> 2. dsRNA를하고 siRNA를 디자인 및 제작 <ol><li> 디자인 프라이머는 관심의 유전자에 특정 긴 dsRNA를 템플릿을 구성합니다. E-RNAi의 도구 (25)를 사용합니다. 같은 또는 선택의 방법에 따른 dsRNA를 생산 이전에 설명 19. <ol><li> 음성 대조군의 경우, 녹색 형광 단백질 (GFP), β - 갈 락토시다 제 (β-GAL), 또는 모기로 표현하지 다른 유전자의 서열에 해당하는 dsRNA (19)를 합성. 목적한다면, 다음과 같이 요약 대표적인 결과를 생성하기 위해 이용 된 dsRNA (19)은 양성 대조군으로 사용할 수있다. 스토어 dsRNA를 -80 °에서 RNAse가없는 물에 용해 된; C가 필요할 때까지. </li></ol></li><li> 또한, 디자인 유전자 특이 적 siRNA를 표준 실험실 관행에 따라 또는 이전 10 설명했다. 모든 모기 유전자에 대응하지 않는 음성 대조군 siRNA의 설계를 최저의 siRNA 시퀀스를 스크램블링. 신뢰할 수있는 공급 업체의 숫자를 통해 사용할 수있는 사용자 정의 된 siRNA를 구입합니다. <ol><li> 목적한다면, 다음과 같이 요약 대표적인 결과를 얻기 위해 이용 된 siRNA (20, 21)은 양성 대조군으로 사용할 수있다. 필요할 때까지 보관 된 siRNA는 -80 ° C에 RNAse가없는 물에 용해시켰다. </li></ol></li></ol> 3. R​​NA 나노 입자를 방해 / 키토산 준비 <ol><li> 미리 만들어진 RT 100 mM 염화나트륨, 황산 수집 (탈 H 2 O 100mm의 나 2 SO 4), 0.1 M 아세트산 나트륨 (탈 H 2 O 중 0.1 M NAC 2 H 3 O 2 -0.1 M 아세트산, pH를 4.5) 버퍼. </li><li> 키토산을 용해 (≥75 %탈 아세틸) 0.1 M 아세트산 나트륨 완충액 () V / w 작업 용액 0.02 %를 만들기 위해 사용하기 전에 실온에서 용액을 유지한다. </li><li> H 2 O 탈 50 μL에서의 dsRNA 또는 siRNA에 용해시키고 50 mM 염화나트륨, 황산 100 ㎕ 당하는 dsRNA / siRNA를 32 μg의의 100 μL 용액을 만들기 위해 100 mM의 황산 나트륨 버퍼에 추가한다. </li><li> dsRNA에 / siRNA의 용액에 키토산 용액 100 μl를 첨가하고, 그 다음 1 분 동안 55 ℃에서 수욕에서 혼합물을 가열한다. 키토산 용액 100 ㎕ 50 mM 염화나트륨, 황산 100 ㎕를 첨가함으로써 제어를 설정하고 동일한 절차를 따른다. </li><li> 나노 입자의 형성을 촉진하기 위해 RT에서 고속 텍싱 30 초 동안 즉시 용액을 섞는다. </li><li> 펠릿 볼 수 있어야 그 이후 실온에서 10 분 동안 13,000 XG에서 혼합물을 원심 분리기. 새로운 1.5 ML 튜브에 뜨는을 전송합니다. 모기 음식을 준비하기 위해 그것을 사용하기 전에 실온에서 ≈10 분 펠렛을 공기 - 건조. </li><li> M상청액에 남아하는 dsRNA / siRNA와의 총량을 계산하는 블랭크로서 대조군 (3.5 참조)를 사용하여 상등액 상등액의 dsRNA /의 siRNA 농도를 easure. 초기의 dsRNA / siRNA의 양 및 나노 입자에 포획 된 dsRNA / siRNA의 백분율을 계산하는 상청액에 남아있는 양의 차이를 사용한다. 이 적재 효율은 일반적으로 90 % 이상이다. </li><li> 나노 입자가 더 필요한 경우 동일한 절차를 반복한다. 즉시 건조 된 나노 입자를 사용합니다. 사용하기 전에 입자의 저온 저장의 영향은 평가되지 않았다. </li></ol> 4. 모기 음식 RNA 나노 입자를 방해 / 키토산을 포함하는 시스템 준비 <ol><li> 증류수에 아가 로스를 용융 (V / w) 아가로 오스 2 % 용액 1 ㎖를 제조하고, 사용 전에 55 C 수욕에서 용융 아가로 오스 용액을 유지한다. </li><li> 물고기 음식 부스러기를 혼합 (47 % 조단백질, 분. 조지방 10 %, 최대. 조섬유 3 %)에서 건조 효모(w / w) 1 : 2의 비율. 박격포와 유 봉 (50 위 미국 표준 시험 체를 통해 무난)와 작은 입자의 혼합물을 갈기. 중 즉시, 색상 갈색이어야한다 분쇄 된 음식물을 사용하거나 몇 주 동안 4 ℃에서 밀봉 된 용기에 보관하십시오. </li><li> 1.5 ML 튜브에서, 이쑤시개와 3.7 절에서 건조 된 나노 입자로 분쇄 된 음식물의 6 mg의 혼합. </li><li> 식품 나노 입자 혼합물을 2 % 예비 용융 아가 로스 겔 용액 30 μL를 추가; 이쑤시개 또는 피펫 팁을 사용하여 즉시 철저하게 약동하십시오. </li><li> 바로 모기 유충을 공급하는 음식과 나노 입자를 포함하는 겔을 사용합니다. 젤이 완전히 실온에서 응고되면 다른 방법으로, 4 ° C에서 젤을 저장하고 사용 다음날, 또는 나중에 사용하기 위해 -80 ° C에서. </li></ol> 5. 식품 RNA 나노 입자를 방해 / 키토산을 함유 모기 유충을 먹이 <ol><li> 수유 A. gambiae 모기 유충 : <ol><li> 시 제거이쑤시개를 사용하여 깨끗한 면도날이나 이쑤시개를 사용하여 6 동등한 조각으로 잘라 1.5 ML 튜브에서 ngle 젤 펠릿. </li><li> 100 ml의 탈 이온수를 함유하는 500 ml의 배양 접시에 20 번째 전송 령 유충. </li><li> 페트리 접시에 한 번 사일을위한 하루 겔 펠렛 (미세하게 작은 조각으로 잘게) 한 조각을 추가하여 모기 유충을 먹이. 크게 크기가 감소 또는 다음날에 의해 완전히 결석해야 펠릿에 애벌레 먹이를 준수해야합니다. 시간이 지나면 모기 늦게 넷째 령 애벌레로 발전 할 것이다. </li><li> 실험 기간 동안 눈에 보이는 표현형의 변화를 기록한다. 네 일의 기간이 종료 6 절에 설명 된대로 성적 수준과 다른 표현형의 변화를 검사합니다. </li></ol></li><li> 수유 A. aegypti 모기 유충 : <ol><li> 깨끗한 면도날이나 이쑤시개를 사용하여 6 동등한 조각으로 젤 펠렛을 잘라. </li><li> 달걀 부화 Fi를 한 후 50 세 동기 24 시간 배치탈 이온수 ≈40 용액에 배양 접시에 첫 령 유충. </li><li> 하루 중 나머지 1 접지 물고기 음식 부스러기와 건조 효모 : 다음이 정기적으로 애벌레 다이어트로 다시 유충을 전송, 4 시간 / 일에 대한 페트리 접시 당 유충 한 조각을 넣습니다. 매일 네 령 유충에 걸쳐 절차를 반복합니다. </li><li> 원하는 발달 시점에서 6 절에 설명 된대로 성적 수준과 다른 표현형의 변화를 검사합니다. </li></ol></li></ol> 유전자 넉다운 6. 확인 <ol><li> A.에 QRT-PCR에 의해 상대 성적 증명서 정량 aegypti 및 A. gambiae 애벌레. <ol><li> 수행하고 11,19,20 설명, 또는 표준 실험실 절차에 따라 같은 실험 유충 대 이상 10 풀링 제어 세 생물학적 복제물에 QRT-PCR 분석을 분석 할 수 있습니다. 대표 결과가 들어 있습니다. </li><li> 특정 조직 유형 / 신체 일부 (예., 뇌 또는 안테나), PERFO의 분석을 위해관심있는 조직을 복구하기 위해 다음의 6.1.1에 기재된 바와 같이 절개 RM QRT-PCR은 (예를 들면, 소르 등. 21). </li></ol></li><li> 현장 하이브리드 화에 의한 최저의 확인 <ol><li> digoxygenin 표지 안티센스를 합성 표준 실험실 연습에 또는 26 설명 된대로 따라 제어 리보 프로브를 감지. </li><li> 이전에 11,20을 설명하고 아래의 대표적인 결과 섹션에서 논의 된 바와 같이 최저의 확인을 위해 저자 Haugen 등. (27) 프로토콜에 따라 현장 하이브리드에 실행합니다. </li></ol></li><li> 면역 조직 화학 염색에 의한 최저의 확인 <ol><li> 표적 유전자의 단백질 생성물에 대한 항체를 사용할 수있는 경우 이전에 기술 된 바와 같이 28, representati에 예시 된 바와 같은 표현형 분석에서 20 녹다운의 최대 수준의 개인 식별을 용이하게, 면역 조직 화학 법을 수행아래의 결과 섹션을했습니다. </li></ol></li></ol>

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The authors have nothing to disclose.

본원에 기술 된 키토산 / RNA 간섭 나노 방법론 효과적으로 A.에서 유생 중에 유전자를 대상으로 사용되어왔다 gambiae (도 2, 3) 및 A. aegypti (도 4, 5, 6, 표 1, 2). 키토산 나노 입자는 본원에 기재된 대표적인 결과에 의해 입증되는 바와 같이 모기에서 성공적으로 사용되어왔다 둘 또는 긴 dsRNA에 siRNA를, 어느 제조 될 수있다. dsRNA에의 합성 siRNA를 구매보다 저?...

Anopheline과 Aedine 장군의 혈액 공급 벡터 모기는 인류 최악의 재앙의 몇 가지에 대한 책임 질병을 유발하는 에이전트를 전송합니다. 추정 34억명는 매년 전 세계에 걸쳐 절반 만 죽음에 대한 책임이 말라리아, 계약에 대한 위험이 있습니다. 말라리아 특검팀에 의해 감염 말라리아 결과. 주요 아프리카 벡터과 아노 펠 레스 gambiae (를 포함하여 얼룩 날개 속의 감염된 모기의 물린 상처를 통해 사람에게 전송되는 기생충, <a href="http://www.who.int/topics/malaria/en/">http://www.who.int/topics/malaria/en/</a> 2014) 1. Aedes aegypti는 뎅기열, 세계에서 가장 광범위하고 중요한 arboviral 질병 비특이적 열성 질환을 일으키는 뎅기 바이러스에 대한 기본 모기 벡터이다. 뎅기열 바이러스는 매년 inciden으로, 현재 열대&gt; 25억명에 대한 위협이다24,000 사망자 매년 ~의 결과로 약 5천만가지 경우의 CE ( <a href="http://www.cdc.gov/dengue/">http://www.cdc.gov/dengue/</a> 2014) 2. 인간의 건강에 모기 매개 질병의 파괴적인 글로벌 충격에도 불구하고, 예방 및 질병 치료의 효과적인 수단이 부족하다. 모기 제어는 현재 질병 예방의 가장 좋은 방법입니다. 벡터 곤충의 유전자 조작에 의해 절지 동물 매개 질병을 제어하기위한 잠재력은 지난 4 년간 3 인정을 받고있다. A.의 형질 전환 균주 최근 형질 전환 벡터 제어 전략을 현실로 4-6를 사용하기위한 가능성을 만든 aegypti repressible 여성 고유의 날지 표현형을 가지고 설계. 이러한 발전은 벡터 및 벡터 제어 모기 유전자 기능을 조작하기위한 수단을 추가로 신규 한 유전자 표적을 식별하기 위해 연구에 도전했다. 유전자 발현 (D)의 변경을여성이 날지 모기 4의 경우가 있다는 uring 개발, 새로운 벡터 제어 전략의 해명을 촉진 할 수있다. 그러나, 대부분 기술적 과제를 극소수의 유전자 기능의 개발 과정 A. 특성화되었다 gambiae, A. aegypti의, 또는 다른 모기 종. C.에서의 발견 이후 엘레 7은, 동물, 식물, 미생물에 보존되는 RNA 간섭 (RNAi에)는, 광범위 곤충 8,9 포함한 생물체의 다양한, 기능성 유전 연구에서 사용되어왔다. RNAi의 경로는 순서 특정 간섭 RNA의 기능을 짧은 20 ~ 25 염기 길이의 siRNA를로 다이 서 (Dicer), 절단 긴 dsRNA에 의해 시작됩니다. 성적 증명서 회전율, 분열, 번역 (9)의 중단을 촉진하여 순서에 상보적인 siRNA를 침묵 유전자. particula을 대상으로 긴 dsRNA를 분자 (일반적으로 300 ~ 600 BP) 또는 사용자 정의 된 siRNAR 열은 임의의 관심있는 유전자 사일런 싱에 대한 연구 실험실에서 사용될 수있다. RNA가 전달을 방해 할 때 관리함으로써, 연구진은 유전자에 동수를 침묵 시간을 제어 할 수 있습니다. 그것은 생산과 유전적인 돌연변이, 아직 모든 곤충 종에 존재하지 않는 비용과 노동 집약적 인 프로세스를 베어링 균주의 유지를 방해 할 수 있습니다 이러한 발달 치사 또는 불임 등의 문제를 극복하는 데 사용할 수있는 이러한 장점에 유용합니다. RNAi에 의한 유전자 침묵의 과정은 동물에게 유전자를 조직의 조직 및 동물 유전자에서 달라질 수 있지만, RNAi를 널리 모기 및 다른 곤충 8,9에서 유전자의 기능 분석을 위해 사용된다. 세 간섭 RNA 배달 전략은 모기에 사용되었습니다 미세 주사, 전신 / 국소 응용 프로그램 및 섭취. 자세한 역사와 곤충이 세 가지 기술의 사용의 비교를 위해, 유 등의 알 8을 참조하시기 바랍니다. WE 성공적 A.에서 발달 유전자를 표적으로 siRNA를 전달하는 수단으로서 마이크로 인젝션 10을 사용한 aegypti 배아, 유충, 번데기 11-14. 그러나이 노동 집약적 인 배달 전략은 미세 주사 설정 및 숙련 된 손 모두를 필요로한다. 또한, 미세 주입은 행동 표현형이 부과됩니다 특히, 생물, 교란 요인에 스트레스. 마지막으로, 마이크로 인젝션 배달 벡터 제어를위한 필드로 확장 될 수 없다. 편리하고 작은 장비 또는 노동을 필요로하는 대신, RNA 간섭을 용액에 담그는 유기체 또한, 유전자 침묵을 유도하는 인기 수단이되었다. 잠기기는 주로 곤충 세포 라인에 적용된 것은 8을 연구하지만, 최근의 연구에서, 최저는 A에 달성되었다 aegypti 유충 동물이 섭취하는 dsRNA 것으로 나타났다 (15)의 용액에 침지. 그러나, 여러 experimenta의 분석과 관련된 연구L 그룹이나 표현형은 몸을 담근 채 오히려 비용이 많이 든다. 로페즈 마르티네스 등. 16의 RNAi, RNA 간섭을 함유하는 물을 한 방울과 식염수 탈수 재수 관련된 RNA 전달을 방해하기위한 새로운 접근 방식을 재수 구동을 설명했다. 이 접근법은 전체 동물의 침지와 관련된 비용을 절감하지만 미세 주입보다 더 비싼 높은 삼투압을 허용 할 수있는 종의 적용이 제한 될 수있다 않는다. 또한, 침지 또는 탈수 / 재수 침지 방법이 필드 벡터 제어하도록 적응 될 수있는 방법을 구상하는 것이 곤란하다. 이런 이유로, 이후의 배아 연구를 들면 음식 섭취 RNA 간섭의 배달 대안 전략이다. 섭취 기반 전략이 모든 종의 곤충, 아마도 특히 초파리에서 작동하지 않지만, 음식과 혼합 RNA 간섭의 구두 전달은 유전자의시를 승진시켰다곤충 8,17의 다양한 lencing 포함 A. aegypti 성인 18. 우리는 A에 키토산 나노 입자 매개 RNAi의 설명 gambiae 유충 (19) 성공적으로 A의 유전자 발현의 감소를위한이 방법을 적용했습니다 aegypti 애벌레 (20, 21). 여기서, 고분자 키토산 RNA 간섭의 포획이 포함 RNAi의 절차를위한 방법은, 상세하다. 키토산 / 간섭 RNA 나노 입자 키토산의 아미노기의 양전하와 RNA 19 간섭의 백본에 인산기에 의해지지 마이너스 전하 사이의 정전기력을 통해 RNA 간섭과 폴리 양이온의 자기 조립에 의해 형성된다. 설명 된 절차는 긴 dsRNA를 분자 (이하의 siRNA로 지칭)을 가닥의 siRNA (이하 dsRNA를 라 함) 또는 이중 모두와 호환된다. 다음 합성, 키토산 / 간섭 RNA 나노 입자는 애벌레 음식과 혼합에게 전달됩니다경구 섭취를 통해 유충. 이 방법론은, 비교적 저렴 작은 장비 및 인력 (19)이 필요하며, 동작 (20, 21)의 분석을 포함하는 다수의 표현형의 높은 처리량 분석을 용이하게한다. 다른 질병 벡터 및 곤충 농업 ​​해충 포함한 다른 곤충에서 유전자 침묵 과정에 대해 적응 될 수있다이 방법은, 잠재적으로 다른 동물 종의 다양한 유전자 침묵을 위해 사용될 수있다. 또한, 키토산, 저렴 무독성 생분해 성 고분자 (22)는 잠재적으로 종 특이 모기 제어를위한 분야에서 이용 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1429">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Reagent/Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:177px;">Catalog Number</td>
			<td style="width:663px;">Comments/Descriptions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR20</td>
			<td>for resuspension of interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR200</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-120</td>
			<td>for aliquoting resuspended interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml pipetteman</td>
			<td>Rainin</td>
			<td>PR1000</td>
			<td>for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml graduated tube&#160;</td>
			<td>Fischer Scientific</td>
			<td>05-408-046</td>
			<td>for preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Sodium Acetate, pH 4.5</td>
			<td>TEKnova</td>
			<td>S0299</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic Acid, AIRSTAR. ACS, USP, FCC Grade</td>
			<td>BDH</td>
			<td>VWR BDH3092-500MLP</td>
			<td>to prepare sodium acetate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose Genetic Technology Grade</td>
			<td>MP Biomedicals LLC.</td>
			<td>800668</td>
			<td>to coat prepared interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf AG</td>
			<td>5415D</td>
			<td>to pellet interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, from shrimp shells</td>
			<td>Sigma-Aldrich</td>
			<td>C3646-25G</td>
			<td>to combine with interfering RNA for prepararation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Yeast</td>
			<td>Universal Food Corp</td>
			<td>NA</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-RNAi tool</td>
			<td>German Cancer Research Center</td>
			<td>NA</td>
			<td>for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3//</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goldfish food</td>
			<td>Wardley</td>
			<td>Goldfish FLAKE FOOD</td>
			<td>to prepare mosquito larval food with interfering RNA/nanoparticles</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Heated water bath</td>
			<td>Thermo Scientific</td>
			<td>51221048</td>
			<td>to heat the interfering RNA/nanoparticles at 55oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>for thawing interfering RNA and preparation of interfering RNA/nanoparticles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>02-591-44</td>
			<td>for storage of ice used during the procedure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">liver powder</td>
			<td>MP Biomedicals LLC.</td>
			<td>900396</td>
			<td>to feed mosquito larvae post interfering RNA/nanoparticle treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microwave oven</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>to prepare 2% agarose solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">petridish (100X15 mm)</td>
			<td>Fischer Scientific</td>
			<td>875713</td>
			<td>interfering RNA/nanoparticle feeding chamber for larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td>for preparation of buffers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blade</td>
			<td>Fischer Scientific</td>
			<td>12-640</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">siRNA</td>
			<td>Thermo Scientific/Dharmacon</td>
			<td>custom</td>
			<td>for preparation of siRNA/nanoparticles</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sodium Sulfate, Anhydrous (Na2SO4)</td>
			<td>BDH/distributed by VWR</td>
			<td>VWR BDH0302-500G</td>
			<td>to prepare 50mM sodium sulfate solution in which the interfering RNA will be resuspended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermometer</td>
			<td>VWR</td>
			<td>61066-046</td>
			<td>to measure the water bath temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tooth picks</td>
			<td>VWR</td>
			<td>470146-908</td>
			<td>for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ultralow freezer</td>
			<td>A variety of vendors</td>
			<td>not applicable</td>
			<td>for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex mixer</td>
			<td>Fischer Scientific</td>
			<td>02-216-108</td>
			<td>for preparation of chitosan/interfering RNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Weight paper</td>
			<td>Fischer Scientific</td>
			<td>NC9798735</td>
			<td>to divide the interfering RNA/nanoparticle pellet for feedings</td>
		</tr>
	</tbody>
</table>

chitosan/interfering rna nanoparticle mediated gene silencing in disease vector mosquito larvae

벡터 모기는 다른 생물보다 더 인간의 고통을 입힐 - 이상 백만명 매년 죽인다. 모기 게놈 프로젝트는 기본 아프리카 말라리아 벡터과 아노 펠 레스 gambiae 및 뎅기열과 황열병 벡터 Aedes aegypti의 유전학적인 연구를 포함하여 모기 생물학의 새로운 측면에서 연구를 촉진. RNA 간섭 - (RNAi-) 매개 유전자 침묵은 이러한 질병 벡터 모기 종 모두에 대한 관심의 유전자를 대상으로 사용되어왔다. 여기서는 음식 합하고 유충 섭취 RNA 간섭 나노 입자 / 키토산의 제조 과정을 설명한다. 긴 이중 가닥 RNA (dsRNA에) 또는 작은 간섭 RNA (siRNA의) 분자와 호환이 기술적으로 간단 높은 처리량 및 비교적 저렴한 방법은, A. 다른 유전자의 숫자의 성공적인 최저 사용되어왔다 gambiae 및 A. aegyptiQRT-PCR을 통해 또는 현장 하이브리드에 확인 유충. 애벌레의 먹이에 따라, 최저는, 적어도 후반 번데기를 유지할 수 있습니다. 이 방법론은 모기 및 농업 해충 곤충을 포함하는 다른 종의 다양한뿐만 아니라 다른 비 유기체 모델에 적용될 수있다. 연구 실험실에서 유용성 외에도 향후, 키토산, 저렴하고, 비 독성 및 생분해 성 중합체, 잠재적 분야에서 이용 될 수있다.

<html> A. gambiae : 키토산 / 나노 dsRNA에 의한 키토산의 아미노기와의 dsRNA의 인산기 간의 정전 상호 작용으로 형성된다. 용액으로부터의 dsRNA의 고갈에 의해 측정하는 나노 입자에 혼입하는 dsRNA의 효율은 일반적으로 90 % 이상입니다. 원자력 현미경 이미지 표시하는지 100-200 nm의 (도 1)에 이르는 직경 키토산 dsRNA에 입도 140 nm의 평균. <p class="jove_c...

Watch this Scientific Journal Video about Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae at JoVE.com

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

여기서 우리는 유충 섭취 RNA 나노 입자 / 키토산의 사용을 통해 질병 벡터 모기 유전자 기능을 억제 간섭하는 절차를 설명한다.

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 ...

chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Research

JoVE Journal

Biology

14.2K 조회수.  Kansas State University.  여기서 우리는 유충 섭취 RNA 나노 입자 / 키토산의 사용을 통해 질병 벡터 모기 유전자 기능을 억제 간섭하는 절차를 설명한다. 

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

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

바이러스 유도 진 입을 (VIGS)에 Nicotiana benthamiana 토마토

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

연구

생물학

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다 2 + ER 막의 누출 채널과 규제 메커니즘

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

커튼에서 Agrobacterium - 중재 바이러스 유발 유전자 입을 분석

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribosome 바운드 초기 Polypeptides의 분리<em&gt; 체외에서</em&gt; mRNA 함께 Translational 일시 중지 사이트를 식별하려면

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

모기 내장 Metagenomic RNA-seq에 대한 Ribosomal RNA의 고갈

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

류신 리치의 신진 대사 라벨링 방사성 인산과 키니 아제 1과 2를 반복

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

siRNA를 매개 음소거로 마우스 난 모세포의 감수 스핀들 평가

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

초파리 유충에 있는 Oenocytes의 해부 그리고 지질 물방울 염색

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...