작품은 부여없이 지원했습니다. &quot;Bundesministerium FÜR Forschung 놀이 Technologie (BMBF)의 01GU0615.

shRNAs 1) 세균성 배달 <ol><li> 세균성 문화와 시작하기 전에, 하나는 LB - 미디엄과 LB - 한천을 준비한다. </li><li> LB - 매체는 효모 추출물 (0.5 % W / V), bacto tryptone (1.0 % W / V) 및 NaCl (0.6 % W / V)을 무게와 아쿠아 bidest에서 이러한 구성 요소를 희석하십시오. 준비 솔루션은 압력솥에 소독을해야하고 사용할 수 있습니다. </li><li> LB - 한천 플레이트에 대한 bacto 한천 (1.5 % W / V)은 살균 이전 LB - 매체에 추가되어야합니다. </li><li> LB - 한천이 완전히 해산 때까지 전자 레인지에 LB - 한천을 가열. 병가 불에받지 않고 쉽게 감동 수있을 때까지 솔루션은 진정하자. 추가 단계에 긍정적인 클론의 선택에 대한 kanamycin을 (100 MG / ML) 추가합니다. </li><li> 10 cm 페트리 - 요리로 LB - 한천 솔루션 20 ML을 pipetting하여 층류 벤치 (무균 조건)에 LB - 한천 플레이트를 준비합니다. 액체가 고체가 될 때까지 플레이트가 진정하자. 접시는 이제 사용할 수 있습니다 4 ° C에 저장할 수 있습니다. </li><li> ShRNA 인코딩 표현 벡터는 관할 E.로 변환 아르 CaCl 2 프로 시저를 사용하여 열 충격에 의해 대장균 ceq221. </li><li> 플레이트 100 μg / ML의 kanamycin이 포함된 LB - 한천 플레이트에 변형 박테리아가, 뚜껑을 닫으 parafilm으로 접시를 봉인하고 37 거꾸로 육성 ° 밤이여 C. </li><li> 치아 선택과 성장 식민지를 채취하여 긍정적인 클론과 박테리아 미니 문화의 예방. 치아가 100 MG / ML kanamycin과 ° C 박 200 rpm으로 쉐이크에서 37 육성을 포함하는 신선한 LB - 매체의 7.0 ML로 선택 전송합니다. </li><li> 100 ML의 예방 신선한 ° C 박 200 rpm으로 쉐이크에서 37여 밤 미니 1리터 Erlenmeyer 플라스크에 문화, 부화 100 MG / ML kanamycin 1:100이 포함된 LB - 중간. </li><li> 시드 25 X 10 4, 6에서 잘마다 인간의 위장 암종 세포 (EPG85 - 257RDB) (씨앗을 품고 세포의 숫자에 따라 세포주의 세포 성장의 속도에 따라 달라집니다 confluency 24 시간 시딩 후 ~ 70-80%해야합니다) - 물론 요리와 37 이들을 품어 ° C, 밤 동안 5 % CO 2, 수증기 포화 분위기 인치 </li><li> 암 세포 감염으로 이전, TK RNAi 벡터가 포함된 E.의 밤 문화를 통해 대장균은 OD 600 결정하는 광도계로 측정하고 있습니다. 0.5의 광학 밀도까지 박테리아 솔루션을 희석하면 (OD 600 = 0.5 1.6 X 10 8 박테리아 세포 / ML와 동일)에 도달입니다. </li><li> FCS없는 중간 공동 배양 세균 이전에 30 분 대한 암종 세포의 FCS 함유 세포 배양 매체를 교체합니다. </li><li> 1 X PBS로 두 번 박테리아를 씻어하고, 혈청이없는 Leibovitz L - 15 중간에 희석. </li><li> 37 암 원하는 뫄에서 세포 (암 세포 당 세균 세포의 감염의 다중성은 = 숫자)와 공동으로 품어 박테리아와 2 시간 동안 암세포에 희석 세균을 추가 ° C. </li><li> 공동 배양 암세포 2 시간 100 U / ML 페니실린, 100 μg / ML 스트렙토 마이신, 2.5 μg / ML amphotericin, 150 μg /로 보충 혈청 함유 Leibovitz L - 15 중간과 함께 한 1X X PBS로 두 번 세탁하고 나서 ML gentamicin, 100 μg / ML의 kanamycin. </li><li> 37 암 세포를 배양 ° C를 5 % CO 2, 수증기 포화 분위기에서 진행합니다. </li><li> 세균성 치료의 효과는 mRNA 레벨에서 정량 실시간 RT PCR로 측정 형광 현미경, 의해와 단백질 수준에서 서부 얼룩 분석하여 시각 수 있으며, 외과 분석 및 세포 증식의 assays 같은 기능 assays로 표시하실 수 있습니다. (원한다면, 다음 절차가 자세히 설명되어 있습니다.) </li></ol> 2) 대표 결과 프로토콜이 올바르게 수행하면, 결과는 아래의 것들과 비교해야합니다. 형광 현미경 그림 1은 세 시간 박테리아 치료 후 인간의 위장 암종 세포를 (A) 치료 세포 (B)에 비해 보여줍니다. 취급 세포의 핵 주위, 박테리아 발견하실 수 있습니다. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="그림 1" /> 그림 1 : 세균성 치료 (1:500) 후 형광 현미경 인간 위장 암종 세포와 치료 인간의 위장 암종 세포 제어로, DAPI - 얼룩, DAPI 밴드 필터 (Λem가 = 640 NM), 40x 목적. 정량 실시간 RT PCR 그림 2 약 70 %의 MDR1 mRNA의 아래 규정 (블랙 빔)은 치료 박테리아 치료 후 볼 수 있습니다. 부모 전지 MDR1 overexpression의 부족으로 인해 긍정적인 제어 역할을합니다. 257RDB p170가 포함된 셀 라인 플라스미드 백신 MDR1 shRNAs의 다른 R로 transkingdom RNAi 기술의 직접적인 비교로 찍은 표현나이는 전략을 입을. 치료 내성 세포주 EPG85 - 257RDB overexpressing MDR1, 플라스미드 표현 shRNA를 부족한 박테리아와 치료 같은 세포 라인이 세포주 플라스미드 표현을 방지 MRP2 shRNAs 들고 치료 박테리아 치료는 긍정적인 컨트롤로 이동되었습니다. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="그림 2" /> 그림 2 : 치료 E. 치료 후 정량적 실시간 PCR RT MDR1 mRNA의 발현 안티 MDR1 shRNAs를 (500 뫄 1) 표현 대장균 ceq221. 정규화는 가사 유전자 aldolase를 사용하여 수행되었습니다. 세포주 EPG85 - 257RDB의 치료 세포의 MDR1/aldolase 비율 100 %를 설정했다. P - 값이 학생의 T - 테스트를 사용하여 계산되었다 (* = P &lt;0.05, ** P = &lt;0.005, ** P = &lt;0.001). 서양 얼룩 분석 그림 3은 규제 아래 MDR1 또한 세균성 치료 후 단백질 수준에서 일어나는 보여줍니다. 그것은 약물에 민감한 부모 세포주 (EPG85 - 257P), 치료 약물 모성 세포주 (EPG85 - 257RDB)의 MDR1 표현과 처리 시료의 MDR1 표현의 부족 MDR1 표현을 보여줍니다. bacterially 취급 세포의 MDR1의 취소 규정은 아래로 볼 수 있습니다. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="그림 3" /> 그림 3 : 서양 얼룩 분석 치료 약물에 민감한 EPG85 - 257P의 MDR1 표현 수준, E. 공동 배양 후 치료 약물 모성 EPG - 257RDB 및 EPG85 - 257RDB니다. 대장균 ceq221 + p43 MDR1. 기본 AB C219 1:100, 및 안티 굴지 1시 5분 000, 보조 AB 방지 마우스 1시 10분 000. 세포 독성 분석 그림 4에 표시된 세포 독성 분석과 같은 기능 분석도 transkingdom RNAi의 기능에 대한 지표입니다. 부모의 세포 라인과 플라스미드 표현 anit - MDR1 shRNA를 포함하는 세포 라인은 daunorubicin 어떤 저항을 보여주지 않습니다. 내성 세포주 EPG85 - 257RDB 두 더욱 컨트롤 Daunorubicin에 대한 저항이 90 %로 되돌릴 수있는 백신 MDR1 shRNA 표현 박테리아로 처리 샘플에 비해 저항에 큰 변화를 표시하지 않습니다. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="그림 4" /> 그림 4 : 세포 독성 분석. 의약품 특정 세포 생존에 대한 세포 독성 분석에 의해 결정 IC50 - 값. P - 값이 학생의 T - 테스트를 사용하여 계산되었다 (* = P &lt;0.05, ** P = &lt;0.005, ** P = &lt;0.001). Anthracycline 축적 분석 그림 5에서와 같이 bacterially 취급 샘플 낮춘 저항 (그림 4)에 의하면, 안티 MDR1 shRNA 취급 세포의 anthracycline 축적은 90 % 정도 증가합니다. 부모 세포주 및 세포주 257RDB p170은 100 %로 강한 daunorubicin 축적을 보여줍니다. 내성 변종은 거의 모든 축적을 보여줍니다 않습니다. 안티 MDR1 shRNA 표현 박테리아로 치료 세포는 90 %의 anthracyline 축적 증가를 보여줍니다. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="그림 5" /> 그림 5 : Anthracycline 축적 분석. 육일 유동세포계측법으로 측정 박테리아 치료 후 암 세포의 Anthracycline의 축적. P - 값이 학생의 T - 테스트를 사용하여 계산되었다 (* = P &lt;0.05, ** P = &lt;0.005, ** P = &lt;0.001).

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

휴대폰 번호는 감염 씨앗과 해당 뫄 사용되는, 비판적으로 세포 관찰하에 라인과 성장을 자신의 속도에 따라 달라집니다. 시딩을위한 최적의 휴대 전화 번호를 찾으려면, 사전 실험 성장의 속도를 결정하는 것이 좋습니다. 이 외에, 다른 MOIs는 세포가 스트레스 죽어없이 박테리아 침공 서있을 수있는 어떤 확장으로 인해 제한을 테스트해야합니다. 관찰에서 유전자의 아래 규정이 다를 수 있습니?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bacterial delivery of rnai effectors: transkingdom rnai

RNA 간섭 (RNAi)는 mRNA의 표현의 구체적인 억제를위한 높은 효과적인 메커니즘을 나타냅니다. 강력한 실험실 도구로서의 가능성이 외에도, RNAi 경로는 치료 활용에 대한 가능성 것으로 보인다. RNA 간섭 (RNAi) 기반 요법, 인도의 개발을위한 RNAi - 중재 대상 세포에 대리인 것이 가장 큰 장애물 중 하나입니다. 이 장애물을 극복하기 위해 소설 전략 transkingdom RNAi (TK RNAi)입니다. 이 기술은 생산과 RNAi를 유도하는 대상 세포로 치료 짧은 헤어핀 RNA (shRNA)를 제공하는 이외의 병원성 세균, 예를 들어 대장균을 사용합니다. 첫 세대 TK RNAi - 중재 벡터, 여행, 관심의 치료 shRNA의 표현 규제에 대한 박테리오 파지 T7 프로 모터가 포함되어 있습니다. 또한, 여행 β1 - 인테 - 긍정적인 포유 동물 세포 listeriolysin O.이 효소를 생산 리스테리아 monocytogenes의 HlyA 유전자를 입력하는 자연 비침 투 박테리아를 허용 invasin을 인코딩 Yersinia의 pseudotuberculosis에서 인보이스 로커스을 가지고하면 치료 shRNA가에서 탈출하실 수 있습니다 항목은 대상 세포의 세포질 내에 vesicles. 여행 구조가 shRNAs의 T7 프로 모터 구동 합성에 필요한 T7 RNA 효소를 인코딩 능력 이외의 병원성 대장균 스트레인로 소개하고 있습니다. 다양한 RNAi 전략에 대해 잘 특징 암 관련 대상 분자가 ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp)입니다. 마약 압출 펌프 및 mediates의 &quot;고전&quot;ABCB1 - 중재 multidrug 저항 (MDR) 특정 교차 저항 패턴이 특징입니다 인간의 암 세포의 표현형으로 ABC - 전송 역할을합니다. 다른 ABCB1 - 표현 MDR 암 세포는 안티 - ABCB1 shRNA 벡터 표현 베어링 E. 취급했다 대장균. 이 절차는 암 세포 내에서 RNAi 경로의 활성화와 ABCB1 인코딩 mRNA의 상당한 아래 규정뿐만 아니라 해당 약품 압출 펌프 결과. 따라서, 약물 축적이 간직한 약물 모성 암 세포의 향상되었으며 MDR의 표현형이 반대했습니다. 이 모델에 의해 데이터가 TK RNAi 인간 암 세포에서 암 관련 요인, 예를 들어 ABCB1의 변조에 적합한 것을 증명 개념을 제공합니다.

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA 간섭 (RNAi) 기반 요법의 개발을위한, 소설 전략은 transkingdom RNAi (tkRNAi)를 개발하였습니다. 이 기술은 생산과 대상 세포로 치료 짧은 헤어핀 RNA (shRNA)를 제공하는 비 병원성 박테리아를 사용합니다. 여기 tkRNAi가 성공적으로 암 세포의 고전 ABCB1 - 중재 multidrug 저항 (MDR)의 반전을 위해 적용되었다.

RNAi Effectors의 세균성 배송 : Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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13.4K 조회수.  Charité Campus Mitte. RNA 간섭 (RNAi) 기반 요법의 개발을위한, 소설 전략은 transkingdom RNAi (tkRNAi)를 개발하였습니다. 이 기술은 생산과 대상 세포로 치료 짧은 헤어핀 RNA (shRNA)를 제공하는 비 병원성 박테리아를 사용합니다. 여기 tkRNAi가 성공적으로 암 세포의 고전 ABCB1 - 중재 multidrug 저항 (MDR)의 반전을 위해 적용되었다.

비디오: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

성인 모기 (A. gambiae)에서 RNAi의 Assays을위한 프로토콜

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

연구

생물학

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

현지화된 RNAi과 약용 거머리의 이소성 유전자 발현

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

성장 저해 Phenotypes의 확인은 효모에서 세균의 종류 III Effectors 표현에 의해 유도된

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

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

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

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

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

RNAi는 Necromenic 선충류의 Microinjection에 의해 유전자 최저 및 Transgenesis을 중재 Pristionchus pacificus</em

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi - 중재 유전자 최저 및<em&gt; VIVO에서</em성인 여자의&gt; 이뇨 분석<em&gt; Aedes aegypti</em&gt; 모기

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

설치류 부고환 정자의 포스 포 분석

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...