우리는 다양한 개발 시간 지점에서 동물의 숫자를 제공하기 위해 제임스 클락을 감사드립니다. C. 본 연구의 elegans 변종은 연구 자원에 대한 NIH 국립 센터 (NCRR)에 의해 지원됩니다 Caenorhabditis 유전학 센터에서 얻은되었습니다. 이 작품은 CUNY에서 JL과 CSD에 CIRG 1817에 의해 지원되었다, 그리고 NIH 1R15GM073678-01와 CSD로 1R15GM097692-01에 의해 우리는 원고에 대한 의견에 박사 윌리엄 J 쌀, 박사 Nathalia Holtzman, 그리고 멜리사 Silvestrini 감사드립니다. 이 작품은 뉴욕시 대학 (S. Xiong)의 대학원 센터에서 박사 학위에 대한 요구 사항의 일부 이행 실시되었다.

1. 대상 유전자 시퀀스을 지니고 RNAi 수유 플레이트 준비 <ol><li> 상업적으로 이용 가능한 RNAi 라이브러리 (예를 들면 소스 BioScience LifeSciences에서) 사용하는 경우, 1.4 단계로 진행합니다. 또한, 표준 복제 프로토콜, 벡터 L4440, 8 RNAi 플라스미드 일반적으로 사용되는 웜으로 대상 유전자 시퀀스를 복제. </li><li> 박테리아 변종 HT115 (DE3), 25 μg / ML carbenicillin과 LB 한천 플레이트에 문화를 및 12.5 μg / ML의 테트라 사이클린에 재조합 플라스미드를 변환하고, 향후 사용을위한 단일 클론을 선택합니다. </li><li> 한편, 실험에 대한 제어로 사용하기 위해 박테리아 변종 HT115에 빈 벡터 L4440을 변환 할 수 있습니다. </li><li> 37 번 하루 아침에 100 μg / ML 암피실린 1 ML LB의 국물 ° C.의 문화 재조합과 빈 모두 L4440 운반 세균 테트라 사이클린은이 RNAi 효율을 감소한다는 사실로 인해 추가되지는 않습니다. </li><li> 밤새 cultu에 100 μg / ML 암피실린과 함께 앞으로 5 ML의 LB의 국물을 추가다시, 37에서 다른 4-6 시간에 품다 ° C. </li><li> 종자 0.5 ML 재조합 또는 빈 L4440 운반 RNAi 웜 접시에 박테리아 문화를. 클론 이름으로 모든 접시를 붙입니다. </li><li> 접시를 선택 37에서 하룻밤 배양 ° C 세균 잔디를 성장하기 위해, 이것들은 목표 유전자 순서에 상관없이 RNAi 판입니다. 최소 2 플레이트는 각 조건에 대해 준비해야합니다. </li></ol> 2. RNAi 수유 접시에 문화 웜 <ol><li> 불꽃은 백금 와이어 웜 피크의 끝을 소독. 6-10 네번째 애벌레의 단계 (L4)이 중 재조합 또는 빈 L4440를 포함 각 RNAi 판에 나온 것 데리러 선택 웜을 사용, 동물 식품 소스로 박테리아를 사용합니다. </li><li> 광고에 나온 것 ... 20 ° C (C. elegans에 대한 표준 문화 조건) 밤에 성장하자, 그들은 다음 날 젊은 성인이 될 것입니다. </li><li> 새로운 상응 표시 및 준비 RNAi 판에 6-10 청소년을 전송합니다. </li><li> 하자성인 4-6 시간에 알을 낳고, 다음 판의 모든 성인을 제거,이 단계는 자손을 동기화하는 것입니다. 어머니가 제거되면 시간을 계산하기 시작합니다. 이 시간 제로입니다. </li><li> 20 접시를 길러 ° C 동물​​이 특정 발달 단계에기를,이 프로토콜에, 우리는 phenotypic 분석을위한 청년를 선택합니다. 정상적인 속도로 개발 knockdowns를 들어, 72 시간의 배양은 젊은 성인이되는 동물 충분합니다. 그러나, 여러 유전자가 다른 동물의 개발에 영향을 미칩니다. RNAi가 다른 속도로 성장 동물을가 발생하면, 젊은 성인은 자궁에 완료 외음부 개발 및 2-6 배아 (비옥 한 경우)과 L4를지나 그 더 이상 24 이상의 시간으로 식별 할 수 있습니다. 다른 발달 단계를 확인하려면 수사관이 가이드로 생식샘과 외음부 개발을 사용해야합니다. </li></ol> 3. RNAi의 점수 바디 크기 Phenotypes이 웜을 치료 <ol><li> 유리 슬라이드에 색상 라벨 테이프의 두 레이어를 놓습니다. 의 두 개SE 유리 슬라이드. 그런 다음 테이프 두 슬라이드 사이에 새 유리 슬라이드를 놓습니다. 물에 2% 아가로 오스를 녹여, 새로운 유리 슬라이드의 중심에 한 방울을 적용 한 후 아가로 오스는 이전과 같이 구 설명 2 %의 얇은 층을 만들기 위해 상단에 두 번째 유리 슬라이드를 누릅니다. </li><li> 아가로 오스가 확정되면, 상단 유리 덮개를 제거, 아가로 오스 패드와 슬라이드 웜을로드 할 준비가되어 있습니다. 클론 이름 라벨 슬라이드. </li><li> 아가로 오스 패드에 10 μl 25 MM 앤 3 추가,이를 고정하는 할머니 3 솔루션으로 30-40 동물을 선택 한 후 상단에 coverslip을 넣어, 재조합과 빈 모두 RNAi - 처리 동물을 L4440-수행하기위한 반복. </li><li> 이미지 2.5x 목적 렌즈를 사용하여 해부 현미경으로 웜, 여기에 우리가 소프트웨어 Qcapture을 지원과 함께 Leica 디지털 카메라를 사용합니다. </li><li> 교정을 위해 먼저 표준 마이크로 미터 통치자의 이미지를 캡처합니다. 그런 다음 이미지 Pro 소프트웨어에서 이미지를 열고 선택한 다음 &quot;측정&quot;을 클릭하고 &quot;보정&quot;을 선택&quot;공간 보정 마법사&quot;. 선택 &quot;활성화 된 이미지를 보정&quot;으로, &quot;다음&quot;을 클릭하십시오. 입력 보정을위한 이름과 공간 참조 단위 마이크로 미터로 설정되어 있는지 확인합니다. 그런 다음 &quot;만들기 참조 보정&quot;버튼을 확인하고 &quot;다음&quot;을 클릭합니다. 을 클릭 &quot;그리기 기준선.&quot; 참조 규모 줄이 나타납니다. 마이크로 미터의 끝과 일치 할 수있는 규모의 바 위치를 변경 한 다음 참조가 1,000 대를 나타냅니다 나타냅니다. , &quot;OK&quot;, &quot;다음&quot;, 그리고 &quot;마침&quot;을 클릭합니다. </li><li> 소프트웨어가 교정되면, 웜 이미지를 엽니 다. 그런 다음, &quot;선택 공간&quot;을 클릭 한 다음 &#39;보정&#39;을 선택하고 &#39;측정&#39;메뉴에서 선택합니다. 풀다운 메뉴에서 마이크로 미터 교정을 위해 만든 파일 이름을 선택합니다. 그런 다음 &#39;측정&#39;메뉴에서 &#39;측정&#39;을 선택합니다. 열리는 창에서 자유 그리기 도구를 선택하고 컴퓨터를 마우스 (그림 1)과 꼬리에 머리에서 동물 몸의 중심을 통해 라인을 그린다. 길이는 창에보고됩니다. 지금대상 유전자 RNAi와 빈 L4440 벡터로 처리 제어 동물로 치료 동물의 몸 길이 : 당신이 데이터의 두 그룹이 있습니다. </li><li> Microsoft Excel 파일, 또는 다른 적절한 통계 소프트웨어로 길이 측정을 보냅니다. 각 샘플에 대한 평균과 표준 편차를 계산하여 데이터를 분석 할 수 있습니다. 그런 다음 학생의 t-테스트를​​ 사용하여 샘​​플을 비교합니다. </li></ol>

<ol>
	<li>Melnyk, C. W., Molnar, A., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+and+systemic+movement+of+RNA+silencing+signals.">Intercellular and systemic movement of RNA silencing signals.</a> Embo J. 30, 3553-3563 (2011).</li><li>Wall, N. R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA:+can+RNA+interference+be+exploited+for+therapy.">Small RNA: can RNA interference be exploited for therapy.</a> Lancet. 362, 1401-1403 (2003).</li><li>Kalantidis, K., Schumacher, H. T., Alexiadis, T., Helm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+movement+in+plants.">RNA silencing movement in plants.</a> Biol. Cell. 100, 13-26 (2008).</li><li>Shim, M. S., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+targeted+delivery+of+siRNA+in+vivo.">Efficient and targeted delivery of siRNA in vivo.</a> Febs. J. 277, 4814-4827 (2010).</li><li>Amarzguioui, M., Rossi, J. J., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+chemically+synthesized+siRNA+and+vector-mediated+RNAi.">Approaches for chemically synthesized siRNA and vector-mediated RNAi.</a> FEBS Lett. 579, 5974-5981 (2005).</li><li>Kamath, R. S., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screening+in+Caenorhabditis+elegans.">Genome-wide RNAi screening in Caenorhabditis elegans.</a> Methods. 30, 313-321 (2003).</li><li>Savage-Dunn, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screen+for+small+body+size+mutants+in+C.+elegans+reveals+many+TGFb+pathway+components.">Genetic screen for small body size mutants in C. elegans reveals many TGFb pathway components.</a> Genesis. 35, 239-247 (2003).</li><li>Timmons, L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+by+ingested+dsRNA.">Specific interference by ingested dsRNA.</a> Nature. 395, 854 (1998).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Wang, J., Tokarz, R., Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+TGF&#946;+signal+transducers+in+the+hypodermis+regulates+body+size+in+C.+elegans.">The expression of TGF&#946; signal transducers in the hypodermis regulates body size in C. elegans.</a> Development. 129, 4989-4998 (2002).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130, 6453-6464 (2003).</li><li>Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling.">TGF-&#946; signaling.</a> WormBook. , 1-12 (2005).</li><li>Simmer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+putative+RNA-directed+RNA+polymerase+RRF-3+makes+C.+elegans+hypersensitive+to+RNAi.">Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.</a> Curr. Biol. 12, 1317-1319 (2002).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+misexpression+of+germline+P+granules+and+enhanced+RNA+interference+in+retinoblastoma+pathway+mutants.">Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.</a> Nature. 436, 593-597 (2005).</li><li>De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo:+variations+on+ancestral+themes.">Evo-devo: variations on ancestral themes.</a> Cell. 132, 185-195 (2008).</li><li>Timmons, L., Court, D. L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+bacterially+expressed+dsRNAs+can+produce+specific+and+potent+genetic+interference+in+Caenorhabditis+elegans.">Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.</a> Gene. 263, 103-112 (2001).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 프로토콜에서는, 우리는 C.의 몸 크기 결함이있는 돌연변이의 식별을위한 우리의 방법을 설명 RNAi 먹이로 elegans. 이 방법은 TGF-β 신호 구성 요소의 식별에 적용됩니다. 이러한 구성 요소가 매우 진화 15 일까지 보존되어 있으므로, 이러한 화면은 metazoans에서 TGF-β 신호의 분자 메커니즘을 elucidating에 관련이 있습니다. 이러한 화면 설계의 중요한 고려 사항은 시작 변형입니?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> RNAi 웜 판 17.7 g 웜 매체 믹스 60g의 한천 3리터에 H 2 O를 추가합니다. 압력솥. 3 ML 1M IPTG (살균), 3 ML 25 MG / ML Carbenicillin을 (무균)을 추가 한 후 60mm 배양 접시에 부어. 웜 판 17.7 g 웜 매체 믹스 0.6 g의 스트렙토 마이신의 황산 60g의 한천 3리터에 H 2 O를 추가합니다. 압력솥. 60mm 배양 접시에 넣어. 웜 매체 믹스 55g 트리스-CL 24g 트리스-OH 310g Bacto 펩톤 800 MG의 콜레스테롤 200g NaCl 철저하게 섞어서 덩어리를 방지해야합니다,이 가루 혼합물을 사용하기 전에 몇 개월 동안 저장 될 수 있습니다. 2퍼센트 아가로 오스 0.5 g 아가로 오스 광고D 25 ML DH 2 O. 전자 레인지의 열 용해 될 때까지. 1M 이소 프로필 β-D-1-thiogalactopyranoside (IPTG) 2g IPTG in10 ML DH 2 O를 해산 1M 앤 3 6.5 g 앤 3 100 ML에 DH 2 O를 추가합니다 25 MM 앤 3 2.5 ML 1M 앤 3 100 ML에 DH 2 O를 추가합니다 Caenorhabditis 유전학 센터에서 Caenorhabditis elegans 플라스미드 L4440 (암피실린 저항 유전자를 운반) 16, 박테리아가 HT115 (DE3)을 (또한 테트라 사이클린 - 저항 유전자를 운반 RNAse III 유전자 (dsRNAse)에 대한 결함 IPTG inducible T7의 효소 포함) 변형 60mm 페트리 요리 100mm 배양 접시 14 ML 둥근 바닥 팔콘 문화 관 유리 슬라이드 유리 coverslips 1-20 μl pipettor 20-200 μl pipettor 200-1000 μlpipettor 1-200 μl pipet 팁 200-1000 μl pipet 팁 1.5 ML Eppendorf 튜브 백금 와이어 웜 픽 </td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

두 번 스트랜드 RNA로 인한 간섭 (RNAi)는 대상 유전자 표현 1-3를 비판 할 효과적인 전략이다. 그것은 식물, 무척추 동물 및 vertebrates 등 많은 모델 시스템에 적용되었습니다. 생체 4,5에서 RNAi를 달성하기위한 다양한 방법이 있습니다. 예를 들어, 대상 유전자는 RNAi 벡터로 변환 한 후 영구적 또는 transiently 셀 라인 또는 유전자 최저 효과를 달성 할 기본 세포로 변환 될 수 있으며, 대안 특정 대상 유전자 (RNAi oligos)에서 두 번 스트랜드의 oligonucleotides를 합성 transiently 수 있습니다 대상 유전자를 침묵하는 세포 라인 또는 기본 세포로 변환, 또는 합성 두 번 좌초 RNA 분자는 생명체로 microinjected 할 수 있습니다. 선충류 C. 이후 elegans는 박테리아가 대상 유전자에 대한 이중 가닥 RNA을 표현과 함께 동물 먹이, 식품 소스로 박테리아를 사용하는 가능한 전략 6 제공합니다. 여기 RNAi 먹이를 제공방법은 몸 크기 표현형의 점수를합니다. C. 몸 크기 elegans는 주로 TGF-β에 의해 규제되어 있습니다 - 같은 리간드 DBL-1,이 분석은 TGF-β 신호 구성 요소를 7 식별에 적합합니다. 우리는 RNAi 먹이 실험을 반복하는 두 RNAi 지나치게 긴장 등의 다양한 변종을 사용. 우리의 결과는 rrf-3 스트레인가 우리에게 가장 예상 RNAi의 표현형 준 것으로 나타났다. 방법은 수행 할 수 쉽게 재현, 쉽게 계량입니다. 또한, 우리의 프로토콜은 특수 장비의 사용을 최소화하기 때문에 작은 실험실 또는 주로 대학 기관에서 사람들에게 적합합니다.

우리의 연구에서, 우리는 DBL-1/TGF-β 경로의 몸 크기 조절에 중점을두고 있습니다. 야생 형 동물 7,10,11에 비해 짧은 몸 길이 등의 작은 몸 크기 DBL-1 경로 결과의 함수의 손실. C.에 따라서 화면 elegans의 몸 크기 돌연변이 TGF-β 신호 구성 요소와 수식을 식별 할 수 있습니다. DBL-1 신호는 세포 표면 수용체와 세포 Smad 신호 변환기 12 포함 보존 TGF-β 신호 전달 경로에 의해 ?...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

우리는에서 대상 유전자와 점수 몸 크기 표현형을 노크 RNAi 먹이 기술을 사용하는 방법을 보여줍니다<em&gt; C. elegans</em&gt;. 이 방법은 같은 DBL-1/TGF-β 신호의 몸 크기 조절에 관여 것과 같은 관심의 잠재적 유전자 구성 요소를 식별하기 위해 대규모 화면에 사용 될 수 있습니다.

선충류의 바디 크기 규정에 참여 유전자에 대한 화면으로 RNA로 인한 간섭 수유 전략을 사용하여<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

using-rna-mediated-interference-feeding-strategy-to-screen-for-genes

Research

JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

12.2K 조회수.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). 우리는에서 대상 유전자와 점수 몸 크기 표현형을 노크 RNAi 먹이 기술을 사용하는 방법을 보여줍니다 C. elegans. 이 방법은 같은 DBL-1/TGF-β 신호의 몸 크기 조절에 관여 것과 같은 관심의 잠재적 유전자 구성 요소를 식별하기 위해 대규모 화면에 사용 될 수 있습니다.

비디오: Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

잠재적인 파킨슨병의 유전자 검증을위한 C. 엘레간스 도파민 신경 세포의 변성 분석의 응용

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

연구

생물학

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

항체 염색법에<em&gt; C. 엘레</em&gt; &quot;동결 크래킹 사용&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

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

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

방법은 근육의 세포 내 구획을 평가하기 위해<em&gt; C. 엘레</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

의 발달 프로세스 추적 및 정량화<em&gt; C. 엘레</em&gt; 오픈 소스 도구를 사용하여

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. 예쁜꼬마선충에서 RAN 펩타이드 독성 측정

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

조직별 샤페론 상호 작용을 위해 검사하기 위해 Caenorhabditis elegans를 사용

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...