저자 분사 마커에 대한 dbl-1 cDNA와 닥터 크리스토퍼 Rongo (Waksman 연구소, Rutgers 대학, 뉴저지)의 선물 박사 릭 Padgett (Waksman 연구소, Rutgers 대학, 뉴저지)에 감사드립니다. 닥터 바스 그랜트의 연구실에서 GFP-태그를 dbl-1 구조의 낮은 사본 번호 통합을위한 유전자 총기 포격을 수행. 르네 가르시아 연구실 texIs100의 창출 동안 기술 지원을 제공했습니다. 르네 가르시아, 로빈의 Lints 및 Hongmin 진 실험실 생산적인 조언을 제공했습니다. 이 작품은 분자 및 세포 의학 TAMHSC학과 시동 기금에 의해 자금 지원되었다. 화합물 범위와 회전 디스크 공촛점는 부서와 딘의 의학 사무소의 TAMHSC 대학에서 제공하는 자금으로 구입했습니다.

<ol>
	<li>Giacomotto, J., Segalat, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+small+animal+models,+where+are+we.">High-throughput screening and small animal models, where are we.</a> Br. J. Pharmacol. 160, 204-216 (2010).</li><li>Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+approaches+in+C.+elegans.">Functional genomic approaches in C. elegans.</a> Methods in Molecular Biology. 351, 127-138 (2006).</li><li>Boutros, M., 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=The+art+and+design+of+genetic+screens:+RNA+interference.">The art and design of genetic screens: RNA interference.</a> Nat. Rev. Genet. 9, 554-566 (2008).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157, 1217-1226 (2001).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimethylpsoralen+induces+small+deletion+mutations+in+Caenorhabditis+elegans.">Trimethylpsoralen induces small deletion mutations in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 1381-1385 (1994).</li><li>Frokjaer-Jensen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-copy+insertion+of+transgenes+in+Caenorhabditis+elegans.">Single-copy insertion of transgenes in Caenorhabditis elegans.</a> Nature Genetics. 40, 1375-1383 (2008).</li><li>Giordano-Santini, R., Dupuy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectable+genetic+markers+for+nematode+transgenesis.">Selectable genetic markers for nematode transgenesis.</a> Cell. Mol. Life. Sci. , (2011).</li><li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Stable+Transgenic+C.+elegans+Using+Microinjection.">Generation of Stable Transgenic C. elegans Using Microinjection.</a> J. Vis. Exp. (18), e833 (2008).</li><li>Mello, C., 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=DNA+transformation.">DNA transformation.</a> Methods in Cell Biology. 48, 451-482 (1995).</li><li>Simmer, F. <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>Zhuang, J. J., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specificity+of+Caenorhabditis+elegans+enhanced+RNAi+mutants.">Tissue-specificity of Caenorhabditis elegans enhanced RNAi mutants.</a> Genetics. , (2011).</li><li>Samuelson, A. V., Klimczak, R. R., Thompson, D. B., Carr, C. E., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Caenorhabditis+elegans+genes+regulating+longevity+using+enhanced+RNAi-sensitive+strains.">Identification of Caenorhabditis elegans genes regulating longevity using enhanced RNAi-sensitive strains.</a> Cold Spring Harbor Symposia on Quantitative Biology. 72, 489-497 (2007).</li><li>Wang, D. <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>Fraser, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+C.+elegans+chromosome+I+by+systematic+RNA+interference.">Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.</a> Nature. 408, 325-330 (2000).</li><li>Kamath, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Peters, K., McDowall, J., Rose, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+bli-4+(I)+locus+of+Caenorhabditis+elegans+disrupt+both+adult+cuticle+and+early+larval+development.">Mutations in the bli-4 (I) locus of Caenorhabditis elegans disrupt both adult cuticle and early larval development.</a> Genetics. , 129-195 (1991).</li><li>Thacker, C., Peters, K., Srayko, M., Rose, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bli-4+locus+of+Caenorhabditis+elegans+encodes+structurally+distinct+kex2/subtilisin-like+endoproteases+essential+for+early+development+and+adult+morphology.">The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases essential for early development and adult morphology.</a> Genes &#38; Development. 9, 956-971 (1995).</li><li>Byerly, L., Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+the+nematode+Caenorhabditis+elegans.+I.+Wild-type+growth+and+reproduction.">The life cycle of the nematode Caenorhabditis elegans. I. Wild-type growth and reproduction.</a> Dev. Biol. 51, 23-33 (1976).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</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=Genetic+screen+for+small+body+size+mutants+in+C.+elegans+reveals+many+TGFbeta+pathway+components.">Genetic screen for small body size mutants in C. elegans reveals many TGFbeta pathway components.</a> Genesis. 35, 239-247 (2003).</li><li>Qu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+analysis+of+the+Ahringer+Caenorhabditis+elegans+RNAi+feeding+library:+a+guide+for+genome-wide+screens.">Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens.</a> BMC Genomics. 12, 1471-2164 (2011).</li><li>Nelson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bow-Tie+Genetic+Architecture+for+Morphogenesis+Suggested+by+a+Genome-Wide+RNAi+Screen+in+Caenorhabditis+elegans.">A Bow-Tie Genetic Architecture for Morphogenesis Suggested by a Genome-Wide RNAi Screen in Caenorhabditis elegans.</a> PLoS Genetics. 7, e1002010 (2011).</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>Sarin, S., Prabhu, S., O'Meara, M. M., Pe'er, I., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+mutant+allele+identification+by+whole-genome+sequencing.">Caenorhabditis elegans mutant allele identification by whole-genome sequencing.</a> Nature Methods. 5, 865-867 (2008).</li><li>Lewis, J. A., Fleming, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+culture+methods.">Basic culture methods.</a> Methods in Cell Biology. 48, 3-29 (1995).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

우리는 공개 할게 없다.

여기서 제시 RNAi 심사 방법은 정상적인 (또는 형질) postembryonic 표현형에 필요한 유전자 제품의 민감하고 빠른 분석을 가능하게합니다. 표시 예제 fluorescently 태그가 단백질의 subcellular 지방화에 관련된 유전자에 대한 화면입니다. 그러나,이 프로토콜은 관심사의 다른 postembryonic phenotypes에 영향을 미치는 유전자를 식별하기 위해 수정할 수 있습니다. 이 방법은 RNAi 라이브러리?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> NGM 한천 </td><td> 선충류 성장 매체 </td><td> IPM 과학, INC </td><td> NGM 한천 프로토콜 25 다음 준비할 수 </td></tr><tr><td> M9 중간 </td><td> 22mM KH 2 PO 4, 42mM 나 2 HPO 4, 86mM NaCl, 1 ㎜ MgSO 4 </td><td></td><td> 26 </td></tr><tr><td> 한천 - 한천 </td><td> EMD 화학 주식 회사 </td><td> 1.01614.1000 </td><td> NGM 플레이트 물에 2 %. 현미경 슬라이드 패드를위한 물 4 % (초기 및 이후 녹기 전자 레인지 압력솥). </td></tr><tr><td> Bacto 펩톤 </td><td> Becton 디킨슨 - Difco CP </td><td> 211,677 </td><td> 0.25 % </TD&gt; </tr><tr><td> IPTG </td><td> 연구 제품 국제 주식 회사 </td><td> I56000-5.0 </td><td> 1 ㎜ 최종 농도 </td></tr><tr><td> carbenicillin </td><td> 연구 제품 국제 주식 회사 </td><td> C46000-5.0 </td><td> 50 μg / ML 작업 희석 </td></tr><tr><td> LB 국물 레녹스 </td><td> Becton 디킨슨 - Difco CP </td><td> 240,230 </td><td> 20g / 리터 </td></tr><tr><td> 테트라 사이클린 </td><td> 시그마 </td><td> 268,054 </td><td> 12.5 μg / ML 작업 희석 </td></tr><tr><td> 나트륨 차아 염소 산염 </td><td> 모든 브랜드 </td><td> 5 % 가정용 표백제 </td><td> 신선한 표백제를 사용하십시오. </td></tr><tr><td> 수산화 나트륨 </td><td> 모든 브랜드 </td><td> CAS 1310-73-2 </td><td> 5 N 주식 </td></tr><tr><td> M9 배지 </td><td> Wormlab 제조법 도서 </td><td> <a href="http://130.15.90.245/wormlab_recipe_book.htm#Commonlab">http://1</a>30.15.90.245/wormlab_recipe_book.htm # Commonlab </td><td> 26 </td></tr><tr><td> levamisol </td><td> 시그마 </td><td> 31,742 </td><td> 100 μm의 - 1 MM 근무 희석 </td></tr><tr><td> 나트륨 azide </td><td> 피셔 과학 </td><td> S227 </td><td> M9 근무 희석 10 MM </td></tr><tr><td> 24 잘 플레이트 </td><td> Greiner 바이오 원 </td><td> 662,160 </td><td> VWR 분배기 </td></tr><tr><td> 현미경 슬라이드 </td><td> 모든 브랜드 </td><td> 75 X 25 X 1mm </td><td></td></tr><tr><td> 현미경 커버 전표 </td><td> 모든 브랜드 </td><td> 22 X 22mm No.1.5 </td><td> 현미경 제조 업체에서 권장하는 두께를 사​​용하십시오. </td></tr><tr><td> 복합 현미경 </td><td> 칼 자이스 혈구 주식 회사 </td><td> A1m </td><td> 실험의 요구에 맞게 목표 및 필터를 사용하십시오. </td></tr><tr><td> 미디어 펌프</td><td> Manostat Varistaltic 펌프 </td><td> 케이트 모델 # 72-620-000 </td><td> 관 그리고 기계에 맞는 설정을 사용 </td></tr></tbody></table>

rnai screening to identify postembryonic phenotypes in c. elegans

C. elegans는 많은 유전자와 유전자 경로 1의 발견과 기능적 특성에 대한 유용한 모델 시스템 것으로 입증되었습니다. 보다 정교한 도구와이 시스템의 연구를위한 자원이 더 미묘한 phenotypes이나 역할과 유전자의 지속적인 발견을 촉진하고 있습니다. 여기에서 우리는 C를 식별 맞게 일반화된 프로토콜을 제시 RNAi 2를 사용 흥미 postembryonic phenotypes와 elegans의 유전자. 이 절차는 쉽게 여부 해부 또는 복합 현미경의 빛 또는 형광 광학에 의해 검정 선택의 표현형으로 수정됩니다. 이 심사 프로토콜은 유기체의 물리적 자산 및 분자 도구 C.에 capitalizes elegans 연구 커뮤니티하였습니다. 예를 들어, 우리는 이것의 일반적인 로컬 라이제이션에 필요한 유전자를 식별하기 위해 RNAi 화면에 형광등 제품을 표현하는 통합 transgene의 사용을 보여줍니다늦은 무대 애벌레와 어른의 제품입니다. 첫째, 우리는 전체 길이 cDNA 삽입으로 상용 게놈 RNAi 라이브러리를 사용했습니다. 이 라이브러리는 후보 유전자 산물의 RNAi 감소하여 여러 후보자의 급속한 식별을 용이하게합니다. 둘째, 우리는 RNAi를 구분 배경에 관심이 우리의 fluorecently 태그가 단백질을 표현 통합 transgene을 올렸습니다. 셋째, RNAi에 부화 동물을 노출하여,이 화면은 특별히 관심 단백질을 규제에서 사후 배아 역할을 숨길 수있는 중요한 배아 역할을 유전자 제품의 식별을 허용합니다. 마지막으로,이 화면은 단세포 결의안에 대한 갖춘 복합 현미경을 사용합니다.

Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

우리의 단백질 표현 및 현지화의 postembryonic 규제를 식별할 수 sensitized 방법을 설명<em&gt; C. elegans</em&gt; RNAi 기반 게놈 화면과 기능, fluorescently 태그가 단백질을 표현 통합 transgene을 사용합니다.

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 ...

rnai-screening-to-identify-postembryonic-phenotypes-in-c-elegans

Research

JoVE Journal

Biology

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

16.9K 조회수.  Texas A&M University System Health Science Center. 우리의 단백질 표현 및 현지화의 postembryonic 규제를 식별할 수 sensitized 방법을 설명 C. elegans RNAi 기반 게놈 화면과 기능, fluorescently 태그가 단백질을 표현 통합 transgene을 사용합니다.

비디오: RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

연구

생물학

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 ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

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 ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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. ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

에서 Hypoxia의 효과를 연구하기 위해 정의된 기체 환경 만들기<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

에 산소 결핍 유도 일시 중지 된 애니메이션을 시각화하기 위해 시간 경과에 현미경을 사용<em&gt; C. elegans</em&gt; 태아

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans의 주 화성 분석

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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 ...