우리는 그들의 공헌에 대해 D. Braendle, CL Kurz와 F. Montañana-Sanchis 감사드립니다. 이 프로젝트 ANR과 Conseil 지역 PACA와 INSERM과 CNRS의 기관 자금 지원에서 교부금에 의해 지원되었다.

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Microbiol. 76, 5710-5717 (2010).</li></ol>

Ewbank 연구실 연합 Biometrica 및 Modul-바이오와 협력.

<p class="jove_content"&gt; 현재의 프로토콜은 대규모 RNAi 심사에 허용<em&gt; C. elegans</em&gt; 형광 리포터 유전자의 표현의 수준을 같이 읽고 아웃 이용한 고체 매체에. 그것은, 효율적인 신뢰성과 재현성 방식으로, 샘플 다수의 쉽고 빠른 처리를 허용하는 높은 처리량 assays를 촉진하기 위해 자동화된 도구에 최적화되었습니다. 다른 방법은 자동화된 액체 시료 처리에 너무 크게 의존하지 않는 것이 설명되었습니다<sup&gt; 8</sup&gt;.</p&gt;<p class="jove_content"&gt; 프로토콜에 대한 하나의 중요한 조직 양상은 회사 ModulBio와 협력 RNAi 화면 프로 시저로 상용 MBioLIMS 소프트웨어를 채택하여 샘플 추적의 강력한 방법의 개발되었습니다. 절차의 모든 단계에서, 접시는 이제 LIMS 내에 생성된 바코드에 의해 식별됩니다. LIMS는 또한 각각의 접시에 각각 우물에 클론의 신원을 기록으로 우물에서 putative 유전자에가는 것은 간단합니다. 그리고이 넘어, 커뮤니티 데이터베이스 WormBase에 LIMS 링크는 특정 유전자에 대한 분자 및 기능적 데이터에 직접 액세스할 수 있도록합니다. LIMS는 분석이 판 또는 염색체와 전체를 게놈 수준에서 실시하기 용이하게합니다. 이러한 분석은 데이터 등 COPAmulti와 같은 이전에 설명된 도구와 완전히 호환되는 방식으로 저장됩니다<sup&gt; 16</sup&gt;.</p&gt;<p class="jove_content"&gt; 메서드에 하나의 중요한 실험적 포인트는 벌레의 숫자에 상대적인 음식의 수량의 조정이다. 많은 assays 들어, 웜은 실험 끝에 굶어하지 않는 것이 중요하지만 그것은 시작에 너무 많은 음식을 필요하지 않는 것도 중요합니다. 우리는 음식의 높은 밀도는 P를 옮기는 ​​벌레를 사용하여 프로토콜에 형광 유도를 감소 것을 관찰했습니다<em&gt; nlp-29</em&gt; :: GFP 기자. 벌레가 (학사되지 않은 결과) 너무 많은 박테리아에서 배양해 때이 줄었 감염 효율이 반영됩니다. 이 dsRNA가 최적의 수준에서 생산 여부를 결정 이후 동시에 RNAi 박테리아의 문화 조건도 중요합니다. 대규모 RNAi에 대한 몇 가지 프로토콜은 액체 배양을 퍼뜨리고하기 전에 한천 플레이트에 RNAi 클론의 문화를 필요로<sup&gt; 8</sup&gt;. 우리는이 단계를 생략 강력한 유전자 입을를 취득. 우리는 동질적인 결과를 보장하기 위해 각 문화의 과잉 성장을위한하기로했다. 사실에서 설명한 프로토콜은 병렬로 2 개 또는 3 개의 화면을 수행할 수 있도록 충분한 자료를 제공합니다. 가장 강력한 결과를 얻기의 관심에서 우리의 스크린을 실시하지만, 선호 웜, RNAi 박테리아 및 곰팡이 포자의 독립적인 문화를 사용하여 중복.</p&gt;<p class="jove_content"&gt; 또 다른 중요한 단계는 너무 습해도 너무 건조하지도 매체를 사용하는 것에 관련이있다. 접시가 충분히 건조되지 않으면 RNAi 박테리아가 제대로 건조되지 않고 웜은 분석 중에 수영 것입니다. 이것은 문제가 발생할 수 있습니다. 예를 들어, 웜이 효과적으로 감염되지 않습니다<em&gt; 디 coniospora</em&gt; 액체의 (C. Couillault, 게시되지 않은 결과). 한편, 건조 접시에 배지 증가 osmolarity. 이것은 웜 생리 기능에 직접적인 영향을 가지고<sup&gt; 17</sup&gt;, 등 일부 항균 펩타이드 유전자의 유도를 포함<em&gt; nlp 29</em&gt;<sup&gt; 18</sup&gt;. 웜 및 포자를 분배하는 데 사용되는 볼륨은 효과적이고 신속한 건조를 허용하는 것이 중요합니다. 액체 중 10 개 이상 μL가 신선한 접시에 추가되면, 우물은 건조 많은 시간이 걸릴 수 있습니다. 이것은 접시에 벌레를 추가하는 로봇 방법을 사용하기 어렵다는 것을 의미합니다. 예를 들어, 각각의 벌레는 액체의 주위에 1 μL의 Biosort에 의해 적절합니다. 그것은 또한 우리가 침전에 대한 솔루션 웜의 경향을 극복할 수있는대로 저희는 현재 수동으로 벌레를 배포합니다.</p&gt;<p class="jove_content"&gt; 우리는 이전에 한 96 - 웰 플레이트가 36분에서 분석을 할 수 있도록 Biosort의 조정보고<sup&gt; 19</sup&gt;. 기계 제조 업체 유비는 이제 더 이상 분석 시간을 줄일 수있는 Biosort의 수정을 제공합니다. &quot;FastReFlex&quot;로 샘플을 직접 버블 트랩 필터를 거치지 않고 분석을위한 흐름 전지를 통해 라우트됩니다. 데이터 수집을위한 시간을 크게 감소의이 결과는, 전체 플레이트가 하루에 치료할 수 있도록 20 접시 주위에있게 22 분 안에 분석할 수 있습니다. 접시는 냉동하지 않은 경우 단, 처음이자 마지막 사람 사이의 8 시간 간격이있을 것입니다. 단지 18 시간 지속되는 감염 실험의 비교적 짧은 기간을 감안할 때, 이것은 번호판에 걸쳐 허용되지 않는 변수를 소개합니다. 냉동 번호판은이 문제를 해결 분석을 이전하지,하지만, 2 사람이 쉽게 50 접시 주일까지 처리할 수있는 현재의 프로토콜 및 표준 액체 처리 로봇과로 그것은 또한 높은 처리량 분석의 맥락에서 중요하다뿐만 아니라. 현재 우리가 일상적으로 화면을 30 접시 주 전체를 게놈 스크린에게 있습니다이하보다 2 개월 만에 한 번 실시합니다.</p&gt;<p class="jove_content"이 프로토콜의&gt; 하나 재미있는 기능은 액체 문화에서 수행할 수없는 assays 수 있도록, 고체 한천을 사용한다는 사실입니다. 웜 생리학은 문화 조건에 따라 다릅니다로서<sup&gt; 20,21</sup&gt;, 고체 배지에 assays는 또한 일반적인 액체 기반의 화면을 보완 수행할 수있는. 특정 phenotypes (예 : 운동) 내용 분석 96 - 웰 고체 배지 플레이트와 직접 진행하실 수 있습니다. 현재 화면의 경우 그러나, Biosort으로 고체 배지 플레이트에서 맛볼 수없는, 웜 먼저 동결되기 전에 액체로 전송됩니다. 한편, COPAS Biosort는 여러 phenotypes의 측정과 호환 multiparametric 정량 분석​​이 이루어질 수 있습니다. 그것은 특정 화면에 대한 모든 측정 매개 변수가 기본 읽기있을 가능성이 높습니다. 그럼에도 불구하고, 이러한 측정치는 여러 가지 이유로 유용할 수 있습니다. 그들은 하나가 검정이 올바르게 실행 여부를 확인할 수 있습니다. 예를 들어, 하나는 각 우물에 벌레의 숫자와 크기를 확인하실 수 있습니다. RNAi의 클론 성장 또는 복제를 차단해야만이 곳 예상 값의 편차가 있어야합니다. 20 명 미만 웜 또는 / 30 % 더 작은 동물 (TOF)와 웰스 스탄에 비해dard 성장에 취지만 제어 (<em&gt; 예 GFP (RNAi</em&gt;))는 이후 분석에서 제외시킬 수 있습니다. 그들의 개발은 물론 고급 후 이러한 클론 기능은 웜은 RNAi 세균에 노출되는 어떤 점에서 다른 프로토콜을 사용하여 해결할 수<sup&gt; 15</sup&gt;. 다른 매개 변수는 오직 녹색 형광지만 빨간색이 아니라 영향을 미치는 사람을 히트곡을 제한, 예를 들어 후보 유전자 (조회수)을 선택, 수정하는 데 사용할 수 있습니다. 안타를 선택 선택한 정확한 방법은 화면의 종류와 정확한 읽기 아웃에 따라 달라집니다. 양적 게놈 - 와이드 스크린에서 생성된 데이터의 복잡성을 감안할 때, 통계와의 협력이 필요할 수 있습니다.</p&gt;<p class="jove_content"&gt; 우리가 설명하는 프로토콜은 쉽게 assays 다른 유형의 적응하실 수 있습니다. 예를 들어, 감염 단계에서<em&gt; 디 coniospora</em&gt; 고체 미디어에 감염될 수있는 다른 병원균에 의해 대체될 수 있습니다. 우리가 액체 문화 Serratia marcescens 감염에 중요한 독성 요소의 많은 고체 매체 (E. Pradel, 개인 통신)에 감염하는 동안 역할을하지 않는 것으로 나타났습니다로서 이것은 잠재적으로 유용합니다. 프로토콜은 또한 화학 화합물의 시험과 완벽하게 호환<sup&gt; 22-24</sup&gt; 또는 박테리아의 다른 라이브러리를 사용 bioactivity와 화합물을 식별하는 방법<sup&gt; 25</sup&gt; 그래서의 일반 유틸리티를 발견해야<em&gt; C. elegans</em&gt; 연구 커뮤니티입니다.</p

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> BactoAgar </td><td> BD 진단 시스템 </td><td> 214,010 </td><td></td></tr><tr><td> 암피실린 </td><td> 시그마 - 올드 리치 </td><td> A9518 </td><td></td></tr><tr><td> BactoPeptone </td><td> BD 진단 시스템 </td><td> 211,677 </td><td></td></tr><tr><td> CaCl 2 </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> AeraSeal 접착 필름 </td><td> Dutscher </td><td> 760,214 </td><td></td></tr><tr><td> 이소 프로필 β-D-Thiogalactopyranoside (IPTG) </td><td> 써모 과학 </td><td> AB-0481 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> MgSO 4 </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> NaCl </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> 나 2 HPO 4 </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> NaOH </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> 96 깊은 우물을 판 </td><td> 써모 과학 </td><td> AB-0932 </td><td></td></tr><tr><td> 96 잘 플랫 플레이트 </td><td> 매 </td><td> 353,072 </td><td></td></tr><tr><td> 96도 회전 플레이트 </td><td> 매 </td><td> 353,077 </td><td></td></tr><tr><td> 테트라 싸이클린 </td><td> 시그마 - 올드 리치 </td><td> T8032 </td><td></td></tr><tr><td> 트리톤 X-100 </td><td> 모든 공급 업체 </td><td></td><td></td></tr><tr><td> TECAN 로봇 </td><td> TECAN </td><td></td><td></td></tr><tr><td> Liquidator96 TM </td><td> 비가 </td><td></td><td></td></tr><tr><td> COPAS Biosort </td><td> 연합 Biometrica </td><td></td><td></td></tr><tr><td> LIMS </td><td> Modul - 바이오 </td><td></td><td></td></tr></tbody></table>

quantitative and automated high-throughput genome-wide rnai screens in c. elegans

RNA 간섭은 전체를 게놈 규모와 양적 맥락에서 실시한 특히 유전자 기능을 이해하는 강력한 방법입니다. C로 elegans은 유전자 기능은 특정 유전자 하나에 해당하는 dsRNA을 표현 세균과 벌레를 먹이로 간단하고 효율적으로 무너뜨렸다 수 있습니다. RNAi의 클론의 라이브러리의 생성 C.의 대부분을 커버하면서 elegans 게놈 2,3 진정한 기능적 게놈 연구 (예 : 4-7에 대한 참조)를 위해 길을 열었습니다, 대부분의 설립 방법 힘드는 있습니다. Moy와 동료들은 게놈 - 와이드 스크린에게 8을 용이하게 세미 자동화된 프로토콜을 개발했습니다. 접근 방식은 미세한 이미징 및 이미지 분석에 의존합니다. 여기서 우리는 대안 세균성 RNAi 클론, COPAS Biosort (연합 Biometrica (유비)을)를 사용하여 정량 분석​​의 로봇 핸들링을 바탕으로 높은 처리량 게놈 - 와이드 스크린을위한 프로토콜, 그리고을 설명통합 소프트웨어 : MBioLIMS (Modul-바이오의 실험실 정보 관리 시스템) 데이터 관리 및 샘플 추적을위한 향상된 처리량을 제공하는 기술입니다. 방법은 화면이 고체 배지 플레이트에 실시 할 수 있습니다. 이것은 C.에 해당 주소 호스트 병원체 상호 작용과 같은 몇 가지 연구를 위해 특히 중요 elegans, 특정 미생물을 효율적 액체 문화 웜 감염되지 않습니다 때문입니다. 우리는 메서드가 C.의 안티 - 에게선 타고난 면역에 유전자의 중요성을 계량하는 데 사용할 수있는 방법을 보여 elegans. 이 경우, 접근 방식에 constitutively 표현된다 항균 펩타이드 유전자 nlp 29 발기인과 빨간색 형광 기자의 통제하에 GFP으로 표피 감염-inducible 형광 리포터 유전자를 나르는 유전자 변형 변형의 사용에 의존 표피. 후자는 표피의 기능 무결성을위한 내부 통제를 제공그리고 특이 현상 transgene은 9 입을. 제어 웜은 균류에 감염되면 그들은 녹색 형광. RNAi 감염 후 감소된 형광의 nlp 29 표현 결과에 필요한 유전자에 의해 아래로 들렸습니다. 현재이 프로토콜은 3,000 명이 넘는 RNAi의 클론이 덜보다 2 개월 만에 전체 게놈을 분석해의 가능성을 열어, 주당 테스트 및 분석 수 있습니다.

Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

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

우리가 사용하는 프로토콜을 설명<em&gt; C. elegans</em이러한 형광, 크기와 인구에서 개별 웜의 투명도 등 여러 매개 변수의 자동 측정을 허용&gt;과 RNAi 먹이 라이브러리. 우리의 안티 - 에게선 타고난 면역에 관여 유전자를 식별하는 화면 중 하나를 예를 들면요<em&gt; C. elegans</em&gt;.

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. ...

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Biology

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

17.6K 조회수.  Université de la Méditerranée. 우리가 사용하는 프로토콜을 설명 C. elegans과 RNAi 먹이 라이브러리. 우리의 안티 - 에게선 타고난 면역에 관여 유전자를 식별하는 화면 중 하나를 예를 들면요 C. elegans.

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

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

zebrafish 화학 화면을위한 자동화 이미징 및 분석의 개발.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

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생물학

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

이소형 특정 유전자 목표를 식별 칩을 사용하여 게놈 차원의 분석

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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 ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

자동을 사용하여 염색체의 높은 처리량 물리적 매핑<em&gt; 현장에서</em&gt; 하이브리드화

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

게놈 전체 유전자 삭제할에<em&gt; 연쇄상 구균 sanguinis</em&gt; 높은 처리량 PCR에 의한

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

에서 지방 질량의 생화학 및 높은 처리량 미세 평가<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).