C. 엘레간스 transgenes은 Caenorhabditis 유전학 센터 (미네소타, 미네 아 폴리스 대학, MN)에 의해 제공되었다. 이 작품은 NIH R21 부여 KS로 NS0667678 - 01에 의해 지원되었다. 모든 저자는 수집, 분석 및 데이터 해석에 참가했습니다. CKL, KS 및 KPC는 원고를 작성하고 수정에 참여했습니다.

1. 박테리아 웜 음식 준비 1 일 <ol><li> 포화 E. 5 ML 추가 대장균 박테리아 OP50 문화 500 ML 끝내주는 국물은 50 μg / ML 스트렙토 마이신과 보충 37에 떨고 인큐베이터 (225 RPM) 숙박 성장 ° C. </li></ol> 날 2 <ol start="2"><li> 20 분 동안 2,500 RCF에서 냉장 원심 분리기에서 만 50 ML 튜브 및 원심 분리기 박테리아에 하루 박테리아 문화를 분할. </li><li> LB의 국물에서 가만히 따르다 액체 선충류의 성장 미디어 (NGM)의 10 ML 각 박테리아 펠렛을 resuspend. 박테리아를 resuspend 15 분 동안 바닥 통에 수평으로 흔들어. NGM 버퍼를 만들려면, 1 패 탈이온수 및 압력솥에 3g NaCl를 추가합니다. 55 쿨 ° C 및 주문에 다음 멸균 솔루션을 추가 : 1 M CaCl 2, 1 M MgSO 4 개 중 1 개 ML, 1 M의 칼륨 나트륨, 산도 6.0의 25 ML 1 ML합니다. </li><li> 20 분 동안 2,500 RCF에서 냉장 원심 분리기에 박테리아 문화를 원심 분리기. </li><li> NGM 버퍼에서 가만히 따르다 및 박테리아 펠렛의 무게. </li><li> 산탄, 나누어지는 박테리아 3 ML -20 15 ML 튜브, 그리고 가게에 집중 ° C.를 resuspend하는 NGM 버퍼의 동일한 볼륨 추가 </li></ol> 2. 대규모 C. 엘레간스 액체 문화 우리는 유전자 변형 라인을 사용 VP596 (dvIs19 [pAF15 (GST - 4 : GFP : : NLS)]; vsIs33 [dop - 3 : RFP]) 2 형광 구성 운반 : SKN - 1을 모니터링하는 GFP 14 : Pgst -4를 : 활동 및 Pdop - 3 : RFP 17 웜 번호 정상화를위한 표준으로 제공합니다. 1 일 <ol><li> 150 ML NGM 버퍼, 1.5 ML의 LB의 국물, 5 MG / ML 콜레스테롤 150 μl (에탄올)을, 100 MG / ML 스트렙토 마이신의 75 μl를 섞는다. 혼합 필터 - 소독 및 멸균 1리터 플라스크에 추가할 수 있습니다. 참고 : NGM 버퍼에 1 % LB의 국물을 추가하는 것이 유리하고 plasticware에 고집에서 벌레를 방지하는 데 도움이 있지만,이 금액은 박테리아의 성장을 지원하기 위해 충분하지 않습니다. </li><li> 표준 염소산 절차 18 벌레를 동기화할 수 있습니다. 최대 gravid 웜 0.5 ML에이 염소산 솔루션 5 ML (3.75 ML 멸균 물 1 ML 가정용 표백제, 250 μl 10 N NaOH)과 하나의 15 ML 튜브에서 처리할 수 있습니다. 멸균 물 출시 달걀을 씻고 NGM 버퍼 10 ML 그들을 resuspend. </li><li> 계란 NGM 버퍼 100 배 (예, 10 ML 100 μl)의 샘플을 희석 세 별도 5 μl aliquots에 달걀의 개수를 계산합니다. 여러 200,000 (100 희석 요인이 X 2,000)의 평균 숫자는 계란의 총 수를 측정합니다. </li><li> NGM 버퍼와 술병에 200,000에 2,000,000 계란 추가 20 100 RPM에서 문화를 흔들 ° C. </li></ol> 날 2 <ol start="5"><li> 적어도 16 H 술병에 달걀을 추가한 후, 정지 웜 문화의 약 0.5 ML를 제거하는 살균 serological pipet을 사용합니다. 페트리 접시의 멸균 뚜껑에 Pipet 세 5 μl 방울. 벌레를 마비로 1~2분위한 -20 ° C의 냉장고에서 뚜껑을 놓습니다. 부화 웜의 총 수를 추정 30000 (150 ML / 5 μl) 5 μl를 여러 당 사는 부화 웜의 평균 개수를 계산합니다. </li><li> 냉동 OP50 세균성 문화 (프로토콜 1.6)의 튜브를 해동하고 500,000 부화 웜 당 웜 문화에 50% OP50 3 ML를 추가합니다. 20 100 RPM에서 술병을 흔들어 ° C. 웜이 대략 51 시간 L4 유충 젊은 성인으로 개발할 것입니다. 박테리아의 OD600 처음에 2.200에 대해해야합니다. 흡광도에 의해 박테리아의 양을 모니터링하여 OD 600 이하 0.900에 떨어뜨리면 더 추가합니다. </li></ol> 3. 웜 수집 및 분배 일 사 <ol><li> 표준 NGM의 한천 플레이트에 정지 웜 문화의 약 0.5 ML를 전송하는 살균 serological pipet을 사용합니다. 대부분의 L4 유충이나 젊은 성인되었는지 확인하기 위해 stereomicroscope있는 벌레를 볼 수 있습니다. L4 유충은 신체의 중앙에 복부 분명 자리를합니다. 청소년 약간 크고 분명 자리를 없을 것입니다. </li><li> 무균 50 ML 튜브에 웜 문화를 붓고하고 benchtop에서 테스트 튜브 랙에 튜브를 놓으십시오. 웜은 약 10 분 동안 정착을 허용합니다. 흡인이나 pipetting하여 표면에 뜨는를 제거합니다. 참고 : 그들이 느린보다 L4 유충 젊은 성인을 해결하기 때문에이 단계는 안깐 계란이나 개발하지 않았 웜을 제거하는 것이 중요합니다. </li><li> 1 % LB의 국물 (NGM + LB) 박테리아를 제거하는 3-4 회와 NGM 버퍼와 단일 50 ML 튜브 및 세척에 모든 벌레를 수집합니다. 참고 : 이것은 최고 30 초 500 RCF에서 벌레를 수집하고 신선한 NGM + LB 버퍼로 뜨는을 대체하여 스윙 - 양동이 원심 분리기에서 이루어집니다. </li><li> 세척 후, 50 ML에 + NGM와 LB 버퍼를 튜브를 작성 및 사용자 정의 벌레 분배 플라스크에 붓는다. 작은 저어 표시줄을 추가하고 벌레가 정지 계속 저어. 일 카운트세 5 μl 방울의 벌레의 전자 번호는 위에서 지적했다. 당신이 5 μl 드롭 (~ 2.5-3.0 웜 / μl) 당 12-15 벌레를 얻을 때까지 NGM + LB로 희석. 참고 : 20 ML (60,000 웜)의 최소 분배 술병과 디스펜서 카세트 죽음의 공간을 채우기 위해 필요합니다. 각 384 잘 플레이트는 약 35,000 웜 또는 중지 웜의 11.5 ML이 필요합니다. </li><li> 10 μl 분배 카세트를로드하고 70 % 에탄올과 프라이밍하여 소독. 멸균 물을 마중물하여 에탄올을 씻어. </li><li> 단지 그 움직임을 방해하지 않고 교도소 표시줄 위에 있도록 술병에 분배 카세트의 끝에 삽입합니다. 벌레가 전체 술병에 걸쳐 중단 남아 있는지 확인합니다. 프로그램 디스펜서 2 사전 펄스 낮은 속도로 분배합니다. 프라임 및 정지 웜 중 적어도 10 ML을 실행합니다. 튜브에 정착에서 벌레를 방지하기 위해 신속하게 필요한 번호판을 입력합니다. </li><li> 숨 테이프로 번호판을 밀봉합니다. </li><li> 귀하의 분석에 적합한 온도 인큐베이터에서 흔들림 플랫폼에서 접시를 놓습니다. 접시를 쌓아하지 마십시오. </li></ol> 4. 형광 분석 <ol><li> 적절한 방출 및 여기 파장 (; RFP 540/25ex 590/35em GFP 485/20ex 528/20em 우리의 분석을위한 필터)와 각 우물의 형광 강도를 측정하기위한 microplate 판독기에 대상 접시를 놓습니다. </li><li> GFP / RFP의 비율을 계산하고 개별 치료에서 파생 형광 강도의 정확한 배 차이를 결정하는 제어 우물 (활성화 화합물 제외)의 수치로 정상화. </li></ol> 5. 대표 결과 : 우리 SKN - 1 검정은 chromosomally 통합 듀얼 기자 변형 (VP596)를 사용합니다. 그림 1에서와 같이, 웜의 숫자가 잘 적절 볼륨에 상관 있습니다. 뿐만 아니라 당 그림 2A와 2B, 총 GFP 및 RFP 형광을 표시하는 것은 우물에서 384 잘 접시에 잘 걸쳐 매우 재현할 수 있습니다. 384 우물에 걸쳐 RFP : GFP 형광은 선형 Pdop - 3 상호는 :으로 취소 유발 Pgst - 4 그림 2C에 표시됩니다. : 다양성을 줄이기 위해 RFP 기자 : GFP / RFP의 비율로 표현하면, 형광은 Pdop - 3의 능력을 보여주는 384 잘 접시 (2A에 2D 그림에서 변이 계수를 비교)에 걸쳐 우물에서 잘 고도로 재현할됩니다. 그림 3과 RFP에 GFP의 상대의 유도에 표시된 SKN - 1 생체 이물 활성화가 (38 μm의 juglone) 강력하고 384 잘 접시에 걸쳐 고도로 재현할 수있다. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="그림 1"/> 그림 1. 볼륨 대 벌레 수시키지. 웜 약 2/μl로 희석하고 stereomicroscope와 함께 수동으로 계산을위한 24 잘 접시에 적절했다. N 볼륨 당 = 8 웰스. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="그림 2"/> 그림 2. 384 잘 접시에 적절 웜 총 형광 다시 재현할 수 있습니다. 웜가 약 2.5/μl 30 μl에 희석되었습니다 것은 384 자 판 매 자로 적절했다. 모든 웰스의 GFP (A)와 RFP (B) 형광 9 % 이하의 유사 계수를했다. (C) GFP 형광은 매우 RFP 형광을 서로 관련됩니다. (D) RFP에 GFP의 비율을 계산하면 6% 아래 편차의 계수를 감소 (A, B, 및 D) 고체 라인 수단을 표시하고 깨진 라인이 3 개의 표준 편차 평균 위 또는 아래를 나타냅니다. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="그림 3"/> . Pgst - 4 그림 3 활성화 : GFP는 강력하고 일관성이다. 약 75 L4 벌레가 384 잘 접시와 38 μm의의 juglone의 모든 우물에 적절했다하는 것은 다른 모든 항목에 추가되었습니다. GFP 및 RFP 형광은 부화 21 H 후에 측정되었다. 말은 상대 형광 모든 컨트롤의 비율 (1.0)와 juglone 웰스 (8.9)은 고체 라인으로 표시됩니다. 컨트롤 위에 3 개의 표준 편차 뜻과 의미 juglone 아래 깨진 라인으로 표시됩니다.

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관심 없음 충돌 선언하지 않습니다.

우리는 culturing위한 방법을 제시하고 유전자 변형 nematodes 많은 숫자를 분배. culturing 벌레에 사용되는 장비는 분자 복제를 수행 실험실을위한 표준이며, 액체 처리 및 형광 장비 microplates 많은 숫자를 처리 실험실에 대한 표준입니다. 라이브 C. 다수의 분배의 다른 방법 엘레간스 장비 19 정렬 비싼 입자가 필요합니다. Pgst - 4 : GFP 분석은 뚜껑 &#39;N&#39;칼라 전사 요소의 작...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> LB의 수프 </td><td> 연구 제품 국제 주식 회사 </td><td> L24066 </td><td></td></tr><tr><td> 끝내주는 국물 </td><td> 연구 제품 국제 주식 회사 </td><td> T15100 </td><td></td></tr><tr><td> 시너지 ™ HT 멀티 모드 Microplate Reader를 </td><td> BioTek </td><td></td><td> 필터 : GFP 485/20ex 528/20em; RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo 선택 디스펜서 </td><td> BioTek </td><td></td><td></td></tr><tr><td> 웜 분배 플라스크 </td><td> 남부 과학 주식 회사, 미캐노피, FL </td><td></td><td> 사용자는 (352) 284-2531를 조립 </td></tr><tr><td> 384 microplates </td><td> 그라 이너 바이오 원 </td><td> 5678-1209 </td><td></td></tr><tr><td> 통풍 씰링 테이프 </td><td> Nunc </td><td> 241205 </td><td></td></tr><tr><td> (5 - 히드록시 - P - naphthoqinone) Juglone </td><td> ACROS </td><td> 121640010 </td><td> Juglone은 DMSO에 녹아있다 </td></tr><tr><td> DMSO </td><td> 시그마 - 알드리치 </td><td> D - 5879 </td><td></td></tr><tr><td> C. 엘레간스 형질 변형 </td><td> 저자의 연구실 </td><td> VP596 </td><td> Pgst - 4 : GFP 및 Pdop - 3 : RFP </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

하이 스루풋 스크리닝 (HTS)은 생물 학적 과정의 화학 변조기를 식별에 대한 강력한 접근 방식이다. 그러나, 세포 배양 모델을 사용하여 화면에서 확인된 많은 화합물은 종종 생체내 1-2에 독성이나 pharmacologically 비활성으로 볼 수 있습니다. 전체 동물 모델에서 상영하는 것은 이러한 함정을 피하는데 도움이 및 약물 개발에 대한 경로를 합리화 수 있습니다. C. 엘레간스 잘 HTS 적합 다세포 모델 생물이다. 그것은 작은 (&lt;1mm)과 경제적 교양과 액체에 적절하게 사용하실 수 있습니다. C.가 엘레간스 또한 약물 모드의 액션 3 신속하고 상세한 신분증을 허용 가장 실험적으로 다루기 쉬운 동물 모델 중 하나입니다. 우리는 culturing에 대한 프로토콜을 설명하고 C의 형광 변종을 분배 화학 라이브러리 또는 특정 유전자의 표현을 변경할 환경 오염 물질의 검출에 높은 처리량 검사 엘레간스. 발달 동기 웜의 큰 번호, 액체 문화에 재배 수확, 씻어, 그리고 정의 밀도에서 중단됩니다. 웜는 다음 연동 액체 디스펜서를 사용하여 검정, 평평한 바닥 384 잘 접시에 추가됩니다. 화학 라이브러리 또는 테스트 샘플 (예, 물, 음식, 또는 토양)에서 작은 분자는 벌레와 우물에 추가할 수 있습니다. 생체내에서 실시간 형광 강도가 형광 microplate 판독기로 측정됩니다. 이 방법은 C.에 inducible 유전자에 적용할 수 있습니다 적당한 기자 사용할 수있는 엘레간스. 많은 inducible의 스트레스와 발달 transcriptional 경로는 잘 C.에 정의되어 있습니다 엘레간스와 GFP 유전자 변형 기자의 종자는 이미 4 많은 존재합니다. 적절한 형질 기자와 함께하면, 우리의 방법은 경로의 모듈 레이터를위한 화면이나 환경 오염에 대한 강력한 바이오 센서 assays을 개발하는 데 사용할 수 있습니다. 우리는 C를 증명 엘레간스 문화와 분배 프로토콜은 HTS 분석과 함께 우리는 C를 모니터하기 위해 개발된 엘레간스 모자 &#39;N&#39;칼라 전사 인자 SKN - 1. SKN - 1과 포유류의 homologue의 Nrf2 5-10 산화 스트레스와 생체 이물 중 cytoprotective 유전자를 활성화해야합니다. Nrf2는 암, neurodegeneration, 그리고 만성 염증과 같은 다양한 연령과 관련된 질환에서 포유류를 보호하고 주요 chemotherapeutic 대상 11-13되고있다. 우리의 분석은 -4 SKN - 1 대상 유전자 GST 14 GFP 유전자 변형 기자를 기반으로, 이것은 글루타티온 - S transferase 6 인코딩합니다. GST -4 기자도 SKN - 1을 활성화 및 아크릴 아미드와 메틸 수은과 같은 오염 물질의 15-16 낮은 수준을 감지하는 데 사용할 수있는 생체 이물 및 산화 화학 바이오 센서이다.

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

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

의 액체 기반 culturing 및 분배를위한 절차<em&gt; C. 엘레간스</em형광 기자 단백질을 표현&gt; 종자는 고가의 정렬 장비를 필요로하지 않는다는 설명이다. 이 접근법은 많은 inducible에 적용할 수 있습니다<em&gt; C. 엘레간스</em&gt; 오염 물질의 약물 발견을위한 유전자 또는 biosensing.

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 ...

high-throughput-screening-biosensing-with-fluorescent-c-elegans

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JoVE Journal

Biology

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

17.9K 조회수.  University of Florida. 의 액체 기반 culturing 및 분배를위한 절차 C. 엘레간스 종자는 고가의 정렬 장비를 필요로하지 않는다는 설명이다. 이 접근법은 많은 inducible에 적용할 수 있습니다 C. 엘레간스 오염 물질의 약물 발견을위한 유전자 또는 biosensing.

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

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

생산 C. 엘레간스</em함께 Recombineering 통해> Transgenes galK 선택 마커

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

연구

생물학

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

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

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

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

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

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

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

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

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

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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 Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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 ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

높은 처리량 검열을 사용 키나제 기판 쌍의 식별


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Methods Article

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스