이 작품은 존스 홉킨스에​​서 ALS 연구 팩커드 센터 (ADG), NIH 원장의 새로운 이노 수상 1DP2OD004417 - 01 (ADG), NIH R01 NS065317 (ADG), 리타 앨런 재단 학술 상에서 교부금에 의해 지원되었다. ADG는 퓨 자선 트러스트가 지원하는 의생명 과학 퓨 학술 있습니다.

1. 효모 변환을위한 준비 이 프로토콜은 열 96 - 웰 플레이트를 위해 설계되었습니다하지만, 이에 따라 위 또는 아래로 확장할 수 있습니다. 우리는이 프로토콜은 변화 라운드 당보다 20 96 - 웰 플레이트에 대해 제대로 작동하지 않는 것으로 확인되었습니다. 전체 변환 절차 (단계 I.3에서) 약 여덟 시간이나 걸립니다. <ol><li> 나누어지는 Biorobot의 RapidPlate 액체 처리기와 함께 96 - 웰 플레이트 왕복 아래의 각 우물에 효모 ORF FLEXGene 라이브러리에서 플라스미드 DNA (μl / 100 NG)의 5 10μL. 장소 건조 하룻밤 클린 벤치 펌 모자 96 - 웰 플레이트를 발견. 우리는 센와 2μ 효모의 plasmids과 유사한 변환 효율성을 발견했다. </li><li> 500mL 당황하게 Erlenmeyer 플라스크에서 쿼리 변형과 효모 펩톤 덱스 트로 오스 (YPD) 미디어 (10g / L 효모 추출물, 20g / L 펩톤, 20g / L의 포도당)의 200mL의 예방. (200 RPM) 흔들림 30 ° C에서 밤새 품어. 필요한 경우, 선택적 미디어 이러한 스타터 문화 사용할 수 있습니다. </li><li> 다음날 아침, = 0.1 YPD의 2L에서 OD 600 쿼리 긴장을 희석. 30 ° C (200 RPM) 문화 OD 600 = 0.8-1.0에 도달할 때까지에서 4-6시간 위해 흔들어. 최적의 성장을위한 별도의 2.8 L 당황하게 flasks 두 1L 문화를 성장. 필요한 경우, 선택적 미디어도 YPD 대신 사용할 수 있습니다. </li></ol> 2. 효모 변환 <ol><li> 원심 분리하여 세포를 수확. 테이블탑 원심 분리기 5 분 (Eppendorf 5810R)에 대한 3000rpm (~ 1800 XG)에서 효모 문화와 스핀없이 네 일회용 225mL 원뿔 튜브를 입력합니다. 뜨는을 붓고 모든 세포를 수확 때까지 반복합니다. </li><li> autoclaved 소주 H 2 O.의 200mL의 세포 와시 흔들어 또는 vortexing하여 Resuspend 세포 펠릿. 한 225mL 원뿔 튜브에 세포를 결합합니다. 5 분 3000rpm에서 스핀. 뜨는 제거합니다. </li><li> 0.1MLiOAc/1XTE 솔루션 (0.1M LiOAc, 10mM 트리스, 1mM EDTA (에틸렌 다이아 민 테트라 초산), autoclaved 증류수에 산도 8) 준비합니다. 100mL 0.1MLiOAc/1XTE의 세포를 씻으십시오. 5 분 3000rpm에서 스핀. 뜨는 제거합니다. </li><li> 70mL 0.1M LiOAc/1xTE에 Resuspend 세포 펠릿. 흔들어 및 / 또는 소용돌이 펠렛 완전히 resuspended되도록합니다. </li><li> 30 분 (200 RPM) 30 ° C에서 잡고 알을 품다. </li><li> 0.1M로 β - 메르 캅 토 에탄올을 추가합니다. 30 분 (200 RPM) 30 ° C에서 잡고 알을 품다. 참고 : β - 메르 캅 토 에탄올로 구분 효모 종자를 사용하는 경우이 단계는 생략할 수 있습니다. 우리는이 단계가 성공적으로 변환을 위해 필수적 있지 않은 것으로 나타났습니다, 그러나 그것은 변환 효율을 개선 않습니다. </li><li> 15 분 동안 sonicated 연어 정자 DNA (10mg/mL)의 2mL를 삶아 후 얼​​음에 진정해. </li><li> 세포 혼합물에 삶은 연어 정자 DNA의 2mL를 추가합니다. 주의 : 침전물은 세포의 혼합물로 연어 정자 DNA의 형태에 따라 또한 수 있습니다. pipetman 200 μl 팁을 사용하여, 신중하게이 하류 pipetting을 방해할 수 있기 때문에 어떤 형태의 침전물을 제거합니다. </li><li> BioRobot의 Rapidplate를 사용하여 96 - 웰 플레이트의 각 잘하는 세포 혼합물 나누어지는의 50μL은 (또한 멀티 채널 피펫을 사용할 수 있습니다.) 혼합하지 마십시오. </li><li> 떨지 않고 30 분 동안 상온에서 알을 품다. </li><li> 200mL 40 %에게 PEG-3350/10 % DMSO/0.1M LiOAc을 (160mL 50 % PEG - 3350, 20mL DMSO, 20mL 1M LiOAc) 준비합니다. 바로 사용하기 전에 솔루션을 준비합니다. </li><li> 각 잘하는 PEG / LioAc / DMSO 용액 125μL를 추가합니다. RapidPlate로 8 번 aspirating 및 세포 현탁액을 분배하여 섞는다. </li><li> 떨지 않고 30 분 동안 상온에서 알을 품다. </li><li> 열충격 42 세포 ° C 건조한 인큐베이터에서 15 분. 접시를 쌓아하지 마십시오. 참고 :이 열 충격 단계가 성공적으로 변환을 위해 필수적 있지 않은 것으로 나타났습니다. 특정 효모 종자 (예를 들어, 온도에 민감한 돌연변이)의 경우, 열 충격 해로운 것입니다. 우리는 이전에 1 분 정도 가열 충격 시간을 단축하고 성공적으로이 프로토콜을 사용하여 ypt1 온도에 민감한 돌연변을 품고 효모 긴장을 변환 할 수있다. </li><li> 96 - 웰 플레이트를 수용하기 위해 원심 분리기 어댑터를 사용하여 5 분 2500rpm (~ 1100 XG)에서 번호판을 돌려. 폐기물 양동이를 통해 반전 접시로 PEG 용액을 제거합니다. 잔류 액체를 제거하기 위해 종이 타월에 반전 번호판을 가릴 (전지 우물의 바닥에 남아있게됩니다). </li><li> RapidPlate 각 잘하는 (예를 들어 SD / - 우라) 최소 미디어 200μL를 추가하여 세포를 씻어. </li><li> 5 분 2500rpm에서 번호판을 스핀과 반전을 통해 폐기물 양동이와 종이 타월에 모래 바닥에서 뜨는 제거합니다. </li><li> RapidPlate 각 잘하는 최소한의 미디어 200μL (예 : SD / - 우라) 추가합니다. </li><li> 떨지 않고 2 일 30 ° C에서 알을 품다. 이일 후, 세포의 작은 식민지를 성공적으로 잘 변형 각의 하단에있는 양식을 시작합니다. </li></ol> 3. 분석 구경하기 <ol><li> 각 우물에 raffinose을 기반으로 최소한의 미디어 (예 : SRaf / - 우라)의 200μL를 분배새로운 평면 바닥 96 - 웰 플레이트. </li><li> Rapidplate로 8 번 aspirating 및 세포 현탁액을 분배하여 변환 플레이트의 각 잘 섞는다. </li><li> 변환 접시에서 세포 혼합물 5μL와 SRaf 접시의 예방. </li><li> 하루 30 ° C에서 알을 품다. 작은 식민지 언젠가는 이후 각 우물의 하단에 제시해야합니다. </li><li> SD / - 우라와 모자 SGal / - 우라 플레이트에 스팟 식민지. 불타는하여 96 볼트 복제 (frogger)을 소독. 선택적 미디어 접시에 야생 전에 frogger과 식민지를 섞습니다. 관찰 후, 플레이트 5-10 분 동안 모자 건조 수 있습니다. </li><li> 2-3 일동안은 30 ° C에서 알을 품다. </li></ol> 디지털 카메라와 사진의 접시는 시각 쿼리 스트레인 독성이 (빠른 성장 / 더 밀도 식민지) (느린 성장 / 이하 밀도 식민지) 또는 표시되지 향상된되는 식민지를 찾을 수 SGal / - 우라 플레이트에 식민지의 성장을 비교합니다. 4. 대표 결과 : <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="그림 1"/> 그림 1. 효모 플라스미드 overexpression 화면 TDP - 43 독성의 진압 및 강화를 식별합니다. TDP - 43은 루게릭병 (루게릭 병)의 pathogenesis에 연루되어있는 인간의 단백질이다. TDP - 43의 세포질 집계는 두뇌와 ALS 환자 17 척수 신경에 축적. 집계 및 세포 독성 18 효모 세포 결과에 TDP - 43를 표현. 우리는 TDP - 43 19,20 독성의 메커니즘을 정의하기 위해이 모델 시스템을 사용하고 있습니다. 그림은 우리의 누룩 TDP - 43 독성 수정자 화면에서 접시의 repesentative 예입니다. 이 번호판은 FLEXGene ORF 표현 라이브러리에서 plasmids과 통합 갈락토 오스 - inducible TDP - 43 플라스미드 또한 변형과​​ 식민지를 표시합니다. 왼쪽에있는 플레이트는 TDP - 43 또는 FLEXGene의 plasmids의 표현을 represses 포도당을 포함하고 있습니다. 오른쪽 접시는 TDP - 43의 표현과 FLEXGene의 plasmids의 ORFs를 유도 갈락토 오스를 포함하고 있습니다. 녹색 화살촉은 TDP - 43의 독성을 억제하는 플라스미드로 변환 식​​민지를 나타냅니다. 빨간 화살촉은 TDP - 43의 독성을 강화하는 플라스미드로 변환 식​​민지를 나타냅니다.

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관심 없음 충돌 선언하지 않습니다.

여기 우리는 효모에서 높은 처리량 플라스미드 overexpression 화면을 수행하는 프로토콜을 제시한다. 이러한 접근 방식은 여러 세포 phenotypes의 유전자 조절에 대한 신속하고 불편 검사 수 있습니다. 이 방법을 사용하여 연구원이 주 만에 효모 게놈의 상당 부분을 스크린 수 있습니다. 이러한 불편 접근법은 또한 이전의 연구 결과에 따라 예측되지 않았을 수 있습니다 수정자의 식별을 위해 수 있습니...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9,000,490 </td></tr><tr><td> 96 볼트 레플리케이터 (frogger) </td><td> V &amp; P 과학 </td><td> VP404 </td></tr><tr><td> FLEXGene ORF 도서관 </td><td> Proteomics 연구소, 하버드 메디컬 스쿨 </td><td></td></tr><tr><td> 테이블탑 원심 분리기 </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500mL 플라스크 실패하는 </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L 트리플 당황하게 Fernbach 플라스크 </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate 피펫 팁 </td><td> Axygen </td><td> ZT - 100 - RS </td></tr><tr><td> 200μL Rapidplate 피펫 팁 </td><td> Axygen </td><td> ZT - 200 - RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

신진 효모, Saccharomyces cerevisiae의는 인간의 질병에 직접 관련성이있는 사람 등 많은 중요한 세포 과정의 기본 메커니즘을 정의하는 강력한 모델 시스템입니다. 워낙 짧은 세대 시간과 잘 특징 게놈의 효모 모델 시스템의 주요 실험 이점은 주어진 과정에 관련된 유전자 및 경로를 식별하는 유전자 화면을 수행할 수있는 능력입니다. 지난 30 년 동안 같은 유전자 화면은 세포주기, 분비 경로와 진핵 세포 생물학 1-5의 많은 높은 보존 측면을 명료하게하다하는 데 사용되었습니다. 지난 몇 년 동안, 효모 종자와 plasmids 여러 genomewide 라이브러리는 60-10을 생성되었습니다. 이 컬렉션은 이제 이득과 손실 함수 접근법 11-16을 사용하여 유전자 기능의 체계적인 심문을 위해 수 있습니다. 여기 우리는 5500 효모 plasmids의 못하였 느니라 라이브러리를 사용하여 플라스미드 overexpression 화면을 수행하는 액체 처리 로봇과 높은 처리량 효모 변환 프로토콜의 사용에 대한 자세한 프로토콜을 제공합니다. 우리는 집합을 일으키는 인간 neurodegenerative 질병 단백질의 축적과 관련된 독성의 유전자 조절을 식별이 화면을 사용하고 있습니다. 여기에 제시 방법은 관심의 다른 세포 phenotypes의 연구에 쉽게 적용할 수 있습니다.

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

여기에 플라스미드 overexpression 화면 설명<em&gt; Saccharomyces cerevisiae</em&gt; 액체 처리 로봇과 못하였 느니라 플라스미드 라이브러리 및 높은 처리량 효모 변환 프로토콜을 사용합니다.

높은 처리량 효모 플라스미드 Overexpression 화면

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

high-throughput-yeast-plasmid-overexpression-screen

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JoVE Journal

Biology

16.3K 조회수.  University of Pennsylvania School of Medicine. 여기에 플라스미드 overexpression 화면 설명 Saccharomyces cerevisiae 액체 처리 로봇과 못하였 느니라 플라스미드 라이브러리 및 높은 처리량 효모 변환 프로토콜을 사용합니다.

비디오: High-throughput Yeast Plasmid Overexpression Screen

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

연구

생물학

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

리그노셀룰로오스성 자료에 대한 높은 처리량 당화 분석

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

선호도 태그 재조합 단백질의 높은 처리량 정화

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

높은 처리량 SHAPE를 사용하여 RNA 이차 구조 예측

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

성게 접합자의 높은 처리량 Microinjections

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

루시 페라 제 발현 재조합 바이러스의 높은 처리량의 적정

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

높은 처리량<em&gt; 다니오 레 리오 (rerio)</em&gt; 에너지 지출 분석

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...