의학의 알버트 아인슈타인 대학에서 시작 자금,이 작품은 부분적으로 국민 건강의 연구소 (RDS에 3U54AI057158 - 06S1 PW로 GM093282)에 의해 지원되었다.

1. 에그 수집 및 Dechorionation <ol><li> 수집 및 35mm 페트리 접시에 zebrafish 달걀을 전송, 가능한 한 많은 물을으로 제거한 다음 E3 배아 매체 1 MG / ML Pronease 전자 (5 MM NaCl, 0.17 MM KCl, 0.33 MM CaCl 2 · 2 시간 2 O, 0.33 MM MgSO을 추가 chorion을 소화하기 위해 4, 산도 = 7.4). </li><li> 3~5분 후, 생선 물 (리터 60 MG &quot;인스턴트 오션 &#39;소주 H 2 O)로 가득 비커에 요리를 병합하고, 부드럽게 비커에 달걀을 전송하고 계란 물을에&quot;가을 &quot;수 있습니다. </li><li> 생선 물 세 번와 계란을 씻어. 대부분의 달걀들은 chorion에서 발표됩니다. </li><li> 불타는 광택 유리 파스퇴르 피펫을 사용하여 E3 배아 매체 가득 아가로 오스 - 코팅 배양 접시에 dechorionated 달걀을 전송합니다. </li></ol> 2. GDP - FucAl로 Microinjection <ol><li> 보고된 7 번 프로토콜 다음과 같은 사출 요리를 준비합니다. </li><li> E3 배아 매체 가득 주입 요리에 dechorionated 계란을 전송합니다. </li><li> 바늘을 준비, 2 μL 주입 솔루션을로드합니다. 이 솔루션은 추적기 0.2 M KCl에 페놀 레드 로딩 염료 (0.1 % W / V)와 같은 20 GDP - FucAl가 chemoenzymatically 8 합성 MM하거나, 알렉사 플루어 594 - dextran (5 % W / V)가 포함되어 있습니다. 대조군으로 주입 용액에 GDP - fucose와 GDP - FucAl를 교체하십시오. </li><li> 바늘을 부수고 1 NL 드롭 9 얻을 수있는 분사 압력 및 시간을 조정합니다. </li><li> 두 솔루션 1 NL와 계란을 주사. </li><li> E3 배아 매체 가득 아가로 오스 - 코팅 배양 접시에 계란을 전송합니다. </li><li> 28 ° C에서 알을 품어하고 배입니다 3-4시간 이내 unfertilized 계란을 제거합니다. </li></ol> 3. BTTES - 잘라내기 (I) 촉매 클릭 화학 반응 <ol><li> 배아가 발달 단계 (예 : 후반 gastrula, 조직 세분화 및 초기 유충), 코트 아가로 오스와 96 - 웰 플레이트의 기반을 원하는 도달했을 때. </li><li> 각 잘하는 92 μL E3 배아 매체를 추가, (H 2 O의 2.5 MM 주식에서) 4 μL 알렉사 플루어 - 488 azide를 추가하여 다음 2 μL BTTES - CuSO 4 6시 1분 복잡하고 섞어 부드럽게 발라줍니다. </li><li> 잘 불타는 광택 유리 파스퇴르 피펫을 사용하여 클릭 화학 시약을 포함하는으로 배아를 전송합니다. 각 잘 다섯 배아 미만을 포함해야합니다. </li><li> 클릭 반응 3 시작 2.5 μL 신선한 나트륨 아스코 브를 (H 2 O에서 100 MM 재고에서) 추가합니다. 각 시약의 최종 농도 : 알렉사 플루어 - 488 azide : 100 μm의, CuSO 4 : 50 μm의; BTTES : 300 μm의, 나트륨 아스코 브 : 2.5 MM. </li><li> 3 분 후, 100 μL E3의 배아 매체 즉시 희석 후 반응을 담금질하기 위해, 2 μL bathocuproine sulphonate (H 2 O 50 MM 재고), biocompatible 구리 chelator를 추가합니다. </li><li> 유리 페트리 접시에 배아를 전송 15 ML E3 배아 매체로 취급 태아에게 2 번 씻는다. </li></ol> 4. 이미징 <ol><li> MatTek 유리 바닥 microwell 요리에 ultralow 용융 점 아가로 오스 (E3 배아 매체에서 1.2 % (W / V))의 방울 장소, 아가로 오스 드롭으로 배아를 놓으십시오. </li><li> dorsally 또는 옆으로 배아를 위치와 아가로 오스 드롭을 응고 5 분 얼음 접시를 놓습니다. 그것은 아가로 오스 드롭을 커버 때까지 음식을 부드럽게 E3 배아 매체를 추가합니다. </li><li> 형광 및 명시야 이미지는 순차적으로 공촛점 현미경을 사용하여 획득하고 있습니다. 모든 배아 이미지는 5 μm의 단계 간격을 사용하는 인수입니다. 복합 수치는 ImageJ 소프트웨어를 사용하여 준비가되어 있습니다. </li></ol> 5. 대표 결과 그림 1은 우리 두 단계 라벨링 전략의 흐름을 보여줍니다. 그림 2는 9.5 시간 게시물 수정 (HPF)에서 BTTES - 중재 CuAAC 통해 zebrafish 배아의 라벨을 보여줍니다. BTTES는 트리스 (triazolylmethyl) 아민 기반 리간드이다. 그것은 현장에서 생성된 잘라내기 (I)로 조정하면 극적으로 CuAAC 가속하고, 명백한 독성없이 생활 시스템에 빠르게 cycloaddition 반응을 촉진합니다. 즉시 3 분 클릭 반응 다음, 우리는 국내 총생산 (GDP) - FucAl 취급 배아의 강력한 라벨 (그림 2, 왼쪽 패널)을 감지할 수 있습니다. 단지 배경 형광은 GDP - fucose (그림 2, 오른쪽 패널)과 microinjected 제어 배아의 발견입니다. <img src="/files/ftp_upload/2686/2686fig1.jpg" alt="그림 1" /> 그림 1. zebrafish 배아의 뒤덮었 레이어 fucosylated glycans를 라벨링의 전략. <img src="/files/ftp_upload/2686/2686fig2.jpg" alt="그림 2" /> 그림 2. BTTES - 잘라내기 (I) 촉매를 클릭 화학 통해 zebrafish embryogenesis 동안 fucosylated glycans의 생체내 이미징에서. 한 세포 단계의 배아 zebrafish는 GDP - FucAl의 단일 복용과 함께 microinjected과 devel 수있게됩니다9.5 HPF로 조합. 배아는 다음 알렉사 플루어 BTTES - 잘라내기 (I)에 의해 488 - azide의 촉매와 반응하고 있습니다. 반응 배아는 공촛점 현미경을 사용하여 몇 군데 있습니다. 명시야 (하단 패널), 알렉사 플루어 488 형광 (어퍼 패널)의 최대 강도 Z - 프로젝션 이미지. 스케일 바 : 100 μm의. 문제 해결 <table border="1"><tbody><tr><td> 문제 </td><td> 원인 </td><td> 치료제 </td></tr><tr><td> 배아는 건강에 해롭습보세요 </td><td> microinjected 솔루션은 오염 물질을 포함하고 </td><td> 뉴클레오 티드 설탕의 순도 85 %보다 커야한다. 소스를 확인합니다. </td></tr><tr><td> 반응 후 아무 라벨링이 없습니다 </td><td> 구리 농도는 아래 30 μm의 수 있습니다 </td><td> 배아를 추가할 때 구리 농도가 아래에 30 μm의 때 반응 속도가 크게 방울로 초과 E3의 배아 미디어를 추가하여 반응 용액을 희석하지 않도록주의하십시오. </td></tr><tr><td> 배아는 반응 후 다이 </td><td> 배아의 부적 절한 처리 </td><td> pipet 불타는 광택이며 반응 용기는 0.5 % 아가로 오스의 매우 얇은 레이어로 코팅되어 있는지 확인합니다. </td></tr><tr><td> 이미지가 낮아요보세요 </td><td> 시약은 제대로 배아를 추가하기 전에 혼합하지 않습니다 </td><td> 시약에 대한 이외의 권장 순서를 따라, 그리고 클릭 화학 시약 제대로 배아를 추가하기 전에 혼합되었는지 확인하십시오. </td></tr><tr><td> 이미지가 배아가 손상보기 </td><td> 반응 동안 힘차게 손해를 흔들림하면 배아 </td><td> 배아는 솔루션에 한번 부드럽게 미만 10 초 동안 흔들. </td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

<ol>
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관심 없음 충돌 선언하지 않습니다.

<p class="jove_content"&gt; 이미징 biomolecules<em&gt; 생체내에</em&gt; 자신의 모국어 환경에서 자신의 생물 학적 활동의 중요한 통찰력을 제공합니다. 이 비디오에서는, 우리는 zebrafish 배아의 뒤덮었 계층에 fucosylated glycans의 라벨이 BTTES - 중재 biocompatible CuAAC 통해 GDP - FucAl 하나의 세포 단계의 배아와 두 번째 단계 형광단의 활용을 microinjecting하여 실현하는 방법을 보여줍니다<sup&gt; 3</sup&gt;. 강력한 라벨이 2~3분 이내에 얻을 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 구리 (II) 황산염의 pentahydrate </td><td> 시그마 - 알드리치 </td><td> 203165 </td></tr><tr><td> 알렉사 플루어 488 azide </td><td> Invitrogen </td><td> A10266 </td></tr><tr><td> dextran, 알렉사 플루어 594 </td><td> Invitrogen </td><td> D - 22913 </td></tr><tr><td> (+) - L - 아스코 브 산 나트륨 </td><td> 시그마 - 알드리치 </td><td> A7631 </td></tr><tr><td> Bathocuproinedisulfonic 산성 </td><td> Acros의 생명체 </td><td> 164060010 </td></tr><tr><td> 유리 바닥 microwell 요리 </td><td> MatTek </td><td> P35G - 1.5-14 - C </td></tr></tbody></table>

imaging glycans in zebrafish embryos by metabolic labeling and bioorthogonal click chemistry

생체내의 이미징 glycans 최근 azide 또는 alkyne - 태그 단당류 1, 2 세포 또는 유기체를 처리하여 bioorthogonal 화학 기자 전략을 사용하여 활성화되었습니다. glycan의 biosynthetic 기계에 의해 처리 수정 단당류는, 세포 표면 glycoconjugates에 통합됩니다. bioorthogonal azide 또는 alkyne 태그 다음 시각화를위한 형광 프로브와 공유 결합 결합을 허용하거나, 농축 및 glycoproteomic 분석을위한 화성 탐사선과. 의 뒤덮었 레이어 fucosylated glycans를 라벨 GDP - 5 - alkynylfucose (GDP - FucAl), 2 하나의 세포 단계의 배아 1) microinjection) :이 프로토콜을 포함하여 일반적으로 zebrafish 배아에서 fucosylated glycans의 비침 투 이미징에 사용되는 절차를 설명 공촛점 현미경 3 biocompatible 잘라내기 (I) 촉매 azide - alkyne cycloaddition의 (CuAAC), 3) 영상을 통해 azide - 복합 fluorophores와 zebrafish 배아. 여기에서 설명한 방법은 쉽게 glycans의 다른 클래스, sialic 산성 4 N - acetylgalactosamine 5, 6를 포함하는 예 glycans 개발 zebrafish과 다른 살아있는 생물에을 시각화하기 위해 확장될 수 있습니다.

Watch this Scientific Journal Video about Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry at JoVE.com

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

zebrafish 배아에서 alkyne - 태그 glycans의 급속한, 비침 투, 그리고 강력한 라벨을 허용 클릭 화학 기반 방법을 설명합니다. 후반 gastrulation 단계에 zebrafish 배아의 뒤덮었 레이어 Fucosylated glycans이 연구에서 몇 군데 있었다.

신진 대사 라벨링 및 Bioorthogonal 클릭 화학에 의해 Zebrafish 태아의 영상 Glycans

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

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Research

JoVE Journal

Biology

13.6K 조회수.  Albert Einstein College of Medicine, Yeshiva University. zebrafish 배아에서 alkyne - 태그 glycans의 급속한, 비침 투, 그리고 강력한 라벨을 허용 클릭 화학 기반 방법을 설명합니다. 후반 gastrulation 단계에 zebrafish 배아의 뒤덮었 레이어 Fucosylated glycans이 연구에서 몇 군데 있었다.

비디오: Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Zebrafish 태아의 mRNA와 Morpholino의 안티 센스 Oligonucleotides의 Microinjection.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

연구

생물학

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

라이브 zebrafish의 배아에서 두 광자의 axotomy하고 시간이 경과 공촛점 영상

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

이식에 의해 키메라 Zebrafish의 배아를 생성

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

의 이식 GFP - 표현 키메라 Zebrafish의 태아의 망막과 두뇌 개발의 라이브 영상에 대한 Blastomeres을

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

신진 대사 레이블 및 유동세포계측법와 세포주기 진행의 동력학 측정

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Zebrafish 태아의 대사 프로필 분석

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

류신 리치의 신진 대사 라벨링 방사성 인산과 키니 아제 1과 2를 반복

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

제브라 피쉬 배아에서 산화 스트레스의 분석

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish의 태아의 조직 특이 형광의 반 자동화 이미징

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...