우리는 기꺼이 디스커버리 그랜트뿐 아니라 연구 도구 및 자연 과학, 캐나다 공학 연구 협의회 (NSERC)에서 악기 부여를 통해 BHR 지원을 인정합니다. 또한 H.오다와 예제 라이브 영상 시퀀스에 사용되었습니다 유전 주식을 제공하는 블루밍턴 Drosophila 주식 센터를 인정합니다.

준비 <ol><li> 표준 포도 - 쥬스 한천 플레이트 4 배아를 수집합니다. 그것은 자동 Drosophila 에그 콜렉터 (Flymax 첨단 장비 회사)를 이용하시면 편리합니다. 동기를 사용하면 배아 컬렉션 원하는 발달 단계에서 배아의 충분한 숫자를 얻기 위해 수행되어야합니다 dechorionations의 수를 줄일 수 있습니다 개최. </li><li> 현미경 슬라이드에 양면 테이프의 조각을 부착하여 dechorionation 슬라이드를 준비하며 테이프에서지지를 제거합니다. </li><li> 챔버의 잘 적응하기 위해 조직의 일부를 절단하여 라이브 영상 챔버를 준비합니다. 우물에 조직을 넣고 증류수로 젖었어. </li><li> 1시 1분의 비율로 혼합 할로 카본 오일 (할로 카본 제품 주식 회사) 점도 시리즈 700 및 시리즈 56. 혼합물은 저장하고 무기한 사용할 수 있습니다. </li><li> coverslip (22 X 40mm, 제 2)의 표면에 할로 카본 오일의 여러 작은 방울을 넣으십시오. 볼륨 중요한 것은 아니지만 20-40 μl의 순서에 있어야합니다. </li></ol> 절차 <ol><li> stereomicroscope의 도움으로 중 보석의 포셉 한 쌍의 (제 5) 또는 아주 좋은 페인트 브러쉬를 사용하여 포도 주스 - 한천 플레이트에서 배아를 수집합니다. 배아는 서로와 포셉 &#39;팁에 충실하는 경향이, 그들은 쉽게 대단히 짧은 시간에 모여 있습니다. </li><li> stereomicroscope의 도움으로 부드럽게 dechorionation 슬라이드에있는 테이프의 표면에 포셉의 조언을 준수해야 태아의 대단히 짧은 시간을 낮추십시오. </li><li> 다시 stereomicroscope를 사용하여 부드럽게 안쪽 vitelline 막 (vitelline 막이 터진 경우 배아가 파열됩니다) rupturing없이 외부 밀랍 chorion을 열고 침입하기 위해 포셉의 측면으로 배아를 슬쩍 찌르다 또는 뇌졸중. </li><li> 외부 chorion가 파열되면 chorion에서 배아를 애타게, dechorionated 배아는 포셉의 표면을 준수하는 경향이있다. 테이프의 표면에 dechorionated 배아를 만지지 않도록 조심 해요. </li><li> 마자 배가 dechorionated이므로, 빨리 coverslip에 할로 카본 오일의 이전 준비 드​​롭로 전송할 수 있습니다. 할로 카본 오일 드롭의 표면에 dechorionated 배아를 가지고 포셉의 팁을 터치 - 이것은 기름에 포셉에서 배아를 d​​islodges. </li><li> 오일 드롭 배아의 전송을 확인합니다. 이것은 당신이 검정색 배경을 통해 업무와 경사 각도에서 설정 광섬유 조명 소스를 사용하는 제공된 육안으로 수행할 수 있습니다. </li><li> 다른 배아를 d​​echorionate하기 전에, 포셉로부터 기름을 청소하십시오. 기름에 코팅 집게는 dechorionation 더 어렵게 chorion 표면에 적은 구입했습니다. </li><li> Dechorionated 배아는 할로 카본 기름에 부력이 있습니다. 포셉 또는 페인트를 사용하고 stereomicroscope를 통해 보는 동안, 오일 드롭의 하단에 배아를 밀고하고 원하는 위치에 배열. </li><li> 신속 라이브 이미징 챔버 잘 통해 coverslip을 반전. 태아가 원하는 방향으로 coverslip에 대한 쉬고 있는지 확인합니다. 기름에서 부력으로 인해 태아는 이제 coverslip의 낮은 표면으로부터 떠 것입니다. 테이프와 라이브 이미징 챔버로 coverslip을 수정. 테이프가 coverslip의 끝에와 라이브 이미징 챔버 잘의 끝 사이의 간격을 펼쳐져있는 지역의 테이프에 구멍을 절단하여 챔버를 환기. </li><li> 직립 형광 현미경 또는 형광 stereomicroscope 이제 이미지 배아를 사용할 수 있습니다. </li></ol>

<ol>
	<li>Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L., Montague, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forces+contribute+to+cell+sheet+morphogenesis+for+dorsal+closure+in+Drosophila.">Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.</a> J. Cell Biol. 149, 471-490 (2000).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+processes+associated+with+germ+band+retraction+in.">Cellular processes associated with germ band retraction in.</a> , 248-2429 (2002).</li><li>Reed, B. H., Wilk, R., Schock, F., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+apposition+of+Drosophila+extraembryonic+membranes+promotes+morphogenesis+and+prevents+anoikis.">Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis.</a> Curr. Biol. 14, 372-380 (2004).</li><li>Wieschaus, E. p., Nusslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a practical approach. , 199-227 (1986).</li></ol>

우리는 우리가 매달려 드롭 프로토콜을 호출 라이브 영상 분석에 대한 Drosophila의 배아를 준비하는 새로운 방법을 설명합니다. 불행히도, 거꾸로 현미경으로 작업하는 경우 매달려 놓기 프로토콜을 사용하는 것은 불가능합니다. 이 경우에는 sandwiching 기술은 (같은 추상적 위에서 설명한) 사용하고 배아의 압축 우려 남아 있어야합니다. 세포 모양과 크기가 morphogenetic 운동과 ?...

현미경 슬라이드, coverslips (22 X 40mm, 번호 2), 양면 테이프, 보석의 포셉 (더몬트 스타일 번호 5 : 표준 장비 및 매달려 드롭 프로토콜을 사용하여 라이브 영상을위한 배아를 준비하는 데 필요한 자료는 다음과 같은 항목을 포함 ), 할로 카본 오일 시리즈 56 및 시리즈 700 (할로 카본 제품 주식 회사), 유틸리티 나이프 단일 에지 면도날, 조직 종이, 가위, 증류수, 범용 테이프, 그리고 20-40 μl를 전달하기위한 적합한 pipet. 라이브 영상 프로토콜은 또한 사용자 정의 만든 라이브 영상 챔버가 필요합니다. 이것은 5mm가 표준 현미경 슬라이드 (75 X 25mm)의 크기에 폴리 카보 네이트 플라스틱 시트를 생각 절단 및 슬라이드 (20 X 55mm)에 3mm 깊은 우울증을 생성하기 위해 로터를 사용하여 준비가되어 있습니다. stereomicroscope (현미경을 해부) 및 광섬유 조명 소스도 필요에 액세스할 수 있습니다.

the preparation of drosophila embryos for live-imaging using the hanging drop protocol

그린 형광 단백질 (GFP) timelapse 라이브 영상 그러한 조직 morphogenesis, 세포 - 세포 부착, 또는 세포 사망과 같은 동적 프로세스의 유전자 조절을 연구위한 강력한 기법입니다 기반. GFP를 표현 Drosophila의 배아는 즉시 중 입체 또는 공촛점 현미경을 사용하여 몇 군데 있습니다. 살아있는 영상 프로토콜의 목표는 탈수와 hypoxia로 해로운 영향을 최소화하는 것입니다. 라이브 영상 분석을위한 Drosophila의 배아를 준비 이전 프로토콜 할로 카본의 기름에 dechorionated 배아를 삽입하고 할로 카본 가스 투과 막과 coverslip 사이에 그들을 sandwiching 참여했습니다<sup&gt; 1-3</sup&gt;. 이러한 방식으로 장착 배아를 통해 압축의 도입은 morphogenesis의 biomechanical 기반의 분석을위한 바람직하지 않은 복잡을 나타냅니다. 우리가 매달려 드롭 프로토콜, 라이브 영상 중에 태아의 생존에 좋은 결과를 전화 배아를 압축하는 것이 필요하지 않습니다 우리의 방법. 간단히, 매​​달려 드롭 프로토콜은 습기 챔버를 통해 위치에 고정 차례입니다 coverslip,에서 일시 중지되었습니다 할로 카본 오일 드롭 태아의 위치를​​ 포함합니다. 가스 교환을 제공하고 탈수를 방지하는 것 외에도,이 협정 timelapse 취득하는 동안 위치에서 벗어나 표류에서 그들을 방지하기 위해 할로 카본 오일에서 태아의 부력을 활용합니다. 이 동영상은 매달려 드롭 프로토콜을 사용하여 라이브 영상에 대한 Drosophila의 배아를 수집하고 준비하는 방법을 자세히 설명합니다. 이 프로토콜은 stereomicroscopy 또는 수직 복합 형광 현미경을 사용하여 영상 dechorionated 배아에 적합합니다.

Watch this Scientific Journal Video about The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol at JoVE.com

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

라이브 영상 분석을위한 Drosophila의 배아를 준비, 간단한 저렴하고 효과적인 방법이 제공됩니다. 우리 프로토콜은 습도와 가스 교환을 제공하고 Drosophila의 배아를 압축하지 않습니다. 이 방법은 stereomicroscope 또는 수직 복합 현미경을 사용하여 Drosophila의 배아의 GFP 기반의 라이브 영상에 적합합니다.

의 준비 Drosophila</em공중 드롭 프로토콜을 사용하여 라이브 영상을위한> 태아

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

15.0K 조회수.  University of Waterloo. 라이브 영상 분석을위한 Drosophila의 배아를 준비, 간단한 저렴하고 효과적인 방법이 제공됩니다. 우리 프로토콜은 습도와 가스 교환을 제공하고 Drosophila의 배아를 압축하지 않습니다. 이 방법은 stereomicroscope 또는 수직 복합 현미경을 사용하여 Drosophila의 배아의 GFP 기반의 라이브 영상에 적합합니다.

비디오: The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrula 스테이지 Drosophila의 태아에서 문화의 연결 준비

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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생물학

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

공중 드롭 방법을 사용하여 마우스 배아 줄기 (MES) 세포의 체외 차별에

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Drosophila 아이 Imaginal 디스크에 Glial 세포 이미징 마이 그 레이션 라이브

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

개발 및 성인의 준비 Drosophila 두뇌와 라이브 영상을위한 Retinae

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

개별 준비 Drosophila</em라이브 이미징을위한> 계란 회의소

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...