이 프로토콜은 현재 저희 실험실에서 사용되고 DRS 데이비드 샤프, Mijung 권, Patrizia Sommi, Dhanya Cheerambathur를 포함한 많은 사람들이 지난 몇 년 동안 정제되었습니다. 우리는이 시스템에서 우리의 작업이 시작 받고 초기 Drosophila의 배아의 조작과 microinjection (ref. 13)에 대한 훌륭한 조언으로 우리를 제공 박사 빌 설리반 (UCSC)를 주셔서 감사합니다. 우리는 스칼리 연구소의 모든 구성원 감사합니다. Drosophila에서 유사 분열에 우리의 작품은 NIH 부여 GM55507에 의해 지원됩니다.

요리법 : 포도 주스 플레이트 : <ul><li> 5.5g bacto 한천 </li><li> 14.5 g 덱스 트로 오스 또는 포도당 </li><li> 7.15 g의 자당 </li><li> 45 ML의 포도 주스 농축물 (100 % 주스). </li><li> 204.5 ML H 2 0 </li><li> 625 μl 10N NaOH </li></ul> 끓는까지 모든 재료와 전자 레인지를 섞는다. 2.8 ML의 산성 믹스 (20.9 ML 프로피 온산, 2.1 ML의 인산 27 ML H 2 0 산성 믹스) 추가 믹스와 35mm 페트리 요리에 부어. 한 이틀 동안 실온에서 고체화 보자. 접시가 곧 사용할 수 않을 경우, 4 parafilm과 인감 유지 ° C (사용하기 전에 RT로 평형 수 있습니다.) 효모 붙여넣기 : 두꺼운 붙여넣기를 형성 효모 중 한 티스푼 (시그마 YSC2, Saccharomyces cerevisiae 유형 II에서 효모) 물에 대한 용해. 사용하기 직전 각 포도 주스 판에이의 작은 금액을 넣으십시오. G - PEM 버퍼 : <ul><li> 80 MM 나 - 파이프, 산도 6.9 </li><li> 1 ㎜ MgCl 2 </li><li> 1 ㎜ EGTA </li><li> 1 ㎜ GTP </li></ul> 사출 버퍼 : <ul><li> 150 MM K - Aspartate </li><li> 10 MM K - 인산 </li><li> 20 MM 이미다졸, 산도 7.2 </li></ul> 헵탄 접착제 : 100ml 병 두 번 끈적끈적한 테이프 및 장소를 풀다, 50ml 헵탄에 대한 추가 며칠 동안 병, 그리고 바위 인감. 접착제가 너​​무 두꺼운 경우 사용하는 경우, 헵탄 추가할 수 있습니다. 탈수 챔버 : , 100mm 페트리 접시를 타고 &quot;표&quot;를 만들기 위해 35mm 요리 내부의 한 부분을 넣고 Drierite의 높이가 &quot;테이블&quot;및 커버보다 높은 어떤 있도록 주위 Drierite을 (무수 황산 칼슘) 추가하지 않습니다. 배아와 coverslip은 사출하기 전에 탈수이 &quot;테이블&quot;에 배치됩니다. 그것은 적어도 몇 나타내는 Drierite를 사용하고 색상을 변경했을 때 그것을 변경하는 것이 좋습니다. 프로토콜 : 이 프로토콜은 Drosophila syncytial 배아에 거의 모든 용해 시약의 주입에 사용할 수 있습니다 : 예를 들어, 형광 관찰이나 대상 단백질 억제제 단백질 또는 둘 다. 모든 준비하기 : , 플라스틱 병 (6온스, 응용 과학 Drosophila에서 라운드 바텀)를 타고 반대편에 두 개의 작은 구멍을 (2 1cm 정도) 컷 : 1 .- 실험 전에 2-3일는 포도 주스 번호판을 확인하고 설정 케이지하다 병 및 면화의 조각 (이것은 공기 흐름 수)로 기입은 병으로 파리를 누르고 포도 주스 플레이트와 커버. 25 유지 ° C (또는 상온), 파리는 약 10 일 동안 배아 누워해야합니다. 실험 주사 바늘을 뽑아 2 .- 최소한 1 일 전에, 우리는 Kopf 니들 / 피펫 끌어당기는 모델 720 및 얇은 벽의 유리 필라멘트 (세계 정밀 계측기 tw100f)를 사용합니다. 형광 tubulin의 주입을 위해, 4 바늘을 유지 ° C가 주사 바늘에 온도 유도 중합을 방지하기 위해. Tubulin 준비 : 참고 : tubulin과 함께 모든 작업이 4 이루어져야 ° C가 중합을 방지하기 위해. <ol><li> 냉동고의 표시 tubulin을 꺼내 5 ~ 15 분 정도 얼음에 넣어. </li><li> G - PEM 버퍼 (4 10 배 이상이 요구되는 강도에 따라)로 희석. </li><li> 4 13k rpm으로 원심 분리기 ° 10-15분를위한 C. </li><li> 로드 1 - 바늘에 2 μl. </li></ol> 항체 또는 화학 억제제 : 적절한 농도 (이것은 테스트해야 할 수도 있습니다)를 사용하여, tubulin에 대해서는 바늘을 준비합니다. 항체는 사출 버퍼 G - PEM 버퍼 또는 PBS 수 있습니다. 다른 버퍼가 필요한 경우,이 버퍼는 버퍼 자체에 아무런 손상을 초래하지 않도록 제어로 주입해야합니다. 참고 : 억제제와 형광 tubulin이 혼합 및 농도가 허용하는 경우 함께 주입 수 있습니다. 그렇지 않으면, 형광 tubulin을 삽입하고 억제제를 주사하기 전에 5-10분을 기다립니다. 적은 배아가 정상적으로 복구하기 때문에 이중 주사를 수행하면, 더 많은 배아와 함께 시작합니다. 배아 컬렉션 <ol><li> 라이 새장에 새 포도 주스 플레이트 놔. </li><li> 일시간 후에이 판을 제거, 이것이 오늘의 첫 컬렉션이며, 대부분 매우 좋지 않습니다. 그것은 삭제할 수 있습니다. </li><li> 일시간 각 수집 배아를 유지하기 위해 판 매 시간마다를 변경합니다. </li></ol> Coverslip 준비 : <ol><li> 현미경 슬라이드의 한쪽에 50 X 22mm coverslip 놔. 슬라이드 테이프 네 모퉁이가 움직이지 않도록. </li><li> 커튼을 사용하면, 작은 주걱을 밀고 coverslip (접착제는 점성하지 않아야하고 몇 초 만에 건조해야하고, 그렇지 않으면 더 헵탄 추가) 한 줄에 헵탄 접착제 중 하나가 레이어를 넣어. </li><li> 한 조각을 넣어coverslip의 측면 방향으로 슬라이드에 끈적끈적한 테이프를 두 번. </li></ol> 배아 준비 : 참고 : 태아가 컬렉션의 시작 후 2 시간 정도 군데해야하므로 충분한 시간이 시간 프레임 내에이를 완료 수 있도록 다음 단계를 시작합니다. <ol><li> moistened 브러시로 조심스럽게 슬라이드를 두 번 끈적거리는 테이프에 포도 주스 판과 장소에서 배아를 선택하십시오. </li><li> chorion (외부 멤브레인)가 열려있는 휴식까지 족집게의 바깥쪽 부분을 사용하여 두 끈끈한 테이프 통해 배아를 롤. </li><li> 그것은 트위터에 막대기와 coverslip의 긴 측면에 배아 병렬의 긴 면을 coverslip에 헵탄 접착제에 배치하므로 부드럽게 chorion을 통해 그것을 압연하여 배아를 선택하십시오. 행 하나에 10-20 배아를 설정합니다. </li><li> coverslip을 제거하고 3-8분 (이것은 객실과 주입하는 금액의 습도에 따라 다릅니다)의 탈수 챔버에 놓으십시오. </li><li> 진공 그리스와 금속 챔버에 놓습니다. </li><li> 더 탈수 방지하기 할로 카본 오일 700으로 배아를 커버. 배아는 이제 주사에 대한 준비가되어 있습니다. </li></ol> 배아 주입 : <ol><li> 16X 목표 아래 배아를 찾습니다. </li><li> 멀리 배아를 이동하고, 초점 비행기를 이동하지 않고 바늘을 찾으십시오. </li><li> 센터 바늘과 X 또는 Y 방향으로 이동하지 않고 그것을 이동하십시오. </li><li> 바늘이 열려 있지 않은 경우보기의 분야에서 coverslip의 가장자리를 넣어하지만 바늘이되지 않습니다 어디에, 그리고 같은 초점 비행기에 바늘을 낮추십시오. 그것은 바늘을 명중하고 부드럽게 열어 휴식까지 매우 조심스럽게 coverslip 이동합니다. 바늘이 열려 있으면 5 단계로 이동합니다. (니들이 작성 전에 불화 수 소산​​ 산성과 열 수 있습니다.) </li><li> 보기에서 배아 (하지만 바늘이 거기에있을 것이다)을 넣어, 기름에 바늘을 절감하고 사용자가 바늘에서 좋은 액체 방울을 얻을 있는지 확인하십시오. </li><li> 조심스럽게하지만 꾸준히 바늘로 배아를 이동하고 배아에 드롭을 삽입하고 멀리 배아를 이동합니다. (또는 분사 장치의 지침을 따르십시오.) </li><li> 모든 배아가 주입되고 나면, 그들은 공촛점 현미경에서 관찰하기 위해 준비가되어 있습니다. </li></ol> 이미징 : 60 100X 목적으로 공촛점 현미경에서 이미지 배아를 : 참고 : <ol><li> 배아의 유사 분열은 매우 빠른이기 때문에 시간 경과 시리즈에 시간 간격은 공부하는 정확한 절차에 따라, 대부분의 몇 초 있어야합니다. </li><li> 3D 시리즈 또는 하나의 비행기는 관심의 과정에 따라 취득하실 수 있습니다. </li><li> 빨리 연기 억제제의 경우, 주사 현미경에서 이미징 현미경을 전송 빨리되어야한다. </li></ol><img src="/files/ftp_upload/1382/1382fig1.jpg" alt="그림 1" /> 그림 1 : Drosophila syncytial 배아에서 유사 분열 연구. 초기 배아의 핵은 13 핵 양식 피질에서 monolayer 통해 사이클 10 중, cytokinesis없이 빠른 부문을 받고있다.가. 대뇌 피질의 핵과 초기 배아의 개략도. 난자는 표시된 단백질 GFP 및 / 또는 RFP를 표현할 수있는 다른 표시 단백질 및 / 또는 억제제와 함께 microinjected 수 있습니다. 사이클 11 GFP - tubulin과 극 - 극 분리의 RFP - 히스톤. C. 플롯을 표현 배아의 B. 시간 저속 이미징 , 야생 입력 배아에 12 13. 스핀들 길이는 사시 길이 매우 일관성과 재현성 아르 연신율 기간의 기간을 나타냅니다. 스핀들 microtubules의 D. 역학. rhodamine tubulin의 낮은 농도의 주입은 미세 소관 역학을 연구하는 데 사용할 수있는 몇 가지 tubulin의 subunits의 형성 speckles로 연결됩니다.

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이 프로토콜은 그러나 각 단계는 배아가 손상되지 않도록 연습을 필요로 상대적으로 간단합니다. 주의 제어 실험은 항상 신뢰할 수있는 결과가 취득되는 것을 보장하기 위해 수행해야합니다. 이것을 시작하는 한 가지 좋은 방법은 유사 분열은 배아 표면 (사이클 13를 통해 10)의 모든 사이클을 통해 정상적으로 진행되었는지 확인하기 위해 GFP - tubulin을 표현하는 제어 배아에 버퍼 또는 rhodamine tubul...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

이 프로토콜은 유사 분열 1 공부에 대한 Drosophila melanogaster syncytial 배아의 사용을 설명합니다. Drosophila는 합성의 게놈과 유용한 유전자를 가지고 있으며, 쉽게 유지하고 조작하실 수 있습니다. 많은 mitotic의 돌연변이가 존재하고, (예 : GFP) 찬란 기능적 표현 형질 파리 - 태그 mitotic 단백질이되었으며 생성되고 있습니다. 타겟 유전자 발현은 GAL4/UAS 시스템 2를 사용 가능합니다. Drosophila 초기 배아는 (세포주기 기간, ≈ 10 분) 매우 빠르게 여러 mitoses를 수행합니다. 사이클 10-13 동안 핵 막 피질 아래 단일 monolayer의 배 (胚)의 표면에 cytokinesis을 개입하지 않고 빠르게 분열과 동기 때문에 잘, 영상 유사 분열에 적합합니다. 이러한 급속하게 나누어 핵은 아마도 다른 세포와 같은 mitotic 기계를 사용하지만, 그들은 속도에 최적화된되며 검사점 일반적으로 그의 부재 강한 검문소와 세포 세포주기 체포를 일으킬 것입니다 mitotic 단백질의 연구를 수 있도록 엄격한되지하는 생각입니다 . 또는 GFP 표시된 단백질을 표현 배아는 찬란 표시된 단백질과 microinjected 쉽게 라이브 역학 (그림 1)에 따라 몇 군데 있습니다. 또한, 배아들은 기능 3의 손실 또는 섭동의 효과를 연구하기 위해 특정 단백질의 기능을 차단 항체 또는 억제제와 microinjected 수 있습니다. 이 시약은 억제제의 농도의 그라데이션, 돌연변이의 allelic 시리즈에 비해 결함의 기울기에 차례 결과 인치를 생산하기 위해 많은 스핀들에 도달, 배 (胚)에 걸쳐 확산 수 있습니다 이상적으로, 대상 단백질이 찬란 표시된 경우 억제의 기울기는 4 직접 시각하실 수 있습니다. 그것이 공식적으로 항체가 적용되어야합니다 드물 때문에 엄격한 컨트롤 및주의 해석에 지배적인 영향을 수있는 가능성을 제외하기 힘들지만 그것은 가장 강한 표현형가 NULL 표현형 비교는 것을 가정합니다. 멀리 사출 사이트에서, 단백질의 기능은 부분적으로 타겟 단백질의 다른 기능은 분명이 될 수 있도록 손실됩니다.

Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

이 프로토콜은에 microinjection과 고해상도 이미지의 사용에 대해 설명합니다<em&gt; Drosophila melanogaster</em유사 분열을 공부&gt; syncytial 배아.

의 유사 분열 유학을위한 Microinjection 기법 Drosophila melanogaster Syncytial 엠브료

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

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Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

14.0K 조회수.  University of California, Davis. 이 프로토콜은에 microinjection과 고해상도 이미지의 사용에 대해 설명합니다 Drosophila melanogaster syncytial 배아.

비디오: Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila Melanogaster에서 애벌레 CNS의 해부

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

연구

생물학

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

성인 Drosophila Melanogaster의 로코의 고해상도 비디오 추적

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

에 Electrophysiological 녹음 Drosophila 엠브료

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

애벌레의 모니터링 심장​​ 기능 Drosophila melanogaster 생리 연구

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

생활에서 보여주는 동적 Kinetochore 단백질 행동과 염색체 모션으로 Mitotic 검사 점 유학<em&gt; Drosophila</em&gt; Syncytial 태아

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

의 수명 측정<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

의 번데기 케이스를 통해서 영상<em&gt; 초파리</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

작은 RNA 종의 북부 오 탐지의 향상된<em&gt; 초파리 Melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.