Оригинальная конструкция компенсатора крутящего момента берет свое начало с Карлом Гец. Конкретной установки в данной презентации, в значительной степени от кредитов и был первоначально разработан Мартин Гейзенберга и Райнхард Вольф. Я особенно признателен этих двух лиц за их постоянную поддержку, поощрение и опыта.

Fly среда Состав летать пищи имеет решающее значение для обучения (Guo и др., 1996.): <ul><li> Вода 1000 мл </li><li> Кукурузной муки 180 г </li><li> Соя 10 г </li><li> Дрожжи 18,5 г </li><li> Агар 7,5 г </li><li> Патока 40 г </li><li> Сироп (сахарной свеклы) 40 г </li><li> Нипагин 2,5 г </li></ul> Каждый флакон снабжен прикосновение свежей, живой пасты дрожжей и кусочек фильтровальной бумаги, чтобы обеспечить дополнительную поверхность для мух и куколок. Fly разведения и проведения Следующая процедура проводится каждый день, что приводит к точно поставил животных, выращенных при соответствующей плотности. Все вновь ecclosed мух с момента последней процедуры в предыдущий день собираются для размножения и экспериментов. Старейших флаконах без каких-либо оставшейся жизни куколки, отбрасываются. Четыре дня старой мухи добавляются свежие флакон для откладывания яиц за одну ночь. Плотность самок должно быть около 20 для каждого флакона, с поправкой на размер флакона и напряжение используется. Идеальная плотность это тот, который достаточно высок для летают средних жидкое состояние во время личиночной стадии и достаточно низко, так, что все личинки pupated до первого ecclose мух. Откладки яиц мух от предыдущего дня, удаляются и удаляются. Fly подготовки Мухи держатся на стандартных фуражное зерно / патоки среду, как описано выше при 25 ° С и влажности 60% с 12hr light/12hr темно режима. После краткого иммобилизации 24-48 часов старых пролетает холодной анестезии, мух приклеены (суперклей УФ стекла клей, 505127A, Pacer технологии, Кукамонга, Ca., США) с головы и грудной клетки, чтобы треугольник-образных медных трубки (диаметром 0,05 мм ) за день до эксперимента. Животные затем держали индивидуально на ночь в небольшой влажной камерах, содержащих несколько крупинок сахара. Аппарат Основным устройством настройки является крутящий момент компенсатора (крутящий момент метр) (Гетц, 1964). Он измеряет момент импульса мухи вокруг своей вертикальной оси тела, вызванного предполагаемых маневрах полета. Летать, приклеены к крючок, как описано выше, прикрепляется к крутящий момент метра через зажим для выполнения полета в стационарном центре цилиндрической панорамы (арены, диаметр 58 мм), который является однородно освещенной сзади. Источник света 100Вт, 12В вольфрамо-йод лампочки. Для зеленого и синего освещения арены, свет проходит через монохроматический широкая полоса Kodak Wratten фильтры желатин (# 47 и # 99, соответственно). Фильтры могут быть заменены быстро соленоида в течение 0,1 с. Кроме того, арена освещается &quot;дневного света&quot;, пропустив его через сине-зеленый фильтр (Роско &quot;surfblue&quot; № 5433), или без фильтра вообще. Спектр пропускания Rosco сине-зеленый фильтр, используемый в данном исследовании, эквивалентной BG18 фильтр (Шотт, Майнц) и представляет собой промежуточное между Kodak синий и зеленый фильтры (Brembs и Хемпель де Ибарра, 2006; Лиу и др. ., 1999). Арене может поворачиваться вокруг лету с помощью управляемых компьютером электромотор. В такой «полет-симулятор&quot; ситуацию, угловая скорость арене пропорциональна, но направлено против рыскания момент мухи (коэффициент связи K =- 11 ° / с • 10-10 нм). Это позволяет летать по стабилизации панорамы и контролировать его угловой ориентации. Этот виртуальный &quot;направление полета&quot; (т. е. арене позиции) записывается непрерывно с помощью круговых потенциометров (Novotechnik, A4102a306). Аналог карты цифрового преобразователя (PCL812; Advantech Co) каналы позиции арене и рыскания сигнала крутящего момента в компьютер, который хранит следы (частота дискретизации 20 Гц) для последующего анализа. Наказание достигается путем применения тепла из регулируемых инфракрасного лазера (825 нм, 150 мВт), режиссер сзади и выше на голову мухи и грудной клетки. Лазерный луч импульсного (около 200 мс ширина импульса при ~ 4 Гц) и его интенсивность снижается, чтобы обеспечить выживание летать. Эксперименты План обучения Для традиционной модели обучения, эксперимент (укроп и Гейзенберг, 1995; укроп и др., 1993, 1995;. Лю и др., 2006;. Лю и др., 1998;. Лю и др., 1999;. Вольф и Гейзенберг, 1991) , четыре черных, Т-образные модели переменного ориентации (то есть, двух вертикальных и двух перевернутой) являются равномерно расположенных на арене стен (шаблон ширина ψ = 40 °, высота θ = 40 °, ширина баров = 14 °, как видно с позиции лету). Компьютерная программа делит 360 ° арене в 4 виртуальных 90 квадрантах °, центры которых обозначаются узоры. Мух контроля углового положения модели с ее рыскания момент (ситуация имитатор полета). Во время обучения, тепло наказания производится смежных с появлением одного из картины ориентации в лобной поля зрения. Усиление каждого шаблонавсегда выравнивается внутри групп. Во время теста, тепло постоянно выключен, и узор предпочтение мухи зарегистрированы. Цвет обучения Цвет обучения осуществляется, как описано выше (Brembs и Гейзенберг, 2000; Brembs и Хемпель де Ибарра, 2006; Brembs и Винер, 2006; Вольф и Гейзенберг, 1997). Арена делится на четыре виртуальных 90 квадрантах °, центры которых обозначаются четыре одинаковых вертикальных полос (ширина ψ = 14 °, высота θ = 40 °). Муха контроля углового положения четырех одинаковых полос с его крутящий момент рыскания, как описано для Т-образной модели выше. Цвет подсветки целом арене меняется, когда один из виртуальных границ квадранта проходит точку перед летать. Во время обучения, тепло наказания производится в зависимость от одного из двух цветов. Во время тестирования, тепло постоянно выключен, и цвет предпочтение мухи зарегистрированы. Конечно, цвета могут быть объединены с узорами для соединения кондиционирования (Brembs и Гейзенберг, 2001). Yaw крутящий момент обучения Yaw крутящий момент обучение осуществляется как описано ранее (Brembs и Гейзенберг, 2000; Гейзенберга и Вольф, 1993). Спонтанного момента рыскания мухи диапазон делится на &quot;левых&quot; и &quot;право&quot; домена, примерно соответствующей левой или правой повороты. Есть не узоры на арене стен. За время обучения, тепло применяется всякий раз, когда крутящий момент рыскания мухи находится в одном домене и отключается, когда крутящий момент переходит в другую. В этапов испытания, тепло постоянно выключен, и выбор мухи рыскания области крутящий момент записывается. Композитный обучения Композитный обучения является расширение обучения крутящий момент рыскания, как описано выше (Brembs и Гейзенберг, 2000). В основном, отклонение от курса обучения крутящего момента и цвет обучения объединены в эксперименте с эквивалентными оперантного (рыскания крутящего момента) и классических (цветов) предикторов. Во время обучения летать нагревается, когда крутящий момент рыскания мухи проходит в область связана с наказанием. Всякий раз, когда летают переключатели области рыскания крутящего момента, а не только температуры, но и ареной окраска изменилась (от зеленого до синего или наоборот). Таким образом, отклонение от курса домена крутящего момента и цвет служить предикторами эквивалент тепла. В этапов испытания, тепло постоянно выключен, и единственный выбор мухи рыскания крутящего момента домены / цвета записывается. Обсуждение Это экспериментальная установка сочетает в себе превосходный контроль над экспериментальных условиях с передовой генетической модель организма. Использование процедур, описанных в данной презентации, молекулярной и нейробиологических основ различных поведенческих черт могут быть исследованы, в том числе, но не ограничиваясь, механизмов спонтанного зарождения поведения, оперантного и классического обусловливания, распознавание образов, цветовое зрение или курс управления .

<ol>
	<li>Guo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila;+Dependence+on+age,+practice+and+diet.">Conditioned visual flight orientation in Drosophila; Dependence on age, practice and diet.</a> Learn. Mem.. 3, 49-59 (1996).</li><li>Goetz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optomotorische+Untersuchung+des+visuellen+Systems+einiger+Augenmutanten+der+Fruchtfliege+Drosophila.">Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila.</a> Kybernetik. 2, 77-92 (1964).</li><li>Liu, L., Wolf, R., Ernst, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+in+Drosophila+visual+learning+requires+the+mushroom+bodies.">Context generalization in Drosophila visual learning requires the mushroom bodies.</a> Nature. 400, 753-756 (1999).</li><li>Brembs, B., Hempel de Ibarra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+parameters+support+generalization+and+discrimination+learning+in+Drosophila+at+the+flight+simulator.">Different parameters support generalization and discrimination learning in Drosophila at the flight simulator.</a> Learn. Mem. 13, 629-637 (2006).</li><li>Liu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+memory+traces+for+two+visual+features+in+the+Drosophila+brain.">Distinct memory traces for two visual features in the Drosophila brain.</a> Nature. 439, 551-556 (2006).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+of+operant+behavior+as+revealed+in+Drosophila+flight+orientation.">Basic organization of operant behavior as revealed in Drosophila flight orientation.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 169, 699-705 (1991).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila+melanogaster+abolished+by+benzaldehyde.">Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde.</a> Pharmacol Biochem Behav. 61, 349-355 (1998).</li><li>Dill, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+memory+without+shape+recognition.">Visual pattern memory without shape recognition.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 349, 143-152 (1995).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+recognition+in+Drosophila+involves+retinotopic+matching.">Visual pattern recognition in Drosophila involves retinotopic matching.</a> Nature. 365, 751-753 (1993).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+analysis+of+Drosophila+landmark+learning+in+the+flight+simulator.">Behavioral analysis of Drosophila landmark learning in the flight simulator.</a> Learn. Mem. 2, 152-160 (1995).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Space+from+Visual+Motion:+Turn+Integration+in+Tethered+Flying+Drosophila.">Visual Space from Visual Motion: Turn Integration in Tethered Flying Drosophila.</a> Learn. Mem. 4, 318-327 (1997).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+operant+and+the+classical+in+conditioned+orientation+in+Drosophila+melanogaster+at+the+flight+simulator.">The operant and the classical in conditioned orientation in Drosophila melanogaster at the flight simulator.</a> Learn. Mem. 7, 104-115 (2000).</li><li>Brembs, B., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+and+occasion+setting+in+Drosophila+visual+learning.">Context generalization and occasion setting in Drosophila visual learning.</a> Learn. Mem. 13, 618-628 (2006).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+with+compound+stimuli+in+Drosophila+melanogaster+in+the+flight+simulator.">Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.</a> J Exp Biol. 204, 2849-2859 (2001).</li><li>Heisenberg, M., Wolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensory-motor+link+in+motion-dependent+flight+control+of+flies.">The sensory-motor link in motion-dependent flight control of flies.</a> Rev. Oculomot. Res. 5, 265-283 (1993).</li></ol>

The authors have nothing to disclose.

Это экспериментальная установка сочетает в себе превосходный контроль над экспериментальных условиях с передовой генетической модель организма. Использование процедур, описанных в данной презентации, молекулярной и нейробиологических основ различных поведенческих черт могут быть исследованы, в том числе, но не ограничиваясь, механизмов спонтанного зарождения поведения, оперантного и классического обусловливания, распознавание образов, цветовое зрение или курс управления .

operant learning of drosophila at the torque meter

Для экспериментов на метр крутящего момента, мух хранятся на стандартной среде летать на 25 ° С и влажности 60% с 12hr light/12hr темно режима. Стандартизированного режима разведения обеспечивает надлежащее личиночной плотности и возраста из когорт. Холодный наркозом мухи склеиваются с головы и грудной клетки, чтобы треугольный крюк день до эксперимента. В приложении к крутящий момент метра через зажим, предназначенный маневры мухи полета измеряется как угловой момент вокруг вертикальной оси тела. Летать находится в центре цилиндрической панорамы для достижения стационарного полета. Аналого-цифровой преобразователь карты отправляет сигнал рыскания крутящий момент в компьютер, который хранит след для последующего анализа. Компьютер также контролирует различные раздражители, которые могут быть взяты под контроль мухи, закрыв обратную связь между этими стимулами и следа рыскания крутящего момента. Наказание достигается путем применения тепла из регулируемых инфракрасного лазера.

Watch this Scientific Journal Video about Operant Learning of Drosophila at the Torque Meter at JoVE.com

Operant Learning of Drosophila at the Torque Meter

Измерение крутящего момента рыскания привязных дрозофилы с крутящим моментом метр позволяет невролог изысканный контролю стимула ситуации экспериментальных животных. Вместе с уникальной генетической инструментов, доступных в плодовой мушки, эта парадигма используется для широкого спектра нейробиологических исследований.

Оперантное обучения дрозофилы на метр крутящего момента

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

operant-learning-of-drosophila-at-the-torque-meter

Research

JoVE Journal

Biology

13.4K Views.  Free University of Berlin. Измерение крутящего момента рыскания привязных дрозофилы с крутящим моментом метр позволяет невролог изысканный контролю стимула ситуации экспериментальных животных. Вместе с уникальной генетической инструментов, доступных в плодовой мушки, эта парадигм?...

Video: Operant Learning of Drosophila at the Torque Meter

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Загрузка дрозофилы нервными окончаниями с кальцием Индикаторы

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 опосредованная стимуляция синаптические потенциалы в Drosophila Нервно-мышечная переходов

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Флуоресцентная маркировка Drosophila Структур сердца

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

В естественных изображений интактного личинки дрозофилы на субклеточном Резолюцию

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Количественное сравнение СНГ-Регуляторного элемента (CRE) Деятельность в Трансгенные Дрозофилы</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

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.

Живая съемка апоптоза клеток во время акции<em&gt; Drosophila</em&gt; Эмбриогенеза

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

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

MultiBac белкового комплекса добывающей платформы на EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering Гомологичная рекомбинантные конструкции в<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Культивирование носа человека эпителиальных клеток в Военно-воздушной жидкость

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Вскрытие и Монтаж<em&gt; Drosophila</em&gt; Куколочные глаз Диски

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...