Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1 АТФ-зависимых нуклеосомных Ремоделирование Анализы Для измерения АТФ-зависимой нуклеосом ремоделирования деятельности, иммунологически INO80 или INO80 подкомплексы инкубируют с АТФ и mononucleosomal подложки, которая содержит одну нуклеосомы, расположенную на одном конце 216 п.н., 32 Р-меченного фрагмента ДНК. Реакционные продукты затем подвергали электрофорезу в нативных поли-акриламида гелей. <ol><li> Для создания 32 P-меченых, &#39;601&#39; фрагмента ДНК, амплификации из pGEM-3Z-601 4 фрагмент ДНК 216 б.п., содержащий 601 нуклеосом последовательность позиционирования конечного расположенный, с использованием олигонуклеотидов 5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG и 5&#39;-AATACTCAAGCTTGGATGCCTGCAG как прямого и обратного праймеров. <ol><li> Установка ПЦР с 100 мкл, как описано в таблице 1. </li><li> Выполните реакции ПЦР в термоциклере используя следующую программу: 1 мин при 96 ° C, followeD на 45 сек при 94 ° С, 30 сек при 57 ° С, 60 сек при 72 ° С в течение 30 циклов; заканчивая 7 мин при 72 ° С. </li><li> После того как реакция завершена ПЦР, удалить некорпоративные нуклеотидов путем передачи продуктов реакции в два раза через нуклеазы без центрифужных колонок. </li><li> Запуск 5 мкл очищенного продукта ПЦР в 1,2% агарозном геле, чтобы подтвердить, что реакция методом ПЦР нужный фрагмент ~ 216 п.н. ДНК. </li><li> Развести 5 мкл очищенного продукта ПЦР в 20 раз, и измерения концентрации ДНК с помощью УФ-спектрофотометра. Средняя урожайность составляет ~ 40 нг / мкл. </li><li> Измеряют радиоактивность 1 мкл очищенного продукта ПЦР в сцинтилляционном счетчике. Оцените эффективность маркировки путем расчета CPM / нг. Успешный реакция маркировки ожидается выход ~ 15 000 CPM / нг 601 фрагмента ДНК. </li></ol></li><li> Передача клеток HeLa нуклеосомы на меченого 601 ДНК с помощью метода серийных разведений 5. <ol><li> Смешайте 2 пмоль32 Р-меченого ДНК-фрагмент 601 с 6 мкг Hela нуклеосом (полученный, как описано 5) в 50 мкл буфера, содержащего 1,0 М NaCl, 10 мМ Трис-HCl, рН 8,0, 1 мМ ЭДТА, 0,1 мМ PMSF и 1 мМ ДТТ и инкубируют при 30 ° С в течение 30 мин. </li><li> Последовательно разбавляя смесь до 0,8 М, 0,6 М и 0,4 М NaCl разбавлением 12,5 мкл, 20,8 мкл, и 41,6 мкл, соответственно, 10 мМ Трис-HCl (рН 8,0), 1 мМ ЭДТА, 0,1 мМ PMSF, и 1 мМ ДТТ, с 30-минутной инкубации при 30 ° С после каждого разведения. </li><li> Кроме того разбавленной смеси до 0,2 М, а затем 0,1 М NaCl добавлением 125 мкл, а затем 250 мкл того же самого буфера, содержащего 0,1% Nonidet P-40, 20% глицерина, и 200 мкг / мл БСА, с 30-минутной инкубации при 30 ° С между двух заключительных разведения. Храните mononucleosome подложку при 4 ° С в течение 3 месяцев. </li></ol></li><li> Выполнение АТФ-зависимые нуклеосом скольжения реакции в общем объеме 10 мкл. REAКомпоненты ие приведены в таблице 2. Поскольку оптимальная концентрация NaCl в течение INO80 нуклеосом ремоделирования активности ~ 50 мМ NaCl (неопубликованные результаты), регулировать общую концентрацию NaCl в каждой реакции до 50 мм, с учетом количества NaCl, содержащейся в препараты нуклеосом и фермента. <ol><li> Перед началом настроить анализов, бросить родные поли-акриламид гели (18 х 16 см). Чтобы подготовить единый гель, содержащий 5% акриламида (акриламид: бис-акриламид 37,5: 1), 0.5x КЭ (45 мМ Трис борат, 1 мМ ЭДТА), 0,01% персульфата аммония (APS), и 0,001%, N, N, N, N&#39;тетраметилэтилендиамина (TEMED), смешивают ингредиенты, перечисленные в таблице 3. Разрешить гель для полимеризации, по крайней мере, 2 ч при комнатной температуре. </li><li> Между тем, для каждой реакции должна быть выполнена, сочетают в предварительно охлажденной силиконизированный 1,5 мл микроцентрифужных трубки ~ 20 нМ или INO80 INO80 подкомплексом (полученного, как описано 3) с некоторым количеством EB100 буфера ( <strОнг&gt; Таблица 4) достаточно, чтобы дать объем 4,75 мкл. Незамедлительно заморозить оставшиеся INO80-содержащие фракции в порошкообразной сухой лед или жидкий азот. </li><li> Настройка мастера коктейль с остальными ингредиентами, расширения с коэффициентом &#39;X&#39; (где X = общее число реакций +3). Количество каждого ингредиента, необходимого для одной реакции 10 мкл представлены в таблице 5; Рецепт должен быть расширен в зависимости от количества реакций, чтобы быть выполненной. Все хорошо перемешать, нажав на трубу или с помощью пипетки вверх и вниз с Pipetman и спин трубку на несколько секунд в настольной микроцентрифуге. </li><li> Разливают 5,25 мкл мастера коктейль, чтобы каждый из реакционных труб, установленных на шаге 1.3.2. Все хорошо перемешать с помощью пипетки вверх и вниз. Запуск реакции путем передачи реакционные трубы до 30 ° С тепла блока или водяной бане и инкубируют в течение 2 часов. </li><li> Между тем, готовить &#39;удаления смеси&#39; коктейль, содержащий конкурента ДНКи нуклеосомы, расширения на коэффициент X (X = общее количество реакций и 4). Количество каждого ингредиента, необходимого для подготовки 1,5 мкл смеси для удаления одной реакции приведены в таблице 6; Рецепт должны быть расширены в зависимости от количества анализов должны быть выполнены. </li><li> Завершить реакции добавлением 1,5 мкл удаления смеси. Хорошо перемешать, спин вниз, и инкубируют при 30 ° С в течение еще 30 мин. </li><li> В то же время, предварительно запустить родной полиакриламидном геле в вертикальном блоке электрофореза при 100 V в течение 30 мин при 4 ° С, с использованием 0.5x TBE буфер, как работает с помощью магнитной мешалки внутри нижней камеры для поддержания постоянной циркуляции буфера. </li><li> Чтобы загрузить образец, добавить 2,5 мкл красителем, содержащим 3x КЭ, 30% глицерина, 0,25% бромфеноловый синий и 0,25% ксилолцианола FF. Все хорошо перемешать, кратко вращаться образцы, и загрузить на гель, используя советы загрузки. </li><li> Запуск гель при 200 V в течение 4,5 ч при 4 ° С с циркуляцией буфера.</li><li> Чтобы обнаружить сигнал, перевести гель для стопку двумя листами фильтровальной бумаги. Оберните бумагу фильтра с гелем на вершине, используя прозрачную пластиковую обертку, а затем выставить его на экране хранения люминофора при 4 ° С в течение требуемого времени. </li><li> Сканирование экран с системой сканера изображений изотопов и проанализировать данные с помощью соответствующего программного обеспечения. </li></ol></li></ol> 2 Mononucleosome Анализы связывания Для анализа сродство связывания данного INO80 комплекса для mononucleosomes, выполнить электрофоретической подвижности клавишу Shift Анализ (EMSA), используя mononucleosomal субстрат сгенерированный на шаге 1.2. <ol><li> Настройка реакция смесей для анализов связывания, как описано для нуклеосомных ремоделирования анализов, но опустить АТФ и удаление смесь из реакций; инкубировать при 30 ° С в течение 30 мин. </li><li> Добавить 2,5 мкл загрузочного красителя к каждой реакционной смеси, и распространяется на родном полиакриламидном геле, содержащем 3,5% акриламида (акриламид: бис 37,5: 1), 1% глицерина, 0.5x КЭ, 0,01% АПС и 0,001% тетраметилендиамин. </li><li> Использование 0.5x TBE буфер, как работает, запустить гель при 200 V в течение 2,5 ч при 4 ° С с циркуляцией буфера и подвергают к экрану хранения люминофора. </li></ol> 3 ДНК-и нуклеосом-зависимые АТФазы Анализы Выполнение анализов АТФазы в 5 мкл реакционной смеси, содержащей 20 мМ Трис-HCl (рН 7,5), 60 мМ NaCl, 6,6 мМ MgCl 2, 0,8 мМ ЭДТА, 0,015% Nonidet P-40, 2,5% глицерина, 0,1 мг / мл BSA, 1 мМ ДТТ, 0,1 мМ ФМСФ, 2 мМ АТФ, 2 мкКи [α- 32 P] АТФ (3000 Ки / ммоль). Для каждого INO80 комплекса или количества INO80 комплекса, который надо анализировать созданы три параллельные реакции, один, содержащий EB100 буфер для измерения ДНК-или нуклеосом-независимый АТФазы, одна из которых замкнутой кольцевой ДНК плазмиды (5000 б.п., ~ 30 нм) для измерения ДНК- зависит АТФазы, и один, содержащий HeLa oligonucleosomes (~ 185 нм) для измерения нуклеосом-зависимой АТФ-азы. Настройте все реакции на льду. <oл&gt; <li> Для каждой реакции, сочетают 10-50 нм иммунологически INO80 или INO80 подкомплексов с количеством EB100 буфера, достаточном для получения объема 2,2 мкл в предварительно охлажденные смазкой 1,5 мл микроцентрифужных пробирках. Незамедлительно заморозить любые INO80-содержащие фракции в порошкообразной сухой лед или жидкий азот. </li><li> Настройка мастера коктейль. Количество каждого ингредиента, необходимого для одной реакции приведены в таблице 7. Масштабирование рецепт путем расширения с коэффициентом 3 (Х 2) 1, где Х = количество INO80 препаратов для анализа. </li><li> Для подготовки &#39;суб-коктейли », содержащий буфер только, ДНК, или нуклеосом, обойтись 2,5 (X + 2) мкл мастер коктейля на три отдельных труб. Добавить 0,3 (X + 2) мкл либо EB100, замкнутую круговую ДНК плазмиды (1,5 мкг / мкл), или HeLa oligonucleosomes (1,5 мкг / мкл) и хорошо перемешать. </li><li> Разливают 2,8 мкл соответствующего суб-коктейль с ферментом, содержащих реакционных труб, установленных в стEP 3.1. Аккуратно пипетки вверх и вниз, чтобы смешать; избежать введения пузырьков. </li><li> Для начала реакции, передать реакционные трубы до 30 ° C тепла блока с. </li><li> После 5, 15, 30, и 60 мин инкубации, определить 0,5 мкл каждой реакционной смеси на полиэтиленимин целлюлозы тонкослойной хроматографии (ТСХ) пластины (20 х 10 см) в прямой линии по меньшей мере, 1,5 см от нижнего края . Сразу вернуться реакционные трубы в 30 ° C тепла блока, так что несколько моментов времени можно взять от одной пробирке. После появление пятен, насухо ТСХ пластины, используя сушилку удара. </li><li> Перенесите ТСХ-пластин в стеклянную камеру, содержащую достаточно 0,375 М фосфата калия (рН 3,5), чтобы позволить нижние 0,5 см ТСХ пластины, которые будут погружен в раствор. </li><li> Накройте камеру, и развивать, пока на передней жидкой фазы не достигнет верхней части ТСХ-пластин. Немедленное высушить тарелки тщательно с использованием фена. </li><li> Защиту высушенных пластин ТСХ на экране накопительный люминофор при комнатной температуре.Сканирование экран с системой сканера изображений изотопов и определить количество радиоактивного субстрата АТФ и АДФ продукта. </li><li> Чтобы рассчитать количество АТФ гидролизуется, умножить% АТФ гидролизуется в количестве настоящее АТФ в исходной реакционной смеси с использованием следующей формулы: пмоль АТФ гидролизуется = 10 пмоль АТФ в исходной реакционной х [АДФ / (АТФ + АДФ)] </li></ol>

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The authors declare that they have no competing financial interests.

Чтобы гарантировать, что нуклеосом ремоделирование и деятельность АТФазы мы наблюдаем в анализах зависит от каталитической активности INO80 комплексов, а не на загрязнение ремоделирования и / или ферменты, АТФазы, мы обычно анализ нуклеосом реконструкции и АТФазы каталитически неактив?...

SNF2 семья хроматина комплексы включают центральную SNF2-как АТФазную субъединицы 1,2. Некоторые SnF2-АТФазы, как функция в виде отдельных субъединиц ферментов, в то время как другие функционировать в качестве каталитической субъединицы крупных мульти-субъединицы комплексов. Выяснение молекулярных механизмов, с помощью которых каждый из субъединиц хроматина комплексов способствовать их деятельности требует способность выполнять биохимические анализы, которые рассекают процесс ремоделирования. АТФ-зависимой нуклеосом ремоделирование человеческим INO80 комплекса и других ферментов хроматина могут быть предусмотрены как многоступенчатый процесс, который начинается с связывания фермента ремоделирования к нуклеосом, с последующей активацией его ДНК-и / или нуклеосом-зависимой АТФ-азы, транслокация ремоделирования фермента на нуклеосомной ДНК, и в конечном итоге репозиционирования нуклеосом 1,2. Понимание молекулярных детали АТФ-зависимый процесс ремоделирования хроматина гequires рассечение реакции ремоделирования в его отдельных этапов и определения вклада отдельных субъединиц хроматина комплекса к каждому стадии реакции. Такие анализы требуют умение анализировать нуклеосом ремоделирования и другие мероприятия с использованием определенных молекулярных субстратов в пробирке. В предыдущем протоколе богу, мы описали процедуры, используемые для создания INO80 хроматина комплексов и подкомплекса с определенными субъединиц композиций 3. Здесь мы представляем три биохимические анализы, которые позволяют количественный анализ нуклеосоме связывания, ДНК-и нуклеосом-активируется АТФ-азы, и нуклеосом деятельности ремоделирования связанных с такими комплексами.

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biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Цифры показывают репрезентативные результаты биохимических анализов, используемых для характеристики INO80 деятельности, в том числе нуклеосоме скольжения (рисунок 1) и связывания (рисунок 2) анализов и ДНК-или нуклеосомных-зависимой АТФазы анализов (рисунок 3).</stron...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Биохимические анализы для анализа деятельности АТФ-зависимых ферментов хроматина

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

biochemical-assays-for-analyzing-activities-atp-dependent-chromatin

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JoVE Journal

Biology

14.7K Views.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Video: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Хроматин Пробирной по правам ткани мозга

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Данио рерио желудочков мозга инъекций

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Фотообесцвечивания Анализы (FRAP и FLIP), чтобы измерить хроматина Динамика белков в живых эмбриональных стволовых клеток

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Забор крови для биохимического анализа у взрослых данио рерио

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Биохимические и высокая пропускная способность Микроскопическая оценка жировой массы в<em&gt; Caenorhabditis Элеганс</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Биохимический Титрование гликогена<em&gt; В пробирке</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Генерация и очистка человека INO80 хроматина комплексов и подкомплексов

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Выделение конкретных областей генома и идентификации Associated молекул enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, следующего поколения Секвенирование и геномную ДНК Mapping ретровирусных интеграции сайты

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...