Авторы хотели бы поблагодарить Стивена Экипажи для dysfusion кДНК, DYS антител и W 1118 мух.

1. Эмбрион коллекции <ol><li> Настройка клетки при 25 ° С с использованием 2-4 дневных ш 1118 пластин сок flies.Grape меняются каждый час в течение суток для синхронизации сбора яиц в течение 1-2 дней до сбора </li><li> Сбор эмбрионов в 1 час при 25 ° С </li><li> Отрежьте прямоугольный кусок виноградного сока агар, вырезать слегка в середине с лезвием, оставьте строку в агар </li><li> Используйте металлический зонд для передачи эмбрионов из виноградного сока пластины Вашей части виноградного сока агар, выстроить их в прямой линии с длинной оси эмбриона на 45 градусов к линии в середине агара. Получите прямой около 50 эмбрионов. Убедитесь, что все эмбрионы указывают в одном направлении, с задним концом направлен от линии. </li><li> Отрежьте прямоугольный кусок двойной ленты палки, и поместить его в 18x18mm скольжения покрытия. </li><li> Возьмите эмбрионов с двойной ленты палки на покровное стекло </li><li> Место покровным стеклом, чтобы стекло, сухие эмбриона в воздухе около 10 мин. </li><li> Обложка эмбриона с тонким слоем масла Хладон 700, сейчас он готов для инъекции. </li></ol> 2. Тяговая иглы Сделать микроинъекции иглы, потянув стеклянные капилляры. Мы используем иглы съемник от Всемирного точный инструмент (модель ПУЛ-1). Иглы спецификации съемник программы являются: тепло: 1, задержка 4. 3. Заполнение иглы Вернуться нагрузки иглы использованием Eppendorf p20 пипетки оснащены Эппендорф советы Microloader, как правило, нагрузка 1.5-2 мкл дсРНК 4. Перерыв иглы <ol><li> Перед тем как игла, место покровное на слайде. </li><li> Подготовка слайдов с каплей масла в середине слайда </li><li> Включите Picospritzer III picopump для регулировки давления воздуха </li><li> Перерыв заполнен иглу к краю покровным стеклом, создавая острие. </li><li> Когда кончик сломан, шаг на педали из Picospritzer III picopump, некоторые дсРНК будет вытекать из иглы. </li><li> Выньте слайд с покровным. </li><li> Введите иглу под жировая капля в середине слайд подготовлена ​​раньше. </li><li> Отрегулируйте настройку Picospritzer III picopump, чтобы получить 100-200pl дсРНК когда вы выходите на педали. дсРНК можно рассматривать как небольшой капли. </li></ol> 5. Инъекция <ol><li> Принесите иглы к задней части первого эмбриона бластодерма в ту же фокальной плоскости и ввести эмбрион от центра около 30-50% длины яйца. </li><li> После того как вы шаг на педали из Picospritzer III picopump, небольшое количество дсРНК будет выглядеть как маленькая точка в месте инъекции. </li></ol> 6. Фенотипический анализ <ol><li> После инъекции место вставляются покрыты влажной камере (пластиковые чашки Петри) при 18 ° С до эмбрионы развиваются до желаемого зачаточном состоянии. </li><li> Используйте зонд, чтобы удалить из эмбрионов двусторонний скотч и подтолкнуть их к краю крышки скольжения. Вымойте эмбрионов за пределы слайда в коллекции флакон с нейлоновая сетка на дне использованием гептан, промыть 3 раза PBT. Dechorionate эмбрионов в 50% отбеливателя в течение 5 минут, и промыть 3 раза PBT. Трансфер эмбрионов из коллекции флакон на кусок нейлоновой сетки, а затем падение нейлоновая сетка в фиксирующий раствор (5 мл гептана, 4,5 мл PBS, 0,5 мл 37% формальдегида), чтобы освободить эмбрионов. Fix эмбрионов в фиксирующий раствор в течение 30 минут. Devitellinize эмбрионов с метанолом, трансфер эмбрионов трубки Эппендорф, и immunostain эмбрионов с использованием стандартных протоколов. </li><li> Эмбрионы могут также разработать соответствующие личиночной стадии. </li><li> Подготовка слайдов с каплей масла хладон 700 в середине </li><li> Передача один личинок на слайд, положить покровное стекло на верхнем </li><li> Соблюдайте личинки при ярком поле микроскопа. </li></ol> 7. Представитель Результаты: Примером дсРНК сбить dysfusion (дисфункции) генов в эмбрион Drosophlia показан на Рис1. GFP-дсРНК вводят эмбрионы отрицательного контроля. Дисфункции является транскрипцией bHLH PAS фактор выражается в клетках трахеи синтеза. GFP-дсРНК вводят эмбрионы демонстрируют нормальное трахеи синтеза, а DYS белок присутствует в области термоядерного синтеза клетками (рис. 1А). С другой стороны, дисфункции дсРНК вводят эмбрионы не удалось показать филиала синтеза, а DYS белки нет в слиянии клетки (рис. 1В). Когда дсРНК вводят эмбрионы развиваются до личиночной стадии, дисфункции дсРНК вводят личинок показали удалось филиала синтеза (Fig.1D) по сравнению с GFP-дсРНК вводят отрицательного контроля (рис. 1в). Эти результаты показали, эффективное сбить эндогенной экспрессии генов дисфункции по дсРНК инъекции в эмбрионы дрозофилы <img src="/files/ftp_upload/2477/2477fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Удаление дисфункции функции дисфункции RNAi показывает, трахеи слияние гвидеоэффекты. Бластодерма эмбрионов (W 1118) вводили либо GFP-дсРНК (отрицательный контроль) или дисфункцией дсРНК и анализировали на трахеи дефектов. () Этап 16 эмбрион вводят GFP-дсРНК и окрашивали α-DYS и МАБ 2A12, что пятна трахеи просвет. Заметно показан боковой ствол, который расплавился. Стрелки указывают на боковой ствол DYS-положительных клеток синтеза. (В) Этап 16 эмбриона вводят дисфункции дсРНК и окрашивали α-DYS и МАБ 2A12. Эмбрионов показало отсутствие бокового ствола слияния (звездочка означает нормальное расположение бокового ствола синтеза в контрольных эмбрионов). Нет DYS-положительных клеток наблюдалось, что свидетельствует о дисфункции РНК по сути упразднил DYS белка. (От С до D) второго возраста личинки либо с GFP-дсРНК или дисфункции дсРНК были рассмотрены светлого поля микроскопию для трахеи дефектов. (C) Сайты плавления обозначены звездочкой в ​​GFP-дсРНК вводят контроль личинок. (D) бокового ствола не удалось соединить в дис-РНК вводили личинки, (*) указывает нормальное расположение боковых слияние ствола.

<ol>
	<li>St Johnston, D., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+pattern+and+polarity+in+the+Drosophila+embryo.">The origin of pattern and polarity in the Drosophila embryo.</a> Cell. 68, 201-219 (1992).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+gene+manipulation+in+Drosophila+melanogaster.">Emerging technologies for gene manipulation in Drosophila melanogaster.</a> Nat. Rev. Genet. 6, 167-178 (2005).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95, 1017-1026 (1998).</li><li>Misquitta, L., Paterson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+gene+function+in+Drosophila+by+RNA+interference+(RNA-i):+a+role+for+nautilus+in+embryonic+somatic+muscle+formation.">Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 1451-1456 (1999).</li><li>Horowitz, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis.">Branching morphogenesis.</a> Circ. Res. 103, 784-795 (2008).</li><li>Hogan, B. L., Kolodziej, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis:+molecular+mechanisms+of+tubulogenesis.">Organogenesis: molecular mechanisms of tubulogenesis.</a> Nat. Rev. Genet. 3, 513-523 (2002).</li><li>Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis+of+the+Drosophila+tracheal+system.">Branching morphogenesis of the Drosophila tracheal system.</a> Annu. Rev. Cell Dev. Biol. 19, 623-647 (2003).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfusion+transcriptional+control+of+Drosophila+tracheal+migration,+adhesion,+and+fusion.">Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion.</a> Mol. Cell. Biol. 26, 6547-6556 (2006).</li><li>Jiang, L., Crews, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+dysfusion+basic+helix-loop-helix+(bHLH)-PAS+gene+controls+tracheal+fusion+and+levels+of+the+trachealess+bHLH-PAS+protein.">The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein.</a> Mol. Cell. Biol. 23, 5625-5637 (2003).</li></ol>

Нет конфликта интересов объявлены.

Метод дсРНК инъекции присутствует здесь дает очень чувствительный и быстрый анализ функций генов дрозофилы трахеи развития. Этот метод потенциально может быть применена для анализа функции гена и для других тканей и органов развитии.

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<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
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<td>Picospritzer III picopump</td>
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<td>Parker Precision Fluidics</td>
<td>051-0500-900</td>
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<td>Micro-pipettes</td>
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<td>Fisher</td>
<td>21170M</td>
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<td>Microloaders</td>
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rnai interference by dsrna injection into drosophila embryos

Генетический скрининг является одним из самых мощных методов, доступных для получения взглянуть на сложный биологический процесс, 1. За эти годы много улучшений и инструментов для генетических манипуляций стали доступны у дрозофилы 2. Вскоре после первоначального открытия Фри и Мелло 3 видно, что двухцепочечной РНК могут быть использованы для нокдаун активности отдельных генов в Caenorhabditis Элеганс, РНК-интерференция (RNAi) было показано, что обеспечивает мощный обратный генетический подход к анализу функций генов в развитии дрозофилы орган 4, 5. Многие органы, в том числе легких, почек, печени, сосудистой системы, состоят из трубчатых разветвленных сетей, транспорта жизненно важные жидкости или газы, 6, 7. Анализ дрозофилы трахеи образование обеспечивает отличную модель системы для изучения морфогенеза других трубчатых органов 8. Беркли дрозофилы генома проекта выявил сотни генов, которые выражаются в трахеи системы. Для изучения молекулярных и клеточных механизмов трубки образования, задача состоит в том, чтобы понять роль этих генов в развитии трахеи. Здесь мы описали подробный метод дсРНК инъекции в эмбрион дрозофилы нокдаун отдельных экспрессии генов. Мы успешно сбил эндогенных dysfusion (дисфункции) экспрессии генов с помощью инъекции дсРНК. Дисфункции является bHLH-PAS белка выражается в клетках трахеи синтеза, а это необходимо для трахеи филиала слияние 9, 10. дисфункции RNAi полностью устранить дисфункции выражение и в результате трахеи дефект синтеза. Этот сравнительно простой метод обеспечивает инструментом для выявления генов requried для tissure и развития органов у дрозофилы.

Watch this Scientific Journal Video about RNAi Interference by dsRNA Injection into Drosophila Embryos at JoVE.com

RNAi Interference by dsRNA Injection into Drosophila Embryos

РНК-интерференция была доказана очень эффективно анализировать функции генов в<em&gt; Drosophila</em&gt; Трахеи развития. Подробное протокол, используемый Цзян лаборатории, чтобы придать дсРНК в летают эмбрионов нокдауна экспрессии генов иллюстрируется. Эта технология обладает потенциалом для скрининга генов, необходимых для развития тканей и органов в<em&gt; Drosophila</em&gt;.

RNAi Вмешательство инъекций в дсРНК Drosophila Эмбрионы

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

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RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K Views.  Oakland University. РНК-интерференция была доказана очень эффективно анализировать функции генов в Drosophila Трахеи развития. Подробное протокол, используемый Цзян лаборатории, чтобы придать дсРНК в летают эмбрионов нокдауна экспрессии генов иллюстрируется. Эта технологи...

Video: RNAi Interference by dsRNA Injection into Drosophila Embryos

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Инъекция дсРНК в женский А. Комары aegypti

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Живая Препарирование Drosophila Эмбрионы: Упрощенная методы скрининга Мутант Коллекции антител Окрашивание

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

В естественных условиях центрифугирования эмбрионов дрозофилы

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Бактериальные Доставка RNAi создания эффектов: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Внутрибрюшинного введения в взрослых данио рерио

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Вертикальная съемка Drosophila Эмбрионы

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Первичных клеточных культур из Drosophila Гаструла Эмбрионы

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Выделение и очистка от кинезин Drosophila Эмбрионы

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Перенос генов в старых куриных эмбрионов на<em&gt; Бывший ово</em&gt; Электропорация

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi Trigger поставки в<em&gt; Anopheles gambiae</em&gt; Куколки

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...