Авторы хотели бы выразить признательность биологический факультет Сиракузского университета и доктор Мэрилин Керр за их роль в управлении Сравнительный курс биологии позвоночных. Albertson лаборатории поддержан грантом R21DE019223 из Национального института здоровья / Национального института стоматологических и черепно-лицевых исследований, а также предоставлять R01AG031922 из Национального института здоровья / Национальный институт по проблемам старения.

<ol>
	<li>Albertson, R. C., Yelick, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+for+fgf8+signaling+in+left-right+patterning+of+the+visceral+organs+and+craniofacial+skeleton.">Roles for fgf8 signaling in left-right patterning of the visceral organs and craniofacial skeleton.</a> Dev. Biol. 283, 310-321 (2005).</li><li>Aramaki, M., Kimura, T., Udaka, T., Kosaki, R., Mitsuhashi, T., Okada, Y., Takahashi, T., Kosaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+expression+profile+of+chicken+CHD7,+the+ortholog+of+the+causative+gene+for+CHARGE+syndrome.">Embryonic expression profile of chicken CHD7, the ortholog of the causative gene for CHARGE syndrome.</a> Birth Defects Res A Clin Mol Teratol. 79, 50-57 (2007).</li><li>Begemann, G., Schilling, T. F., Rauch, G. J., Geisler, R., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+neckless+mutation+reveals+a+requirement+for+raldh2+in+mesodermal+signals+that+pattern+the+hindbrain.">The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain.</a> Development. 128, 3081-3094 (2001).</li><li>Brand, M., Heisenberg, C. P., Jiang, Y. J., Beuchle, D., Lun, K., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D. A., Kelsh, R. N., Mullins, M. C., Odenthal, J., van Eeden, F. J., Kane, D. A., N&#252;sslein-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=Mutations+in+zebrafish+genes+affecting+the+formation+of+the+boundary+between+midbrain+and+hindbrain.">Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain.</a> Development. 123, 179-190 (1996).</li><li>Clark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ Hybridization: Laboratory Companion. , (2003).</li><li>D'Costa, A., Shepherd, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+genetics:+Introducing+undergraduates+to+developmental+biology+and+genetics+in+a+large+introductory+laboratory+class.">Zebrafish development and genetics: Introducing undergraduates to developmental biology and genetics in a large introductory laboratory class.</a> Zebrafish. 6, 169-177 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Emerging Model Organisms: A Laboratory Manual, Volume 1. , (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Emerging Model Organisms: A Laboratory Manual, Volume 2. , (2010).</li><li>Geetha-Loganathan, P., Nimmagadda, S., Scaal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+limb+organogenesis.">Wnt signaling in limb organogenesis.</a> Organogenesis. 4, 109-115 .</li><li>Geetha-Loganathan, P., Nimmagadda, S., Scaal, M., Huang, R., Christ, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+somite+development.">Wnt signaling in somite development.</a> Ann Anat. 190, 208-222 .</li><li>Huelsken, J., Behrens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+signaling+pathway.">The Wnt signaling pathway.</a> J Cell Sci. 15, 3977-3978 (2002).</li><li>Krauss, S., Concordet, J. P., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functionally+conserved+homolog+of+the+Drosophila+segment+polarity+gene+hh+is+expressed+in+tissues+with+polarizing+activity+in+zebrafish+embryos.">A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos.</a> Cell. 75, 1431-1444 (1993).</li><li>Oates, A. C., Mueller, C., Ho, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+function+of+deltaC+and+her7+in+anterior+segment+formation.">Cooperative function of deltaC and her7 in anterior segment formation.</a> Dev. Biol. 280, 133-149 (2005).</li><li>Pizard, A., Haramis, A., Carrasco, A. E., Franco, P., L&#243;pez, S., Paganelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridization+and+detection+of+RNAs+in+vertebrate+embryos+and+isolated+organs.">Whole-mount in situ hybridization and detection of RNAs in vertebrate embryos and isolated organs.</a> Curr Protoc Mol Biol. Chapter 14, (2004).</li><li>Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier, D. Y., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+is+mutated+in+zebrafish+acerebellar+(ace)+mutants+and+is+required+for+maintenance+of+midbrain-hindbrain+boundary+development+and+somitogenesis.">Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis.</a> Development. 125, 2381-2395 (1998).</li><li>Schmoldt, A., Forecki, J., Hammond, D. R., Udvadia, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+differential+gene+expression+in+zebrafish+to+teach+basic+molecular+biology+skills.">Exploring differential gene expression in zebrafish to teach basic molecular biology skills.</a> Zebrafish. 6, 187-199 (2009).</li><li>Schoenwolf, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Studies of Vertebrate and Invertebrate Embryos: Guide and Atlas of Descriptive and Experimental Development. , (2008).</li><li>Stickney, H. L., Barresi, M. J., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somite+development+in+zebrafish.">Somite development in zebrafish.</a> Dev Dyn. 219, 287-303 (2000).</li><li>Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A., Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J., Riggleman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+zebrafish+MyoD+in+wild-type,+no+tail+and+spadetail+embryos.">Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos.</a> Development. 122, 271-280 (1996).</li><li>Yelon, D., Horne, S. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+expression+of+cardiac+myosin+genes+reveals+regulated+aspects+of+heart+tube+assembly+in+zebrafish.">Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.</a> Dev. Biol. 214, 23-37 (1999).</li></ol>

Нет конфликта интересов объявлены.

ЖЕЛАЕМ был использован в сравнительных Биология лаборатории позвоночных, чтобы помочь учащимся понять роль генетики в анатомическом развития через визуализацию известных моделей экспрессии генов. Для первой части курса студенты осуществляется вскрытия на организмы, представляющих несколько различных таксонов хордовых, что позволяет им достаточно времени, чтобы изучить, понять, сравнить, и анатомии позвоночных контраст. В качестве введения второй части курса студенты были даны формальные лекции описывающих развитие данио и анатомии. Методы и ожидаемые результаты WISH эксперимента были также обсуждены. Студенты затем давали жить данио на сомитогенеза и чопорная-6 стадий развития, и на 2 и 5 дней после оплодотворения (DPF) для изучения под микроскопом вскрытии. Это должно было дать студентам лучше понять, что данио эмбрионы выглядят и типов морфологических изменений, которые происходят в течение онтогенеза. В следующей сессии лаборатории, студенты получили эмбрионы рыбок данио, в которой желание было ранее выполнялись. Им было предложено изучать и описывать закономерности экспрессии генов для каждого гена (riboprobe используется). Эмбрионы для WISH были получены из вязки между членами гетерозиготных линий ace/fgf8a. На основании менделевской наследование, 25% эмбрионов из вязки ace/fgf8a должны были быть гомозиготной мутантов и проявлять дефектов во многих анатомических структур особое внимание в этом курсе. На основе опубликованных отчетов и неопубликованные наблюдения в Albertson лаборатории, дефекты головного мозга и неправильное цикла сердца ожидали, а также дефекты в сомитов (Brand и др., 1996;. Albertson и Yelick, 2005; личных наблюдений). Студентам было предложено, чтобы изучить все образцы, дикого типа (гетерозиготных животных неотличимы от диких братьев и сестер типа на ранних стадиях развития) и гомозиготных мутантов, для каждого шаблона экспрессии генов представлены. Затем их попросили написать лаборатории доклады с описанием их результаты, и на основе их знания анатомии и генетики, как дефектных генов, возможно, осажденный анатомических пороков развития. Студенты, казалось, получить эту лабораторию упражнение с волнением и любопытством. Большинство из них никогда не использовал WISH до и были крайне заинтересованы в этой части курса. Студенты обнаружили различные модели экспрессии генов в эмбрионах данио интригующим, а некоторые даже описаны модели окрашивания визуализированы, ассоциируя их с известными дизайн и символы, такие как смайлик. В результате отчеты лаборатории показали студенты общее понимание протокола желание и экспрессии генов в специфических анатомических структур. Студенты также должны были понимать специфические функции генов изучены с помощью WISH в лаборатории (Стикни и др., 2000;. Huelsken и др., 2002;. Гита-Loganathan и др., 2008a;.. Гита-Loganathan и др., 2008b ). Видно было, однако, что некоторые студенты имели ограниченные знания фоне сигнальных путей и гены, имеющие интерес. Более подробную информацию об этих понятиях в WISH вводная лекция может быть полезным дополнением к использованию WISH в будущем Сравнительный курсы биологии позвоночных. Так как протокол обычно занимает от четырех дней подряд, в зависимости от графика, конечно, студенты могут только быть в состоянии завершить часть эксперимента в классе и инструктор должен нести ответственность за оставшуюся часть. В нашей сравнительной биологии позвоночных класса, студенты завершили окрашивание реакции в лаборатории, в то время как ассистент выполнили все предыдущие шаги. Если он предпочитал иметь студенты выполняют WISH в классе, протокол может быть разделена на подразделения, которые могут быть выполнены в течение нескольких сессий лаборатории, в зависимости от сроков класса. Если это не возможно для студентов, чтобы выполнить весь протокол, так как он был здесь в связи с лабораторией встреча только раз в неделю, студенты могут добавить красящим раствором в начале класса и, в зависимости от riboprobe используются, имеют полное окрашивание в течение часа. Время, необходимое для окраски развиваться значительно варьируется с каждым riboprobe и различных экспериментальных условиях, и должны быть заранее определены до начала занятий. Примечательно, что если студенты будут только развивающихся пятно в лаборатории, преподаватели будут отвечать за все предыдущие шаги, которые потребуют значительного времени и усилий за пределами классной комнаты. При желании, короче альтернатива WISH может быть иммуногистохимии с использованием антител, этикетки для визуализации белка локализации, однако в это время, данио-специфические антитела для развития гены не являются легкодоступными. Другим вариантом было бы выполнять WISH на различные виды позвоночныхой у студентов сравнить картин экспрессии тех же генов у разных организмов (Pizard и др., 2004;. Арамаки и др., 2007;. Новые модельных организмов, 2008; Новые модельных организмов, 2010). Главной целью использования в WISH Сравнительный курс биологии позвоночных было продемонстрировать студентам, как молекулярно-биологические методы используются для изучения анатомического развития. Она также предоставила возможность для студентов размышлять о том, как изменить экспрессию генов может привести не только к пороки развития, но и эволюционные изменения. Формализованные как эволюционная биология развития (часто упоминается как &quot;Evo-Devo&quot;), этой быстро развивающейся области исследований должна стать связующим звеном генотип и фенотип за счет развития, а также для выяснения потенциальных баз механистической эволюционных изменений. С появлением этой области, более экологи, организменном биологов, физиологов и применяют молекулярные методы в своих исследованиях. Мы утверждаем, что использование WISH в сравнительной курс биологии позвоночных поможет сохранить программу в курсе текущих технологических и концептуальных достижений в области исследований, а также способствовать лучшему горизонтального выравнивания верхней курсы биологии уровня путем объединения биологических полей. Более того, это интегративный подход обеспечит студентам возможность учиться ассортимент биологических методов исследования в один курс, что приводит к более диверсифицированной образования и содействие перспективным междисциплинарных научных исследований.

<ul><li> Автоклав </li><li> Микро-центрифуга </li><li> 37 ° C инкубатора (с шейкером) </li><li> 4 ° С микро-центрифуга </li><li> Водяная баня </li><li> Вихревой </li><li> Аппарат Гель-электрофорез </li><li> -20 ° C морозильника </li><li> -80 ° С морозильной камерой </li><li> Рокер / шейкер </li><li> 4 ° C холодильник </li><li> Пройдя через микроскоп (желательно с экрана обучения или камеры) </li></ul>

using whole mount in situ hybridization to link molecular and organismal biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Watch this Scientific Journal Video about Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology at JoVE.com

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Всего горе<em&gt; На месте</em&gt; Гибридизации (желание) был использован в верхнем уровне бакалавриата Сравнительный курс биологии позвоночных, в дополнение к позвоночным вскрытия. Это дало студентам возможность учиться моделей экспрессии генов, а также валовой анатомии, связывая изучение молекулярных и организменном биологии в течение одного курса.

Использование Всего горе На месте Гибридизация в ссылку молекулярной биологии и организменном

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

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Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

21.5K Views.  Syracuse University. Всего горе На месте Гибридизации (желание) был использован в верхнем уровне бакалавриата Сравнительный курс биологии позвоночных, в дополнение к позвоночным вскрытия. Это дало студентам возможность учиться моделей экспрессии генов, а также валовой а...

Video: Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Дважды Всего горе на месте гибридизация раннего куриных эмбрионов

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Метод полного окрашивания антител горе в Чик

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Данио рерио Всего горе высокого разрешения двумя люминесцентными В Ситу Гибридизация

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Всего горы<em&gt; На месте</em&gt; Гибридизация E8.5 в E11.5 эмбрионов мыши

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Высокая пропускная способность физического картирования хромосом использованием автоматизированных<em&gt; На месте</em&gt; Гибридизация

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Хромосома Репликация времени в сочетании с Флуоресцентные<em&gt; В месте</em&gt; Гибридизация

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Всего горы РНК Флуоресцентные<em&gt; На месте</em&gt; Гибридизация<em&gt; Drosophila</em&gt; Эмбрионы

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

РНК<em&gt; В месте</em&gt; Гибридизации эмбрионов Всего в гору и сотовых гистологии адаптированный для морских хрящевых рыб

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Визуализации и анализа молекул мРНК с помощью флуоресцентной<em&gt; В Ситу</em&gt; Гибридизация в<em&gt; Saccharomyces CEREVISIAE</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Высокое разрешение Всего горе<em&gt; В Ситу</em&gt; Гибридизация в Эмбрионы рыбок данио изучить экспрессию генов и функции

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...