Работы, проводимые в лаборатории Лекьюайер поддерживается финансирование от Национального наук и инженерного совета Канады (NSERC), Канадский институт исследований в области здравоохранения (CIHR) и Фон-де-Recherche ан Здоровье Квебека (FRSQ). Фабио Алексис Лефевра и Гаэль Moquin-Бодри поддерживается NSERC студентов studentships исследования, в то время как Кэрол Iampietro поддерживается Angelo Pizzagalli докторской стипендии.

<ol>
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Cytochem. 41, 1725-1733 (1993).</li><li>Tautz, D., Pfeifle, 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+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98, 81-85 (1989).</li><li>Lecuyer, E., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+gene+expression+universe.">Mapping the gene expression universe.</a> Curr Opin Genet Dev. 18, 506-512 (2008).</li><li>Tomancak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+patterns+of+gene+expression+during+Drosophila+embryogenesis.">Global analysis of patterns of gene expression during Drosophila embryogenesis.</a> Genome Biol. 8, 2007-208 (2007).</li><li>Thisse, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+expression+of+the+zebrafish+genome+by+large-scale+in+situ+hybridization+screening.">Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening.</a> Methods Cell Biol. 77, 505-519 (2004).</li><li>Quiring, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+expression+screening+by+automated+whole-mount+in+situ+hybridization.">Large-scale expression screening by automated whole-mount in situ hybridization.</a> Mech. Dev. 121, 971-976 (2004).</li><li>Pollet, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+atlas+of+differential+gene+expression+during+early+Xenopus+embryogenesis.">An atlas of differential gene expression during early Xenopus embryogenesis.</a> Mech. Dev. 122, 365-439 (2005).</li><li>Lein, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445, 168-176 (2007).</li><li>Lecuyer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Imai, K. S., Levine, M., Satoh, N., Satou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+blueprint+for+a+chordate+embryo.">Regulatory blueprint for a chordate embryo.</a> Science. 312, 1183-1187 (2006).</li><li>Bell, G. W., Yatskievych, T. A., Antin, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEISHA,+a+whole-mount+in+situ+hybridization+gene+expression+screen+in+chicken+embryos.">GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos.</a> Dev. Dyn. 229, 677-687 (2004).</li><li>Wilkie, G. S., Shermoen, A. W., O'Farrell, P. H., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcribed+genes+are+localized+according+to+chromosomal+position+within+polarized+Drosophila+embryonic+nuclei.">Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei.</a> Curr. Biol. 9, 1263-1266 (1999).</li><li>Rothwell, W. F., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+embryo+dechorionation.">Drosophila embryo dechorionation.</a> CSH Protoc. , (2007).</li><li>Stapleton, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+gene+collection:+identification+of+putative+full-length+cDNAs+for+70%+of+D.+melanogaster+genes.">The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes.</a> Genome Res. 12, 1294-1300 (2002).</li><li>Rubin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+complementary+DNA+resource.">A Drosophila complementary DNA resource.</a> Science. 287, 2222-2224 (2000).</li><li>Tomancak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+determination+of+patterns+of+gene+expression+during+Drosophila+embryogenesis.">Systematic determination of patterns of gene expression during Drosophila embryogenesis.</a> Genome Biol. 3, (2002).</li><li>Terashima, J., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+available+food+into+the+number+of+eggs+laid+by+Drosophila+melanogaster.">Translating available food into the number of eggs laid by Drosophila melanogaster.</a> Genetics. 167, 1711-1719 (2004).</li><li>Lecuyer, E., Parthasarathy, N., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+protocols+in+Drosophila+embryos+and+tissues.">Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.</a> Methods Mol. Biol. 420, 289-302 (2008).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fluorescent+in+situ+hybridization+in+Drosophila.">High resolution fluorescent in situ hybridization in Drosophila.</a> Methods Mol. Biol. 714, 31-47 (2011).</li><li>Kosman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846 (2004).</li></ol>

Нет конфликта интересов объявлены.

Для шагов зонд синтеза, мы обычно генерируют стоки антисмысловых РНК-зондов в пробирке транскрипции от полной длины кДНК Drosophila амплифицировали из плазмиды найден у дрозофилы ген Collection (РСК), ресурс подробно описаны на сайте: <a href="http://www.fruitfly.org/DGC/index.html" target="_blank">http://www .fruitfly....

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии (по желанию) </td></tr><tr><td> T7, Т3 или SP6 РНК-полимеразы </td><td> Fermentas Life Sciences </td><td> EP0101, EP0111, EP0131 </td><td> Комплекты содержат реакции буфера. </td></tr><tr><td> DIG РНК маркировки Mix </td><td> Roche Applied Science </td><td> 11 277 073 910 </td><td></td></tr><tr><td> RNAguard </td><td> Amersham Biosciences </td><td> 27-0816-01 </td><td></td></tr><tr><td> 3М ацетата натрия </td><td></td><td></td><td></td></tr><tr><td> Холодные 100% этанола. </td><td></td><td></td><td></td></tr><tr><td> Холодный 70% этанола. </td><td></td><td></td><td></td></tr><tr><td> Хлор раствором хлорной извести разводят водой в соотношении 1:1. </td><td></td><td></td><td></td></tr><tr><td> Гептан </td><td></td><td></td><td></td></tr><tr><td> Метанол </td><td></td><td></td><td></td></tr><tr><td> протеиназы К </td><td> Sigma Aldrich Oakville, ON, Канада </td><td> Инвентарный номер P2308 </td><td></td></tr><tr><td> 40% раствор формальдегида, свежеприготовленного </td><td></td><td></td><td></td></tr><tr><td> PBS-Tween решение (PBT) </td><td></td><td> 1xPBS, 0,1% Tween-20 </td><td></td></tr><tr><td> Глицин решения </td><td></td><td> 2 мг / мл глицина в PBT </td><td></td></tr><tr><td> HRP-конъюгированных моноклональных антител мыши-DIG </td><td> Джексон ImmunoResearch Laboratories Inc </td><td> 200-032 - 156 </td><td> (1/400 разведении 1 мг / мл маточного раствора в PBTB </td></tr><tr><td> HRP-конъюгированных овец моноклональных анти-DIG </td><td> Roche Applied Scienсе, Laval, QC </td><td> 1 207 733 </td><td> 1/500 разбавление исходного раствора в PBTB </td></tr><tr><td> Биотин-сопряженных мышиных моноклональных анти-DIG </td><td> Джексон ImmunoResearch Laboratories, Inc, West Grove, PA, USA </td><td> 200-062-156 </td><td> (1/400 разведении 1 мг / мл маточного раствора в PBTB </td></tr><tr><td> Стрептавидин-HRP сопряженных </td><td> Молекулярные зонды, Евгений Орегон, США </td><td> S991 </td><td> (1/100 разбавление 1 мкг / мл складе </td></tr></tbody></table>

whole mount rna fluorescent in situ hybridization of drosophila embryos

Оценка экспрессии генов, а также субклеточном свойства локализации его транскрипции РНК, являются ключевыми для понимания особенностей его биологической функции в процессе разработки. РНК в гибридизация (РНК-ISH) представляет собой мощный метод, используемый для визуализации свойств РНК распределения, будь то на организменном, клеточном и субклеточном уровнях 1. РНК-ISH основан на гибридизации меченого зонда нуклеиновой кислоты (например, антисмысловую РНК, олигонуклеотиды), комплементарный последовательности мРНК и некодирующих РНК объектом интереса 2. Так как процедура требует первичной информации о последовательности только для генерации последовательности конкретных датчиков, он может применяться универсально для широкого круга организмов и образцов тканей 3. Действительно, ряд крупномасштабных ISH исследования были реализованы к документу экспрессии генов и РНК локализации динамики в различных модельных организмов, которые привели ксоздание важных ресурсов сообщества 4-11. В то время как различные маркировки и обнаружения зонда стратегии были разработаны на протяжении многих лет, в сочетании использование флуоресцентно-меченных обнаружения реагентов и ферментативных реакций усиления сигнала предлагают значительные улучшения чувствительности и разрешающей процедуры 12. Здесь мы описываем оптимизированы флуоресцентные на месте методом гибридизации (FISH) с использованием tyramide усиления сигнала (TSA) для визуализации экспрессии РНК и локализации динамику в постановке эмбрионов дрозофилы. Процедура проводится в 96-луночный ПЦР планшет формата, что значительно облегчает одновременную обработку большого количества образцов.

Когда выполняется успешно, эта процедура предлагает поразительно повышения уровня детализации в пространственно-временной анализ экспрессии генов и динамику локализации мРНК во время раннего эмбриогенеза дрозофилы. Действительно, как показано на рисунке 3А для класси?...

Watch this Scientific Journal Video about Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos at JoVE.com

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Здесь мы опишем весь монтаж флуоресцентных<em&gt; На месте</em&gt; Гибридизации (FISH) протокол для определения экспрессии и локализации свойств РНК, высказанные в ходе эмбриогенеза дрозофилы,<em&gt; Дрозофилы</em&gt;.

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 ...

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Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

18.2K Views.  Institut de Recherches Cliniques de Montréal (IRCM). Здесь мы опишем весь монтаж флуоресцентных На месте Гибридизации (FISH) протокол для определения экспрессии и локализации свойств РНК, высказанные в ходе эмбриогенеза дрозофилы, Дрозофилы.

Video: Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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 ...

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 ...

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.

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

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

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 ...

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 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Обнаружение вирусной РНК с помощью флуоресцентного<em&gt; На месте</em&gt; Гибридизация (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Анализ одноклеточных транскрипции гена РНК-флуоресцентный<em&gt; В Situ</em&gt; Гибридизация (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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 ...

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 ...

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 ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Анализ апоптоза у эмбрионов данио рерио по целом монтажа иммунофлуоресценции для обнаружения активированного каспазы 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...