Name: chromosome replicating timing combined with fluorescent in situ hybridization
Uploaded: 2012-12-10T13:00:00
Description: Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Эта работа была поддержана грантом Национального института рака, CA131967.

1. BrdU регистрации (Терминал маркировки) <ol><li> Пластина клеток до 70% слияния в 150 мм ткань культуры блюдо 24 часа до добавления BrdU. </li><li> Заменить СМИ со свежей полной среды, содержащей 20 мкг / мл BrdU (Sigma) в соответствующие моменты времени до сбора урожая. Время, клетки культивируют в средах с Brd...

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	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation+induces+frequent+translocations+with+delayed+replication+and+condensation.">Ionizing radiation induces frequent translocations with delayed replication and condensation.</a> Cancer Research. 64, 8231-8238 (2004).</li><li>Smith, L., Plug, A., Thayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Replication+Timing+Leads+to+Delayed+Mitotic+Chromosome+Condensation+and+Chromosomal+Instability+of+Chromosome+Translocations.">Delayed Replication Timing Leads to Delayed Mitotic Chromosome Condensation and Chromosomal Instability of Chromosome Translocations.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13300-13305 (2001).</li><li>Stoffregen, E. P., Donley, N., Stauffer, D., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autosomal+locus+that+controls+chromosome-wide+replication+timing+and+mono-allelicexpression.">An autosomal locus that controls chromosome-wide replication timing and mono-allelicexpression.</a> Hum. Mol. Genet. 20, 2366-2378 (2011).</li><li>Breger, K. S., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+translocations+with+delayed+replication:+evidence+for+cis+control+of+chromosome+replication+timing.">Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing.</a> Hum. Mol. 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V., 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+deletion+at+the+mouse+Xist+gene+exposes+trans-effects+that+alter+the+heterochromatin+of+the+inactive+X+chromosome+and+the+replication+time+and+DNA+stability+of+both+X+chromosomes.">A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.</a> Genetics. 174, 1115-1133 (2006).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Schlesinger, S., Selig, S., Bergman, Y., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+inactivation+of+rDNA+loci.">Allelic inactivation of rDNA loci.</a> Genes Dev. 23, 2437-2447 (2009).</li><li>Branzei, D., Foiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+checkpoint+response+to+replication+stress.">The checkpoint response to replication stress.</a> DNA Repair (Amst). 8, 1038-1046 (2009).</li></ol>

Подготовка хромосомы спредов является важным шагом для успешной репликации сроков анализа, описанного здесь. Включение колцемид стадии предварительной обработки до гипотоническому лечение может помочь в частоте и распространения делящихся клеток. Мы обычно предоставляют клетки colce...

<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> Anti-BrdU-FITC </td><td> Roche Millipore </td><td> 11202593001 MAB326F </td><td> 50 мкг / мкл </td></tr><tr><td> Ник Перевод Kit </td><td> Abbott молекулярной (Vysis) </td><td> 07J00-0001 </td><td></td></tr><tr><td> Спектр оранжевой дУТФ </td><td> Abbott молекулярной (Vysis) </td><td> 02N33-050 </td><td></td></tr><tr><td> КЭП </td><td> Abbott молекулярной (Vysis) </td><td> Варьируется </td><td></td></tr><tr><td> LSI / ВКП гибридизации буфер </td><td> Abbott молекулярной (Vysis) </td><td> 06J67-011 </td><td></td></tr><tr><td> КЭП гибридизации буфер </td><td> Abbott молекулярной (Vysis) </td><td> 07J36-001 </td><td></td></tr><tr><td> Хромосома пайНТС </td><td> Метасистем группы </td><td> D-14NN-050-TR </td><td></td></tr><tr><td> Olympus BX61 флуоресцентного микроскопа </td><td> Олимп </td><td> BX61TRF-1-5 </td><td></td></tr><tr><td> Микроскоп изображений программное обеспечение системы </td><td> Прикладная изображений </td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td> Цифровые камеры </td><td> Олимп </td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> РЕЦЕПТЫ НА МЕСТЕ ГИБРИДИЗАЦИИ Решения Формамид 70% Formamide/2x SSC 35 мл Формамид * (Sigma) 10 мл 10x SSC 5 мл D 2 H 2 0 рН до 7,0 с помощью HCl (Sigma) * Важно использовать формамид, которые хранились при температуре -20 ° C. Длительное хранение комнатной температуре будет генерировать муравьиной кислоты и рН Wплохо быть слишком низкой. 50% Formamide/2x SSC 25 мл формамида (Sigma) 10 мл 10x SSC 15 мл дН 2 O рН до 7,0 с помощью HCl (Sigma) 20x SSC, 4 л 702 г NaCl (Sigma) 358 г цитрата натрия (Sigma) дН 2 O в объеме PN Buffer [0,1 M NaP0 4 0,1% NP_40 (Sigma)] Сделайте 0,1 М раствора каждого из фосфата натрия (Filter стерилизовать и хранить в 500 мл аликвоты). 0,1 М NaH 2 P0 4, 1 л 13,8 г NaH 2 P0 4 (Sigma): дН 2 O в объеме 0,1 М NaH 2 P0 4 1 L </strОнг&gt; 14,2 г NaH 2 P0 4 (Sigma) дН 2 O в объеме. PN: Отрегулируйте рН 0,1 М Na 2 HP0 4 до рН 8,0 с 0,1 М NaH 2 P0 4. Фильтр стерилизуют и добавляют 1 мл NP-40. PNM 50 мл 1,25 г сухого обезжиренного молока (Sigma) PN 25 мл буфера (рецепт выше) Смешайте в течение 15-20 мин при постоянном помешивании. Spin 2 раза при 400 х г в течение 10 мин. Используйте супернатант, и убедитесь, что не беспокоить осажденные белки молока. </td></tr></tbody></table>

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.

Пример анализа репликации сроки человеческой хромосомы 6, показано на рисунке 2. Клетки, содержащие удаление гена ASAR6 4, расположенный на 6q16.1, подвергались BrdU в течение 5 часов, собирали для митотических клеток и обрабатываются для рыбы с хромосоме 6 краску зонда (Vysis) и для в...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Количественный метод для анализа хромосом времени репликации описано. Метод использует BrdU включение в сочетании с флуоресцентными<em&gt; На месте</em&gt; Гибридизации (FISH) для оценки времени репликации хромосом млекопитающих. Эта техника позволяет прямое сравнение переставить и ООН-переставить хромосом в пределах одной ячейки.

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 ...

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Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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 ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Fluorescence in situ hybridization FISH Protocol in Human Sperm

Флуоресценция На месте Гибридизации (FISH) протокола в человеческой сперме

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

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

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

In Situ Hybridization for the Precise Localization of Transcripts in Plants

In Situ Hybridization for the Precise Localization of Transcripts in Plants

В гибридизация для точной локализации стенограммы в растениях

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

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 ...

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

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

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 ...

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

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

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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 ...

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

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

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 ...