Este trabalho foi financiado pelo NIH Grant GM59354 atribuído a Sharon E. Bickel. Agradecemos Huy p. Nguyen assistência no desenvolvimento do protocolo para a geração de sondas fluorescentes braço, Ann Lavanway para ajuda com microscopia confocal e J. Emiliano Reed para assistência técnica. Agradecemos também a numerosos colegas na Comunidade da drosófila para discussões úteis e conselhos.

<ol>
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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=Disjunction+of+homologous+chromosomes+in+meiosis+I+depends+on+proteolytic+cleavage+of+the+meiotic+cohesin+Rec8+by+separin.">Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.</a> Cell. 103 (3), 387-398 (2000).</li><li>Bickel, S. E., Orr-Weaver, T., Balicky, E. 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+sister-chromatid+cohesion+protein+ORD+is+required+for+chiasma+maintenance+in+Drosophila+oocytes.">The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes.</a> Curr. Biol. 12 (11), 925-929 (2002).</li><li>Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMC1beta-deficient+female+mice+provide+evidence+that+cohesins+are+a+missing+link+in+age-related+nondisjunction.">SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction.</a> Nat Genet. 37 (12), 1351-1355 (2005).</li><li>Freeman, S. 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=The+National+Down+Syndrome+Project:+design+and+implementation.">The National Down Syndrome Project: design and implementation.</a> Public Health Rep. 122 (1), 62-72 (2007).</li><li>Nagaoka, S. I., Hassold, T. J., Hunt, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+cohesin+is+associated+with+age-dependent+misaligned+chromosomes+in+oocytes.">Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes.</a> Reprod Biomed Online. 16 (1), 103-112 (2008).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+predisposes+oocytes+to+meiotic+nondisjunction+when+the+cohesin+subunit+SMC1+is+reduced.">Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced.</a> PLoS Genet. 4 (11), e1000263 (2008).</li><li>Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+weakened+centromere+cohesion+is+a+leading+cause+of+age-related+aneuploidy+in+oocytes.">Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes.</a> Curr. Biol. 20 (17), 1522-1528 (2010).</li><li>Lister, L. 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=Age-related+meiotic+segregation+errors+in+mammalian+oocytes+are+preceded+by+depletion+of+cohesin+and+Sgo2.">Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2.</a> Curr. Biol. 20 (17), 1511-1521 (2010).</li><li>Duncan, F. 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Os autores declaram não concorrentes interesses financeiros.

O uso de peixes sondas para avaliar o estado da coesão de braço em prometafase I e metáfase eu Drosophila oócitos é um avanço significativo no campo da drosófila meiose. Historicamente, a drosófila pesquisadores foram limitados ao testes genéticos para inferir a perda prematura da coesão de braço em oócitos maduros11,18,19. Agora, com os métodos apresentados aqui, o estado da coesão do br...

Adequada segregação dos cromossomos durante a mitose e meiose exige que a irmã cromátide coesão ser estabelecido, mantido e lançado em uma coordenada1,2. Coesão é estabelecida durante a fase S e é mediada pelo cohesin complexo, que forma ligações físicas que segure a irmã cromátides irmãs juntos. Na meiose, coesão distal de um crossover também funções para armazenar recombinantes homologs juntos e esta associação física ajuda a garantir a adequada orientação do forma bivalente na metáfase eu do eixo (Figura 1)3,4, 5. Lançamento do braço coesão na anáfase permite-lhe as homologs segregar para polos opostos do fuso. No entanto, se a coesão do braço é perdido prematuramente, recombinantes homologs perderá sua conexão física e segregar aleatoriamente, que pode resultar em gâmetas aneuploid (Figura 1).
Em oócitos humanos, erros na segregação do cromossomo são a principal causa de abortos e defeitos de nascimento, como síndrome de Down,6, e sua incidência aumenta exponencialmente com a idade materna7. Coesão de cromatídeos é criada em oócitos fetais e recombinação meiótica é concluída antes do nascimento. Oócitos depois prenda no meio da prófase eu até ovulação e durante esta prisão, associação física continuada de recombinantes homologs baseia-se na irmã coesão de cromátides irmãs. Portanto, precisa segregação durante a meiose e os resultados de gravidez normal exigem que coesão permanecem intactas por até cinco décadas.
Perda prematura de coesão durante a prolongada detenção meiótica de oócitos humanos tem sido proposta para contribuir para o efeito da idade materna e várias linhas de evidência suportam esta hipótese8,9. No entanto, tendo em conta os desafios da coesão meiótica em oócitos humanos a estudar, muito da nossa compreensão deste fenômeno baseia-se sobre a utilização do modelo organismos5,10,11,12, 13de14,,15.
Drosophila melanogaster oócitos oferecem inúmeras vantagens para o estudo da segregação meiótica de coesão e cromossomo. Um ensaio simples genético permite recuperar a descendência dos gâmetas aneuploid e medir a fidelidade do X-segregação de cromossomos em uma grande escala11,16,17. Além disso, um pode também determinar se erros de segregação do cromossomo surgem porque homologs recombinantes missegregate durante a meiose I, um fenótipo que é consistente com a perda prematura de braço coesão11,18, 19. direta observação do estado de coesão meiótica em oócitos de Drosophila também é possível usar a fluorescência em situ da hibridação (peixe). Apesar de oligonucleotídeos fluorescentes que hibridizam às sequências repetitivas de satélite têm sido usados por mais de uma década para monitorar pericentromeric coesão em madura Drosophila oócitos4,20, análise de braço coesão tem sido muito mais desafiador. Visualização do estado de coesão braço requer uma sonda que se estende por uma grande região de cópia única sequências e é brilhante o suficiente para resultar em sinais visíveis para cada irmã cromátides irmãs quando braço coesão está ausente. Além disso, as condições de fixação do oócito e tamanho dos DNAs rotuladas devem facilitar a penetração21 para o grande oócito maduro de Drosophila (200 µm de largura por 500 µm de comprimento). Recentemente, uma sonda de braço com êxito foi utilizado Visualizar cromátides irmãs oócito de Drosophila durante a anáfase, eu, mas os autores afirmou que não podia detectar um sinal em prometafase ou metáfase prendi oócitos22. Aqui nós fornecemos um protocolo detalhado para a geração de X-braço do cromossoma sondas de peixe e as condições de preparação de oócito que nos permitiram a ensaiar para a perda prematura da irmã coesão cromátide em prometafase I e metáfase eu oócitos. Estas técnicas, que nos permitiram identificar produtos de genes que são necessários para a manutenção da coesão meiótica, permitirá que outros a ensaiar para a irmã coesão cromátide defeitos em oócitos maduros de drosófila de diferentes genótipos.

<table><tbody><tr><td>&lt;strong&gt;Kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Midi Prep kit</td><td>Qiagen</td><td>12143</td><td>Prep BAC clone DNA</td></tr><tr><td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td><td>Sigma</td><td>WGA2</td><td>Amplify BAC clone DNA</td></tr><tr><td>ARES Alexa Fluor 647 DNA labeling kit</td><td>Invitrogen</td><td>A21676</td><td>Label BAC clone DNA</td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28104</td><td>Remove non-conjugated dye following labeling of BAC clone DNA</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals &amp;amp; Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Note: &lt;/strong&gt;All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td><td></td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP1600-100</td><td>Prepare 10% stock&lt;br/&gt; Freeze aliquots</td></tr><tr><td>Calcium chloride</td><td>Fisher Scientific</td><td>C75-500</td><td></td></tr><tr><td>DAPI (4&amp;rsquo;, 6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. Prepare 100&amp;micro;g/ml stock in 100% ethanol, store in aliquots at -20 &amp;deg;C. Prepare 1 &amp;micro;g/ml solution in 2X SSCT before use.</td></tr><tr><td>Dextran sulfate</td><td>Sigma</td><td>D-8906</td><td></td></tr><tr><td>Drierite</td><td>Drierite Company</td><td>23001</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Invitrogen</td><td>15508-013</td><td>Prepare 10 mM stock</td></tr><tr><td>dTTP (10 &amp;micro;mol, 100 &amp;micro;l)</td><td>Boehringer Mannheim</td><td>1277049</td><td>Prepare 1 mM stock</td></tr><tr><td>EDTA (Disodium ethylenediamine tetraacetic acid)</td><td>Fisher Scientific</td><td>S311-500</td><td>Prepare 250 mM stock</td></tr><tr><td>100% ethanol (molecular grade, 200 proof)</td><td>Decon Laboratories</td><td>2716</td><td></td></tr><tr><td>Tris (Ultra Pure)</td><td>Invitrogen</td><td>15504-020</td><td></td></tr><tr><td>EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)</td><td>Sigma</td><td>E-3889</td><td></td></tr><tr><td>16% formaldehyde</td><td>Ted Pella, Inc.</td><td>18505</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection</td></tr><tr><td>Formamide</td><td>Invitrogen</td><td>AM9342</td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection</td></tr><tr><td>Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Glycogen</td><td>Roche</td><td>901393</td><td></td></tr><tr><td>HEPES</td><td>Boehringer Mannheim</td><td>737-151</td><td></td></tr><tr><td>Heptane</td><td>Fisher Scientific</td><td>H350-4</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection.</td></tr><tr><td>Hydrochloric acid</td><td>Millipore</td><td>HX0603-4</td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection.</td></tr><tr><td>Hydroxylamine</td><td>Sigma</td><td>438227</td><td>Prepare 3 M stock</td></tr><tr><td>4.9 M Magnesium chloride</td><td>Sigma</td><td>104-20</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;Yuml; 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S373-500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&amp;Yuml; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>Poly-L-lysine (0.1mg/ml)</td><td>Sigma</td><td>P8920-100</td><td></td></tr><tr><td>Potassium acetate</td><td>Fisher Scientific</td><td>BP364-500</td><td></td></tr><tr><td>Sodium acetate</td><td>Fisher Scientific</td><td>S209-500</td><td>Prepare 3M stock</td></tr><tr><td>Sodium cacodylate</td><td>Polysciences, Inc.</td><td>1131</td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection. Prepare 400mM stock</td></tr><tr><td>Sodium citrate</td><td>Fisher Scientific</td><td>BP327-1</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>Sodium chloride</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>10% Tween 20</td><td>Thermo Scientific</td><td>28320</td><td>Surfact-Amps</td></tr><tr><td>10% Triton X-100</td><td>Thermo Scientific</td><td>28314</td><td>Surfact-Amps</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Note: &lt;/strong&gt;All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td><td></td><td></td><td></td></tr><tr><td>TE buffer</td><td></td><td></td><td>10 mM Tris, 1 mM EDTA, pH = 8.0</td></tr><tr><td>20X SSC (Saline Sodium Citrate)</td><td></td><td></td><td>3 M NaCl, 300 mM sodium citrate</td></tr><tr><td>2X cacodylate fix solution</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA</td></tr><tr><td>1.1X Hybridization buffer</td><td></td><td></td><td>3.3X SSC, 55% formamide, 11% dextran sulfate</td></tr><tr><td>Fix solution</td><td></td><td></td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution</td></tr><tr><td>PBSBTx</td><td></td><td></td><td>1X PBS, 0.5% BSA, 0.1% Trition X-100</td></tr><tr><td>PBSTx</td><td></td><td></td><td>1X PBS, 1% Trition X-100</td></tr><tr><td>Extraction buffer (PBSTx + Rnase)</td><td></td><td></td><td>1X PBS, 1% Trition X-100, 100 &amp;micro;g/mL RNase</td></tr><tr><td>2X SSCT</td><td></td><td></td><td>2X SSC, 0.1% Tween 20</td></tr><tr><td>2X SSCT + 20% formamide</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide</td></tr><tr><td>2X SSCT + 40% formamide</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide</td></tr><tr><td>2X SSCT + 50% formamide</td><td></td><td></td><td>&lt;strong&gt;Toxic:&lt;/strong&gt; wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AluI</td><td>New England Biolabs</td><td>R0137S</td><td></td></tr><tr><td>HaeIII</td><td>New England Biolabs</td><td>R0108S</td><td></td></tr><tr><td>MseI</td><td>New England Biolabs</td><td>R0525S</td><td></td></tr><tr><td>MspI</td><td>New England Biolabs</td><td>R0106S</td><td></td></tr><tr><td>RsaI</td><td>New England Biolabs</td><td>R0167S</td><td></td></tr><tr><td>BfuCI</td><td>New England Biolabs</td><td>R0636S</td><td></td></tr><tr><td>100X BSA</td><td>New England Biolabs</td><td></td><td>Comes with NEB enzymes</td></tr><tr><td>10X NEB buffer #2</td><td>New England Biolabs</td><td></td><td>Restriction enzyme digestion buffer. 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Comes with TdT enzyme</td></tr><tr><td>RNase A (10 mg/mL)</td><td>Thermo-Scientific</td><td>EN0531</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cytology Tools etc.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>Dumont</td><td></td><td>#5 INOX, Biologie</td></tr><tr><td>9&amp;rdquo; Disposable glass Pasteur pipettes</td><td>Fisher</td><td>13-678-20C</td><td>Autoclave to sterilize</td></tr><tr><td>Shallow glass dissecting dish</td><td></td><td></td><td>Custom made</td></tr><tr><td>Deep well dish (3 wells)</td><td>Pyrex</td><td>7223-34</td><td></td></tr><tr><td>Fisherfinest Premium microscope slides</td><td>Fisher Scientific</td><td>22-038-104</td><td>Used to cover deep well dishes</td></tr><tr><td>Frosted glass slides, 25 x 75 mm</td><td>VWR Scientific</td><td>48312-002</td><td></td></tr><tr><td>Glass slides, 3 x 1 in, 1 mm thick</td><td>Thermo-Scientific</td><td>3051</td><td></td></tr><tr><td>Coverslips, 10 x 10 mm, No. 1.5</td><td>Thermo-Scientific</td><td>3405</td><td></td></tr><tr><td>Tungsten needle</td><td></td><td></td><td>homemade</td></tr><tr><td>Prolong GOLD mounting media</td><td>Molecular Probes</td><td>P36930</td><td></td></tr><tr><td>Compressed-air in can</td><td>Various</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PCR machine</td><td>Various</td><td></td><td></td></tr><tr><td>Nanodrop 2000, spectrophotometer</td><td>Thermo-Scientific</td><td></td><td>microvolume spectrophotometer</td></tr><tr><td>Vortexer</td><td>Various</td><td></td><td></td></tr><tr><td>Table top microfuge at room temperature</td><td>Various</td><td></td><td></td></tr><tr><td>Table top microfuge at 4 &amp;deg;C</td><td>Various</td><td></td><td></td></tr><tr><td>Heat block</td><td>Various</td><td></td><td></td></tr><tr><td>Hybridization oven or incubator with rotator</td><td>Various</td><td></td><td></td></tr><tr><td>Nutator</td><td>Various</td><td></td><td></td></tr><tr><td>A1RSi laser scanning confoal</td><td>Nikon</td><td></td><td>40X oil Plan Fluor DIC (NA 1.3)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables etc.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>Various</td><td></td><td></td></tr><tr><td>15 mL conical tubes</td><td>Various</td><td></td><td></td></tr><tr><td>1.5 mL microfuge tubes</td><td>Various</td><td></td><td></td></tr><tr><td>500 &amp;micro;l microfuge tubes</td><td>Various</td><td></td><td></td></tr><tr><td>200 &amp;micro;l PCR tubes</td><td>Various</td><td></td><td></td></tr><tr><td>Plastic container with tight fitting lid</td><td>Various</td><td></td><td>To hold Drierite</td></tr><tr><td>Kimwipes</td><td>Various</td><td></td><td>disposable wipes</td></tr><tr><td>Parafilm</td><td>Various</td><td></td><td>paraffin film</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HPLC purified 5&#039;-labeled oligonucleotides</td><td>Integrated DNA Technologies</td><td></td><td>Cy3-labeled probes that recognize the 359 bp satellite repeat of the &lt;em&gt;X&lt;/em&gt; chromosome</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>Version 6.3</td></tr></tbody></table>

using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes

Em humanos, erros de segregação do cromossomo em oócitos são responsáveis para a maioria dos abortos espontâneos e malformações congénitas. Além disso, como as mulheres envelhecem, o risco de conceber que um feto aneuploid aumenta dramaticamente e este fenómeno é conhecido como o efeito da idade materna. Um requisito para a segregação do cromossomo precisos durante as divisões meióticas é manutenção da irmã coesão de cromátides irmãs durante o período de prófase estendida que a experiência de oócitos. Citológicas em seres humanos e organismos modelo sugerem que a coesão meiótica deteriora-se durante o processo de envelhecimento. Além disso, erros de segregação em oócitos humanos são mais prevalentes durante a meiose I, consistente com a perda prematura da coesão do braço. O uso de organismos modelo é fundamental para desvendar os mecanismos subjacentes a idade-dependente perda de coesão. Drosophila melanogaster oferece diversas vantagens para estudar o Regulamento de coesão meiótica em oócitos. No entanto, até recentemente, apenas testes genéticos estavam disponíveis a ensaiar para a perda de coesão de braço em oócitos de genótipos diferentes ou em diferentes condições experimentais. Aqui, um protocolo detalhado é fornecido usando fluorescência em situ hibridação (peixe) para visualizar diretamente os defeitos na coesão de braço em prometafase I e metáfase prendi Drosophila oócitos. Gerando uma sonda de peixe que se para o braço distal do cromossomo X e coletando pilhas de Z confocal, um pesquisador pode visualizar o número de sinais individuais de peixes em três dimensões e determinar se irmã cromátide braços são separados. O procedimento descrito torna possível quantificar defeitos de coesão braço em centenas de oócitos de Drosophila . Como tal, este método oferece uma importante ferramenta para investigar os mecanismos que contribuem para a manutenção da coesão, bem como os fatores que levam à sua extinção durante o processo de envelhecimento.

A Figura 5 apresenta imagens obtidas com uma sonda de braço que se a região citológica 6E-7B no cromossomo X . Este resulta da sonda em um sinal de que co localiza com o de DAPI, é facilmente distinguível do fundo e tem sido usada com sucesso para quantificar defeitos de coesão braço em diferentes genótipos de19. Quantificação de defeitos de coesão se restringia a prometafase I e metáfase eu estágios; oócitos ant...

Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

Usando FISH de hibridização in situ por fluorescência para monitorar o estado da coesão do braço em oócitos de Drosophila em prometáfase e metáfase I | Desenvolvimento | Biologia | Vídeo

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Uso de hibridização in situ de fluorescência (FISH) para monitorar o estado da coesão do braço em oócitos de Drosophila na prometáfase e na metáfase I

Este manuscrito apresenta um método detalhado para a geração X-cromossomo braço sondas e realizando fluorescência situ hibridação in (peixe) para examinar o estado da irmã coesão cromátide em prometafase e metáfase prendi Drosófila ovócitos. Este protocolo é apropriado para determinar se a coesão de braço meiótica é intacta ou interrompidos em diferentes genótipos.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Meiosis

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11.3K visualizações.  Dartmouth College. Este manuscrito apresenta um método detalhado para a geração X-cromossomo braço sondas e realizando fluorescência situ hibridação in (peixe) para examinar o estado da irmã coesão cromátide em prometafase e metáfase prendi Drosófila ovócitos. Este protocolo é apropriado para determinar se a coesão de braço meiótica é intacta ou interrompidos em diferentes genótipos.

Vídeo: Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

Alta resolução hibridação fluorescente In Situ em embriões da drosófila e tecidos usando amplificação de sinal Tyramide

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Genética

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Uso de uma única molécula fluorescente hibridação In Situ (FISH-SM) para Quantify e Localize os mRNAs em oócitos murino

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Migração Nuclear no Oócito de Drosophila

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Biologia do Desenvolvimento

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Estudar checkpoint mitótico por Ilustrando comportamento dinâmico cinetocoro Proteína e Movimento de cromossomos em Viver<em&gt; Drosophila</em&gt; Os embriões Sincicial

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Biologia

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Imagens ao vivo de Proteínas GFP-rotulados em<em&gt; Drosophila</em&gt; Oócitos

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

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.

Todo o Monte RNA Fluorescente<em&gt; In situ</em&gt; A hibridação de<em&gt; Drosophila</em&gt; Embriões

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

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Citológica Análise da espermatogênese: Preparados ao vivo e fixo<em&gt; Drosophila</em&gt; Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Técnicas de imagem Prometáfase e Metaphase da meiose I em Fixed<em&gt; Drosophila</em&gt; oócitos

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

Neurociência

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

Mancha imunofluorescente para visualização de proteínas associadas à heterocromatina em glândulas salivares drosophila

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...

Uso de hibridização in situ de fluorescência (FISH) para monitorar o estado da coesão do braço em oócitos de Drosophila na prometáfase e na metáfase I

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