Questo lavoro è stato supportato da NIH Grant GM59354 assegnato a Sharon E. Bickel. Ringraziamo Huy d. Nguyen per assistenza nello sviluppo del protocollo per la generazione di sonde fluorescenti braccio, Ann Lavanway per aiuto con microscopia confocale e J. Emiliano Reed per l'assistenza tecnica. Ringraziamo anche numerosi colleghi nella comunità della drosofila per utili discussioni e consigli.

<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. 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=Chromosome+cohesion+decreases+in+human+eggs+with+advanced+maternal+age.">Chromosome cohesion decreases in human eggs with advanced maternal age.</a> Aging Cell. 11 (6), 1121-1124 (2012).</li><li>Yun, Y., Lane, S. I., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+dyad+separation+in+meiosis+II+is+the+major+segregation+error+with+maternal+age+in+mouse+oocytes.">Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes.</a> Development. 141 (1), 199-208 (2014).</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=Heterochromatin-mediated+association+of+achiasmate+homologues+declines+with+age+when+cohesion+is+compromised.">Heterochromatin-mediated association of achiasmate homologues declines with age when cohesion is compromised.</a> Genetics. 181, 1207-1218 (2009).</li><li>Jeffreys, C. A., Burrage, P. S., 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=A+model+system+for+increased+meiotic+nondisjunction+in+older+oocytes.">A model system for increased meiotic nondisjunction in older oocytes.</a> Curr. Biol. 13 (6), 498-503 (2003).</li><li>Weng, K. A., Jeffreys, C. A., 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=Rejuvenation+of+Meiotic+Cohesion+in+Oocytes+during+Prophase+I+Is+Required+for+Chiasma+Maintenance+and+Accurate+Chromosome+Segregation.">Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation.</a> PLoS Genetics. 10 (9), e1004607 (2014).</li><li>Perkins, A. T., Das, T. M., Panzera, L. C., 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=Oxidative+stress+in+oocytes+during+midprophase+induces+premature+loss+of+cohesion+and+chromosome+segregation+errors.">Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors.</a> Proc Natl Acad Sci U S A. 113 (44), E6823-E6830 (2016).</li><li>Dernburg, A. F., Sedat, J. W., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+a+role+for+heterochromatin+in+meiotic+chromosome+segregation.">Direct evidence of a role for heterochromatin in meiotic chromosome segregation.</a> Cell. 86, 135-146 (1996).</li><li>Dernburg, A. F., Sedat, J. 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Gli autori non dichiarano concorrenti interessi finanziari.

L'uso di pesce sonde per valutare lo stato di coesione braccio in prometafase io e metafase ho Drosophila ovociti è un progresso significativo nel campo della drosofila meiosi. Storicamente, i ricercatori della Drosophila sono stati limitati a test genetici per dedurre la perdita prematura di coesione braccio in ovociti maturi11,18,19. Ora, con i metodi qui presentati, lo stato di coesione del braccio...

Corretta segregazione dei cromosomi durante la mitosi e meiosi richiede che la sorella cromatidio coesione essere stabilito, mantenuto e rilasciato in un modo coordinato1,2. Coesione è stabilito durante la fase S ed è mediato dalla coesina complessa, che forma legami fisici che tengono la sorella cromatidi insieme. Nella meiosi, coesione distale di un crossover funziona anche per tenere insieme gli omologhi ricombinanti e questa associazione fisica contribuisce a garantire il corretto orientamento di bivalente su metafase mandrino (Figura 1)3,4, 5. Rilascio del braccio coesione a anafase permette l'omologhi segregare ai poli opposti del mandrino. Tuttavia, se la coesione del braccio è perso prematuramente, ricombinanti omologhi perderanno loro connessione fisica e segregare in modo casuale, che può provocare gameti aneuploid (Figura 1).
In ovociti umani, errori nella segregazione del cromosoma sono la principale causa di aborti e malformazioni alla nascita, come la sindrome di Down6, e la loro incidenza aumenta esponenzialmente con l'età materna7. Coesione di cromatidio della sorella è stabilito negli ovociti fetali e ricombinazione meiotic viene completata prima della nascita. Ovociti quindi arrestare in Mid-Profase I fino a quando l'ovulazione e durante questo periodo di arresto, l'associazione fisica costante degli omologhi ricombinanti si basa sulla sorella coesione tra cromatidi fratelli. Di conseguenza, accurata segregazione durante la meiosi e gli esiti di gravidanza normale richiedono che coesione rimangono intatti per fino a cinque decenni.
La perdita prematura di coesione durante l'arresto prolungato meiotic di ovociti umani è stata proposta per contribuire all'effetto dell'età materna e linee multiple di prova supportano questa ipotesi8,9. Tuttavia, date le sfide di studiare meiotica coesione in ovociti umani, gran parte della nostra comprensione di questo fenomeno si basa sull'uso di modello organismi5,10,11,12, 13,14,15.
Drosophila melanogaster ovociti offrono numerosi vantaggi per lo studio della coesione e cromosoma meiotic di segregazione. Una semplice analisi genetica permette di recuperare la progenie da gameti aneuploid e misurare la fedeltà di X-segregazione cromosomica su una larga scala11,16,17. Inoltre, si può anche determinare se gli errori di segregazione del cromosoma sorgono perché missegregate ricombinanti omologhi durante la meiosi I, un fenotipo che è coerenza con la perdita prematura di braccio coesione11,18, 19. diretta osservazione dello stato di coesione meiotica in ovociti di Drosophila è anche possibile mediante ibridazione in situ di fluorescenza (pesce). Anche se oligonucleotidi fluorescenti che ibridano a sequenze ripetitive satellite sono stati utilizzati per oltre un decennio per monitorare pericentromeric coesione maturo Drosophila ovociti4,20, analisi del braccio coesione è stato molto più difficile. Visualizzazione dello stato di coesione braccio richiede una sonda che si estende su una vasta regione di copia singola sequenze ed è abbastanza luminosa per provocare segnali visibili per singolo sorella cromatidi quando braccio coesione è assente. Inoltre, il condizioni di fissazione degli ovociti e le dimensioni delle autorità nazionali designate con etichettate deve facilitare la penetrazione21 nell'ovocita maturo grande di Drosophila (200 µm larga di 500 µm lungo). Recentemente, una sonda di braccio è stato correttamente utilizzato per prevedere i cromatidi oocita di Drosophila durante l'anafase io, ma gli autori ha dichiarato che potrebbe non rilevare un segnale in prometafase o metafase sono arrestato ovociti22. Qui forniamo un protocollo dettagliato per la generazione di X-braccio del cromosoma FISH sonde e le condizioni di preparazione degli ovociti che ci hanno permesso di analizzare per la perdita prematura della sorella coesione cromatidio in prometafase io e metafase ho ovociti. Queste tecniche, che hanno permesso di identificare prodotti genici che sono necessari per il mantenimento della coesione meiotica, permetterà ad altri di dosaggio per sorella coesione cromatidio difetti in ovociti maturi di Drosophila di genotipi differenti.

<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. Comes with NEB enzymes</td></tr><tr><td>Terminal deoxynucleotidyl transferase (TdT) 400 U/&amp;micro;l</td><td>Roche/Sigma</td><td>3333566001</td><td></td></tr><tr><td>TdT buffer</td><td>Roche/Sigma</td><td></td><td>Comes with TdT enzyme</td></tr><tr><td>Cobalt chloride</td><td>Roche/Sigma</td><td></td><td>&lt;strong&gt;Toxic: &lt;/strong&gt;wear appropriate protection. 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using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes

Negli esseri umani, gli errori di segregazione del cromosoma in ovociti sono responsabili della maggior parte di aborti e malformazioni congenite. Inoltre, come le donne invecchiano, il loro rischio di concepire che un feto aneuploide aumenta notevolmente e questo fenomeno è noto come l'effetto dell'età materna. È un requisito per la segregazione del cromosoma accurata durante le divisioni meiotiche manutenzione della sorella coesione tra cromatidi fratelli durante il periodo di profase estesa esperienza di ovociti. Prova citologica in entrambi esseri umani e gli organismi modello suggerisce che coesione meiotica si deteriora durante il processo di invecchiamento. Inoltre, gli errori di segregazione in ovociti umani sono più prevalenti durante la meiosi I, coerente con la perdita prematura di coesione di braccio. L'uso di organismi modello è fondamentale per dipanare i meccanismi che sottendono la perdita età-dipendente di coesione. Drosophila melanogaster offre diversi vantaggi per studiare il regolamento di meiotic coesione negli ovociti. Tuttavia, fino a poco tempo, solo i test genetici erano disponibili test per la perdita di coesione del braccio in ovociti di genotipi diversi o in diverse condizioni sperimentali. Qui, un protocollo dettagliato è fornito per l'utilizzo di fluorescenza in situ ibridazione (pesce) per visualizzare direttamente difetti nella coesione del braccio in prometafase io e metafase sono arrestato ovociti di Drosophila . Generando una sonda di pesce che ibridizza con il braccio distale del cromosoma X e raccogliendo gli stack Z confocale, un ricercatore può visualizzare il numero di singoli segnali di pesce in tre dimensioni e determinare se sorella cromatidio braccia sono separati. La procedura descritta permette di quantificare i difetti di coesione braccio in centinaia di Drosophila ovociti. Come tale, questo metodo fornisce un importante strumento per indagare i meccanismi che contribuiscono alla coesione manutenzione, nonché i fattori che portano alla sua scomparsa durante il processo di invecchiamento.

Figura 5 presenta le immagini ottenute con una sonda di braccio che ibridizza con citologico regione 6E-7B sul cromosoma X . Questo risultati di sonda in un segnale che co-localizza con quello di DAPI, è facilmente distinguibile dallo sfondo ed è stato usato con successo per quantificare i difetti di coesione del braccio in diversi genotipi19. Quantificazione dei difetti di coesione è stata limitata a prometafase io e metaf...

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

Utilizzo del FISH con ibridazione in situ a fluorescenza per monitorare lo stato di coesione del braccio negli ovociti di Drosophila prometafase e metafase I | Sviluppo | Biologia | Video

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

Mediante ibridazione In Situ di fluorescenza (pesce) per monitorare lo stato di coesione braccio in prometafase e metafase I Oocytes di Drosophila

Questo manoscritto presenta un metodo dettagliato per la generazione di X-cromosoma braccio sonde e performanti in situ ibridazione di fluorescenza (pesce) per esaminare lo stato della sorella coesione cromatidio in prometafase e metafase sono arrestato Drosofila ovociti. Questo protocollo è adatto a determinare se la coesione meiotica braccio è intatto o perturbato in differenti genotipi.

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

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11.3K Views.  Dartmouth College. Questo manoscritto presenta un metodo dettagliato per la generazione di X-cromosoma braccio sonde e performanti in situ ibridazione di fluorescenza (pesce) per esaminare lo stato della sorella coesione cromatidio in prometafase e metafase sono arrestato Drosofila ovociti. Questo protocollo è adatto a determinare se la coesione meiotica braccio è intatto o perturbato in differenti genotipi.

Video: 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.

Ibridazione fluorescente In Situ ad alta risoluzione in embrioni della drosofila e tessuti mediante amplificazione del segnale 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 ...

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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 di singola molecola fluorescente ibridazione In Situ (SM-pesce) per quantificare e Localize mRNAs in ovociti murini

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.

Migrazione nucleare nell'ovocita di Drosophila

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

Biologia dello sviluppo

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.

Studiare Checkpoint mitotica illustrando un comportamento dinamico delle proteine ​​cinetocoro e Motion cromosoma in Living<em&gt; Drosophila</em&gt; Embrioni sinciziale

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.

Vivo Imaging delle proteine ​​GFP-etichettati in<em&gt; Drosophila</em&gt; Gli oociti

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.

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

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;

L&#39;analisi citologica della spermatogenesi: Preparati Live e fisse di<em&gt; Drosophila</em&gt; Testicoli

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.

Tecniche di Imaging Prometafase e metafase della meiosi I nel fisso<em&gt; Drosophila</em&gt; ovociti

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 ...

Neuroscienze

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.

Colorazione immunofluorescente per la visualizzazione delle proteine associate all'eterocromatina nelle ghiandole salivari della Drosophila

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

Mediante ibridazione In Situ di fluorescenza (pesce) per monitorare lo stato di coesione braccio in prometafase e metafase I Oocytes di Drosophila

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ibridazione fluorescente In Situ ad alta risoluzione in embrioni della drosofila e tessuti mediante amplificazione del segnale Tyramide

Uso di singola molecola fluorescente ibridazione In Situ (SM-pesce) per quantificare e Localize mRNAs in ovociti murini

Migrazione nucleare nell'ovocita di Drosophila

Studiare Checkpoint mitotica illustrando un comportamento dinamico delle proteine cinetocoro e Motion cromosoma in Living

Vivo Imaging delle proteine GFP-etichettati in

Tutta la Monte RNA fluorescente

L'analisi citologica della spermatogenesi: Preparati Live e fisse di

Tecniche di Imaging Prometafase e metafase della meiosi I nel fisso

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

Colorazione immunofluorescente per la visualizzazione delle proteine associate all'eterocromatina nelle ghiandole salivari della Drosophila

Mediante ibridazione In Situ di fluorescenza (pesce) per monitorare lo stato di coesione braccio in prometafase e metafase I Oocytes di Drosophila

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ibridazione fluorescente In Situ ad alta risoluzione in embrioni della drosofila e tessuti mediante amplificazione del segnale Tyramide

Uso di singola molecola fluorescente ibridazione In Situ (SM-pesce) per quantificare e Localize mRNAs in ovociti murini

Migrazione nucleare nell'ovocita di Drosophila

Studiare Checkpoint mitotica illustrando un comportamento dinamico delle proteine ​​cinetocoro e Motion cromosoma in Living

Vivo Imaging delle proteine ​​GFP-etichettati in

Tutta la Monte RNA fluorescente

L'analisi citologica della spermatogenesi: Preparati Live e fisse di

Tecniche di Imaging Prometafase e metafase della meiosi I nel fisso

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

Colorazione immunofluorescente per la visualizzazione delle proteine associate all'eterocromatina nelle ghiandole salivari della Drosophila

Studiare Checkpoint mitotica illustrando un comportamento dinamico delle proteine cinetocoro e Motion cromosoma in Living

Vivo Imaging delle proteine GFP-etichettati in