We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1. Cromosoma Preparazione <ol><li> Germinazione dei semi e la fissazione di apici radicali <ol><li> Germinare 10-20 semi di orzo su due strati di carta da filtro umida in una capsula di Petri in condizioni di oscurità per 2 giorni a 22-24 ° C. Tagliare radici vigorose con la lunghezza di 1-2 cm dal seme usando una lama di rasoio. </li><li> Preparare acqua ghiacciata mettendo una bottiglia di vetro da 500 ml contenente acqua di rubinetto fredda nel ghiaccio tritato-acqua. Aerare l&#39;acqua ghiacciata e immergere apici radicali per 20 ore di aumentare la frequenza di cellule in metafase. </li><li> Trasferire radici da acqua a 50 ml di etanolo: acido acetico (3: 1) fissativo fissarli a temperatura ambiente per 2 giorni. Conservare radici in etanolo appena preparato: acido acetico (3: 1) fissativo a 4 ° C fino al momento dell&#39;uso fino ad un anno. </li></ol></li><li> Lavaggio e trattamento enzimatico <ol><li> Lavare le 10-20 radici con 30 ml acqua di rubinetto ghiacciata per 5 minuti due volte con un bicchiere di vetro da 50 ml. Utilizzare microscopio binoculare. Trasferire radici uno per uno in30 ml di 0,01 M tampone citrato (0,01 M di acido citrico + 0,01 M sodio citrato, pH 4.8) con pinze e lavare agitando il bicchiere di vetro per 5 minuti due volte. Posizionare radici su carta da filtro per rimuovere completamente il liquido e cut-off non-tessuto meristematico indesiderato usando una lama di rasoio. </li><li> Incubare fino a 20 apici radicali in miscela enzimatica 1 ml a 37 ° C per circa 50 minuti per ammorbidire i tessuti vegetali (Tabella 1) in un vetro da orologio. Miscela enzimatico contiene 0,7% cellulasi R10, 0,7% cellulasi, 1% e 1% pectolyase cytohelicase diluito in tampone citrato 0,01 M. Conservare la miscela enzimatica a -20 ° C e riutilizzati fino a cinque volte. </li><li> Rimuovere l&#39;enzima pipettando e lavare le punte delle radici su ghiaccio con 5 ml di 0,01 M tampone citrato due volte per sostituire l&#39;enzima residuale. </li></ol></li><li> Root macerazione <ol><li> Apici radicali Lavare con 1 ml di etanolo al 96% per due volte con attenzione nello stesso vetro dell&#39;orologio. Sostituire etanolo con appena preparato fissativo (75% di acido acetico: 25% etanolo). Usa 10-15fissativo pl per punta della radice. </li><li> Apici radicali trasferimento insieme al fissatore in un tubo da 2 ml e disintegrare meristemi radice con un ago da dissezione o pinze. Premere il tubo 20 volte per risospendere le cellule per ottenere una sospensione di cellule. Conservare la sospensione cellulare a -20 ° C fino a due mesi. </li></ol></li><li> Cadute di sospensione cellulare <ol><li> Mettere 2-3 strati di carta velina imbevuta di acqua su un piatto caldo a 50 ° C. Immergere i vetrini microscopici in acqua di rubinetto ghiacciata in frigo per 30 minuti. E posto scivola sulla parte superiore del tessuto di carta umida. </li><li> Pipettare 7-10 ml di sospensione cellulare e inserirla da una distanza di 20 cm sul vetrino raffreddata posto sulla piastra calda. Pipettare 10 ml di miscela di acidi acetico-etanolo nello stesso luogo come sospensione cellulare sul vetrino e mantenere la slitta sulla piastra calda per ulteriori 2 min. Posizionare il vetrino sulla piastra calda senza il tessuto bagnato e lasciare asciugare per 1 minuto. </li></ol></li><li> Controllo qualità e contene di diapositive <ol><li> Controllare diapositive utilizzando un microscopio a contrasto di fase per il controllo della qualità della diffusione cromosoma. Utilizzare vetrini sia lo stesso giorno o deposito immergendo in 96% di etanolo in una vaschetta Coplin a -20 ° C. </li></ol></li><li> Il pretrattamento di diapositive prima FISH; tutte le fasi vengono eseguite a temperatura ambiente <ol><li> Collocare i vetrini in una vaschetta Coplin contenente 50 ml di 2x SSC (20x SSC contiene 3 M di NaCl e 300 mm citrato trisodico) per 5 min. Utilizzando pinze, trasferimento vetrini in una vaschetta Coplin contenente 50 ml di 45% di acido acetico per 3-10 min. </li><li> Trasferire i vetrini in una vaschetta Coplin contenente 50 ml di 2x SSC per 10 min. Trasferire i vetrini ad una vaschetta Coplin contenente 50 ml di formaldeide al 4% (in 2x SSC) e immergere i vetrini per 10 minuti per riparare i cromosomi. </li><li> Rimuovere formaldeide risciacquando i vetrini 3 volte per 4 min ciascuno, in una vaschetta Coplin contenente 50 ml 2x SSC. Disidratare i vetrini in una vaschetta Coplin per 2 min in serie di 70%, 90% e 100% etanolo, rispettivamente, e scorre secco in verticaleposizione. </li></ol></li></ol> 2. ibridazione in situ fluorescente (FISH) <ol><li> Per ciascun vetrino, preparare una soluzione di ibridazione di 20 microlitri in totale utilizzando 10 ml di formammide deionizzata, 5 ml di tampone 4x ibridazione (200 microlitri di buffer contiene 80 microlitri 20x SSC, 8 microlitri 1 M Tris-HCl pH 8,0, 1,6 microlitri 0,5 M EDTA, 11.2 ml 10 mg / mL di sperma di salmone e acqua priva di DNasi 99,2 ml), 3 ml di sonda e 2 ml di acqua priva di DNasi. </li><li> Aggiungere 20 ml di soluzione di ibridazione per vetrino e coprire con un vetrino 24 x 32 mm ed arrestare la polizza di copertura con il cemento di gomma. Denaturare vetrini con sonde contemporaneamente a 80 ° C per 2 min su una piastra calda. </li><li> Trasferire i vetrini in una camera umida e incubare i vetrini a 37 ° CO / N luce evitando. Rimuovere vetrini risciacquando i vetrini in una vaschetta Coplin con SSC 2x. Collocare i vetrini in una vaschetta Coplin contenente 55-60 ° C 2x SSC e incubare per 20 min. </li><li> Porre i vetrini a 2x SSC in una vaschetta Coplin per 2 minuti a temperatura ambiente. Disidratare i vetrini in una vaschetta Coplin per 2 min in serie di 70%, 90% e 100% etanolo, rispettivamente. </li><li> Asciugare le diapositive e di contrasto con 1 mg / ml di 4 &#39;, 6-diamidino-2-phenylindoline (DAPI) in antifade mezzo di montaggio, evitare la luce intensa. </li></ol> 3. Analisi microscopica e bagagli <ol><li> Analizzare le diapositive utilizzando un microscopio a epifluorescenza. La selezione di filtro dipende dalla fluorocromo usato per la marcatura della sonda. Se necessario, conservare i vetrini a 4 ° C in condizioni di oscurità fino a un anno. </li></ol>

<ol><li>Geber, G., Schweizer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytochemical+heterochromatin+differentiation+in+Sinapis+alba+%28Cruciferae%29+using+a+simple+air-drying+technique+for+producing+chromosome+spreads%5BTitle%5D)+AND+%22Pl+Syst+Evol%22%5BJournal%5D)">Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads.</a> Pl Syst Evol. 158 (2-4), 97-106 (1988).</li>
<li>Andras, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+drop-spreading+technique+to+produce+cytoplasm-free+mitotic+preparations+from+plants+with+small+chromosomes%5BTitle%5D)+AND+%22Chromosome+Res%22%5BJournal%5D)">A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.</a> Chromosome Res. 7 (8), 641-647 (1999).</li>
<li>Rothfels, K. H., Siminovitch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+air-drying+technique+for+flattening+chromosomes+in+mammalian+cells+grown+in+vitro%5BTitle%5D)+AND+%22Stain+Technology%22%5BJournal%5D)">An air-drying technique for flattening chromosomes in mammalian cells grown in vitro.</a> Stain Technology. 33 (2), 73-77 (1958).</li>
<li>Martin, R., Busch, W., Herrmann, R. G., Wanner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+preparation+of+plant+chromosomes+for+high-resolution+scanning+electron+microscopy%5BTitle%5D)+AND+%22Chromosome+Res%22%5BJournal%5D)">Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy.</a> Chromosome Res. 2 (5), 411-415 (1994).</li>
<li>Kato, A., Albert, P. S., Vega, J. M., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive+fluorescence+in+situ+hybridization+signal+detection+in+maize+using+directly+labeled+probes+produced+by+high+concentration+DNA+polymerase+nick+translation%5BTitle%5D)+AND+%22Biotech.+Histochem%22%5BJournal%5D)">Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.</a> Biotech. Histochem. 81 (2-3), 71-78 (2006).</li>
<li>Kirov, I., Divashuk, M., Van Laere, K., Soloviev, A., Khrustaleva, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+easy+%27SteamDrop%27+method+for+high+quality+plant+chromosome+preparation%5BTitle%5D)+AND+%22Mol.+Cytogenet%22%5BJournal%5D)">An easy 'SteamDrop' method for high quality plant chromosome preparation.</a> Mol. Cytogenet. 7, 21(2014).</li>
<li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+chromosome+spreading%5BTitle%5D)+AND+%22Am+J+Med+Genet%22%5BJournal%5D)">Dynamics of chromosome spreading.</a> Am J Med Genet. 61 (4), 387-393 (1996).</li>
<li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synteny+between+Brachypodium+distachyon+and+Hordeum+vulgare+as+revealed+by+FISH%5BTitle%5D)+AND+%22Chromosome+Res%22%5BJournal%5D)">Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH.</a> Chromosome Res. 18 (7), 841-850 (2010).</li>
<li>Kato, A., Lamb, J. C., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+painting+using+repetitive+DNA+sequences+as+probes+for+somatic+chromosome+identification+in+maize%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U+S+A%22%5BJournal%5D)">Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize.</a> Proc. Natl. Acad. Sci. U S A. 101 (37), 13554-13559 (2004).</li>
<li>Pan, W. H., Houben, A., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+effective+cell+synchronization+in+plant-roots+by+hydroxyurea+and+amiprophos-methyl+or+colchicine%5BTitle%5D)+AND+%22Genome%22%5BJournal%5D)">Highly effective cell synchronization in plant-roots by hydroxyurea and amiprophos-methyl or colchicine.</a> Genome. 36 (2), 387-390 (1993).</li>
<li>Kim, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+karyotyping+of+sorghum+by+in+situ+hybridization+of+landed+BACs%5BTitle%5D)+AND+%22Genome%22%5BJournal%5D)">Integrated karyotyping of sorghum by in situ hybridization of landed BACs.</a> Genome. 45 (2), 402-412 (2002).</li>
<li>Lapitan, N. L. V., Brown, S. E., Kennard, W., Stephens, J. L., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FISH+physical+mapping+with+barley+BAC+clones%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">FISH physical mapping with barley BAC clones.</a> Plant J. 11 (1), 149-156 (1997).</li>
<li>Aliyeva-Schnorr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytogenetic+mapping+with+centromeric+BAC+contigs+shows+that+this+recombination-poor+region+comprises+more+than+half+of+barley+chromosome+3H%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Cytogenetic mapping with centromeric BAC contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.</a> Plant J. 84, 385-394 (2015).</li>
</ol>

The authors have nothing to disclose.

L&#39;esperimento preparazione cromosoma è stata effettuata utilizzando radici giovani di cereali appartenenti alla famiglia delle graminacee (Poaceae). Tutte le specie analizzate hanno 14 cromosomi in metafase relativamente lunga mitotico (11-15 micron) nel set genoma diploide e appartengono a specie di grande genoma (5,1-7,9 GBP). Lunghezza di radici germinati non era più di 2 cm per ottenere un massimo di tessuti meristematici. Sincronizzazione di divisione delle cellule è stato raggiu...

Ibridazione in situ fluorescente (FISH) è uno strumento efficace per la mappatura fisica delle sequenze singole e alta copia a livello cromosomico. Il presupposto è la preparazione degli spread cromosomiche di alta qualità. Non c&#39;è cromosoma protocollo preparazione generale che sarebbe ugualmente adatto per cellule animali e vegetali. Preparazione dei cromosomi vegetali è particolarmente difficile a causa della parete cellulare rigida e vari coerenza citoplasma in specie diverse. Uno dei metodi favorevoli per la preparazione di cromosomi pianta è una tecnica cosiddetta goccia anche noto come tecnica di diffusione drop e asciugatura all&#39;aria tecnica 1,2. Questo metodo è stato introdotto nel 1958 da Rothfels e Siminovitch per in vitro cellule di mammifero coltivate 3. Successivamente Martin et al. 4 e Kato et al. 5 adattato questo metodo per le piante. Più di recente, un metodo denominato &#39;SteamDrop &amp;# 39; stato sviluppato che utilizzava vapore acqueo per la preparazione di cromosomi non sovrapposti 6. Anche se, l&#39;influenza positiva di elevata umidità è stato osservato in precedenza 7, &#39;SteamDrop&#39; offre un flusso di lavoro controllata di preparati cromosomici di alta qualità 6. Il trattamento a vapore provoca stiramento dei cromosomi, probabilmente legati ad alcune modifiche di proteine ​​cromosomiche. La qualità del risultato spread metafase è molto alta, anche se di contenimento del numero sufficiente di completo metafase si diffonde per le successive esperimenti di FISH richiede competenze tecniche. Qui vi presentiamo un protocollo per la preparazione di cromosomi mitotici di cereali adatti per la rilevazione FISH di singoli e di alta copia sonde 5,8. Questo metodo è una migliore variante del metodo caduta asciugare descritto da Kato 9 eseguita sotto umidità relativa del 50% -55% (Figura 1). Questo protocollo comprende un numero ridottodi fasi di lavaggio rendendo l&#39;applicazione facile, efficiente e riproducibile. Usando questo protocollo abbiamo ottenuto spread cromosomiche di alta qualità e risultati FISH per Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e Secale cereale.

<table><tbody><tr><td>Hot Plate</td><td>MEDAX GmbH</td><td>12603</td><td></td></tr><tr><td>Cellulase R10</td><td>Duchefa</td><td>C8001</td><td></td></tr><tr><td>Cellulase&amp;nbsp;</td><td>CalBioChem</td><td>219466</td><td></td></tr><tr><td>Pectolyase</td><td>Sigma</td><td>P3026</td><td></td></tr><tr><td>Cytohelicase</td><td>Sigma</td><td>C8274</td><td></td></tr><tr><td>Texas Red-12-dUTP</td><td>Invitrogen</td><td>C3176</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluorecsence microscope</td><td>Olympus BX61</td><td>BX61</td><td></td></tr><tr><td>CCD camera</td><td>Orca ER, Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1200</td><td>fluorecsent dye</td></tr></tbody></table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

Preparazione degli spread dei cromosomi è un prerequisito per la performance di successo di ibridazione in situ fluorescente (FISH). Preparazione di alta qualità spread pianta cromosomiche è difficile a causa della parete cellulare rigida. Uno dei metodi approvati per la preparazione di cromosomi pianta è una cosiddetta preparazione goccia, noto anche come ripartizione goccia o tecnica aria di essiccazione. Qui, presentiamo un protocollo per la preparazione rapida del cromosoma mitotica diffonde adatto al rilevamento di FISH sonde singole e alte DNA copia. Questo metodo è una migliore variante del metodo goccia aria secca eseguita sotto un&#39;umidità relativa del 50% -55%. Questo protocollo comprende un numero ridotto di fasi di lavaggio rendendo l&#39;applicazione facile, efficiente e riproducibile. Evidenti vantaggi di questo approccio sono ben distribuite, non danneggiati e numerosi cromosomi in metafase che servono come presupposto ideale per l&#39;analisi FISH successo. Usando questo protocollo abbiamo ottenuto cromato di alta qualitàosome spread e risultati FISH riproducibili per Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e Secale cereale.

Preparati microscopici con spread metafase mitotico sono stati preparati dalla caduta cromosoma metodo rapido di preparazione asciugare sopra descritto (Suppl. Figura 1). Analisi FISH è stata condotta utilizzando sia, sequenze ripetitive e singola copia. Le immagini sono state ottenute con un microscopio a epifluorescenza dotato di una serie di filtri che permettono di eccitazione di fluorofori corrispondenti e catturato da una telecamera CCD monocromatica ad alta sensibilità. Per l&#39;acquisizione d...

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Un rapido aria secca cadente Cromosoma Metodo di preparazione adatto per i pesci nelle piante

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

a-fast-air-dry-dropping-chromosome-preparation-method-suitable-for

Research

JoVE Journal

Biology

13.2K Views.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Video: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Tempo di replicazione del DNA utilizzando Zebrafish come un sistema di modello In Vivo di profilatura

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Ricerca

Genetica

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipolazione Ploidia di embrioni di zebrafish con Heat Shock 2 Trattamento

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biologia dello sviluppo

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescente<em&gt; In situ</emIbridazione&gt; su cromosomi mitotici di zanzare

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Immunologia e infezioni

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Preparazione delle scime cromosomiche meiotiche da Spermatociti di pesce zebra

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Co-espressione delle proteine chimeriche fluorescenti Fusion Multiple in modo efficiente nelle piante

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chimica

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Pittura cromosoma 2D e 3D nelle zanzare della malaria

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

A High-Throughput Metodo per la crioconservazione dello sperma e Zebrafish In Vitro Fecondazione

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Biologia

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Fare Diploide Zebrafish Gynogenetic di Pressure presto

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Metodo di preparazione delle diapositive per preservare l'architettura tridimensionale della cromatina delle cellule germinali testicolari

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Un rapido aria secca cadente Cromosoma Metodo di preparazione adatto per i pesci nelle piante

Riepilogo

Esplora Altri Video