Name: 3d multicolor dna fish tool to study nuclear architecture in human primary cells
Uploaded: 2020-01-25T13:00:00
Description: Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

Les auteurs reconnaissent l'assistance technique de l'INSTALLATION d'imagerie INGM (Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" (INGM), Milan, Italie), en particulier C. Cordiglieri, pour l'assistance lors des images 3D multicolor DNA FISH Achat. Ce travail a été soutenu par les subventions suivantes à B.B.: EPIGEN programme phare italien, Association Française contre les Myopathies (AFM-Téléthon, subvention nr 18754) et Giovani Ricercatori, ministère italien de la Santé (GR-2011-02349383). Ce travail a été soutenu par la subvention suivante à F.M.: Fondazione Cariplo (Bando Giovani, subvention nr 2018-0321).

1. Procédures de préparation et d'étiquetage de la sonde d'ADN avec traduction de pseudo
<ol><li>Purifiez et nettifiez les chromosomes artificiels bactériens (BAC), les plasmides ou les produits PCR avec des kits spécifiques (Table of Materials), resuspend in ddH2O, vérifier par électrophorèse sur un gel agarose et quantifier.</li>
	<li>Effectuer une traduction de pseudo sur 1,5 à 2 g d'ADN à partir de l'étape 1.1 dans un volume final de 50 L en mélangeant tous les réactifs dans...

<ol>
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H., 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=Extensive+Heterogeneity+and+Intrinsic+Variation+in+Spatial+Genome+Organization.">Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization.</a> Cell. 176 (6), 1502-1515 (2019).</li><li>Zheng, 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=Multiplex+chromatin+interactions+with+single-molecule+precision.">Multiplex chromatin interactions with single-molecule precision.</a> Nature. 566 (7745), 558-562 (2019).</li><li>Solovei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH)+on+tissue+cryosections.">Fluorescence in situ hybridization (FISH) on tissue cryosections.</a> Methods in Molecular Biology. 659, 71-82 (2010).</li><li>Cremer, 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=Multicolor+3D+fluorescence+in+situ+hybridization+for+imaging+interphase+chromosomes.">Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.</a> Methods in Molecular Biology. 463, 205-239 (2008).</li><li>Legant, W. 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La méthode actuelle décrit un protocole étape par étape pour effectuer 3D MULTIcolor DNA FISH sur un large éventail de cellules primaires humaines. Bien que l'ADN FISH soit une technologie largement utilisée, l'ADN 3D multicolore FISH sur les noyaux interphases 3D conservés est encore difficile à réaliser dans de nombreux laboratoires, principalement en raison des caractéristiques des échantillons utilisés23,24.
La traducti...

Les eucaryotes plus élevés doivent condenser et compacter systématiquement une énorme quantité d'informations génétiques dans l'espace 3D minute du noyau1,2,3,4. Aujourd'hui, nous savons que le génome est classé spatialement dans des compartiments et des domaines topologiquement associés5 et que les niveaux multiples de pliage de l'ADN génèrent des contacts entre différentes régions génomiques qui peuvent impliquer la formation de boucle de chromatine6,7. La boucle dynamique 3D de la chromatine peut influencer de nombreux processus biologiques différents tels que la transcription8,9, la différenciation et le développement10,11, réparation de l'ADN12,13, tandis que ses perturbations sont impliqués dans diverses maladies14,15,16 et les défauts de développement15,17,18.
De nombreuses approches ont été développées pour étudier l'organisation du génome 3D. Des technologies basées sur le capture de conformation de chromosome (C-technologies, 3C, 4C, 5C, Hi-C et dérivés) ont été développées pour étudier l'organisation de génome dans les cellules fixes3,4,19,20. Ces approches sont basées sur la capacité de capturer les fréquences de contact entre les loci génomiques à proximité physique. C-technologies, en fonction de leur complexité, attraper l'organisation mondiale du génome 3D et la topologie nucléaire d'une population cellulaire3,4,19,20. Néanmoins, les interactions 3D sont dynamiques dans le temps et l'espace, très variables entre les cellules individuelles composées d'interactions multiplexes, et sont largement hétérogènes21,22.
L'hybridation 3D de fluorescence in situ de l'ADN multicolore (FISH) est une technique qui permet la visualisation de loci génomiques spécifiques à un seul niveau de cellule, permettant une étude directe de l'architecture nucléaire 3D d'une manière complémentaire aux technologies C. Il représente une technologie actuellement utilisée pour valider sans ambiguïté les résultats C. 3D multicolor DNA FISH utilise des sondes étiquetées fluorescentes complémentaires aux loci génomiques d'intérêts. L'utilisation de différents fluorophores et d'équipements de microscopie appropriés permet la visualisation contemporaine de cibles multiples dans l'espace nucléaire23,24. Ces dernières années, FISH a été combiné avec les progrès technologiques dans la microscopie pour obtenir la visualisation des structures à échelle fine à haute résolution25,26 ou avec des approches CRISPR-Cas pour la visualisation des acides nucléiques en imagerie en direct27,28. Malgré une large adoption, l'approche 3D multicolor DNA FISH est encore considérée comme difficile dans de nombreux laboratoires car le matériel biologique utilisé doit être adapté.
Ici, nous fournissons un protocole complet pour 3D multicolor DNA FISH (de la préparation cellulaire / sonde à l'analyse des données) applicable à un large éventail de cellules primaires humaines, permettant la visualisation de loci génomiques multiples et la préservation de la structure 3D des noyaux. Afin d'étudier l'architecture nucléaire, la structure 3D des noyaux doit être préservée. Pour cette raison, contrairement à d'autres protocoles existants29,30,31, nous évitons l'utilisation d'un gradient d'alcool et le stockage des couvertures dans l'alcool qui peut affecter la structure de chromatine32. La méthode est adaptée à partir de protocoles FISH d'ADN 3D conservés24,33 à appliquer à un large éventail de cellules primaires humaines, à la fois isolées ex vivo ou cultivées in vitro. Il existe des paramètres de perméabilisation et de déprotéinisation pour différentes morphologies nucléaires et caractéristiques cytologiques (p. ex., différents degrés de compactage nucléaire, abondance de cytosquelettes)34. Ces paramètres sont souvent généralement décrits dans d'autres protocoles24,33, sans fournir une discrimination claire de la procédure au sein de différents types de cellules. En outre, nous avons développé un outil spécifique nommé NuCLD (localisateur de contacts nucléaires en 3D)16, fournissant des principes pour l'analyse des données qui permettra d'améliorer la proximité 3D entre les différents loci et leur distribution topologique nucléaire dans l'espace nucléaire d'une manière automatisée.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin 488</td>
			<td>DBA</td>
			<td>DI7488</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin 11 d-UTP</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0081</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumine)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverlsips</td>
			<td>Marienfeld</td>
			<td>117500</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 d-UTP</td>
			<td>GE Healthcare</td>
			<td>PA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D21490</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acids single strand from salmon testes</td>
			<td>Sigma</td>
			<td>D7656</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate (powder)</td>
			<td>Santa Cruz</td>
			<td>sc-203917A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin 11 d-UTP</td>
			<td>Roche</td>
			<td>11093088910</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21969-035 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>18010-017</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>AMPD1</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (C-G-A-T)</td>
			<td>Euroclone</td>
			<td>BL0423A/C/G</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644.2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Hyclone</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30109</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutammine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>30721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Human</td>
			<td>Sigma</td>
			<td>I9278-5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>NaAc (Sodium Acetate, pH 5.2, 3 M)</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate-buffered saline)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennycillin/Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070-063 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Biorad</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>PhasePrep BAC DNA Kit</td>
			<td>Sigma</td>
			<td>NA0100-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Gel Extraction &#38; PCR Purification Combo Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K220001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K210010</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2288</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubbercement</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>631-0114</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidina Alexa fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>S21374</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate</td>
			<td>Sigma</td>
			<td>1110379026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P9416-100mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d multicolor dna fish tool to study nuclear architecture in human primary cells

Une question majeure en biologie cellulaire est l'organisation génomique dans l'espace nucléaire et comment l'architecture de chromatine peut influencer des processus tels que l'expression des gènes, l'identité cellulaire et la différenciation. De nombreuses approches développées pour étudier l'architecture 3D du génome peuvent être divisées en deux catégories complémentaires : les technologies basées sur la conformation chromosomique (technologies C) et l'imagerie. Alors que le premier est basé sur la capture de la conformation chromosomique et des interactions d'ADN proximal dans une population de cellules fixes, le second, basé sur la fluorescence de l'ADN in situ hybridation (FISH) sur des noyaux conservés en 3D, permet la visualisation contemporaine de loci multiples à un seul niveau cellulaire (multicolor), examinant leurs interactions et leur distribution dans le noyau (3D MULTIcolor DNA FISH). La technique de l'ADN multicolore 3D FISH a une limitation de visualiser seulement quelques loci prédéterminés, ne permettant pas une analyse complète de l'architecture nucléaire. Cependant, compte tenu de la robustesse de ses résultats, 3D multicolor DNA FISH en combinaison avec la microscopie 3D et la reconstruction d'image est une méthode possible pour valider les résultats basés sur la technologie C et d'étudier sans ambiguïté la position et l'organisation de loci spécifiques au niveau d'une seule cellule. Ici, nous proposons une méthode étape par étape de 3D MULTIcolor DNA FISH adapté à un large éventail de cellules primaires humaines et discuter de toutes les actions pratiques, étapes cruciales, notions d'imagerie 3D et l'analyse des données nécessaires pour obtenir un succès et instructif 3D multicolore DNA FISH dans différents contextes biologiques.

La méthode de l'ADN multicolore 3D FISH décrit dans cet article permet la visualisation contemporaine de différents loci génomiques dans les noyaux 3D conservés (Figure 1B). Ce protocole permet de mesurer les distances entre les allèles et les différents loci génomiques afin d'évaluer leur proximité spatiale et d'évaluer leur emplacement dans l'espace nucléaire (p. ex., distance loci du centroïde ou de la périphérie des noyaux)16. Cepen...

Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D multicolor DNA FISH représente un outil pour visualiser les loci génomiques multiples dans les noyaux préservés 3D, définissant sans ambiguïté leurs interactions réciproques et leur localisation dans l'espace nucléaire à un seul niveau cellulaire. Ici, un protocole étape par étape est décrit pour un large éventail de cellules primaires humaines.

3D Multicolor DNA FISH Outil pour étudier l'architecture nucléaire dans les cellules primaires humaines

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

L’ADN multicolore 3D FISH permet à la visualisation de multiples loci génomiques dans les noyaux préservés de définir de manière ambiguë leur interaction réciproque et leur localisation au niveau d’une seule cellule. 3D multicolore DNA FISH permet l’étude directe de l’architecture nucléaire. En général, il fonctionne en conjonction avec les technologies basées sur la capture chromosomique, faisant de cette technique un outil disponible pour la validation des données C au sein de cellules individuelles.La démonstration de la procédure sera Federica Marasca. C’est un postdoc dans mon laboratoire. Commencez par couver le mélange de traduction nick dans un mélangeur thermique à 16 degrés Celsius selon la longueur du matériel d’ADN de départ.À la fin de l’incubation, vérifiez la taille des sondes sur un gel agarose de 2,2 %. Pour chaque expérience DNA FISH, précipiter la quantité appropriée de sonde selon le matériau d’ADN de départ à partir duquel les sondes ont été produites dans 150 microlitres d’eau distillée double, 20 microgrammes d’ADN de sperme de saumon non étiqueté, 3,5 microgrammes d’ADN spécifique cot-1, trois volumes de 100% d’éthanol, et un 1/10e volume de trois acétate de sodium molaire pendant une heure à moins 80 degrés Celsius. À la fin de l’incubation, centrifuger l’échantillon à vitesse maximale pendant une heure à quatre degrés Celsius.Après avoir jeté le supernatant, laver la pastille deux fois avec 500 microlitres de 70% d’éthanol. Après le deuxième lavage, resuspendez la pastille en deux microlitres de 100% formamide et secouez la sonde pendant 30 minutes à 40 degrés Celsius avant d’ajouter un volume égal de citrate de sodium salin 4X dans du sulfate de 20% dextran. Pour la fixation pré-traitement et la perméabilisation des petites cellules avec de petits noyaux et une faible quantité de cytoplasme, d’abord ajouter deux fois 10 à la sixième cellule en 200 microlitres de PBS directement sur des couvertures de verre dans les puits individuels d’une plaque de 24 puits et permettre aux cellules de se contenter de 30 minutes à température ambiante.À la fin de l’incubation, remplacez rapidement le PBS par 300 microlitres de paraformaldéhyde fraîchement préparés complétés par 0,1 % de Tween 20. Après 10 minutes, laver les cellules fixes trois fois en 300 microlitres de 0,05% TPBS pendant cinq minutes par lavage à température ambiante. Après le dernier lavage, perméabiliser les cellules T avec 300 microlitres de 0,5%TPBS pendant 10 minutes à température ambiante avant de traiter les cellules avec 250 microlitres par puits de cocktail RNAse dilué 1:100 en PBS pendant une heure à 37 degrés Celsius.À la fin de l’incubation, rincer les cellules dans 300 microlitres de PBS et ajouter 300 microlitres de 20% glycérol dans PBS à chaque puits avec une incubation d’une nuit à quatre degrés Celsius. Le lendemain matin, placez le verre sur de la glace sèche pendant 15 à 30 secondes pour congeler les cellules avant de décongeler graduellement les échantillons à température ambiante et de les tremper dans 300 microlitres de 20 % de glycérol dans le PBS pendant 30 secondes. Après le gel/dégel des cellules trois fois de plus comme nous venons de le démontrer, lavez les échantillons dans 300 microlitres de 0,5 % de TPBS pendant cinq minutes à température ambiante, suivis de deux lavages dans 300 microlitres de 0,05 % de TPBS pendant cinq minutes par lavage à température ambiante.Après le dernier lavage, traiter les cellules avec 300 microlitres de 0,1 acide chlorhydrique normal pendant 12 minutes à température ambiante, suivi de deux rinçages dans 300 microlitres de citrate de sodium salin. Puis fixer les échantillons pendant la nuit dans 300 microlitres de 50% formamide en citrate de sodium salin 2X à température ambiante. Pour la fixation, le pré-traitement et la perméabilisation de grandes cellules avec une grande quantité de cytoplasme, fixer, pré-traiter et perméabiliser les cellules de la même manière que démontré pour les cellules T primaires humaines et traiter les échantillons avec 300 microlitres de 0,0025% pepsine en 0,01 acide chlorhydrique normal pendant quelques secondes jusqu’à cinq minutes.Observez les cellules au microscope optique et arrêtez la réaction avec deux lavages de cinq minutes dans 300 microlitres de chlorure de magnésium de 50 millimlaires dès que les noyaux sont exempts du cytoplasme, mais les noyaux sont encore visibles et intacts. Pour les POISSONS d’ADN multicolores 3D, dénominatez les sondes d’hybridation à 80 degrés Celsius pendant cinq minutes et placez immédiatement les sondes sur la glace. Chargez les sondes sur une lame de microscope propre et placez un coverslip des cellules sur chaque goutte de sonde.Sceller les bords de coverslip avec du ciment en caoutchouc. Lorsque le ciment a séché complètement, déliaturez les échantillons sur un bloc chauffant de 75 degrés Celsius. Après quatre minutes, hydrater les échantillons à 37 degrés Celsius pendant la nuit dans une boîte métallique flottant dans un bain d’eau.Le lendemain matin, peler le ciment en caoutchouc et avec les toboggans immergés dans 2X citrate de sodium salin soigneusement décoller les coverslips. Placez chaque coverslip dans des puits individuels d’une assiette de six puits contenant deux millilitres de citrate de sodium salin frais par puits pendant deux lavages de cinq minutes à 37 degrés Celsius avec des secousses à 90 révolutions par minute. À la fin de l’incubation, rincer brièvement les coverslips en deux millilitres de 0,2 %Tween 20 dans le citrate de sodium salin 4X.Pour la détection 3D de poissons d’ADN multicolores, transférez les coverslips dans les puits individuels d’une nouvelle plaque de 24 puits et bloquez toute liaison non spécifique en 300 microlitres de tampon de blocage pendant 20 minutes à 37 degrés Celsius et 20 révolutions par minute. À la fin de l’incubation, traiter les échantillons avec la concentration appropriée d’anti-digoxigenin et/ou de streptavidine diluée dans le tampon bloquant pendant 35 minutes dans une chambre sombre et humide à 37 degrés Celsius. À la fin de l’incubation, transférer les échantillons dans des puits individuels d’une assiette de six puits et laver les échantillons trois fois en deux millilitres de 0,2 % de citrate de sodium salin 20 et 4X pendant cinq minutes par lavage avec secousses à 90 révolutions par minute.Après le dernier lavage, équilibrer les échantillons en deux millilitres de PBS avant de transférer les coverslips dans une nouvelle plaque de 24 puits pour la post-fixation dans 300 microlitres de 2% de formaldéhyde en PBS par puits pendant deux minutes à température ambiante. Lavez les cellules fixes cinq fois brièvement dans 300 microlitres de PBS, suivis de la coloration avec DAPI diluée à un nanogramme par millilitre dans 300 microlitres de PBS pendant cinq minutes à température ambiante. Lavez les coverslips cinq fois de plus dans PBS et montez les échantillons avec un support de montage approprié.Puis acquérir des images 3D avec un système de microscope. 3D multicolore DNA FISH permet la visualisation contemporaine de différents loci génomiques au sein de noyaux 3D préservés. Pour la préparation de la sonde d’ADN, une sonde de moins de 200 paires de base assure une procédure fish 3D multicolore réussie.Les sondes FISH d’ADN sous-optimales produites par la traduction par entaille peuvent être partiellement ou trop digérées. Des sondes surdé digérées entraîneront un signal non spécifique en raison d’une perte de spécificité dans l’hybridation et d’une augmentation conséquente de l’arrière-plan. Pour les lymphocytes T primaires humains et les petites cellules avec de petits noyaux et une faible quantité de cytoplasme, un traitement hydrochlorique normal d’acide chlorhydrique de 12 minutes 0,1 est recommandé pour favoriser l’accessibilité des noyaux aux sondes d’ADN et pour préserver l’intégrité nucléaire.La digestion cytoplasmique de pepsine n’est pas nécessaire pour obtenir un bon signal de FISH d’ADN dans de petites cellules. Pour les myoblastes primaires humains et les cellules qui ont de grands noyaux et cytoplasme abondant, cependant, l’étape de pepsinisation est fondamentale. Une pepsinisation courte et sous-optimale du cytosquelette entravera l’entrée de la sonde dans les noyaux se terminant en l’absence d’un signal FISH ADN.Cependant, si les cellules sont sur pepsinisées, les noyaux ne resteront pas intacts en perdant leur structure 3D. Un FISH d’ADN multicolore 3D réussi se traduira par la présence de signal dans les noyaux. Je suggère de travailler avec du matériel biologique frais, de tester des sondes avant d’effectuer le FISH d’ADN multicolore 3D, de préparer des solutions fraîches et d’être précis avec les temps d’incubation et les températures.N’oubliez pas que le formamide et le HCL sont des substances dangereuses et doivent toujours être utilisés dans un capot de fumée chimique avec des matériaux jetables appropriés.

3d-multicolor-dna-fish-tool-to-study-nuclear-architecture-human

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Mesure parallèle d&#39;expression Circadian Clock génique et la sécrétion d&#39;hormones dans des cultures de cellules humaines primaires

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Recherche

Génétique

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Facteurs artificiels artificiels pour manipuler spécifiquement l&#39;épissage alternatif dans les cellules humaines

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Laser Microirradiation à l’étude In Vivo des réponses cellulaires aux dommages d’ADN simples et complexes

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

Immunostaining des Modifications de l’ADN : Computational Analysis of Images confocales

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

ATAC-seq test ADN Mitochondrial faible contamination de primaires humaines CD4 + Lymphocytes T

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

Écrans un hybride levure améliorée afin d’identifier le facteur de Transcription lie à des séquences de l’ADN humain

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Multi-locus Nombre variable Tandem-répétition Analyse du Bacterium Yersinia ruckeri par Multiplex PCR et Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Naviguer MARRVEL, un outil Web qui intègre la génomique humaine et l'information génétique des organismes modèles

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Étude fonctionnelle In Vivo des variantes humaines rares associées à la maladie utilisant la drosophile

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Caractérisation de la différenciation in vitro des kératinocytes primaires humains par analyse ARN-Seq

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...