Ce travail a été financé par des subventions de recherche des NIH K01AR053210, R01AR061484 et une subvention du National Psoriasis Foundation.

1. Informations générales Les deux hybrides de levure système est une technique puissante génétique utilisé pour découvrir interactions protéine-protéine 16, 17. Plusieurs types de deux systèmes hybrides, tels que les systèmes basés sur lexA, le système de recrutement Sos, et de bactéries ou de mammifères basés sur les cellules 2-hybrides, sont disponibles dans le commerce, ce document se concentre spécifiquement sur les modifications de la plus couramment utilisée basée GAL4 levure 2-hybride. Brièvement, la méthode est basée sur les propriétés de la protéine GAL4 de la levure qui se compose des domaines séparables responsable de l&#39;activation liaison à l&#39;ADN et la transcription. La protéine appât est exprimé comme une fusion de la liaison à l&#39;ADN GAL4 de domaine (ADN-BD), tandis que les protéines proies sont exprimés sous forme fusionnée avec le domaine d&#39;activation GAL4 (AD). L&#39;interaction entre l&#39;appât et la proie des protéines de fusion conduit à des activations de la transcription de GAL4 sites de liaison contenant des gènes rapporteurs qui sont intégrés dans le g de levureenome. Le principe de Y2H est illustré dans la Fig. 1 et la procédure expérimentale est résumée dans la Fig. 2. 2. Matières obligatoires et des solutions <ol><li> Milieu de croissance YPD (un mélange de peptone, extrait de levure et dextrose dans des proportions optimales pour la culture la plupart des souches de Saccharomyces cerevisiae). </li><li> Minimal Bases SD (Minimal synthétiques définis (SD) des bases comprennent une base azotée de levure, sulfate d&#39;ammonium, et une source de carbone, le dextrose. Dropout (DO) des suppléments peuvent être ajoutés à la base minimale SD pour faire une synthèse, un milieu défini manquent l&#39;spécifiée nutriments). </li><li> Leu /-Trp Dropout (DO) supplément (contenant tous les acides aminés essentiels, sauf pour la leucine et de tryptophane) </li><li> Dropout -His/-Leu/-Trp/-Ura (NE) Supplément (contenant tous les acides aminés essentiels, sauf pour la leucine, le tryptophane, l&#39;histidine et uracile) </li><li> Luria Broth (LB) (tryptone 10 g / L, extrait de levure 5 g / L, NaCl 5 g / L) </li><li> X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) solution: préparer un 20 mg / ml de solution concentrée dans le N, N-Diméthylformamide </li><li> 10X TE (100 mM Tris-HCl (pH 7,5), EDTA 10 mM, à l&#39;autoclave) </li><li> Hareng soniqué ou de l&#39;ADN de sperme de saumon, cuit (10 mg / ml) </li><li> LiAc 10X (1 M d&#39;acétate de lithium, à l&#39;autoclave) </li><li> 50% de PEG-3350 solution, stérilisée par filtration </li><li> 3-amino-1, 2, 4-triazole (3AT) </li><li> Kanamycine </li><li> Ampicilline </li><li> ElectroMax cellules DH5a </li><li> Z buffer: 16,1 g de Na2HPO4 7H2O (ou 8,52 g anhydre), 5,5 g de NaH2PO4 H2O (ou 4,8 ganhydrous), 0,75 g de KCl, 0,246 g MgSO4 7H2O (ou 0,12 g anhydre), dissous dans 1 L autoclave, l&#39;eau distillée et ajustée à pH 7,0. </li></ol> 3. La génération d&#39;appâts (pDBLeu-PGRN) Un fragment d&#39;ADNc codant PGRN manque peptide signal (aa21-588) a été cloné dans le directionnellement Sal I-Non, je les sites du vecteur pDBLeu (le ProQuest système double-hybride, Invitrogen),garder la frame même traduction lecture comme domaine liant l&#39;ADN GAL4 pour générer pDBLeu-PGRN. <ol><li> Amplifier le fragment d&#39;ADNc de PGRN décrit ci-dessus par PCR utilisant des amorces oligonucléotidiques conçues pour contenir des sites de restriction (Sal I à l&#39;extrémité 5 et Not I à l&#39;extrémité 3) pour permettre au châssis de fusion. </li><li> Gel purifier le produit de PCR, digestion avec des endonucléases de restriction Sal I et Not I. Préparer la DB vectorielle pDBLeu par digestion de restriction double avec les endonucléases de restriction même. </li><li> Ligaturer les fragments de restriction dans PGRN le vecteur linéarisé pDBLeu et se transforment en DH5a avec sélection pour LB + kanamycine à 25 pg / ml. </li><li> Vérifiez la correction de la construction par séquençage d&#39;ADN. </li></ol> 4. La transformation à petite échelle de l&#39;appât plasmidique <ol><li> Inoculer 5 ml de YPD avec une colonie de Mav203 (Invitrogen), agiter une nuit à 30 ° C. </li><li> Diluer la culture durant la nuit dans 50 ml de YPD. Cultivez un complémentitional 2-4 heures. </li><li> Pellet les cellules à 3000 rpm pendant 5 min à température ambiante. Reprendre le culot dans 40 ml d&#39;autoclave, de l&#39;eau distillée. </li><li> Re-culot cellulaire. Remettre en suspension dans 2 ml de solution I, incuber à température ambiante 10 min. Solution I: 0,5 ml 10xLiAc, 0,5 ml 10xTE, 4 ml de H 2 O </li><li> Distribuer de l&#39;ADN à des tubes: 2-3 pl d&#39;pDBLeu-PGRN (0.25μg/μl) et 10 pi de dénaturé, d&#39;ADN de sperme de saumon cisaillé (10μg/μl). Ajouter 100 ul de cellules de levure, bien mélanger. </li><li> Ajouter la solution 700μl II, bien mélanger. Incuber à 30 ° C pendant 30 min. Solution II: 0,2 ml 10xLiAc, 0,2 ml 10xTE, 1,6 ml de 50% PEG3350. </li><li> Choc thermique à 42 ° C pendant 15 min. </li><li> Pellet pendant 2 min. Jeter le surnageant. Reprendre le culot dans 200 pi autoclave, l&#39;eau distillée. </li><li> Plaque de la suspension sur SD-Leu plaques avec des dilutions successives. Incuber la plaque à 30 ° C pendant 2-3 jours. </li></ol> 5. Validation des appâts Avant d&#39;effectuer yeal&#39;écran er de double-hybride, le test pDBLeu-PGRN pour l&#39;auto-activation et de déterminer les niveaux d&#39;expression basale du gène HIS3 journaliste. Ce test détermine si appâts activer la transcription et si l&#39;auto-activation peut être neutralisé par des inhibiteurs. 3-amino-1, 2, 4-triazole (3-AT) est un inhibiteur compétitif du produit HIS3-gène et peut être utilisé pour titrer le niveau minimum de HIS3 d&#39;expression nécessaires à la croissance sur l&#39;histidine déficientes médias. <ol><li> Transformer le pDBLeu-PGRN dans la levure Mav203 souche, plaque de la transformation sur SD-Leu plaques et incuber pendant 48-72 h à 30 ° C. </li><li> Patch colonies de chaque transformation à l&#39;aide de cure-dents autoclavés sur SD-Leu-His plaques contenant 3-AT à des concentrations de 10 mm, 25 mm, 50 mm, 75 mm et 100 mm. 3-AT est un inhibiteur compétitif de l&#39;enzyme HIS3 et appâts qui présentent uniquement l&#39;auto-activation sera capable de croître dans la présence du 3-AT. </li><li> Souches appât qui poussent sur des plaques containing 100 mM 3-AT ne sont pas adaptés pour une utilisation dans l&#39;écran du double hybride. Pour appâts qui peuvent être utilisées, l&#39;utilisation la plus faible concentration en 3-AT qui inhibe la croissance cellulaire. Dans de nombreux cas, 25 mM de 3-AT est utilisé pour le dépistage. </li></ol> 6. criblage de banques d&#39;ADNc Le plasmide pDBLeu-PGRN est introduit dans MaV203 utilisant une transformation à petite échelle comme décrit ci-dessus. Pour introduire pPC86-bibliothèque (Invitrogen) dans MaV203 (pDBLeu-PGRN), la procédure décrite ci-dessous donne généralement ~ 4 x 10 4 colonies avec 0,5 pg d&#39;ADN plasmidique bibliothèque. Ainsi, 2,5 x 10 6 transformants de levure, il faudra ~ 30,0 mg pPC86-ADNc ADN plasmidique bibliothèque, 25 transformations, et cinquante plaques de 10 cm (SD-Leu-Trp-His-Ura 3 AT). <ol><li> Préparer le nombre approprié de 10 cm de SD-Leu-Trp-His-Ura 3 AT plaques (50 pour la procédure ci-dessous). Également préparer au moins quatre de 10 cm SD-Leu-Trp plaques pour estimer le nombre de transformants. </li><li> Transformez la bibliothèque dansl&#39;MaV203 contenant pDBLeu-PGRN selon la procédure décrite ci-dessous. <ol><li> Suspendre plusieurs colonies isolées de MaV203 (pDBLeu-PGRN) dans ~ 100 ul autoclave, l&#39;eau distillée et de les étaler sur une période de 10 cm SD-Leu assiette. Répétez la procédure pour un deuxième DD-Leu assiette. Incuber les deux plaques pendant 18-24 h à 30 ° C. </li><li> Grattez et complètement cellules en suspension dans 10 ml autoclave, l&#39;eau distillée. Ajouter un volume suffisant de cellules en suspension dans 500 ml de milieu YPD liquide dans un flacon de donner une DO 600 de 0,1 ~. </li><li> Vérifiez que la DO est ~ 0,1 après l&#39;inoculation. </li><li> Agiter à 30 ° C jusqu&#39;à une DO 600 atteigne environ 0,4. </li><li> Préparer une solution fraîche: <ol type="a"><li> 110 ml 1X TE / LiAc en combinant 11 ml 10X TE, 11 ml LiAc 10X, et 88 ml d&#39;eau en autoclave. </li><li> 16 ml de PEG / LiAc en combinant 1,6 ml 10X TE, 1,6 ml LiAc 10X, et de 12,8 ml à 50% de PEG-3350. </li></ol></li><li> Pellet les cellules à 3000g pendant 5 min à température ambiante. </li><li> Jeter tIl surnageants et doucement resuspendre le culot par pipetage haut et en bas de 100 ml en autoclave, de l&#39;eau distillée à température ambiante. </li><li> Centrifuger à 3000 g pendant 5 min à température ambiante. </li><li> Jeter le surnageant des cellules centrifugé et remettre le culot cellulaire dans 50 ml 1X TE / LiAc solution. </li><li> Centrifuger à 3000 g pendant 5 min à température ambiante. </li><li> Retirer les surnageants et remettre le culot dans un volume final de 2,5 ml 1X TE / LiAc solution. </li><li> Effectuer 25 transformations. Combinez 2,5 ml de cellules, 125 pi (10μg/μl) dénaturé, cisaillé d&#39;ADN de sperme de saumon, et 30 mg de bibliothèques d&#39;ADNc. Mélanger doucement par aspiration et refoulement. Ajouter 15 ml de PEG / LiAc solution et mélanger doucement. Aliquoter en 25 autoclave microtubes de 1,5 ml de 700 ul chacun. </li><li> Incuber pendant 30 min dans un bain d&#39;eau à 30 ° C. </li><li> Choc thermique pendant 15 min dans un bain d&#39;eau à 42 ° C. </li><li> Centrifuger à 6000 g pendant 1 min à température ambiante. Retirez délicatement le supersurnageant. Doucement resuspendre chaque culot dans 400 pi autoclave, l&#39;eau distillée par aspiration et refoulement. </li></ol></li><li> Plaque de transformation du mélange 200 pi de chaque transformation sur simple de 10 cm SD-Leu-Trp-His-Ura 3 AT (3AT de concentration: 25 mm) des plaques en utilisant une barre de propagation, donc 25 autoclave de 1,5 ml microtubes peut faire 50 plaques (SD-Leu-Trp-His-Ura 3 AT). </li><li> Incuber les boîtes pendant 5-10 jours à 30 ° C. </li><li> Pour estimer l&#39;efficacité de transformation de la réaction, des dilutions en série d&#39;une réaction (1:40; 1:400 et 1:4000) sont plaquées sur le DD-Leu-Trp assiette. Après une incubation de 3 jours à 30 ° C, le nombre de colonies est compté et le nombre total de transformants calculé. </li></ol> 7. X-gal test <ol><li> Transfert des colonies sur la plaque YPD; incuber à 30 ° C pendant la nuit. </li><li> Placez arrondis membrane de nitrocellulose sur la plaque YPD; s&#39;assurer qu&#39;il n&#39;y ait pas de bulles entre la membrane et la plaque YPD. Après 1-2 min, MAke vous que toutes les colonies sont transférées sur des membranes (papier de nitrocellulose). </li><li> Placez le côté colonie de la membrane dans l&#39;embarcation faite avec du papier aluminium. </li><li> Mettre la membrane dans l&#39;azote liquide, attendre vingt secondes, et plonger dans l&#39;azote liquide pour un couple de minutes. </li><li> Sortez la membrane à dégeler. </li><li> Pendant ce temps mélanger les réactifs suivants: 1.5ml de tampon Z 20 pi X-gal (20 mg / ml) </li><li> Déposer Z buffer / X-gal dans une boîte de Pétri, placer du papier Whatman sur le dessus de cela. </li><li> Placez délicatement le papier de nitrocellulose sur du papier Whatman. </li><li> Incuber à 37 ° jusqu&#39;à ce que les colonies bleues apparaissent. </li></ol> 8. Dosage de retransformation Protéines de fusion Prey (AD-Y) isolée de criblage de la banque devrait conserver l&#39;interaction avec la protéine de fusion d&#39;appât (DB-X) pour induire les gènes rapport, et la retransformation des clones proie et l&#39;appât de construire dans la levure peut encore éliminer les faux positifs et de faciliter ajoutezAnalyse itional. <ol><li> Isoler l&#39;ADN plasmidique à partir de souches de levures contenant potentiellement des protéines qui interagissent. </li><li> Transformez l&#39;ADN dans E. coli DH5a cellules, plaque les transformations sur LB 100 pg / ml d&#39;ampicilline plaques pour isoler sélectivement la bibliothèque pPC86-ADNc, et incuber une nuit à 37 ° C. </li><li> Choisissez plusieurs colonies de la plaque pour mettre en LB 100 pg / ml d&#39;ampicilline bouillon. </li><li> Préparer l&#39;ADN miniprep et examiner par analyse de restriction doubles avec Sal I et Not I. </li><li> Co-transformer les proies plasmide et appâts plasmide dans MaV203, plaque les mélanges de transformation sur SC-Leu-Trp plaques et incuber pendant 2-3 jours à 30 ° C. </li><li> Choisissez trois différentes colonies de la plaque SC-Leu-Trp, exécuter X-gal Assay. </li></ol> 9. Séquençage et analyse bioinformatique Séquence de l&#39;ADN plasmidique a été isolé à partir probablement vrai clones positifs, comparer ces séquences à celles de la GenBank en utilisant la BLAProgramme ST, et d&#39;identifier les clones à deux hybrides qui correspondent à des gènes connus. Les données de séquençage a montré que deux des 12 positifs obtenus ci-dessus ont été la surface des cellules TNFR2 (TNFRSF1B/CD120b; Adhésion # NM_130426). En outre, l&#39;interaction entre PGRN et TNFR a été vérifiée à l&#39;aide de divers tests d&#39;interaction protéine-protéine, y compris in vitro, en phase solide test de liaison, de co-immunoprécipitation, résonance plasmonique de surface d&#39;analyse et de test de cytométrie en flux 14. 10. Les résultats représentatifs L&#39;organigramme du dépistage est indiqué dans la Fig. 3. Typiquement 50-100 candidats clone positif sera obtenu à cette étape. Nous avons d&#39;abord isolées 54 candidats clone positif parmi les 2,5 millions de transformants sélectionnés avec l&#39;appât PGRN. Candidats clone positif ont ensuite été vérifiés en effectuant X-gal dosage. Typiquement, environ 50% de faux clones positifs sont éliminés par X-gal dosage. Nous avons obtenu 23 clones positifs candides<html> Ates à cette étape pour l&#39;appât PGRN (fig. 4). Retransformation des clones proies et de construire des appâts dans la levure encore éliminer les faux positifs et les clones qui continuent d&#39;activer les gènes rapporteurs susceptibles de représenter l&#39;vrais positifs. Typiquement, environ 50% des candidats clone positif sera supprimé. Nous avons finalement isolé 12 clones positifs qui interagissent avec PGRN dans la levure. <img alt="Figure 1" src="/files/ftp_upload/3562/3562fig1.jpg" /> Figure 1. Principe de la levure système double-hybride <img alt="Figure 2" src="/files/ftp_upload/3562/3562fig2.jpg" /> Figure 2. Pipeline d&#39;identifier des partenaires de liaison aux protéines en utilisant des levures système double-hybride <img alt="Figure 3" src="/files/ftp_upload/3562/3562fig3.jpg" /> Figure 3. Organigramme de la levure de dépistage double-hybride en bibliothèque.rge.jpg &quot;target =&quot; _blank &quot;&gt; Cliquez ici pour voir une version pleine grandeur de cette image. <img alt="Figure 4" src="/files/ftp_upload/3562/3562fig4.jpg" /> Figure 4. Bêta-galactosidase dosage des candidats clone positif. Un des clones, obtenus à partir de Positive écran de la bibliothèque ont été transférés à la plaque YPD et incubées à 30 ° C pendant la nuit; B, toutes les colonies sur la plaque YPD ont été transférés à des membranes de nitrocellulose et de la bêta-galactosidase essai effectué.

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Pas de conflits d&#39;intérêt déclarés.

Deux hybrides de levure de dépistage s&#39;est avéré être un outil efficace dans l&#39;identification des interactions protéine 16, 17. Comparé à d&#39;autres approches pour identifier les protéines liant les partenaires, tels que biochimiques co-purification de protéines et de puces, deux hybrides de levure système est une approche sensible génétiques qui peuvent être utilisés pour le dépistage très grand nombre de séquences codantes dans une expérience relativement simple, dans De plus, il...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td></tr><tr><td> ProQuest système double-hybride </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> Milieu de croissance YPD </td><td> Clontech </td><td> 630409 </td></tr><tr><td> Agar YPD </td><td> Clontech </td><td> 630410 </td></tr><tr><td> Minimal SD Base de gélose </td><td> Clontech </td><td> 630412 </td></tr><tr><td> DO-Leu Supplément </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp NE Supplément </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura NE Supplément </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-amino-1 ,2,4-triazole (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> Luria Broth (LB) </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> X-Gal </td><td> Invitrogen </td><td> 15520-034 </td></tr><tr><td> Soniqué l&#39;ADN de sperme de saumon </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> Ampicilline </td><td> AMRESCO </td><td> 0339 </td></tr><tr><td> Sulfate de kanamycine </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> Efficacité sous-clonage ™ ™ DH5a cellules compétentes </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ®, la base </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> L&#39;acétate de lithium </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Acide éthylènediaminetétraacétique </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Membrane de nitrocellulose </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10 cm de boîte de Pétri </td><td> ITI scientifique </td><td> CT-903 </td></tr><tr><td> Incubateur (30 ° C) </td><td> ATR (Ecotron) </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranuline (PGRN), aussi connu comme granulin epithelin précurseurs (GEP), est un facteur de croissance 593 acides aminés autocrine. PGRN est connue pour jouer un rôle critique dans une variété de processus physiologiques et les maladies, y compris l&#39;embryogenèse précoce, une cicatrisation, l&#39;inflammation 2, 3 et 4 défense de l&#39;hôte. PGRN fonctionne également comme un facteur neurotrophique 5, et des mutations dans le gène PGRN résultant en une perte partielle de la démence frontotemporale PGRN provoquer des protéines 6, 7. Nos études récentes ont conduit à l&#39;isolement du PGRN comme un régulateur important du développement du cartilage et de la dégradation 8-11. Bien PGRN, découvert près de deux décennies auparavant, joue un rôle crucial dans de multiples conditions physiologiques et pathologiques, les efforts pour exploiter les actions du PGRN et de comprendre les mécanismes impliqués ont été considérablement entravés par notre incapacité à identifier ses liaison au récepteur (s). Pour répondre à cette question, nous avons développé une modiFied deux hybrides de levure (MY2H) approche fondée sur le plus couramment utilisé GAL4 base 2-hybride. Comparé avec les deux hybrides de levure écran conventionnel, MY2H raccourcit considérablement le processus de l&#39;écran et réduit le nombre de faux clones positifs. En outre, cette approche est reproductible et fiable, et nous avons utilisé avec succès ce système à isoler les protéines de liaison d&#39;appâts différents, y compris les canaux ioniques 12, protéine de la matrice extracellulaire 10, 13 et 14 du facteur de croissance. Dans cet article, nous décrivons cette procédure expérimentale MY2H en détail en utilisant PGRN comme un exemple qui a conduit à l&#39;identification de TNFR2 que le premier connu PGRN-récepteur associé 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Nous avons modifié la levure classique double-hybride en dépistage, un outil efficace dans l&#39;identification génétique d&#39;interactions protéiques. Cette modification raccourcit nettement le processus, réduit la charge de travail, et surtout, réduit le nombre de faux positifs. En outre, cette approche est reproductible et fiable.

Mise à jour de levure-système à deux hybrides pour identifier les protéines qui interagissent avec le facteur de croissance progranuline

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

modified-yeast-two-hybrid-system-to-identify-proteins-interacting

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JoVE Journal

Biology

28.6K vues.  NYU Hospital for Joint Diseases. Nous avons modifié la levure classique double-hybride en dépistage, un outil efficace dans l'identification génétique d'interactions protéiques. Cette modification raccourcit nettement le processus, réduit la charge de travail, et surtout, réduit le nombre de faux positifs. En outre, cette approche est reproductible et fiable.

Vidéo: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Facteur-Coated Croissance Placement billes sur des explants du cerveau antérieur dorsal

Recherche

Biologie

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Placer du facteur de croissance des billes recouvertes sur les premiers embryons de poulet scène

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Préparer T facteur de croissance cellulaire à partir de splénocytes de rat

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Protéomique pour identifier les protéines interagissant avec P2X2 canaux cationiques ligand-dépendants

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-ubiquitine membrane à base de levure double hybride (MYTH) Système: Un outil puissant pour identifier les interactions entre protéines

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - Une méthode pour identifier transcriptome à l&#39;échelle des sites de liaison des protéines liant l&#39;ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Dépistage ARNi pour identifier les phénotypes postembryonnaires dans C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Inverser levure système à deux hybrides pour identifier les mammifères Nuclear Receptor résidus qui interagissent avec des ligands et / ou antagonistes

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captation composé spectrométrie de masse - un outil puissant pour identifier de nouveaux c-di-GMP protéines effectrices

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis comme un modèle pour identifier traduction Dépréciation

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...