L&#39;original GFP-Pem1 était un don du Dr. Li Lei. Ce travail a été soutenu par des subventions du NIH et le Ellison Medical Foundation à VG et AS

Dans ce protocole, nous décrivons la méthode pour l&#39;analyse de l&#39;ADN ORD réparation avec le journaliste chromosomique intégrés construit 1,2, où CDB sont induites in vivo par expression transitoire d&#39;une endonucléase de coupe rares I-Scel 3. Le test intégré offre l&#39;avantage de l&#39;analyse de réparation des CDB dans le contexte chromosomique. Cependant, ce protocole nécessite cellulaire prolongée repiquage, ce qui peut être problématique lorsque l&#39;on travaille avec des cellules primaires. Une approche alternative est un test où les constructions rapporteurs extrachromosomique sont digérées in vitro avec I-Scel ou endonucléases HindIII et ensuite transfectés dans des cellules sous forme d&#39;ADN linéaire. Le protocole de dosage exctrachromosomal 4 est similaire à l&#39;essai décrit ci-dessous intégrées avec les modifications suivantes. Pour le dosage extrachromosomique omettre l&#39;intégration des constructions rapporteurs et au lieu de co-transfecter des cellules avec 0,5 ug de plasmide rapporteur linéarisé NHEJ in vitro avec I-Scel ou HindIII et 0,1 ug de pDsRed2-N1 que le contrôle de la transfection. Pour le dosage de la co-transfecter HR 2 HR Reporter ug de plasmide linéarisé avec I-Scel ou HindIII et 0,1 pg pDsRed2-N1. Analyser les cellules par FACS, trois jours après la transfection. Le test extrachromosomique permet l&#39;analyse de la réparation ORD dans des isolats primaires, en évitant la cellule vaste repiquage, et de faciliter l&#39;analyse des lignées cellulaires multiples. 1. Construit Reporter pour NHEJ et RH La cassette journaliste NHEJ 5 contient un gène de la GFP avec une conçues intron 3 kb du gène Pem1 (GFP-Pem1). L&#39;intron Pem1 contient un exon adénoviraux flanqué par des séquences de reconnaissance pour des endonucléases HindIII et I-Scel (figure 1a) pour l&#39;induction de CDB. Les sites I-Scel sont en orientation inversée. I-Scel a une séquence de reconnaissance nonpalindromic, où deux sites inversé générer des extrémités d&#39;ADN incompatibles (figure 1c). L&#39;ADN incompatibles se termine mieux imiter la CDB d&#39;origine naturelle. La cassette intacte NHEJ est négatif comme la GFP de l&#39;exon adénoviraux perturbe l&#39;ORF GFP. Après l&#39;induction de CDB par I-Scel, l&#39;exon adénovirale est enlevé et NHEJ restaure la fonction du gène de la GFP (figure 2). La caractéristique unique de ce journaliste NHEJ est qu&#39;il peut détecter un large spectre de manifestations depuis le NHEJ intron peut tolérer les suppressions et les insertions. La cassette HR Reporter 1 (figure 1b) est également basé sur la GFP-Pem1. Dans la cassette RH, le premier exon du GFP-Pem1 contient une délétion 22 pb combiné avec l&#39;insertion de trois sites de restriction, I-SceI/HindIII/I-SceI. La suppression assure que la GFP ne peut être reconstitué par un événement NHEJ. Les deux I-Scel sites sont en orientation inversée, de sorte que I-Scel la digestion feuilles fins incompatibles (figure 1c). Le premier exemplaire de la GFP-Pem1 est suivie par une promoter-less/ATG-less premier exon et l&#39;intron de GFP-Pem1. La construction est intacte GFP-négatifs. Après induction d&#39;un ORD par I-Scel la digestion du gène de la GFP fonctionnelle est reconstitué par conversion génique intramoléculaire ou intermoléculaire entre les deux copies mutées de la première GFP-Pem1 exon. Depuis la seconde copie du gène de la GFP est manquant du premier codon ATG et le second exon, traversant ou simple-brin de recuit ne restaure pas l&#39;activité la GFP. Cette conception permet la détection exclusive de conversion génique, qui est la voie RH prédominante dans les cellules de mammifères (figure 3). 2. Intégration des constructions rapporteurs dans le génome <ol><li> Faire une préparation de haute qualité NHEJ ou reporter RH plasmide. Pour de meilleurs résultats Qiagen Endo libres Bluetooth. Mesurer la concentration et la pureté plasmidique par absorbance à 260/280 nm. </li><li> Linéariser 10 ug de plasmide rapporteur en digérant avec 50 U d&#39;Nhel dans 50 ul (volume total de la réaction) pendant 6 heures à 37 ° C incubateur (ne pas utiliser le bloc sec, pour éviter l&#39;évaporation). Purifier l&#39;ADN de la solution de digestion avec Qiagen QiaexII Kit, l&#39;ADN éluer dans 20 ul de 10 mM Tris-HCl pH 8,0. Mesurer la concentration d&#39;ADN et de la pureté par absorbance à 260/280 nm. Habituellement, vous allez perdre 20-30% de l&#39;ADN lors de la purification. Confirmer le rendement et la digestion en exécutant une petite aliquote (2 ul) sur un gel, avec un journaliste non digérées construire. La construction journaliste purifiée linéarisée peut être conservé à 20 ° C. </li><li> En préparation pour la transfection, amener les cellules à la meilleure condition de croissance. Si à partir d&#39;un flacon congelé, fractionner les cellules deux fois avant la transfection. Si à partir d&#39;une plaque de cellules confluentes, les diviser une seule fois. </li><li> Cultivez les cellules à 70-80% de confluence (généralement deux jours, si plaqués 5x10 5 cellules/100 plaque de mm, pour des fibroblastes humains normaux). Il est essentiel pour la meilleure efficacité de la transfection d&#39;apporter les cellules en croissance logarithmique. Culture en croissance active contient des cellules en phase M (considéré comme des cellules arrondies attachées à la surface). </li><li> Transfect les cellules en utilisant les recommandations du fabricant Amaxa suivantes Nucleofector est. Pour utiliser la solution de fibroblastes NHDF et U20 programme. Pour la transfection, la récolte des cellules provenant de deux plaques de 100 mm (~ 2x10 6 cellules). Il est essentiel de ne pas trop trypsiniser les cellules. Arrêtez dès que trypsinisation de 80% des cellules détachées de la plaque. Resuspendre les cellules dans une solution de NHDF. Les cellules sont sensibles à la solution de NHDF, par conséquent, de minimiser le temps d&#39;incubation et mélanger délicatement les cellules. Transfecter des cellules avec 0,5 pg de construire journaliste linéarisé. Ces conditions de transfection, lorsqu&#39;il est utilisé avec des fibroblastes humains normaux résulte de l&#39;intégration d&#39;un seul exemplaire d&#39;un journaliste de construire pour la majorité des intégrants. </li><li> Sélectionnez les cellules avec des constructions rapporteurs chromosomique intégrés en ajoutant 1 mg / mL généticine (G418) 24h après la transfection. Continuer de sélection pour les 7-10 jours. </li><li> Choisissez les colonies résistantes au G418 (vous pouvez choisir des colonies individuelles ou à la piscine les clones résistants). Développer la culture d&#39;environ cinq plaques de 100 mm. Gel de trois plaques pour une utilisation future et d&#39;élargir les deux autres plaques de la cellule numéro désiré (généralement dix plaques de 100 mm sont suffisants pendant cinq transfections). </li><li> Lorsque les cellules atteignent 70-80% de confluence (deux jours après l&#39;étalement 5x10 5 cellules par plaque de 100 mm pour les fibroblastes), les cellules sont prêtes à être transfectées avec I-Scel plasmide exprimant. </li></ol> 3. L&#39;induction de l&#39;ORD par expression transitoire d&#39;I-Scel Endonucléase <ol><li> Transfecter ~ 2x10 6 cellules (deux plateaux) de la culture logarithmique croissante avec un mélange de 5 ug I-Scel plasmide exprimant et 0,1 mg pDsRed2-N1 (Clontech) comme un contrôle de l&#39;efficacité de transfection utilisant Amaxa Nucleofector (utiliser la solution de NHDF et U20 du programme pour les fibroblastes ). </li><li> Dans le même temps de préparer trois contrôles d&#39;étalonnage pour FACS par trasfecting ~ 2x10 6 cellules avec (i) 5 ug EGFP-plasmide exprimant, (ii) 5 ug pDsRed2-N1, (iii) 5 ug d&#39;un plasmide contrôle qui n&#39;exprime pas une fluorescent protein (nous utilisons un plasmide codant HPRT minigène). </li><li> Cultiver les cellules pendant quatre jours pour donner suffisamment de temps pour l&#39;expression de la GFP et les protéines DsRed. </li><li> (Facultatif) Trois jours après la transfection de vérifier l&#39;efficacité de la transfection et l&#39;expression de la GFP et les protéines fluorescentes DsRed par microscope inversé avec des filtres pour la détection de la GFP et DsRed. </li></ol> 4. Analyse de la GFP + + et les cellules DsRed par cytométrie en flux <ol><li> Quatre jours après la transfection de récolte I-Scel cellules transfectées et les cellules de contrôle. Resuspendre les cellules dans 500 ul de PBS et de transfert de cellules pour les tubes FACS. A ce stade, il est essentiel de récolter toutes les cellules et les cellules ont une forme ronde. Pour y parvenir, il est recommandé d&#39;utiliser un plus long temps trypsinisation ou une plus forte trypsine. Confirmez que 99% des cellules sont détachés de la plaque et ont une forme ronde par l&#39;observation des cellules au microscope. Gardez les cellules sur la glace abri de la lumière (couverture seau à glace avec une feuille). Il est possible de stocker les cellules sur la glace pendant quelques heures. Pour les meilleurs résultats d&#39;analyser les cellules immédiatement après la récolte. </li><li> Calibrer FACS avec la GFP, DsRed, et les contrôles négatifs. Ajustez la tension et compensation de couleur afin d&#39;inclure toutes les cellules fluorescentes dans l&#39;analyse (figure 4). Gardez à l&#39;esprit que l&#39;intensité de fluorescence des échantillons expérimentaux seront plus bas que celui des contrôles. </li><li> Analyser I-Scel cellules transfectées par FACS. Nous comptons au moins 20.000 cellules pour chaque traitement. Pour éviter les interférences possibles entre la GFP et DsRed de fluorescence, il est recommandé de garder le pour cent de la GFP + ou + DsRed cellules en dessous de 25%. Si le pourcentage de + DsRed ou GFP + cellules est supérieure réduire la quantité de pDsRed2-N1 ou I-Scel plasmide. Dans nos expériences nous avons seulement besoin d&#39;ajuster la quantité de pDsRed2-N1 plasmide, mais pas du plasmide I-Scel. </li><li> Le pour cent des cellules GFP + correspond à l&#39;efficacité de l&#39;ADN et la réparation de l&#39;ORD pour cent des cellules + DsRed indique l&#39;efficacité de la transfection. Calculer l&#39;efficacité relative de l&#39;ADN ORD réparation comme le ratio de cellules GFP + + aux cellules DsRed. </li><li> Les jonctions fin réparées peuvent être séquencés pour tester l&#39;exactitude de réparation. Les constructions rapporteurs contiennent l&#39;origine de réplication et de E. coli des gènes de résistance à la kanamycine (figure 1). Cela permet le sauvetage des constructions en digérant l&#39;ADN génomique avec enzyme EcoRI, recircularisation, et leur transformation en cellules E. coli compétentes. Les plasmides secourus sont ensuite analysés par digestion de restriction et séquençage. </li></ol> 5. Les résultats représentatifs Les résultats typiques de FACS des transfections de contrôle et de la NHEJ et expériences RH sont illustrés aux figures 4 et 5. Intensité de fluorescence et le nombre de cellules fluorescentes sera plus faible dans les cellules transfectées I-Scel contenant intéed NHEJ et construit RH (figure 5) par rapport aux témoins (figure 4) en raison de la différence dans le nombre de copies de la GFP et les constructions DsRed dans ces cellules. Chaque cellule de l&#39;expérience ne contient qu&#39;un seul exemplaire de la construction rapporteur intégré, les événements de réparation ainsi retenus seront reconstituer le gène de la GFP dans une fraction des cellules. Transfection avec 0,1 pg pDsRed2-N1 dans des fibroblastes humains délivre environ 1 copies du plasmide par cellule. L&#39;efficacité de la NHEJ et RH est calculé comme le rapport de la GFP + / DsRed + cellules. NHEJ est un processus plus efficace que dans les cellules humaines RH 2. Dans les fibroblastes humains de l&#39;efficacité est généralement de 0,6 à 1,3 NHEJ, et l&#39;efficacité des ressources humaines est de 0,05 à 0,3. Depuis l&#39;efficacité ORD est mesurée comme un rapport de la GFP + / DsRed + variations des cellules dans le mélange de I-Scel et DsRed2-N1 plasmides peuvent affecter le résultat. La qualité plasmide peut également affecter les résultats en changeant efficacité de la transfection. Par conséquent, pour obtenir des mesures cohérentes, il est important d&#39;utiliser la même combinaison au sein d&#39;un plasmide expérience. Par ailleurs, l&#39;efficacité de réparation ORD est affectée par le type cellulaire, phase du cycle cellulaire et localisation chromosomique des constructions intégrées. <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Figure 1" /> Figure 1. Reporter construit pour l&#39;analyse de l&#39;ADN ORD réparation par NHEJ (A) et HR (B). (C) l&#39;ADN incompatibles extrémités générées par la digestion de deux inversé les sites I-Scel SD, d&#39;épissage donneur; SA, accepteur d&#39;épissage; places ombragées, des sites de polyadénylation; Neo / Kana, seule ORF contrôlée par deux promoteurs:. SV40 résistance à la néomycine conférant au mammifères cellules, et β-lactamase conférant la résistance kanamicyn dans E. coli; Ori, E. L&#39;origine de réplication pUC Coli. <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Figure 2" /> Figure 2. Reporter construire pour l&#39;analyse de la NHEJ Dans cette construction du gène de la GFP est inactif;. Mais lors de l&#39;induction d&#39;une NHEJ ORD et réussi la construction devient GFP +. Ci-dessous est un produit de réparation de ce journaliste par NHEJ. <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Figure 3" /> Figure 3. Reporter de construire pour l&#39;analyse des RH par conversion génique. Après l&#39;induction de la CDB par I-Scel, RH par le gène de conversion (CG) reconstitue un gène GFP active. Ci-dessous sont des produits de réparation de ce journaliste par les principales voies de réparation d&#39;ADN: GC, crossing-over (X-over), seul brin de recuit (ASS), et NHEJ. Produit de réparation X-over est montrée pour la recombinaison intramoléculaire, la recombinaison exramolecular donnera le même produit, mais situé sur un chromosome. Des gènes exprimant une protéine GFP active (GFP +) sont indiqués par la couleur verte. <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Figure 4" /> Figure 4. Calibrage des paramètres pour l&#39;analyse FACS (A). Fibroblastes transfectés avec le plasmide pHPRT, utilisées comme contrôle négatif de l&#39;exclusion automatique des cellules fluorescentes. (B) fibroblastes transfectés avec le plasmide pEGFP, utilisé pour le réglage du gating pour les cellules GFP + (zone verte). (C) fibroblastes transfectés avec le plasmide pDsRed2-N1, utilisé pour le réglage du gating pour les cellules + DsRed (zone rouge). <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="Figure 5" /> Figure 5. Les résultats typiques de l&#39;analyse de NHEJ (A) et RH (B) dans des fibroblastes humains normaux avec des constructions rapporteurs chromosomique intégré.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

Les fluorescents NHEJ et analyses HR Reporter fournir un moyen quantitatif pour mesurer séparément chaque voie de réparation ORD in vivo. Les tests sont très sensibles, comme FACS peut détecter de façon fiable 10 cellules GFP + dans 20.000 cellules. Les dosages peuvent être adaptés pour mesurer des événements de réparation en &quot;temps réel&quot; en détectant l&#39;apparition de cellules GFP + dans les minutes ou heures après l&#39;induction de CDB 2. Par ailleurs, l&#39;analyse de ce...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
</tr>		</tbody>
		</table>
		</div>

analysis of dna double-strand break (dsb) repair in mammalian cells

L&#39;ADN des cassures double brin sont les lésions de l&#39;ADN le plus dangereux qui peuvent conduire à une perte massive de l&#39;information génétique et la mort cellulaire. CDB à réparer les cellules en utilisant deux voies principales: la fin non homologues (NHEJ) et la recombinaison homologue (RH). Perturbations du NHEJ et RH sont souvent associés au vieillissement prématuré et la tumorigenèse, il est donc important d&#39;avoir un moyen quantitatif de la mesure de chaque voie de réparation ORD. Notre laboratoire a développé des constructions rapporteurs fluorescents qui permettent de mesurer sensible et quantitative du NHEJ et RH. Les constructions sont basées sur un gène de la GFP conçu contenant des sites de reconnaissance pour une rare coupe I-Scel endonucléase pour l&#39;induction de CDB. Les constructions de départ sont GFP négatifs comme le gène de la GFP est inactivé par un exon supplémentaire, ou par des mutations. Réparation réussie des pauses I-Scel induite par NHEJ ou HR restaure le gène de la GFP fonctionnelle. Le nombre de cellules GFP-positives comptées par cytométrie en flux permet une mesure quantitative du NHEJ ou l&#39;efficacité des ressources humaines.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

Cet article décrit la GFP basée sur la fluorescence<em&gt; In vivo</em&gt; Dosages qui part de quantifier la recombinaison homologue et non homologue fin de rejoindre dans les cellules de mammifères.

L&#39;analyse de l&#39;ADN double-brin Break (ORD) de réparation dans les cellules de mammifères

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K vues.  University of Rochester. Cet article décrit la GFP basée sur la fluorescence In vivo Dosages qui part de quantifier la recombinaison homologue et non homologue fin de rejoindre dans les cellules de mammifères.

Vidéo: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfection d&#39;ADN des mammifères muscles squelettiques à l&#39;aide In Vivo L&#39;électroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

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Biologie

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Microinjection d&#39;ADN dans intranucléaires Dissocié neurones de mammifères adultes

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulse-chase Analyse des chaînes de sucre N-glycoprotéines liées à des cellules de mammifères

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Analyse de la cinétique ARNm exportation nucléaire dans les cellules de mammifères par micro-injection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagerie en temps réel de l&#39;unité d&#39;ingénierie des ARN transcrits dans des cellules vivantes utilisant Ratiométrique bimoléculaires Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Mesure des niveaux Heme synthèse dans des cellules de mammifères

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.