Nous sommes reconnaissants au Centre de l&#39;Ohio State University Comprehensive Cancer de fournir un financement pour ce travail.

1. Cell Line avec un substrat recombinaison intégré <ol><li> Les cellules HeLa ont été transformées de façon stable avec le PDR-GFP (plasmide 7 don de M. Jasin, Memorial Sloan-Kettering Cancer Center) et maintenu dans la puromycine pour la sélection des transformants. </li><li> Les cellules sont repiquées dans du DMEM / FBS, contenant 10% de sérum fœtal bovin, 2 mM glutamax, pyruvate de sodium 1 mM, pénicilline / streptomycine (100 UI / mL/0.1 mg / mL), et 1,5 ug / ml de puromycine. </li></ol> Le substrat de recombinaison dans le PDR-GFP plasmide, et dans des transformants stables, contient deux allèles inactifs de la GFP (figure 1). Un allèle est inactif en raison de l&#39;inclusion dans sa séquence codante du site de restriction 18 pb de reconnaissance d&#39;enzymes pour I-Scel. Expression de I-Scel dans ces cellules crée une cassure de l&#39;ADN double-brin (DSB). Il ya un deuxième allèle de la GFP inactif sur cet ADN, mais la partie de sa séquence en corrélation avec le site I-Scel sur le premier allèle est de type sauvage. Cet allèle GFP seconde peut fonctionner comme un donneur de séquence pour la réparation de recombinaison homologue de l&#39;ORD et se traduirait par un allèle GFP active, qui est facilement détectée par cytométrie en flux. Alternativement, l&#39;ORD pourrait être réparé par non homologues fin de rejoindre, ce qui détruirait le site de restriction I-Scel et se traduirait par la GFP cellules négatives. Le niveau de la recombinaison homologue dans une cellule est déterminée en comptant le pourcentage de cellules positives pour la GFP. Cette lignée cellulaire, pHeLa-DR13-9, a été sélectionné parmi tous les clones de zéro du signal GFP de fond et un pourcentage relativement élevé de la GFP de cellules positives à la transfection avec le I-Scel plasmide exprimant, pCBASce. (Le pCBASce et son contrôle des vecteurs, pCAGGS, ont tous deux été fournies par M. Jasin du Centre Memorial Sloan-Kettering Cancer.) La ligne de cellules HeLa-DR13-9 cellules est disponible auprès des auteurs sur demande. 2. La transfection du gène BRCA1 au appauvrissent endogène et Retour Ajouter BRCA1 mutant. <ol><li> Le jour avant la transfection première (généralement un mardi): Commencer avec un plat de 10 cm de cellules HeLa-DR13-9 cellules au moins 90% de confluence. </li><li> Trypsiniser dans 1 ml et arrêter la réaction en ajoutant 9 ml de DMEM / FBS, bien mélanger par pipetage. </li><li> Ajouter 40 ul de cellules trypsinisées par puits d&#39;une plaque à 24 puits dans 0,5 ml de DME / FBS. </li><li> Jour 1 transfections (mercredi): les cellules doivent être confluentes ~ 40-50%, si bien moins recommencer. Transfecter en mélangeant: 5 + 0,3 pmol siARN ug BRCA1 mutantes exprimant l&#39;ADN plasmidique de 12,5 uL Opti-MEM et 0,5 uL Lipofectamine 2000 à 12,5 uL Opti-MEM. Procéder à la transfection selon le protocole Lipofectamine (Invitrogen). </li><li> Remplacer les 4-6 heures plus tard, les médias. Le siRNA nous utilisons pour épuiser BRCA1 est basé sur la séquence spécifiant GCUCCUCUCACUCUUCAGU BRCA1 3&#39;-UTR séquences 80780 au 80798 (numéro d&#39;accession AY273801). </li><li> Jour 2: Transfert des cellules par trypsinisation de la plaque de 24 puits d&#39;une plaque à 6 puits. </li><li> Jour 3 transfections: 25 pmol siARN + 0,75 ug BRCA1 mutantes exprimant l&#39;ADN plasmidique + 0,75 pCBASce ug (ou pCAGGS lutte antivectorielle) dans 62,5 uL Opti-MEM et 2,5 uL Lipofectamine 2000 à 62,5 uL Opti-MEM. Remplacer les 4-6 heures plus tard, les médias. </li></ol> 3. L&#39;analyse de transfectants. <ol><li> Au jour 6, trypsiniser cellules de chaque puits et d&#39;arrêter dans 1 ml de DMEM / FBS. L&#39;analyse des cellules pour expression de la GFP peut être fait sur 5 ou 7 jours, mais nous constatons que 6 jours qui fonctionne le mieux. </li><li> Analyser 10.000 cellules par puits par cytométrie en flux. Nous utilisons un Becton Dickinson FACSCalibur instrument dans l&#39;Ohio State University Comprehensive Cancer Centre de cytométrie analytique ressource partagée. Simple, les cellules vivantes sont déclenchés en utilisant les paramètres de dispersion avant et latérale. GFP-positives cellules sont détectées à l&#39;aide du détecteur de fluorescence de la GFP (FL1), et les résultats sont présentés sous forme d&#39;histogramme (figure 2, en bas). </li><li> Gauche sur les cellules peuvent être réensemencées pour la photographie par microscopie à fluorescence, si désiré. </li></ol> 4. Les résultats représentatifs: BRCA1 régule la voie pour la réparation des cassures double brin par recombinaison homologue 6,11. Les cellules qui ont subi la réparation par recombinaison homologue sont facilement détectés par microscopie à fluorescence (figure 2, en haut). Épuisement des résultats BRCA1 dans une diminution de la GFP-positives détectées cellules (Figure 2, droite). La sortie de la FACSCalibur est un histogramme avec une intensité GFP par cellule sur l&#39;axe X et le nombre de cellules sur l&#39;axe Y (figure 2, en bas). Dans un ensemble typique de résultats d&#39;une expérience unique, 15,5% des cellules GFP-positives ont été, après I-Scel expression et le contrôle ARNi épuisement (figure 3). Par comparaison, l&#39;épuisement des gènes BRCA1 et vecteur de rajouter réduite GFP de conversion à 0,7%. Ajouter-arrière de type sauvage du gène BRCA1 ou du gène BRCA1 (I21V) entièrement restauré recombinaison homologue, détectée par l&#39;obtention de 15-16% de cellules positives GFP. Réintégration des gènes BRCA1 (M18T) mutant (figure 3, ligne 5) a le même effet que l&#39;add-back de la commande vectorielle. Depuis cette variante exprimée à des niveaux similaires à la protéine endogène 9, nous avons donc interpréter les gènes BRCA1 (M18T) variante est non-fonctionnel dans la recombinaison homologue. Nous avons généralement 10-20% des cellules GFP-convertir en positif après transfection l&#39;expression I-Scel plasmide. L&#39;appauvrissement de BRCA1 diminue de façon reproductible la conversion de la GFP-positives cellules par environ 8-10 fois. Bien que les pourcentages réels de la GFP-positives cellules peuvent changer d&#39;une expérience à l&#39;, le ratio de la GFP de cellules positives suite à l&#39;épuisement du gène BRCA1 par rapport à l&#39;épuisement de contrôle au sein d&#39;une expérience est tout à fait cohérente. Nous normalisons les résultats en réglant le taux de conversion désinhibé à 100% et les résultats expriment avec déplétions BRCA1 et ajoutez-dos sous forme de pourcentages. De cette façon, nous pouvons combiner de multiples expériences, et la variation expérimentale n&#39;est pas élevé. Rajouter des mutants BRCA1 ont jusqu&#39;ici donné des résultats robustes: chaque variante du gène BRCA1 est soit entièrement restauré homologues activité de recombinaison ou n&#39;a eu aucun effet. Une considération importante dans l&#39;obtention de ces résultats est que nos conditions de transfection de produire des niveaux de protéine BRCA1 qu&#39;environ égale à la concentration de la protéine endogène dans les cellules non traitées. Trop haut d&#39;une surexpression de même un mutant défectueux du gène BRCA1 peut entraîner une certaine activité de recombinaison homologue positif. Un problème que nous avions besoin pour surmonter était la toxicité de la transfection. L&#39;appauvrissement de BRCA1 se peut ralentir la croissance des cellules, et l&#39;équilibre de l&#39;ADN Lipofectamine-2000 et le plasmide par rapport au nombre de cellules sur le plat doit être maintenue à un niveau constant. L&#39;ADN Trop Lipofectamine ou plasmide réduira le nombre de cellules, et le nombre de cellules seront insuffisants pour obtenir des résultats fiables cytométrie en flux. Le solde du plasmide et réactifs Lipofectamine versus le nombre de cellules transfectées est assez important pour maintenir la viabilité des cellules. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="Figure 1" /> Figure 1. Le substrat de recombinaison. La stratégie qui avait été développé par le groupe de M. Jasin 7,12 est représenté. Le PDR-GFP plasmide contient deux allèles défectueux de la GFP, dont l&#39;un contient un site I-Scel endonucléase de restriction. Une ligne de cellules HeLa dérivé contient ce substrat ADN intégré dans un site unique dans le génome 9 et réparation active de la rupture d&#39;ADN double-brin au site I-Scel par les résultats de recombinaison homologue dans la conversion d&#39;un allèle à la GFP-positives. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="Figure 2" /> Figure 2. Détection de la GFP-positives cellules par microscopie à fluorescence. Les cellules ont été testées selon ce protocole, et le contrôle appauvri cellules ont été actifs dans la recombinaison homologue, détectée par un pourcentage élevé de cellules avec des expression de la GFP (à gauche). L&#39;appauvrissement de BRCA1 par les résultats ARNi dans une forte diminution du nombre de cellules positives pour la GFP (à droite). <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="Figure 3" /> Figure 3. Résultats d&#39;un add-back de mutants faux-sens du gène BRCA1. Les résultats d&#39;une seule expérience sont présentés dans cet histogramme. En l&#39;absence de transfectées I-Scel plasmide exprimant (piste 1) il n&#39;ya pas de cellules GFP-positives détectées. Les résultats dans les couloirs 2-6 ont toutes été prises à partir des cellules dans lesquelles l&#39;I-Scel exprimer plasmide a été transfecté le jour 3. Dans les cellules qui ont été épuisés pour un gène non pertinentes (luciférase) et avec le vecteur add-back, 15,5% des cellules étaient positives GFP (piste 2). L&#39;épuisement des gènes BRCA1 et vecteur add-back révèle l&#39;effet de la perte de BRCA1 sur la recombinaison homologue (piste 3). Les effets de l&#39;appauvrissement des gènes BRCA1 et ajoutez-arrière de type sauvage du gène BRCA1 (piste 4), BRCA1 (M18T) (piste 5), et du gène BRCA1 (I21V) (piste 6) sont présentés. Ces résultats sont issus d&#39;une seule expérience, et serait associée à au moins deux expériences se répètent plus, afin d&#39;obtenir une mesure de la recombinaison homologue soutenu par une protéine BRCA1 mutant donné.

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., 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=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., 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=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. 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L&#39;avantage de cette méthode est que nous pouvons étudier la fonction de la protéine BRCA1 dans la régulation de réparation de l&#39;ADN par recombinaison homologue en utilisant des cellules qui ont de type sauvage du gène BRCA1 endogène exprimé. Nous avons adopté deux séparés des méthodes établies de l&#39;étude des processus de recombinaison homologue et d&#39;utiliser l&#39;ARNi médiation pour obtenir la déplétion nouvelle méthode pour tester des variantes du gène BRCA1 pour la fonction de réparation de l&#39;ADN. La clé de la réussite de cette méthode a été d&#39;établir une lignée cellulaire facilement transfectables, comme les cellules HeLa, avec le substrat de recombinaison dans le génome de telle sorte que nous obtenons des niveaux très élevés de conversion GFP. Nous avions projeté de multiples clones de la transformation originale afin d&#39;obtenir un qui n&#39;avait pas de signal de fond détectable GFP, mais sur la transfection de l&#39;expression I-Scel plasmide avait un niveau très élevé de conversion GFP. Avec cette méthode, nous étudions actuellement d&#39;autres résidus spécifiques du gène BRCA1 acides aminés pour fonctionner dans la recombinaison homologue. Avec les connaissances biologiques à partir de ce travail, ces conclusions peuvent être appliquées à la génétique clinique sur le cancer. Un pourcentage élevé de mutations faux-sens du gène BRCA1 ont inconnus signification clinique car il ya beaucoup de variantes rares et les informations sont insuffisantes pour beaucoup de ces afin de déterminer l&#39;association du cancer par l&#39;analyse de ségrégation génétique 3,10,13,14. En utilisant cette méthode, les conséquences d&#39;une variante spécifique sur la fonction biologique des gènes BRCA1 dans la régulation de la recombinaison homologue pourrait être utilisée pour informer les femmes qui portent l&#39;une de ces variantes dans leur génome.

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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

L&#39;analyse fonctionnelle des mutations faux-sens peut être compliquée par la présence dans la cellule de la protéine endogène. Structure-fonction des analyses des gènes BRCA1 ont été compliquées par l&#39;absence d&#39;un test robuste pour la protéine BRCA1 pleine longueur et les difficultés inhérentes à travailler avec des lignées cellulaires qui expriment la protéine BRCA1 hypomorphes 1,2,3,4,5. Nous avons développé un système dans lequel la protéine BRCA1 endogène dans une cellule a été gravement appauvris par RNAi ciblant le 3&#39;-UTR de l&#39;ARNm du gène BRCA1 et remplacé par co-transfection d&#39;un plasmide exprimant un variant du gène BRCA1. Un avantage de cette procédure est que la réduction au silence aiguë de remplacement BRCA1 et simultanées permettent aux cellules de se développer sans des mutations secondaires ou des adaptations qui pourraient survenir au cours du temps pour compenser la perte de fonction du gène BRCA1. Cet appauvrissement et d&#39;add-back procédure a été fait dans une lignée de cellules HeLa-dérivé qui a été facilement analysés pour l&#39;activité de recombinaison homologue. Le test de recombinaison homologue est basée sur une méthode précédemment publiée par lequel un substrat de recombinaison est intégré dans le génome (figure 1) 6,7,8,9. Ce substrat recombinaison a le rare coupe le site I-Scel enzyme de restriction à l&#39;intérieur d&#39;un allèle GFP inactif, et se trouve en aval d&#39;un allèle GFP secondes inactif. La transfection du plasmide qui exprime I-Scel résultats dans une cassure double brin, qui peuvent être réparées par recombinaison homologue, et si la recombinaison homologue ne réparer la cassure il crée un allèle GFP active qui est facilement obtenu par cytométrie en flux pour l&#39;expression protéique GFP . L&#39;appauvrissement de BRCA1 endogènes conduit à une réduction de 8 à 10 fois dans l&#39;activité de recombinaison homologue, et des add-arrière de type sauvage fonction de plasmide de recombinaison homologue entièrement restauré. Lorsque les mutants point spécifique de BRCA1 pleine longueur ont été exprimés à partir de co-transfecté les plasmides, l&#39;effet du mutant faux-sens spécifique pourrait être marqué. À titre d&#39;exemple, l&#39;expression des gènes BRCA1 (M18T) des protéines, une variante de la signification clinique inconnue 10, a été exprimée dans ces cellules, il a échoué à restaurer BRCA1-dépendant recombinaison homologue. En revanche, l&#39;expression d&#39;une autre variante, également d&#39;importance inconnue, BRCA1 (I21V) entièrement restauré BRCA1-dépendante la fonction de recombinaison homologue. Cette stratégie de test de la fonction des mutations faux-sens du gène BRCA1 a été appliquée à un autre système biologique pour la fonction de titrage centrosome (Kais et al, observations non publiées). Globalement, cette approche est appropriée pour l&#39;analyse des mutants faux-sens dans un gène quelconque qui doit être analysé de façon récessive.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Nous fournissons une méthode pour tester des variantes du gène BRCA1 dans un essai de culture de tissus basées pour la réparation recombinaison homologue des dommages à l&#39;ADN en appauvrissant protéine BRCA1 endogène à partir d&#39;une cellule à l&#39;aide d&#39;ARNi et le remplacer par un mutant BRCA1 points qui contient un changement de codage.

Identifier les effets des mutations du gène BRCA1 sur la recombinaison homologue en utilisant les cellules qui expriment endogène de type sauvage du gène BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

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14.5K vues.  The Ohio State University. Nous fournissons une méthode pour tester des variantes du gène BRCA1 dans un essai de culture de tissus basées pour la réparation recombinaison homologue des dommages à l'ADN en appauvrissant protéine BRCA1 endogène à partir d'une cellule à l'aide d'ARNi et le remplacer par un mutant BRCA1 points qui contient un changement de codage.

Vidéo: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Étudier les effets de matrice de rigidité sur la fonction cellulaire en utilisant l&#39;acrylamide basée hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Recherche

Biologie

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analyse de la fonction des petites GTPases par micro-injection de plasmides dans les cellules épithéliales polarisées

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Une méthode de génotypage basé sur la PCR pour distinguer les variétés de type sauvage et les plantes ornementales de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering homologues constructions de recombinaison<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Mesurer les effets des bactéries sur<em&gt; C. Elegans</em&gt; Comportement aide d&#39;un test de rétention d&#39;oeufs

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

ADNg enrichissement par une technologie basée Transposase-NGS Analyse de la séquence plénier de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, et 9 gènes impliqués dans la réparation des dommages de l&#39;ADN

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Identifier les sites d&#39;interaction protéine-protéine à l&#39;aide de peptides tableaux

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Détermination des acides sialiques dans les échantillons de foie et de lait de type sauvage et<em&gt; CMAH</em&gt; Souris knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Une méthode pour évaluer les effets de la bactériocine sur le microbiota intestinal des souris

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Enquête sur les néfastes effets de basse pression Plasma stérilisation sur la survie de Bacillus subtilis Spores en utilisant Live microscopie des cellules

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...