Ce travail a été soutenu par le Board of Regents de l&#39;État de la Louisiane LEQSF (2009-12)-RD-A-19 (PI: WL Murfee) et l&#39;hypertension Tulane et du Centre rénale d&#39;excellence financé par le NIH subvention P20RR017659-08 (PI : L. Gabriel Navar).

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer+and+other+diseases.">Angiogenesis in cancer and other diseases.</a> Nature. 407, 249-257 (2000).</li><li>le Noble, F. A., Stassen, F. R., Hacking, W. J., Struijker Boudier, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+hypertension.">Angiogenesis and hypertension.</a> J. Hypertens. 16, 1563-1572 (1998).</li><li>Peirce, S. M., Skalak, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+remodeling:+a+complex+continuum+spanning+angiogenesis+to+arteriogenesis.">Microvascular remodeling: a complex continuum spanning angiogenesis to arteriogenesis.</a> Microcirculation. 10, 99-111 (2003).</li><li>Anderson, C. R., Ponce, A. M., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+identification+of+an+extracellular+matrix+scaffold+that+microguides+capillary+sprouting+in+vivo.">Immunohistochemical identification of an extracellular matrix scaffold that microguides capillary sprouting in vivo.</a> J. Histochem. Cytochem. 52, 1063-1072 (2004).</li><li>Ponce, A. M., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+stimulus+determines+the+positioning+of+pericytes+within+capillary+sprouts+in+vivo.">Angiogenic stimulus determines the positioning of pericytes within capillary sprouts in vivo.</a> Microvasc. Res. 65, 45-48 (2003).</li><li>Franzen, L., Ghassemifar, R., Malcherek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mast+cell+activation+improves+connective+tissue+repair+in+the+perforated+rat+mesentery.">Experimental mast cell activation improves connective tissue repair in the perforated rat mesentery.</a> Agents Actions. 33, 371-377 (1991).</li><li>Anderson, C. R., Hastings, N. E., Blackman, B. R., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+sprout+endothelial+cells+exhibit+a+CD36+low+phenotype:+regulation+by+shear+stress+and+vascular+endothelial+growth+factor-induced+mechanism+for+attenuating+anti-proliferative+thrombospondin-1+signaling.">Capillary sprout endothelial cells exhibit a CD36 low phenotype: regulation by shear stress and vascular endothelial growth factor-induced mechanism for attenuating anti-proliferative thrombospondin-1 signaling.</a> Am. J. Pathol. 173, 1220-1228 (2008).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J. Cell. Mol. Med. 10, 588-612 (2006).</li><li>Norrby, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Mesentery+Angiogenesis+Assay.">Rat Mesentery Angiogenesis Assay.</a> J. Vis. Exp. (52), e3078 (2011).</li><li>Murfee, W. L., Rehorn, M. R., Peirce, S. M., Skalak, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+cells+along+venules+upregulate+NG2+expression+during+microvascular+remodeling.">Perivascular cells along venules upregulate NG2 expression during microvascular remodeling.</a> Microcirculation. 13, 261-273 (2006).</li><li>Stapor, C. P., Murfee, L. W. <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+class+III+&#946;-tubulin+as+a+marker+of+angiogenic+perivascular+cells.">Identification of class III &#946;-tubulin as a marker of angiogenic perivascular cells.</a> Microvascular Research. , (2011).</li><li>Yang, M., Aragon, M., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+Mesenteric+Microvascular+Networks+from+Spontaneously+Hypertensive+Versus+Normotensive+Rats.">Angiogenesis in Mesenteric Microvascular Networks from Spontaneously Hypertensive Versus Normotensive Rats.</a> Microcirculation. , (2011).</li><li>Robichaux, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic/Blood+endothelial+cell+connections+at+the+capillary+level+in+adult+rat+mesentery.">Lymphatic/Blood endothelial cell connections at the capillary level in adult rat mesentery.</a> Anat. Rec. (Hoboken). 293, 1629-1638 (2010).</li><li>Amos, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+The+role+of+human+adipose-derived+stromal+cells+in+inflammatory+microvascular+remodeling+and+evidence+of+a+perivascular+phenotype.">IFATS collection: The role of human adipose-derived stromal cells in inflammatory microvascular remodeling and evidence of a perivascular phenotype.</a> Stem Cells. 26, 2682-2690 (2008).</li></ol>

Nous n&#39;avons rien à communiquer.

Le modèle extériorisation a été signalé en 2006 et est adapté des modèles précédents de blessures mécaniques du mésentère de rat de l&#39;angiogenèse 4-7 et produit des résultats similaires à des modèles bien établis injection IP qui tirent parti du rat mésentère 9. Le temps de l&#39;extériorisation 20 minutes a été déterminée expérimentalement pour produire une réponse robuste angiogénique. Bien que cette période pourrait être modifiée, elle permet à une application locale d&#39;inhibiteurs de l&#39;angiogenèse 4 pour des études mécanistiques et l&#39;application directe des cellules exogènes pour les études de lignées cellulaires. De faisabilité de la constitution de cellules dans le remodelage des tissus mésentériques est appuyée par des études préliminaires dans notre laboratoire à l&#39;aide pré-étiquetés cellules de moelle osseuse et les cellules souches mésenchymateuses (Figure 7), et par le succès d&#39;enquêter sur le sort de l&#39;homme tissu adipeux ip injection cellules stromales 14 . Dans notre laboratoire, nous avons utilisé ce modèle pour identifier les phen péricyteschangements otypic au cours du temps de la réponse angiogénique 10 et d&#39;évaluer le potentiel angiogénique dans des conditions pathologiques, telles que l&#39;hypertension 12. La réponse angiogénique et les changements phénotypiques cellulaires associés à ce modèle peut également être observé dans d&#39;autres modèles mésentériques de rat angiogéniques y compris l&#39;exposition hypoxie chronique 10, 11. Une limitation du modèle extériorisation, c&#39;est que les mécanismes exacts de déclenchement de l&#39;angiogenèse ne sont pas connus. Extériorisation du mésentère a été liée à la dégranulation des mastocytes et une augmentation des taux d&#39;histamine 6, mais une enquête plus approfondie est nécessaire pour gagner plus de perspicacité. Le stimulus angiogénique est sans doute multifactorielle, produisant une réponse robuste remodelage dans la hiérarchie d&#39;un réseau microvasculaire. Bien que les mécanismes inconnus restent une critique majeure de ce modèle, sa reproductibilité et la simplicité le rendre attrayant pour identifier la dynamique cellulaire impliqués dans le processus intrinsèquement complexe germination capillaire. La reproductibilité du modèle est soutenu par comparables métriques angiogéniques au cours du temps de la croissance du réseau microvasculaire dans plusieurs souches de rats (mâles Wistar et Sprague-Dawley femelles) dans des études publiées antérieurement dans notre laboratoire 10, 12. Depuis, la majorité des adultes tissus mésentériques de rat sont vascularisés, le modèle permet aussi de multiples tissus à examiner par animal. Malheureusement, ce modèle n&#39;est manifestement pas applicable à des modèles génétiques de souris que les fenêtres mésentériques de souris ont une vascularisation moins natale et, dans notre expérience, manquent souvent observables réseaux de branchement. Les applications futures comprennent l&#39;étude de la fonctionnalité navire au cours de l&#39;angiogenèse en utilisant la microscopie intra-vitale à des moments précis et l&#39;enquête de la dynamique connexes cellulaires impliqués dans la lymphangiogenèse et la neurogenèse. Bien que la mesure de la vascularisation native par la fenêtre mésentérique semble être roughly proportionnelle avec l&#39;âge, nous avons observé de branchement des réseaux microvasculaires chez les rats mâles Wistar aussi jeunes que 4-5 semaines. Ces observations suggèrent que le modèle extériorisation pourrait également être appliqué pour comparer les différences angiogéniques à travers les âges.

<table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires additionnels </td></tr><tr><td> Draper </td><td> Cardinal Health </td><td> 4012 </td><td> 12 &quot;x12&quot; Bio-Shield stérilisation régulière Wraps </td></tr><tr><td> Noyes Micro ciseaux </td><td> Roboz instruments chirurgicaux </td><td> RS-5677 </td><td> Noyes Micro Ciseaux de dissection de printemps; Droites, Sharp-Blunt points; Cutting Edge 13mm; Largeur Astuce 0.25mm, 4 1/2 &quot;Longueur hors tout </td></tr><tr><td> Graefe Pince </td><td> Roboz instruments chirurgicaux </td><td> RS-5135 </td><td> Micro Pinces à disséquer; dentelé; légère courbe; Largeur Astuce 0.8mm; 4 &quot;Longueur </td></tr><tr><td> Graefe Pince </td><td> Roboz instruments chirurgicaux </td><td> RS-5130 </td><td> Micro Pince à dissection; en dents de scie, les droites; Largeur Astuce 0.8mm; 4 &quot;Longueur </td></tr&gt; <tr><td> Suture 4-0 </td><td> ETHICON </td><td> 699g </td><td> (1,5 métrique) monofilament en nylon noir Ethilon Suture </td></tr><tr><td> 5-0 </td><td> ETHICON </td><td> 8556 </td><td> (1,0 métrique) PROLENE Polyprolene suture monofilament Bleu </td></tr><tr><td> 7-0 suture </td><td> ETHICON </td><td> 1647G </td><td> (0,5 métrique) monofilament en nylon noir Ethilon Suture </td></tr><tr><td> Titulaire Castroviejo aiguille Micro </td><td> Outils belle science </td><td> 12060-02 </td><td> Largeur Astuce: 0.6mm De serrage Longueur: 5mm Longueur: 9cm Pointe droite </td></tr><tr><td> Porte-aiguille Castroviejo </td><td> Outils belle science </td><td> 12565-14 </td><td> Forme de la pointe: Droit Largeur Astuce: 1.5mm Longueur de serrage: 10mm Ciseaux: Aucun Lock: Oui Longueur: 14cm Dentelé: Oui </td></tr><tr><td> Manche de bistouri </td><td> Roboz d&#39;instruments chirurgicaux </td><td> RS-9843 </td><td> Scalpel Handle, n ° 3; solide; 4 &quot;Longueur </td></tr><tr><td> Stérile Lame chirurgicale </td><td> Cincinnati chirurgicale </td><td> 0110 </td><td> En acier inoxydable; Taille 10 </td></tr><tr><td> Boîte de Pétri </td><td> Fisher Scientific </td><td> 08-757-13 </td><td> Ridge biseauté, Slippable </td></tr></tbody></table> Table des matières spécifiques chirurgicales et outils. <table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires additionnels </td></tr><tr><td> Beuthanasia </td><td> Schering-Plough Animal Health Corp Union (Commandé par MWI alimentation vétérinaire) </td><td> MWI #: 011168 </td><td> Matière active: par 100 ml, 390 mg de sodium pentobarbital de sodium, 50 mg de phénytoïne </td></tr><tr><td> La kétamine </td><td> Fort Dodge Santé Animale (Commandé par MWI alimentation vétérinaire) </td><td> MWI #: 000680 </td><td> Kateset 100 mg / ml </td></tr><tr><td> La xylazine </td><td> LLOYD. Inc (Commandé par MWI alimentation vétérinaire) </td><td> MWI #: 009307 </td><td> Anased 100 mg / ml </td></tr><tr><td> Saline </td><td> Hospira Inc </td><td> 94-217-JT </td><td></td></tr><tr><td> PBS </td><td> SIGMA </td><td> 011M8207 </td><td></td></tr><tr><td> La saponine </td><td> Sigma </td><td> BCBB4080 </td><td></td></tr><tr><td> PECAM (CD31) </td><td> BD Pharmingen </td><td> 553371 </td><td></td></tr><tr><td> Streptavidine-Cy3 </td><td> Jackson ImmunoResearch </td><td> 016-160-084 </td><td></td></tr><tr><td> BSA </td><td> Jackson ImmunoResearch </td><td> 096555 </td><td></td></tr><tr><td> Vectastain Elite ABC </td><td> Vector Laboratories </td><td> PK-6100 </td><td></td></tr><tr><td> Vecteur Nouvelle-Rouge </td><td> Vector Laboratories </td><td> SK-4800 </td><td></td></tr><tr><td> VectaMount </td><td> Vector Laboratories </td><td> H-500 </td><td></td></tr></tbody></table> Table des réactifs spécifiques

rat mesentery exteriorization: a model for investigating the cellular dynamics involved in angiogenesis

La croissance du réseau Microvacular et le remodelage sont des aspects cruciaux de la cicatrisation des plaies, l&#39;inflammation, la rétinopathie diabétique, la croissance tumorale et les conditions d&#39;autres maladies 1, 2. Croissance du réseau est communément attribué à l&#39;angiogenèse, définie comme la croissance de nouveaux vaisseaux à partir de vaisseaux pré-existants. Le procédé angiogénique est également directement relié à artériogenèse, défini comme l&#39;acquisition d&#39;un capillaire de revêtement cellule périvasculaire et l&#39;élargissement navire. Inutile de dire que, l&#39;angiogenèse est complexe et implique de multiples acteurs au niveau cellulaire et moléculaire 3. Comprendre comment un réseau microvasculaire augmente, il faut identifier les dynamiques spatiales et temporelles ainsi que la hiérarchie d&#39;un réseau au cours du temps de l&#39;angiogenèse. Cette information est essentielle pour le développement de thérapies visant à manipuler la croissance des vaisseaux. Le modèle extériorisation décrit dans cet article représente un modèle simple et reproductible pour stimulerLating l&#39;angiogenèse dans le mésentère de rat. Il a été adapté à partir de cicatrisation des modèles chez le rat mésentère 4-7, et est une alternative à stimuler l&#39;angiogenèse dans le mésentère via des injections ip de pro-angiogéniques 8, 9. Le modèle extériorisation est attrayante parce qu&#39;elle nécessite un minimum d&#39;intervention chirurgicale et produit des augmentations dramatiques, reproductibles dans les germes de la zone capillaire, vasculaire et de la densité vasculaire au cours d&#39;une évolution dans le temps relativement court dans un tissu qui permet de visualiser en deux dimensions des réseaux microvasculaires entières vers le bas pour unique niveau de la cellule. La croissance stimulée reflète naturelles réponses angiogéniques dans un environnement physiologique, sans ingérence des étrangers molécules angiogéniques. En utilisant des méthodes immunohistochimiques étiquetage, ce modèle a été révélée extrêmement utile pour identifier de nouveaux événements cellulaires impliqués dans l&#39;angiogenèse. Les enquêteurs peuvent facilement corréler les mesures angiogéniques au cours du temps de remodelage avec le temps spdynamique, tels que S PROPRES cellulaires changements phénotypiques ou interactions cellulaires 4, 5, 7, 10, 11.

Watch this Scientific Journal Video about Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis at JoVE.com

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Cet article décrit un modèle simple pour stimuler l&#39;angiogenèse dans le mésentère de rat. Le modèle produit des augmentations spectaculaires de la germination capillaire, la zone vasculaire et la densité vasculaire au cours d&#39;une évolution dans le temps relativement court dans un tissu qui permet la visualisation en face de l&#39;ensemble des réseaux microvasculaires vers le niveau de la cellule unique.

Extériorisation Mésentère Rat: Un modèle pour étudier la dynamique cellulaires impliqués dans l&#39;angiogenèse

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

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Biology

19.0K vues.  Tulane University. Cet article décrit un modèle simple pour stimuler l'angiogenèse dans le mésentère de rat. Le modèle produit des augmentations spectaculaires de la germination capillaire, la zone vasculaire et la densité vasculaire au cours d'une évolution dans le temps relativement court dans un tissu qui permet la visualisation en face de l'ensemble des réseaux microvasculaires vers le niveau de la cellule unique.

Vidéo: Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: un modèle in vitro pour l&#39;étude de l&#39;angiogenèse (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

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Biologie

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

Assay Rat angiogenèse Mésentère

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

En utilisant l&#39;ARN médiée par la stratégie d&#39;alimentation interférences à l&#39;écran pour des gènes impliqués dans le règlement de la taille du corps du nématode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal cellulaire attaché patch-clamp enregistrement

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion Technique chargé d&#39;enquêter sur le règlement des microvaisseaux perméabilité au Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Un bioessai simple pour l&#39;évaluation des facteurs de croissance endothélial vasculaire

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblée et l&#39;application des «Shear Anneaux»: A Novel Endothelial Modèle pour Orbital, unidirectionnel et périodique Fluid débit et contrainte de cisaillement

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.