Les auteurs tiennent à remercier le Dr Scott Rizaldy au Mount Sinai Hospital à Toronto, en Ontario, pour son don de la première Rac1 flox / flox fibroblastes embryonnaires de souris. Ce travail a été soutenu par des subventions de fonctionnement à NJ par les Instituts canadiens de recherche en santé du Canada (IRSC, MOP 2009 à 93526) et les sciences naturelles et en génie du Canada (CRSNG, RG 327372-06). SH et MW sont pris en charge par CGSMA et CGS-M prix des IRSC et du CRSNG, respectivement. Steve Wills Hawley et Mélanie ont contribué également à ce document.

La décongélation des cellules <ol><li> Obtenir des fibroblastes de souris embryonnaires congelés (FAE) (généré précédemment à partir de cultures d&#39;embryons individuels primaire) de l&#39;azote liquide. </li><li> Décongelez les cellules en plongeant le flacon dans l&#39;eau 37 ° C en remuant jusqu&#39;à ce contenu est complètement dégelée. </li><li> Ajouter 9 ml de DMEM qui a été complété avec 10% de sérum de veau fœtal et 1% de pénicilline-streptomycine à une plaque de culture cellulaire de 10cm. </li><li> Ajouter la suspension cellulaire décongelée goutte à goutte à la plaque, le rock doucement la plaque pour disperser les cellules et les placer dans une plaque de 37 ° C CO, 5% 2 incubateur de la nuit. Notez que si les cellules sont sensibles à DMSO (à partir du processus de congélation), les médias initiale peut être remplacé par du milieu frais après que les cellules ont adhéré à la plaque (environ quatre heures après plaquage). Notez également que la confluence des cellules après décongélation dépend du nombre de cellules congelées, le taux de récupération des cellules après congélation, et le taux de croissance des cellules. </li></ol> Le repiquage des cellules <ol start="5"><li> Lorsque les cellules se sont développées essentiellement à 100% de confluence (ie: le lendemain), aspirer hors des médias et laver les cellules avec 5 ml de PBS stérile. </li><li> Retirer du PBS et ajouter 1 ml de trypsine EDTA 10mM contenant et laisser reposer la plaque dans l&#39;incubateur pendant environ 5 minutes pour permettre aux cellules de se détacher de la plaque. </li><li> Lorsque les cellules se sont détachées, ajouter 9 ml de médias pour les cellules traitées à la trypsine, une pipette 2-3 fois pour disperser des bouquets, ajouter 1 ml de cette suspension goutte à goutte sur une nouvelle plaque contenant 9 ml de médias, et la place dans le présent 37 ° C durant la nuit incubateur . La présence de SVF dans les médias sera inactiver la trypsine variante, les cellules peuvent être d&#39;abord lavé à diluer la trypsine. </li></ol> Comptage et le placage des cellules sur des lamelles <ol start="8"><li> Retirer les cellules de l&#39;incubateur, les laver et trypsiniser comme avant, sauf que cette fois ajouter 5-6ml média retour aux cellules trypsinisées lieu de 9 ml. </li><li> Cellules pipette soigneusement pour assurer la dissociation complète en une suspension cellulaire unique, et retirer 15μL à combiner avec 15μL du bleu trypan dans un tube de micro séparé. </li><li> Mélanger cette suspension cellulaire / bleu trypan par pipetage de haut en bas plusieurs fois, et d&#39;ajouter 15μL à l&#39;hématimètre. </li><li> Sous le microscope, les cellules mortes apparaissent bleues. Comptez le nombre de cellules claires / incolore (vivant) dans l&#39;une des grilles 4X4 sur l&#39;hématimètre. Répéter cette compter sur les trois grilles 4X4 autres (notés A, B, C et D) et utiliser l&#39;équation suivante pour calculer le nombre de cellules par uL de suspension: <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="L&#39;équation 1" /></li><li> Grâce à l&#39;optimisation du protocole, nous avons déterminé que le placage 30000 résultats dans les cellules densité cellulaire appropriée pour la visualisation à l&#39;issue de l&#39;expérience. </li><li> Pour calculer le volume de suspension cellulaire nécessaire pour 30 000 cellules, utilisez l&#39;équation suivante: <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="Équation 2" /></li><li> Maintenant, placez un bordereau de verre stériles couvrent dans chaque puits d&#39;un plat et ajouter six 2mL des médias dans chaque puits, puis ajouter goutte à goutte à partir de votre suspension cellulaire le volume requis pour 30.000 cellules dans chaque puits. Notez que des lamelles de verre sont utilisés pour cette expérience que nous serons visualisant les cellules en utilisant la microscopie à fluorescence à un stade ultérieur; toutes les applications ne seront nécessairement microscopie, et donc de couvrir glisse peut-être pas nécessaire. </li></ol> Transduction Virus <ol start="15"><li> Après plusieurs heures ou toute la nuit (un temps suffisant pour s&#39;assurer que les cellules adhèrent au lamelles), retirez les médias et laver les cellules avec 1 ml de DMEM sans sérum. </li><li> Ajouter 1 ml de milieu sans sérum dans chaque puits pour l&#39;ajout du virus. Pour cette expérience, les virus adéno-Cre virus est obtenue commercialement à partir Biolabs Vector. </li><li> Optimisation des protocoles antérieurs ont montré que la multiplicité d&#39;infection (MOI) de 500 fournit transduction très efficace dans les primaires FAE avec peu ou aucun effet sur la viabilité cellulaire. MOI peut être calculée comme suit: <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="Équation 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="L&#39;équation 4" /></li><li> Ajouter le volume calculé du virus directement dans chaque puits qui est d&#39;être infectés, secouez légèrement la plaque pour disperser les cellules du virus et le placer dans l&#39;incubateur à 37 ° C pendant la nuit. </li><li> Le lendemain matin, enlever le virus milieux contenant des cellules et laver les cellules dans 1 ml de PBS stérile, puis ajouter 2 mL de médias régulièrement à chaque puits. </li><li> Dans le cas de l&#39;Rac1 cellules illustré dans cette expérience, les changements morphologiques survenus dans les cellules à environ 72 heures après la transduction. </li></ol> Les résultats représentatifs Les cellules ont été de visualiserD par coloration avec la phalloïdine actine colorant (à des fins d&#39;imagerie uniquement) et imagée en utilisant le Leica TCS-SP5 microscope confocal à balayage laser multiphotonique au Centre d&#39;analyse avancée à l&#39;Université de Guelph. La figure 1 montre la morphologie contracté et allongée de la Rac1 flox / flox cellules qui ont été exposés à l&#39;adénovirus Cre virus comparé aux mêmes cellules non exposées au virus. <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Figure 1" /> Figure 1. Comparaison entre Rac1 flox / flox cellules infectées par le virus adéno-Cre (à gauche) par rapport aux cellules non infectées de la même source (à droite).

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements énoncés par le Conseil canadien de protection des animaux.

La vidéo d&#39;accompagnement illustre bien comment un virus recombinant peut être utilisé pour exciser un gène spécifique in vitro en utilisant la recombinase Cre. L&#39;élaboration de ce protocole n&#39;a toutefois révéler certains points particuliers vaut adressage. <ul><li> Procédures de sécurité adéquates doivent toujours être employée lorsque l&#39;on travaille avec des adénovirus. Une blouse et les gants doivent être portés en tout temps tout en travaillant avec le virus. Médias et articles en plastique doivent être traitées avec l&#39;eau de Javel à 10% pendant au moins 30 minutes, et les articles en plastique doivent être autoclavés après. </li><li> Évitez répétés de gel-dégel du stock de virus car cela peut affecter titre du virus et réduire ainsi l&#39;efficacité de transduction. De petites quantités de stock de virus doivent d&#39;abord être répartie dans des tubes séparés. </li><li> MOI allant de 50 à 500 ont été testés pendant le développement de méthodes et les taux de transduction efficaces ont été observées dans la plupart des cas, sans effets négatifs sur la viabilité cellulaire. </li><li> Adéno-GFP a également été utilisé pendant le développement de méthodes pour déceler rapidement les efficacités de transduction. Ce contrôle (ou un vecteur vide) devrait être effectuée durant l&#39;expérience pour assurer que l&#39;infection virale ne suffit pas à entraîner des changements cellulaires. De même, si l&#39;excision du gène d&#39;intérêt floxés dans vos cellules ne résulte pas en des modifications cellulaires (changements de morphologie ou de la viabilité), puis infecter le même type de cellule avec des adéno-GFP est un contrôle important de l&#39;utiliser pour démontrer que la transduction du virus est produisent. </li></ul> Au-delà de l&#39;examen des cascades de signalisation dans la culture, l&#39;approche adéno-CRE a également été employée in vivo pour éliminer les gènes conditionnellement à partir d&#39;animaux expérimentaux 3,4, et d&#39;étiqueter des populations spécifiques de cellules dans un organisme 6. Le système de l&#39;adénovirus peut également être adapté pour introduire des gènes autres que la recombinase Cre dans les cellules hôtes. Deux principaux avantages de l&#39;utilisation de ce vecteur de livraison sont de sa transduction et une efficacité élevées livraison de gènes, et le manque d&#39;intégration avec les ADN de l&#39;hôte. Cependant, la génération de nouveaux adénovirus recombinants peut être beaucoup de travail. Le protocole décrit ici harnais donc la puissance du système en exploitant les adénoviraux précédemment développé adéno-Cre virus, et le couplage que grâce à notre allèle floxés d&#39;intérêt, Rac1. Grâce à ce coup conditionnelle à expérimenter, nous avons confirmé que la transduction utilisant le virus adéno-Cre du MEF portant un allèle Rac1 floxés en effet provoquer l&#39;excision de Rac1 menant à des changements dans la morphologie caractéristique cellulaire de cellules déficientes Rac1 2,5.

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adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

La capacité de supprimer des composants génétiquement spécifiques de diverses cascades de signalisation cellulaire a été un outil essentiel dans l&#39;analyse du signal de transduction moderne. Une méthode particulière pour réaliser cette suppression conditionnelle est via l&#39;utilisation du système Cre-loxP. Cette méthode consiste à flanquant le gène d&#39;intérêt avec des sites loxP, qui sont des séquences de reconnaissance spécifiques de la protéine recombinase Cre. L&#39;exposition de la soi-disant floxés (flanqué de sites loxP) l&#39;ADN de cette enzyme dans les résultats un événement de recombinaison Cre-médiation sur les sites loxP, et l&#39;excision subséquente du gène intervenant 3. Plusieurs méthodes différentes existent pour administrer la recombinase Cre sur le site d&#39;intérêt. Dans cette vidéo, nous démontrons l&#39;utilisation d&#39;un adénovirus contenant le gène de la recombinase Cre pour infecter les fibroblastes primaires de souris embryonnaires (MEF) obtenu à partir d&#39;embryons contenant un floxés Rac1 allèle 1. Notre raison d&#39;être de la sélection Rac1 FAE pour nos expériences est-ce clair des changements morphologiques peuvent être vus sur la suppression de Rac1, due à des altérations du cytosquelette d&#39;actine 2,5. 72 heures après la transduction virale et l&#39;expression de Cre, les cellules ont été colorées en utilisant l&#39;actine colorant phalloïdine et imagées par microscopie confocale à balayage laser. On a observé que FAE qui avaient été exposés au virus adéno-Cre apparu sous contrat et en morphologie allongée par rapport aux cellules non infectées, compatible avec les précédents rapports 2,5. La méthode de la recombinase Cre adénovirus de livraison est avantageux que le virus adéno-Cre est facilement disponible, et une délétion du gène via Cre dans près de 100% des cellules peut être réalisé avec une infection adénovirale optimisé.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Dans cette vidéo, nous utilisons un adénovirus porteur du gène de la recombinase Cre pour infecter les fibroblastes de souris embryonnaires primaires portant un allèle floxés Rac1.

Adénovirus suppression génétique des molécules de signalisation dans les cultures de fibroblastes de souris embryonnaires primaires

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

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JoVE Journal

Biology

24.8K vues.  University of Guelph. Dans cette vidéo, nous utilisons un adénovirus porteur du gène de la recombinase Cre pour infecter les fibroblastes de souris embryonnaires primaires portant un allèle floxés Rac1.

Vidéo: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagerie en temps réel du noyau dense de vésicules cultures primaires neurones de l&#39;hippocampe

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Recherche

Biologie

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Génération de cellules souches pluripotentes induites par la reprogrammation des fibroblastes embryonnaires de souris avec un facteur de transcription Quatre, doxycycline inductible système de transduction lentiviraux

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Dosage de la capacité des molécules de signalisation diffusibles à réorienter embryonnaires épinière axones commissuraux

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Suppression des fibroblastes rapide de haute densité embryonnaires humaines cultures de cellules souches

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Analyse rapide de la génétique des épithélio-mésenchymateuse signalisation lors de la régénération des cheveux

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Organite Transport en culture d&#39;<em&gt; Drosophila</em&gt; Cellules: S2 Cell Line et neurones primaires.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Mesure de l&#39;absorption de glucose et réponse à la stimulation de l&#39;insuline dans<em&gt; In Vitro</em&gt; Myotubes primaires humains cultivés

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

TRANS-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...