Cette étude a été soutenue par le NIH R00HL102154, et un American Society of Hematology prix de savant à P Ji.

Les expériences décrites dans ce protocole ont été réalisées en conformité avec les directives et règlements établis par l&#39;Université Northwestern institutionnel de protection des animaux et l&#39;utilisation Comité. 1 Préparation du milieu de culture <ol><li> Préparer la solution de fibronectine. Ajouter 1 ml d&#39;eau à une fiole de la fibronectine humaine (1 mg). Laissez solution dans la culture de tissus capot pendant 30 minutes sans agitation. Transférer le liquide total à 50 ml de PBS pour obtenir une concentration finale de 20 pg / ml et bien mélanger doucement. Rendre la solution de fibronectine frais avant le revêtement de la plaque (voir ci-dessous). </li><li> Préparer 50 ml de milieu sans Epo (milieu SCF). Combinez les ingrédients énumérés dans le tableau 1. Le milieu peut être conservé à 4 ° C pendant 1 mois quand fait frais. </li><li> Préparer 50 ml de milieu contenant de l&#39;Epo. Combinez les ingrédients énumérés dans le tableau 2. Le milieu peut être conservé à 4 ° C pendant 1 mois quand fait frais. </li></ol&gt; 2. fibronectine plaque recouverte <ol><li> Ajouter 1 ml de solution de fibronectine à chaque puits d&#39;une plaque à 6 puits (ou 0,5 ml à chaque puits d&#39;une plaque à 12 puits). Laisser la plaque dans la culture de tissus capot pendant 1 heure. </li><li> Rincer les puits avec de l&#39;eau stérile et sécher à l&#39;air deux fois la plaque. </li><li> Enveloppez la plaque et enregistrer dans la chambre froide à 4 ° C. </li></ol> 3 Purification de foie fœtal érythroblastes REMARQUE: Utilisez le foie du fœtus à partir de E13 à E15 chronométrés souris gravides (C57BL / 6). foies de E13.5 sont préférées pour optimiser le rendement de progéniteurs CFU-E. Pour obtenir des souris gravides chronométrés, 6 à 8 semaines les souris mâles et femelles ont été placées dans une cage (généralement un mâle avec une ou deux femelles). Bouchon vaginal femmes ont été vérifiés le lendemain matin afin de déterminer si l&#39;accouplement a réussi. Le premier jour de la gestation était le jour après la prise a été trouvé. <ol><li> Préparation des cellules de foie fœtal total <ol><li> Sacrifiez le mousoi par inhalation de CO 2 et la dislocation cervicale. Pulvériser l&#39;abdomen avec de l&#39;éthanol à 70% pour la désinfection. Ouvrez l&#39;abdomen avec dissection des ciseaux pour enlever l&#39;utérus. Laver l&#39;utérus en 1x PBS. </li><li> Disséquer les foetus dans des conditions stériles en utilisant une hotte de culture de tissus et des outils à l&#39;autoclave et laver 1x PBS. </li><li> Disséquer les foies fœtaux des fœtus. Maintenir le corps du fœtus avec une pince, tirez doucement sur le foie fœtal (de couleur rose) à partir du corps avec un autre pince. Nettoyer le foie fœtal avec une pince pour enlever les tissus fibrotiques associées. Placez tous les foies fœtaux en frais 1x PBS contenant 10% de sérum de veau fœtal. </li><li> Perturber mécaniquement le foie du fœtus par aspiration et refoulement. </li><li> On filtre la suspension de cellules à travers un tamis cellulaire de 40 um dans un tube conique de 50 ml. Pellet les cellules à 800 xg pendant 5 min. </li><li> Aspirer le surnageant et remettre en suspension les cellules dans 1 ml de PBS avec 10% de FBS (environ 8 E13.5 foies fœtauxpar ml). </li></ol></li><li> Purification de Ter119 érythroblastes négatifs <ol><li> Bloquer les cellules avec de l&#39;IgG de rat (0,2 mg / ml) sur de la glace pendant 15 min. </li><li> Sans lavage ou centrifugation, ajouter un anticorps biotinylé anti-Ter119 à la suspension de cellules (1 ug / ml). Incuber pendant 15 min sur la glace. </li><li> Laver à 1x PBS avec 10% de FBS (1:10) et centrifuger à 800 g pendant 5 min. Remettre en suspension les cellules dans 1 ml de PBS avec 10% de FBS. </li><li> Ajouter 75 ul streptavidine conjuguée avec des particules magnétiques et incuber pendant 10 minutes sur la glace. </li><li> Ajouter le volume à 2,5 ml avec du PBS contenant 10% de FBS. Transférer la suspension cellulaire dans un 5 ml polypropylene tubes fond rond. </li><li> Insérer le tube dans un appareil de tri cellulaire magnétique et incuber pendant 10 min. Ter119 cellules positives se fixeront sur le côté du tube. Les Ter119 seules érythroblastes négatifs resteront dans la suspension. </li><li> Verser la suspension de cellules dans un nouveau 5 ml polypropylene tubes fond rond. </li><li><html> Répétez les étapes 3.2.6 et 3.2.7 pour éliminer les cellules positives Ter119 résiduelle. </li><li> Pellet les cellules à 800 xg pendant 5 min. Aspirer le surnageant. Remettre en suspension les cellules dans 1 ml de PBS contenant 10% de FBS et compter les cellules vivantes avec du bleu Trypan. </li></ol></li></ol> 4 transduction avec des virus ou ADNc codant shRNA <ol><li> Plaque des cellules de foie foetal négatifs Ter119 purifiée à 3-5 x 10 5 / puits dans une plaque revêtue de fibronectine 12 puits (ajuster le nombre de cellules si vous utilisez une plaque différente). </li><li> Ajouter polybrène à une concentration finale de 10 pg / ml pour faciliter la transduction virale. Spin infecter les cellules avec des lentivirus ou rétrovirus, ADNc codant ou shRNA, à 800 g pendant 1-1,5 heures à 37 ° C. </li></ol> 5. Culture des transduites Ter119 négatif souris foie fœtal érythroblastes <ol><li> Pour tester les fonctions des gènes dans le stade précoce de l&#39;érythropoïèse terminale (Figure 1 pointillés).&quot;&gt; </ol.><ol><li><ol. li=""> Culture des cellules transduites dans un milieu sans Epo (moyen SCF) pour 12 heures afin de permettre l&#39;expression des gènes et de l&#39;entretien de l&#39;état progénitrices transduites. </ol.></li><li> Centrifuger les cellules à 800 xg pendant 5 min. Aspirer le surnageant. Remettre en suspension les cellules dans 1 ml de milieu contenant de l&#39;Epo dans chaque puits d&#39;une plaque à 12 puits. </li><li> Après culture pendant 24 ou 48 heures, la récolte des cellules pour d&#39;autres essais. </li></ol></li><li> fonctions des gènes de test à la fin du stade de l&#39;érythropoïèse terminale (Figure 1 lignes pleines). <ol><li> Culture des cellules immédiatement à l&#39;Epo milieu contenant une transduction. </li><li> Après culture pendant 24 ou 48 heures, la récolte des cellules pour les autres essais. </li></ol></li></ol> 6. coloration des cellules cultivées foie fœtal pour l&#39;analyse par cytométrie en flux <ol><li> Lavez et remettre les cellules en PBS 1x. Récolte 2 x 10 5 cellules en culture pour l&#39;analyse par cytométrie en flux. </li><li> Incuberles cellules avec des anticorps conjugués à un fluorophore de 20 min à température ambiante dans l&#39;obscurité. Pour tester la différenciation, utilisez APC conjugué anti-CD71 et anti-PE Ter119 conjugué. Pour tester l&#39;énucléation, utilisez Hoechst 33342 tache en plus des autres anticorps. </li><li> Laver les cellules avec du PBS 1X. Remettre en suspension les cellules dans 0,5 ml de PBS contenant 1% de FBS et de l&#39;iodure de propidium (PI) (1 pg / ml) pour colorer les cellules mortes et les exclure de l&#39;analyse. </li><li> Conduite flux d&#39;analyse cytrometric. Analyser les cellules de contrôle de couleurs colorées et non colorées simples d&#39;abord pour l&#39;ajustement et la rémunération du cytomètre de flux. </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+cultured+mouse+fetal+erythroblasts+requires+Rac+GTPases+and+mDia2.">Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2.</a> Nature cell biology. 10 (3), 314-321 (2008).</li><li>Chida, D., Miura, O., Yoshimura, A., Miyajima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cytokine+signaling+molecules+in+erythroid+differentiation+of+mouse+fetal+liver+hematopoietic+cells:+functional+analysis+of+signaling+molecules+by+retrovirus-mediated+expression.">Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.</a> Blood. 93 (5), 1567-1578 (1999).</li><li>Patel, V. P., Lodish, H. 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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Nous présentons ici un système unique d&#39;analyser chronologiquement fœtus de souris érythropoïèse terminale du foie. Grâce à l&#39;application de différentes conditions de culture, nous avons disséqué avec succès l&#39;érythropoïèse terminale en début et en fin. Ceci est particulièrement important pour déterminer les mécanismes de gènes ayant des fonctions multiples. Par exemple, GTPases Rac jouent un rôle important dans les différentes étapes de l&#39;érythropoïèse terminale. L&#39;inhibiti...

L&#39;érythropoïèse est le processus de différenciation de cellules souches hématopoïétiques multipotentes à maturité érythrocytes. Ce processus par étapes comprend la formation d&#39;unités engagées en rafale érythroïde formant (BFU-E), les unités formant des colonies érythroïdes à division rapide (CFU-Es), et morphologiquement reconnaissables érythroblastes 1,2. Érythropoïèse terminale à partir de cellules souches CFU-E implique séquentielle érythropoïétine-dépendantes et indépendantes étapes 2,3. Dans le stade précoce de l&#39;érythropoïèse terminale, l&#39;érythropoïétine (EPO) se lie à son récepteur sur la surface cellulaire et induit une série de voies de signalisation en aval qui empêchent l&#39;apoptose cellulaire et favorisent la division cellulaire rapide et l&#39;expression du gène 1,4. À la fin du stade de l&#39;érythropoïèse terminale, érythroblastes subissent sortie de la cellule terminale du cycle, de la chromatine et de noyaux de condensation, et l&#39;extrusion des noyaux fortement condensés 5. Notre compréhension de la borneérythropoïèse s&#39;est grandement améliorée au cours des dernières décennies, ce qui est dû en grande partie à l&#39;utilisation réussie de plusieurs in vitro et dans des modèles murins in vivo 6-9. Parmi ces modèles, la culture in vitro de souris fœtale érythroblastes de foie fournit de nombreux avantages, y compris la facilité de purification des cellules, la prolifération et la différenciation rapide, et un synoptique plus proche de l&#39;érythropoïèse 10,11 humain. Dans ce système, un grand nombre de cellules progénitrices érythroïdes à partir de foie fœtal de souris peuvent être facilement isolés par la purification en une seule étape de Ter119 (un marqueur pour les cellules érythroïdes matures) des érythroblastes négatifs. Au cours de la culture de deux jours des érythroblastes, la différenciation de ces cellules peut être surveillée par une analyse par cytométrie de flux sur la base de l&#39;expression en surface du récepteur de la transferrine (CD71) et l&#39;antigène de Ter119 12. En outre, des érythroblastes énucléation de différenciation terminale peut être détectée par un fabricant de l&#39;ADN (Hoechst 33 342) 13. En outre, les progéniteurs purifiés peuvent être génétiquement modifiées par expression d&#39;ADNc exogène ou de petits ARN en épingle à cheveux (shRNA) pour les gènes d&#39;intérêt, ce qui facilite les études mécanistiques des fonctions de l&#39;expression du gène sur l&#39;érythropoïèse 11,13,14. D&#39;autre part, le taux de croissance de cellule rapide peut être une arme à double tranchant, car il est difficile de caractériser les fonctions des gènes à différents stades de l&#39;érythropoïèse terminale. Dans la plupart des cas, il est difficile de déterminer si une des fonctions des gènes spécifiques à un stade précoce de l&#39;érythropoïèse terminale depuis le temps des ADNc ou shRNA exprimés, les cellules ont déjà passé le stade précoce. Pour résoudre ce problème, nous avons développé un système unique pour disséquer les stades précoces et tardifs de l&#39;érythropoïèse terminale. Pour le stade précoce de l&#39;érythropoïèse terminale, Ter119 érythroblastes négatifs génétiquement modifiés ont été cultivées dans un milieu sans Epo mais contenant du facteur de cellules souches (SCF), l&#39;IL-6 et FLT3ligand de maintenir leur statut de géniteur et laissez les ADNc transduites ou shRNA à l&#39;expression 13. Les cellules ont été modifiées pour l&#39;Epo contenant le milieu après 12 heures pour induire la prolifération et la différenciation cellulaire. De cette façon, lorsque les cellules ont commencé à se différencier, les ADNc ont été transduites ou shRNA déjà exprimé. Pour la phase tardive de l&#39;érythropoïèse terminale, Ter119 érythroblastes négatives ont été cultivées dans un milieu contenant de l&#39;Epo immédiatement après la transduction. Par conséquent, on peut analyser les fonctions des gènes d&#39;intérêt dans la phase tardive de l&#39;érythropoïèse terminale. En résumé, une large application de ce système aiderait les fonctions des gènes disséquer à différents stades de l&#39;érythropoïèse terminale.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

Érythropoïèse implique un processus dynamique qui commence avec des unités engagées en rafale érythroïde formant (BFU-Es) puis en divisant rapidement érythroïde unités formant colonie (UFC-Es). Après CFU-E, les cellules sont morphologiquement reconnaissable et généralement appelée érythroblastes terminaux. L&#39;un des défis pour l&#39;étude de l&#39;érythropoïèse terminale est le manque d&#39;approches expérimentales pour disséquer les fonctions des gènes d&#39;une manière chronologique. Dans ce protocole, nous décrivons une stratégie unique pour déterminer la fonction des gènes dans les stades précoces et tardifs de l&#39;érythropoïèse terminale. Dans ce système, la souris fœtale Ter119 du foie (marqueur des cellules érythroïdes matures) des érythroblastes négatifs ont été purifiés et transduites avec l&#39;expression exogène d&#39;ADNc ou de petits ARN en épingle à cheveux (shRNA) pour les gènes d&#39;intérêt. Les cellules ont ensuite été cultivées dans des facteurs de croissance de milieu contenant d&#39;autres de l&#39;érythropoïétine (Epo) pour maintenir leur stade de progéniteurs pendant 12 heures tout en laissant les ADNc exogène ou shRNAà exprimer. Les cellules ont été modifiées pour Epo milieu après 12 heures pour induire la différenciation et la prolifération cellulaire, tandis que les matériaux génétiques exogènes ont déjà été exprimés. Ce protocole facilite l&#39;analyse des fonctions des gènes dans le stade précoce de l&#39;érythropoïèse terminale. Pour étudier la fin de l&#39;érythropoïèse terminale de stade, les cellules ont été immédiatement mises en culture dans un milieu Epo après transduction. De cette manière, les cellules ont été déjà différenciées à la fin du stade de l&#39;érythropoïèse terminale lorsque les matériaux génétiques transduites ont été exprimés. Nous recommandons une application générale de cette stratégie qui aiderait à comprendre les fonctions des gènes détaillées à différents stades de l&#39;érythropoïèse terminale.

La figure 1 présente les stratégies expérimentales. Le protocole est constitué de deux conditions indépendantes pour viser les fonctions des molécules de signalisation dans les stades précoces et tardifs de l&#39;érythropoïèse terminale. Ter119 négatifs érythroblastes de foie foetal ont été purifiés à partir de fœtus de souris E13.5. Analyse par cytométrie de flux de cellules érythroïdes fœtales du foie avant et après purification a montré que la purification a été efficace <str...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Nous présentons un système in vitro de culture de fœtus de souris des érythroblastes de foie qui dissèque les stades précoces et tardifs de l&#39;érythropoïèse terminale. Ce système facilite l&#39;analyse fonctionnelle des gènes spécifiques à différents stades de développement.

Souris foie fœtal Système de culture à disséquer les fonctions du gène cible dans les stades précoces et tardifs de l&#39;aérogare de l&#39;érythropoïèse

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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Immunology and Infection

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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14.9K vues.  Northwestern University.  Nous présentons un système in vitro de culture de fœtus de souris des érythroblastes de foie qui dissèque les stades précoces et tardifs de l'érythropoïèse terminale. Ce système facilite l'analyse fonctionnelle des gènes spécifiques à différents stades de développement. 

Vidéo: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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Immunologie et infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Utilisation de la souris BLT humanisé comme une tumeur de cellules souches Gene Therapy modèle basé sur

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Multi-cible approche de traitement parallèle pour Gene à la structure Détermination de la polymérase de l&#39;influenza PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Un protocole pour l&#39;analyse de l&#39;hépatite C de réplication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Reporter vaccine virus pour quantifier la fonction virale à toutes les étapes de l&#39;expression des gènes

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Isolement de leucocytes des tissus murins à l&#39;interface materno-foetale

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Cultures placentaire et déciduale organe humain pour l&#39;étude des infections à l&#39;interface materno-fœtale

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Cytométrie de flux à base de test pour la surveillance des cellules NK Fonctions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Génération des variantes d’évasion de neutraliser les anticorps monoclonaux Influenza Virus

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...