Je tiens à souligner Olga De Souza Silva pour l&#39;assistance technique. Ce travail a été soutenu par la Fondation nationale suisse (FNS) Grant 31003A_127029.

La méthode TransFLP (figure 1) est illustrée par trois approches différentes: I) la suppression de deux gènes adjacents codant pour des déterminants de virulence de V. cholerae (par exemple, ΔctxAB), ii) l&#39;élimination d&#39;un îlot de pathogénicité (par exemple, ΔVPI-1), et iii) l&#39;intégration d&#39;une séquence T7 polymérase dépendante de l&#39;ARN promoteur en amont d&#39;un gène d&#39;intérêt, ce qui permet par la suite de son expression artificielle dans le fond souche respective (par exemple, T7-TFox) (figure 2). 1. Préparation des flocons de chitine <ol><li> Poids 50-80 mg de flocons de chitine dans un tube en plastique standard de 1,5 ml. Cela peut être préparé en vrac. Flocons de chitine sont disponibles dans le commerce auprès de Sigma (cat. nombre C9213). </li><li> Autoclaver les flocons de maintien des couvercles des tubes ouverts. </li><li> Immédiatement fermer les paupières après l&#39;autoclave est refroidi. </li><li> Magasin autoclavé flocons de chitineà la température ambiante. </li></ol> 2. Option: Transformation artificielle de V. cholerae avec la recombinase FLP plasmide codant <ol><li> Préparer électrocompétentes V. cellules cholerae utilisant des méthodes standard 11. Stocker des aliquotes de cellules compétentes à -80 ° C. </li><li> Ajouter 1-2 pl d&#39;une mini-préparation régulière de plasmide pBR-flp 5 à la électrocompétentes V. cholerae et transférer les cellules du mélange dans une cuvette d&#39;électroporation (0,2 cm largeur de l&#39;intervalle). </li><li> Appliquer une impulsion de 1,6 kV. </li><li> Ajouter 0,9 ml milieu SOC, doucement transfert de cellule à une norme de tube 14 ml. Incuber non mobile de 2,5 à 3 h à 30 ° C. </li><li> Plaque 100 ul et 300 ul sur des plaques LB contenant 100 ug / ml d&#39;ampicilline et incuber à 30 ° C pendant la nuit. </li><li> Purifier clone unique et l&#39;enregistrer comme stock de glycérol pour des expériences futures TransFLP. </li></ol> 3. Conception oligonucléotide et Polymerase Chain Reaction <ol><li> Au moins six oligonucléotides sont nécessaires pour amplifier la région d&#39;ADN (s) d&#39;intérêt ainsi que la FRT-cassettes antibiotique flanqué par PCR (figure 3). Il est recommandé d&#39;inclure également une paire d&#39;amorçage oligonucléotides en dehors du fragment inséré PCR. Cela permet de vérifier l&#39;intégration et l&#39;excision correcte de la cassette FRT-flanqué de résistance aux antibiotiques (par exemple, décrit comme «chk-up &#39;et les amorces« chk-down »dans les figures 3 à 5). </li><li> Concevoir des oligonucléotides # 2, # 3, # 4 et # 5 avec soin. Ils devraient être en mesure de suffisamment recuit (par exemple, ~ 28 pb) à la matrice d&#39;ADN. La matrice d&#39;ADN est un ADN génomique (ADNg) pour les amorces n ° 2 et n ° 5 et FRT-Kan-FRT contenant l&#39;ADN plasmidique comme pROD17 12 et pBR-FRT-KAN-FRT (cette étude) pour les amorces # 3 et # 4 ( La figure 3A). </li><li> Concevoir des amorces # 2 à # 5 permettant vaste appariement de bases à leur extrémité 5 &#39;entre # 2 / # 3 et # 4 / # 5, respectivement (figure 3A </strong&gt; encadré). Deux cycles de PCR suivre. </li><li> Effectuer trois réactions PCR indépendantes comme un 1 er tour de la PCR. La première sera amplifier la région en amont du gène / ADN région d&#39;intérêt à l&#39;aide d&#39;oligonucléotides N ° 1 et N ° 2. La PCR devrait se traduire par un fragment d&#39;au moins 500 pb de longueur pour permettre la recombinaison homologue dans les cellules. Des fragments plus courts (~ 250 pb) peut être de 8 suffisante, mais ne sont pas recommandés. </li><li> En parallèle effectuer la deuxième PCR qui amplifie la cassette FRT-flanqué antibiotique. Pour les exemples fournis ici, nous avons surtout utilisé aph (kan R). Oligonucléotides utilisation n ° 3 et n ° 4 pour cette réaction (figure 3A). </li><li> De façon concomitante, amplifier la région d&#39;ADN en aval par PCR (troisième échantillon). Le fragment de PCR devrait également être d&#39;au moins 500 pb de longueur. Effectuer la PCR avec ADNg comme matrice et les oligonucléotides # ​​5 et # 6 (figure 3A). </li><li> Après purification PCR de tous les troisfragments, effectuez la 2 ème tour de la PCR. Utilisez un mélange à parts égales de chacun des trois fragments obtenus au premier tour en tant que modèle. L&#39;amplification est catalysée par des oligonucléotides n ° 1 et n ° 6. Le fragment obtenu par PCR (figure 3B) sert de l&#39;ADN transformant dans l&#39;expérience la transformation naturelle (figure 1). </li></ol> 4. La chitine induite par la transformation naturelle <ol><li> Cultivez V. cholerae cellules en aérobiose dans un milieu riche à 30 ° C jusqu&#39;à ce qu&#39;ils atteignent une densité optique à 600 nm d&#39;environ 0,5. </li><li> Récolter les bactéries par centrifugation. Laver à granulés une fois dans un milieu artificiel défini (DASW 6) avant de remettre en suspension les cellules dans 2 volumes de DASW. Ajouter 1 ml de culture à 50-80 mg de flocons stériles chitine (expliqué au point 1). Si les bactéries port plasmide pBR-flp (facultatif point 2), ajouter l&#39;ampicilline (50 ug / ml) à la culture. Incuber à 30 ° C pendant 16-24 heures sans mouvement. </li><li> Ajouter &amp; ge, 200 ng de la 2 e ronde PCR dérivé fragment (expliqué au point 3). Mélanger soigneusement sans beaucoup détacher les bactéries de la surface de chitine. Incuber à 30 ° C pendant 24 heures sans mouvement. </li><li> Vortex de la culture extensive de ≥ 30 sec. Fais 100-300 ul sur des plaques de milieu LB sélectif (par exemple la kanamycine ou des plaques LB contenant de la gentamicine pour les exemples fournis ici). Utilisez deux plaques sélectives si les bactéries abriter le plasmide pBR-flp en ajoutant à l&#39;ampicilline en association aux autres antibiotiques. Incuber les plaques à 30 ° C pendant 16-24 heures ou jusqu&#39;à ce que les colonies sont visibles. </li><li> Isoler transformants simples à partir de plaques sélectives. Ces transformants ont remplacé le lieu d&#39;origine chromosomique par le fragment de PCR en raison d&#39;un événement croisée en double. Ces souches peuvent être directement utilisés pour d&#39;autres expériences dans le cas où le retrait de la cassette antibiotique n&#39;est pas nécessaire. </li></ol> 5. Retrait de la cassette sélectif (s) par Flp-Recombinaison <ol><li> Artificiellement transformer vos transformants avec le plasmide pBR-flp comme décrit au point 2 s&#39;il n&#39;est pas effectué avant l&#39;essai de transformation naturelle. </li><li> Cultiver les bactéries sur des plaques de gélose LB contenant de l&#39;ampicilline à 37 ° C pendant 16-24 heures. Options: changer la température dans l&#39;intervalle de 2-3 heures à 40 ° C comme expression de flp à partir du plasmide pBR-de-flp est réprimée à 5,10 températures plus élevées, bactéries transfert aux plaques fraîches après env. 8 heures d&#39;incubation. </li><li> Test de sensibilité aux antibiotiques (montré ici pour la kanamycine; figure 1) par restreaking les clones en parallèle sur des plaques d&#39;agar contenant des antibiotiques et sans antibiotique. Isoler simples colonies sensibles. Congelez résistant à l&#39;ampicilline clone comme stock de glycerol si vous avez l&#39;intention de modifier encore la souche avec suppression autre / d&#39;insertion en utilisant TransFLP. </li></ol> 6. Curage du plasmide <ol><li> Développer la culture du jour au lendemain dans des conditions aérobies età 30 ° C. Utilisez un milieu riche sans l&#39;addition d&#39;un antibiotique. </li><li> En option: croissance de culture frais pour 3-6 heures par dilution de la culture d&#39;une nuit 1:100 en eau douce milieu sans antibiotique. </li><li> Dilution plaque ou série (s) de la culture sur plaque LB agar plaine (s) et incuber à 30 ° C pendant 8-16 heures (jusqu&#39;à ce que les colonies sont visibles). </li><li> Test de sensibilité à l&#39;ampicilline des clones par restreaking les clones en parallèle sur des plaques de gélose contenant l&#39;antibiotique et sans antibiotique (figure 1). Geler l&#39;ampicilline souche sensible que les stocks de glycérol. </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+fischeri+requires+tfoX+and+tfoY.">Natural transformation of Vibrio fischeri requires tfoX and tfoY.</a> Environ. Microbiol. 12, 2302-2311 (2010).</li><li>Marvig, R. L., Blokesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+cholerae+as+a+tool-optimizing+the+procedure.">Natural transformation of Vibrio cholerae as a tool-optimizing the procedure.</a> BMC Microbiol. 10, 155 (2010).</li><li>Blokesch, M., Schoolnik, G. 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F., Maniatis, T., Ford, N., Nolan, C., Ferguson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory manual. 1, (1989).</li><li>Reyes-Lamothe, R., Possoz, C., Danilova, O., Sherratt, 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=Independent+positioning+and+action+of+Escherichia+coli+replisomes+in+live+cells.">Independent positioning and action of Escherichia coli replisomes in live cells.</a> Cell. 133, 90-102 (2008).</li><li>Karaolis, D. K. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+background+expression+and+improved+plasmid+stability+with+pET+vectors+in+BL21+(DE3).">Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3).</a> Biotechniques. 29, 1234-1238 (2000).</li><li>Faruque, S. M., Zhu, J., Asadulghani, M., Kamruzzaman, J. J., Mekalanos, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+diverse+toxin-coregulated+pilus-positive+Vibrio+cholerae+strains+fails+to+demonstrate+evidence+for+Vibrio+pathogenicity+island+phage.">Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for Vibrio pathogenicity island phage.</a> Infect. 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Aucun conflit d&#39;intérêt déclaré.

La méthode décrite ci-dessus et TransFLP ailleurs 5 a été largement utilisée dans notre laboratoire. Le genre de manipulations génétiques qui sont réalisables comprennent entre autres: la suppression de gènes individuels et groupes de gènes, la suppression des îlots génomiques (par exemple, VPI-1), l&#39;insertion de séquences en amont d&#39;un gène d&#39;intérêt (par exemple, des séquences promotrices) et l&#39;insertion de séquences à la fin d&#39;un gène spécifique (p...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Flocons de chitine </td><td> Sigma </td><td> C9213 </td><td> Autoclavage nécessaire </td></tr><tr><td> OPTION: Micropulser (en boîtier de cassette antibiotique doit être excisée par la recombinase Flp codé sur pBR-flp) </td><td> Biorad </td><td> 165-2100 </td><td> Ou électroporateurs comparables </td></tr></tbody></table>

transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

Plusieurs méthodes sont disponibles pour manipuler les chromosomes bactériens 1-3. La plupart de ces protocoles reposent sur ​​l&#39;insertion des plasmides réplicatifs conditionnelle (par exemple abriter des réplicons pir-dépendants ou sensibles à la température 1,2). Ces plasmides sont intégrés dans les chromosomes bactériens basés sur l&#39;homologie médiée par recombinaison. Ces mutants d&#39;insertion sont souvent utilisés directement dans des cadres expérimentaux. En variante, la sélection pour l&#39;excision du plasmide suivie par la perte peut être effectuée, pour laquelle les bactéries Gram négatif dépend souvent de l&#39;enzyme contre-sélectionnable sucrase lévane codée par le gène sacB 4. L&#39;excision pouvez restaurer le génotype de pré-insertion ou le résultat d&#39;un échange entre le chromosome et la copie plasmidique du gène modifié. Un inconvénient de cette technique est qu&#39;elle prend beaucoup de temps. Le plasmide doit être cloné premier, il nécessite de transfert horizontal V. cholerae la plupart (n<html> otably par accouplement avec un E. coli souche donneuse) ou artificielle transformation de celui-ci, et l&#39;excision du plasmide est aléatoire et peut soit restaurer le génotype initial ou créer la modification souhaitée si aucune sélection positive est exercée. Ici, nous présentons une méthode pour la manipulation rapide de l&#39;V. chromosome cholerae (s) 5 (figure 1). Cette méthode est basée sur TransFLP la chitine récemment découvert à médiation par induction de compétence naturelle dans cette organisme représentatif 6 et d&#39;autre du genre Vibrio tels que V. fischeri 7. Compétence naturelle permet l&#39;absorption de l&#39;ADN libre, y compris des fragments d&#39;ADN générés par PCR. Une fois pris, l&#39;ADN recombine avec le chromosome étant donné la présence d&#39;un minimum de 250-500 pb de la région flanquante homologue 8. Comprenant un marqueur de sélection entre-deux de ces régions adjacentes permet une détection facile des transformants fréquentes. t &quot;&gt; Cette méthode peut être utilisée pour différentes manipulations génétiques de V. cholerae et potentiellement d&#39;autres bactéries naturellement compétentes. Nous fournissons trois exemples de nouvelles sur ce qui peut être accompli par cette méthode, en plus de notre étude précédemment publiée sur les suppressions monogéniques et le plus d&#39;affinité tag séquences 5. Plusieurs étapes d&#39;optimisation concernant le protocole initial de la chitine induite par la transformation naturelle 6 sont intégrés dans le présent protocole TransFLP. Ceux-ci comprennent entre autres le remplacement des fragments de coquilles de crabe en flocons disponible dans le commerce chitine 8, le don de la PCR dérivée d&#39;ADN transformant en matériau 9, et l&#39;ajout de sites cibles FLP-FRT recombinaison () 5. sites FRT permettre l&#39;excision dirigée vers le site du marqueur de sélection par l&#39;intermédiaire de la recombinase Flp 10.

Les résultats représentatifs des trois exemples sont présentés dans les figures 4 à 6. La première approche vise à supprimer les gènes voisins ctxA et ctxB. Ensemble, ils codent le facteur de virulence majeur de V. cholerae, la toxine cholérique. Nous avons conçu des oligonucléotides pour la méthode TransFLP comme décrit ci-dessus et conformément à la figure 3. La souche parentale, la souche intermédiaire hébergeant FRT de la cassette de rési...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

Une méthode rapide pour modifier le génome d&#39;<em&gt; V. cholerae</em&gt; Est décrite. Ces modifications incluent la suppression de gènes uniques, des clusters de gènes et îlots génomiques ainsi que l&#39;intégration de séquences courtes (par exemple des éléments de promoteur ou par affinité tag séquences). La méthode est basée sur la transformation physique et FLP-recombinaison.

TransFLP - Une méthode pour modifier génétiquement<em&gt; Vibrio cholerae</em&gt; Basé sur la transformation naturelle et FLP-recombinaison

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

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Immunology and Infection

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

14.5K vues.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Une méthode rapide pour modifier le génome d' V. cholerae Est décrite. Ces modifications incluent la suppression de gènes uniques, des clusters de gènes et îlots génomiques ainsi que l'intégration de séquences courtes (par exemple des éléments de promoteur ou par affinité tag séquences). La méthode est basée sur la transformation physique et FLP-recombinaison.

Vidéo: TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

La colonisation de Euprymna scolopes Squid par Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

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Immunologie et infection

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Un test Parasite sauvetage et à la transformation pour le dépistage anti-leishmaniose contre intracellulaire<em&gt; Leishmania donovani</em&gt; Amastigotes dans THP1 humaines ligne leucémie aiguë monocytaire cellulaire

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Application clinique de<em&gt; Belle au Bois Dormant</em&gt; Et artificielle cellules présentatrices d&#39;antigènes pour modifier génétiquement des cellules T du sang de cordon ombilical et périphériques

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-résolution de l&#39;imagerie cellulaire Synapse immunologique Natural Killer sur un verre soutenu par Planar lipidique en deux couches

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

La mise en œuvre d&#39;un système d&#39;infection à base Insertion Perméable Membrane pour étudier les effets de sécrétées bactériennes Toxines sur des cellules hôtes mammaliennes

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Développement d&#39;un plus sensible et spécifique chromogénique Agar moyen pour la détection de<em&gt; Vibrio parahaemolyticus</em&gt; Et autres<em&gt; Vibrio</em&gt; espèces

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Une plate-forme de dosages anti-biofilm Adaptés à l&#39;exploration des bibliothèques de composés naturels

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basées sur la fluorescence neuraminidase Essai d&#39;inhibition pour évaluer la sensibilité des virus de la grippe à la neuraminidase Inhibiteur Classe de Antiviraux

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Techniques de laboratoire utilisées pour maintenir et différencier les biotypes de<em&gt; Vibrio cholerae</em&gt; Isolats cliniques et environnementaux

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...