Ich möchte Olga de Souza Silva für die technische Unterstützung zu bestätigen. Diese Arbeit wurde vom Schweizerischen Nationalfonds (SNF) Grant 31003A_127029 unterstützt.

Die TransFLP Methode (Abbildung 1) durch drei verschiedene Ansätze exemplifiziert: I) die Deletion von zwei benachbarten Genen Virulenzeigenschaften V. cholerae (zB ΔctxAB), II) die Entnahme einer Pathogenitätsinsel (zB ΔVPI-1), und III) die Integration eines T7-RNA-Polymerase-abhängigen Promotorsequenz stromauf von einer Gen-von-Interesse, das anschließend erlaubt seinen künstliche Expression in dem jeweiligen Stamm Hintergrund (zB T7-tfox) (Abbildung 2). Ein. Vorbereitung der Chitin Flakes <ol><li> Gewicht 50-80 mg von Chitin Flocken in einem Standard 1,5 ml Plastikröhrchen. Dies kann in Substanz hergestellt werden. Chitin Flocken sind kommerziell erhältlich von Sigma (Kat. Zahl C9213). </li><li> Autoklavieren der Flocken halten die Deckel der Rohre offen. </li><li> Schließen Sie sofort die Deckel nach dem Autoklav abgekühlt hat. </li><li> Shop autoklaviert Chitin Flockenbei Raumtemperatur. </li></ol> 2. Optional: Artificial Transformation von V. cholerae mit Flp-Rekombinase-kodierende Plasmide <ol><li> Bereiten elektrokompetente V. cholerae Zellen unter Verwendung von Standardverfahren 11. Aliquots speichern kompetenter Zellen bei -80 ° C </li><li> Fügen Sie 1-2 ul einer regelmäßigen Mini-Präparation von Plasmid pBR-flp 5 zum elektrokompetente V. cholerae-Zellen und Übertragen der Mischung in eine Elektroporation Küvette (0,2 cm Spaltbreite). </li><li> Tragen Sie einen Puls bei 1,6 kV. </li><li> Fügen Sie 0,9 ml SOC-Medium, übertragen Sie leicht Zelle zu einer Standard-14-ml-Tube. Inkubieren unbewegten für 2,5 bis 3 Stunden bei 30 ° C. </li><li> Tafel 100 ul und 300 ul auf LB-Platten mit 100 ug / ml Ampicillin und inkubieren bei 30 ° C über Nacht. </li><li> Entschlacken einzigen Klon und speichern wie Glycerin Lager für zukünftige TransFLP Experimente. </li></ol> 3. Oligonukleotid Design und Polymerase-Kettenreaktion <ol><li> Mindestens sechs Oligonukleotiden erforderlich sind, um die DNA-Region (en) von Interesse als auch die FRT-Kassette flankiert Antibiotikums durch PCR (Abbildung 3) zu amplifizieren. Es empfiehlt sich, auch ein Paar von Oligonukleotiden Priming außerhalb des PCR-Fragment eingefügt. Dies ermöglicht die Überprüfung der Integration und Excision des korrekten FRT-flankiert Antibiotikaresistenz Kassette (z. B. als &quot;CHK-up&quot; und &quot;CHK-down&quot;-Primer in den 3 bis 5 dargestellt). </li><li> Entwerfen der Oligonukleotide # 2, # 3, # 4 und # 5 mit Sorgfalt. Sie sollten in der Lage sein, ausreichend annealen (zB ~ 28 bp) an die Template-DNA. Der Template-DNA genomische DNA ist (gDNA) zum Primern # 2 und # 5 und FRT-Kan-FRT-enthaltenden Plasmid-DNA, wie pROD17 12 und pBR-FRT-KAN-FRT (diese Studie) zum Primern # 3 und # 4 ( 3A). </li><li> Design Die Primer # 2 bis # 5 ermöglicht eine umfassende Basenpaarung an ihrem 5&#39;-Ende zwischen Nr. 2 / Nr. 3 und Nr. 4 / Nr. 5 bzw. (3A </strong&gt; Kasten). Zwei Runden PCR folgen. </li><li> Führen Sie drei unabhängige PCR-Reaktionen als 1 Runde der PCR. Die erste wird amplifizieren die upstream-Region des Gens / DNA-Bereich-von-Interesse mit Hilfe der Oligonukleotide Nr. 1 und Nr. 2. Das PCR sollte in einem Fragment von mindestens 500 bp in der Länge um eine homologe Rekombination in den Zellen ermöglichen führen. Kürzere Fragmente (~ 250 bp) eine ausreichende 8 sein, aber werden nicht empfohlen. </li><li> Parallel führt die zweite PCR, die die FRT-Kassette flankiert Antibiotikum verstärkt. Für die Beispiele hier bereitgestellten wir hauptsächlich verwendet aph (kan R). Verwendung Oligonukleotiden # 3 und # 4 für diese Reaktion (Abbildung 3A). </li><li> Begleitend verstärken die stromabwärts gelegenen DNA-Bereich durch PCR (drittes Beispiel). Das PCR-Fragment sollte ebenfalls mindestens 500 bp in der Länge sein. Durchführen der PCR mit gDNA als Template und Oligonukleotide # 5 und # 6 (Abbildung 3A). </li><li> Nach Reinigung aller drei PCRFragmente, führen Sie die 2. Runde der PCR. Verwenden Sie die gleiche Mischung aus allen drei Fragmente in der ersten Runde als Vorlage erhalten. Die Verstärkung wird durch Oligonukleotide Nr. 1 und Nr. 6 katalysiert. Das resultierende PCR-Fragment (3B) als transformierende DNA in der natürlichen Transformation Experiment (Abbildung 1). </li></ol> 4. Chitin-induzierte Natural Transformation <ol><li> Wachsen V. cholerae Zellen aerob in Vollmedium bei 30 ° C, bis sie eine optische Dichte erreicht bei 600 nm von etwa 0,5. </li><li> Ernte der Bakterien durch Zentrifugieren. Waschen Pellet einmal in definierten künstlichen Medium (DASW 6) vor Resuspendieren der Zellen in 2 Volumina DASW. 1 ml der Kultur auf 50-80 mg steriles Chitin Flocken (erläutert unter Punkt 1). Wenn Bakterien Hafen Plasmid pBR-flp (optional Punkt 2), Add Ampicillin (50 ug / ml) zu der Kultur. Inkubation bei 30 ° C für 16-24 Stunden ohne Bewegung. </li><li> Add &amp; ge; 200 ng des 2 nd-Runde PCR-abgeleiteten Fragment (erläutert unter Punkt 3). Vorsichtig mischen, ohne ausgiebig Ablösen der Bakterien von den Chitin Oberflächen. Inkubieren bei 30 ° C für 24 Stunden ohne Bewegung. </li><li> Vortex die Kultur ausgiebig für ≥ 30 sec. Verteilt 100-300 ul auf selektiven LB-Medium-Platten (zB Kanamycin oder Gentamicin-haltigen LB-Platten für die bereitgestellten Beispiele hier). Verwenden Sie doppelt selektiven Platten, wenn die Bakterien, die das Plasmid pBR-flp indem Ampicillin gleichzeitig die übrigen Antibiotika beherbergen. Inkubieren Platten bei 30 ° C für 16-24 h oder bis Kolonien sichtbar. </li><li> Isolieren einzelner Transformanten von selektiven Platten. Solche Transformanten haben den ursprünglichen chromosomalen Locus durch die PCR-Fragment durch eine doppelte Crossover-Ereignis ersetzt. Diese Stämme können direkt für weitere Versuche verwendet werden, falls das Entfernen des Antibiotikums Kassette nicht notwendig ist. </li></ol> 5. Entfernung von Selective Cassette (s) durch Flp-Rekombination <ol><li> Künstlich verwandeln Sie Ihr Transformanten mit Plasmid pBR-flp wie unter Punkt 2 beschrieben, wenn nicht vor dem natürlichen Transformation Assay. </li><li> Die Bakterien wachsen auf LB-Agarplatten mit Ampicillin bei 37 ° C für 16-24 Stunden. Optionen: Ändern Sie die Temperatur zwischen 2-3 Stunden auf 40 ° C als Ausdruck flp aus dem Plasmid pBR-flp bei höheren Temperaturen 5,10 de-unterdrückt; Übertragung Bakterien auf frische Platten nach ca. 8 h Inkubation. </li><li> Test für Antibiotika-Empfindlichkeit (hier für Kanamycin gezeigt; Abbildung 1) durch restreaking die Klone parallel auf Antibiotika-haltigen und Antibiotika-freien Agarplatten. Isolieren einzelner sensitive Kolonien. Frieren Ampicillin-resistente Klon als Glycerin Lager, wenn Sie weiter zu modifizieren die Belastung mit anderen Deletion / Insertion mit TransFLP wollen. </li></ol> 6. Plasmid Curing <ol><li> Wachsen Kultur über Nacht unter aeroben Bedingungen undbei 30 ° C. Verwenden Vollmedium ohne Zusatz von Antibiotika. </li><li> Optional: Wachsen frische Kultur für 3-6 Stunden durch Verdünnen der Übernachtkultur 1:100 in frischem Antibiotika-freien Mediums. </li><li> Platte oder Streifen Verdünnung (n) der Kultur auf einfache LB-Agarplatte (en) und Inkubation bei 30 ° C für 8-16 h (bis Kolonien sichtbar sind). </li><li> Test auf Ampicillin-Sensitivität der Klone durch restreaking die Klone, die parallel auf Antibiotikum-haltigen und Antibiotika-freien Agarplatten (Abbildung 1). Frieren Ampicillin-sensitiven Stamm wie Glycerin Lager. </li></ol>

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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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Keine Interessenskonflikte erklärt.

Die TransFLP oben beschriebenen Methode und anderswo 5 wurde ausgiebig in unserem Labor verwendet. Die Art der genetischen Manipulationen, die durchführbar sind, umfassen unter anderem: Deletion einzelner Gene und Gencluster, Deletion Genominseln (zB VPI-1), Insertion von Sequenzen stromaufwärts eines Gens von Interesse (z. B. Promotorsequenzen) und Einführen Sequenzen am Ende eines spezifischen Gens (zB Codierung Affinitätstags). Ein Import Vorausse...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Chitin Flocken </td><td> Sigma </td><td> C9213 </td><td> Autoklavieren erforderlich </td></tr><tr><td> OPTIONAL: Micropulser (bei Antibiotika-Kassette sollte durch Flp Rekombinase auf pBR-flp kodiert herausgeschnitten werden) </td><td> Biorad </td><td> 165-2100 </td><td> Oder vergleichbare electroporators </td></tr></tbody></table>

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

Mehrere Methoden stehen zur Verfügung, um zu manipulieren bakteriellen Chromosomen 1-3. Die meisten dieser Protokolle beruhen auf der Einbringung von konditional replizierende Plasmide (z. beherbergen pir-abhängigen oder temperaturempfindlichen Replikons 1,2). Diese Plasmide sind in bakteriellen Chromosomen auf Homologie-vermittelte Rekombination integriert. Solche Insertions-Mutanten werden häufig direkt in der experimentellen Einstellungen verwendet. Alternativ kann Selektion auf Plasmid Exzision durch seinen Verlust gefolgt durchgeführt werden, die für Gram-negative Bakterien häufig der Zähler wählbare Levansucrase Enzyms durch die sacB Gen 4 codiert wird. Die Excision kann entweder wieder die vor dem Einsetzen Genotyp oder führen zu einem Austausch zwischen dem Chromosom und dem Plasmid-kodierten Kopie des modifizierten Gens. Ein Nachteil dieser Technik ist, dass es zeitraubend ist. Das Plasmid muss zuerst geklont werden, sondern erfordert horizontalen Transfer in V. cholerae (höchstens n<html> otably durch Paarung mit einem E. coli Donor-Stamm) oder künstliche Umwandlung der letzteren, und die Exzision des Plasmids ist zufällig und kann entweder wieder die anfängliche Genotyp oder erzeugen die gewünschte Modifikation, wenn kein positiver Selektion ausgeübt wird. Hier präsentieren wir eine Methode für die schnelle Manipulation der V. cholerae-Chromosom (s) 5 (Abbildung 1). Diese TransFLP Verfahren basiert auf der kürzlich entdeckten Chitin-vermittelte Induktion der natürliche Kompetenz in diesem Organismus 6 und anderen Vertreter der Gattung Vibrio wie V. basierend fischeri 7. Natürliche Kompetenz ermöglicht die Aufnahme von freier DNA einschließlich PCR-generierten DNA-Fragmenten. Sobald aufgenommen, rekombiniert die DNA mit dem Chromosom bei Vorliegen einer minimalen von 250-500 bp der flankierenden homologen Bereich 8. Einschließlich einen Selektionsmarker in-zwischen diesen flankierenden Regionen ermöglicht eine einfache Erkennung von häufig auftretenden Transformanten. t &quot;&gt; Diese Methode kann für verschiedene genetische Manipulationen von V. cholerae verwendet werden und möglicherweise auch andere natürlich kompetente Bakterien. Wir bieten drei neue Beispiele, was man mit dieser Methode zusätzlich zu unseren bereits veröffentlichten Studie auf einzelne Gen-Deletionen und durchgeführt werden Neben der Affinität-tag-Sequenzen 5. Mehrere Optimierungsschritte über die erste Protokoll von Chitin-induzierte natürliche Transformation 6 sind in diesem TransFLP Protokoll aufgenommen. Dazu gehören unter anderem der Austausch von Krabben Granatsplitter durch kommerziell erhältliche Chitin Flocken 8, die Spende von PCR abgeleiteten DNA als transformierenden Materials 9, und die Addition von FLP-Rekombination Zielstellen (FRT) 5. FRT erlauben ortsspezifische Exzision des Selektionsmarkers durch die Rekombinase Flp 10 vermittelt.

Repräsentative Ergebnisse der drei Beispiele sind in den 4 bis 6 gezeigt. Der erste Ansatz, der die Streichung der benachbarten Gene ctxA und ctxB soll. Gemeinsam codieren die wichtigsten Virulenzfaktor von V. cholerae, Cholera-Toxin. Wir entwickelten Oligonukleotiden zur TransFLP Verfahren wie oben beschrieben und gemäß 3. Die elterliche Stamm wurden die Zwischenschicht Stamm, die FRT-flankiert Antibiotikaresistenz Kassette in der ursprün...

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

Eine schnelle Methode, um das Genom zu ändern<em&gt; V. cholerae</em&gt; Beschrieben. Diese Änderungen umfassen das Löschen von einzelnen Genen, Gen-Cluster und genomische Inseln sowie die Integration von kurzen Sequenzen (zB Promotorelemente oder Affinitäts-tag-Sequenzen). Das Verfahren basiert auf der natürlichen Transformation und FLP-Rekombination.

TransFLP - Eine Methode zur genetischen Veränderung<em&gt; Vibrio cholerae</em&gt; On Natural Transformation und FLP-Rekombination

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

transflp-method-to-genetically-modify-vibrio-cholerae-based-on

Research

JoVE Journal

Immunology and Infection

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

14.5K Aufrufe.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Eine schnelle Methode, um das Genom zu ändern V. cholerae Beschrieben. Diese Änderungen umfassen das Löschen von einzelnen Genen, Gen-Cluster und genomische Inseln sowie die Integration von kurzen Sequenzen (zB Promotorelemente oder Affinitäts-tag-Sequenzen). Das Verfahren basiert auf der natürlichen Transformation und FLP-Rekombination.

Serie: 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.

Colonization von Euprymna scolopes Squid durch Vibrio fischeri</em

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

Forschung

Immunologie und Infektion

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.

A Parasite Rescue and Transformation Assay auf leishmanientötende Screening gegen intrazelluläre<em&gt; Leishmania donovani</em&gt; Amastigoten in THP1 Menschliche Akute Monozytenleukämie 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 ...

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.

Klinische Anwendung der<em&gt; Dornröschen</em&gt; Und künstliche Antigen präsentierenden Zellen genetisch zu verändern T Zellen aus dem peripheren und Nabelschnurblut

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-Resolution Imaging der natürlichen Killerzell Immunologische Synapse auf einem Glas-gestützten Planar Lipiddoppelschicht

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.

Implementierung eines Systems durchlässige Membran Insert-basierten Infektion der Auswirkungen von sekretierten bakteriellen Toxins zur Untersuchung auf Säugetierwirtszellen

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.

Entwicklung eines sensitiven und spezifischen chromogenen Agar Medium zum Nachweis von<em&gt; Vibrio parahaemolyticus</em&gt; Und andere<em&gt; Vibrio</em&gt; Arten

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.

Eine Plattform von Assays Anti-Biofilm auf die Erforschung der natürlichen Substanzbibliotheken Geeignet

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.

Fluoreszenz-basierte Neuraminidase-Inhibitionsassay die Empfänglichkeit der Influenza-Viren an die Neuraminidase-Inhibitor-Klasse von Anti-Virus zu beurteilen

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.

Labortechniken zur Aufrechterhaltung und Differenzierung von Biotypen von<em&gt; Vibrio cholerae</em&gt; Klinische und umweltliche Isolate

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.

Ein neuartiges Feeder-freie System für die Massenproduktion von murinen 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 ...