Ben teknik yardım için Olga de Souza Silva kabul etmek istiyorum. Bu çalışma, İsviçre Ulusal Bilim Vakfı (SNSF) Hibe 31003A_127029 tarafından desteklenmiştir.

TransFLP yöntem (Şekil 1) üç farklı yaklaşımlar ile örneklenmiştir: I), V nin ölümcüllüğünün belirleyicilerini kodlayan genin iki bitişik silme cholera (örneğin, ΔctxAB), II) bir patojenisite ada (örn., ΔVPI-1) çıkarılması ve III) T7 RNA polimeraz promotörü sekans bağlı olan entegre bir gen yukan-of-ilgi, daha sonra izin veren, ilgili gerginlik arka planda yapay ifadesi (örneğin, T7-tfoX) (Şekil 2). 1. Chitin Taneli hazırlanması <ol><li> Standart bir 1.5 ml plastik tüp içinde kitin gevreği ağırlığı 50-80 mg. Bu, toplu olarak hazırlanabilir. Chitin pul Sigma (kat. numarası C9213) dan ticari olarak temin edilebilir. </li><li> Tüplerin kapaklarını açık tutarak gevreği otoklavlayınız. </li><li> Otoklav soğuduktan hemen sonra kapaklarını kapatın. </li><li> Mağaza otoklavlanmış kitin gevreğioda sıcaklığında. </li></ol> 2. Opsiyonel: V. Yapay Dönüşüm FLP Rekombinaz kodlama Plazmid ile cholerae <ol><li> Electrocompetent hazırlayın V. standart yöntemler kullanılarak 11 cholerae hücreleri. -80 Yetenekli hücrelerin alikotları saklayın ° C. </li><li> Electrocompetent plazmid pBR-bdp 5 düzenli bir mini hazırlık 1-2 ul ekleyin V. cholera hücresi ve bir elektroporasyon küvetine (0.2 cm boşluk genişliği) karışımı içine aktarın. </li><li> 1.6 kV bir darbe uygulayın. </li><li> 0.9 mL SOC orta ekleyin, hafifçe bir standart 14 ml tüp hücre aktarın. 30 2,5-3 saat süreyle hareketsiz inkübe ° C. </li><li> Levha 100 ul ve 30 ° C&#39;da gece boyunca 100 ug / ml ampisilin ihtiva eden LB ve inkübe plakalar üzerinde 300 ul. </li><li> Gelecekte TransFLP deneyler için gliserol stok olarak tek bir klondan ve mağaza arındırın. </li></ol> 3. Oligonükleotid Tasarım ve Polimeraz Zincir Reaksiyonu <ol><li> En az altı oligonükleotidler ilgilenilen DNA bölge (ler) de olduğu gibi PCR ile FRT yanlı antibiyotik kaseti (Şekil 3) yükseltmek için gereklidir. Aynı zamanda eklenen PCR parçası dış astar oligonükleotidler bir çift eklemek için tavsiye edilir. Bu entegrasyon ve FRT-sardığı antibiyotik direnç kasedi (örn. Şekil 3 ila 5 &#39;chk-up&#39; ve &#39;chk-down&#39; primerleri olarak tasvir) doğru eksizyon için kontrol sağlar. </li><li> Oligonükleotidler # 2, # 3, # 4 ve # 5 dikkatle tasarlayın. Onlar yeterince şablon DNA (örn. ~ 28 bp) tavlama gerekir. Şablon DNA primerleri için genomik DNA (gDNA) gibi pROD17 12 ve pBR-FRT-KAN-FRT (Bu çalışma) primerleri için # 3 ve # 4 (gibi # 2 ve # 5 ve FRT-Kan-FRT-içeren plazmid DNA Şekil 3A). </li><li> Izin # 5 primerler # 2 tasarımı sırasıyla, # 2 / # 3 ve # 4 / # 5 arasındaki 5&#39;-uç taban eşleştirme geniş (Şekil 3A </strong&gt; inset). PCR İki mermi izleyin. </li><li> PCR 1. yuvarlak olarak üç bağımsız PCR reaksiyonları gerçekleştirin. Ilk olarak bir oligonükleotidler # 1 ve # 2 yardımıyla gen / DNA bölge-of-ilgi yukan bölgesi yükseltecektir. PCR hücreleri içinde homolog rekombinasyon izin vermek için uzunluk olarak en az 500 bp&#39;lik bir fragmanı ile sonuçlanmalıdır. Shorter parçaları (~ 250 bp) yeterli 8 olabilir ama tavsiye edilmez. </li><li> Paralel olarak FRT-sardığı antibiyotik kaset yükselterek ikinci PCR gerçekleştirmek. Örnekler burada sağlanan için biz esas aph (kan R) kullanılmıştır. Kullanım oligonükleotidler bu reaksiyon (Şekil 3A) için # 3 ve # 4. </li><li> Eş zamanlı PCR (üçüncü örnek) tarafından aşağı DNA bölgesinde yükseltmek. PCR parçası da uzunluğu en az 500 bp olması gerekir. Şablon ve oligonükleotidler # 5 ve # 6 (Şekil 3A) gibi gDNA ile PCR gerçekleştirin. </li><li> Her üç PCR saflaştırma sonrasındafragmanlar, PCR 2. turda gerçekleştirmek. Şablon olarak ilk turda elde edilen her üç parçaları eşit karışımı kullanın. Amplifikasyon oligonükleotidler # 1 ve # 6 tarafından katalize edilir. Ortaya çıkan PCR parçası (Şekil 3B) doğal transformasyon deneyi (Şekil 1) DNA dönüşüm olarak hizmet vermektedir. </li></ol> 4. Chitin indüklenen Doğal Dönüşüm <ol><li> Büyümek V. 30 zengin besiyerinde aerobik cholerae hücreleri ° C de yaklaşık 0.5 600 nm&#39;de absorbansı ulaşana kadar. </li><li> Santrifüjleme ile hasat bakteri. DASW 2 cilt hücreleri resuspending önce tanımlanan yapay ortam (DASW 6) kez pelet yıkayın. Steril kitin gevreği (nokta 1 altında açıklanan) 50-80 mg kültürü 1 ml ekleyin. Kültür ise bakteriler liman plazmid pBR-bdp (opsiyonel noktası 2), eklenti ampisilin (50 mg / ml). Hareket olmadan 16-24 saat süre ile 30 ° C&#39;de inkübe edin. </li><li> G &amp; Eklee; 2. boyunca PCR-türetilmiş fragmanı (Madde 3 altında açıklanmıştır) 200 ng. Yoğun kitin yüzeylerinden bakteri sökmeden dikkatlice karıştırın. Hareket olmadan 24 saat boyunca 30 ° C&#39;de inkübe edin. </li><li> ≥ 30 sn için yoğun Vorteks kültür. Seçici LB besiyeri yaprakları (burada sağlanan örnekler örneğin kanamisin veya gentamisin içeren LB plakalar) üzerine 100-300 ul yayılır. Bakteri diğer antibiyotiklere beraber ampisilin ekleyerek pBR-bdp plazmid liman eğer çift seçici tabak kullanın. 16-24 saat boyunca 30 ° C&#39;de inkübe plakaları veya koloniler görünür oluncaya kadar. </li><li> Seçici plakaları tek transformantlar ayırın. Böyle transformantlar çift çapraz olay nedeniyle PCR parçası tarafından özgün kromozomal lokus yerini almıştır. Antibiyotik kaset kaldırılması gerekli değildir, ve dolayısıyla bu suşlar direkt olarak başka deneyler için de kullanılabilir. </li></ol> 5. FLP tarafından Seçici Kaset giderimi (ler)-Rekombinasyon <ol><li> Doğal dönüşümü tahlilinden önce yapmadıysanız 2. maddede açıklandığı gibi yapay pBR-bdp plazmid ile transformantlar dönüşümü. </li><li> 16-24 saat boyunca 37 ° C&#39;de ampisilin içeren LB agar plaklarına bakteriler büyümek. Seçenekler: 40 2-3 saat süreyle arasındaki sıcaklık değiştirmek ° C plazmid pBR-bdp den bdp ifadesi olarak yüksek sıcaklıklarda 5,10 de-de bastırılanlardır; yakl taze plakaları transfer bakteri. Inkübasyon 8 sa. </li><li> Antibiyotik içeren ve antibiyotik içermeyen agar plaklarına paralel klonlar restreaking tarafından; antibiyotik duyarlılık (Şekil 1 kanamisin için buraya gösterdi) için test. Tek duyarlı koloniler izole edin. Eğer daha başka silme / TransFLP kullanarak ekleme ile gerginlik değiştirmek istiyorsanız, gliserol stok olarak ampisiline dirençli klon dondurun. </li></ol> 6. Plazmid Kür <ol><li> Aerobik koşullar altında gecede kültür büyütün ve30 ° C&#39;de Herhangi bir antibiyotik ilave olmadan zengin ortamı kullanın. </li><li> Opsiyonel: Taze antibiyotik içermeyen ortamda gecede kültürü 1:100 sulandırarak 3-6 saat süreyle taze kültür büyütün. </li><li> Levha veya çizgi düz LB agar plaka (lar) kültür seyreltme (lar) ve 30 inkübe ° 8-16 saat süreyle C (koloniler görebilir kadar). </li><li> Antibiyotik içeren ve antibiyotik içermeyen agar plakaları (Şekil 1) üzerinde paralel olarak klonlar restreaking ile klon ampisilin-duyarlılık için test edin. Gliserol stok olarak ampisilin duyarlı suşu dondurun. </li></ol>

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Çıkar çatışması ilan etti.

TransFLP yöntemi 5 yoğun laboratuarımızda kullanılmıştır yukarıda ve yerde anlatılmıştır. Mümkün olan genetik manipülasyon tür diğerlerinin yanı sıra şunları içerir: tek genler ve gen kümelerinin silinmesi, genomik adalar silme (örn., VPI-1), ilgilenilen bir gen (örneğin, promoter dizileri) yukarı akış yönünde dizilerin yerleştirilmesi ve ekleme , belirli bir gen (örneğin, kodlama afinite etiketleri) sonunda dizileri. T...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Chitin gevreği </td><td> Sigma </td><td> C9213 </td><td> Gerekli Otoklavlama </td></tr><tr><td> İSTEĞE BAĞLI: Micropulser (durumda antibiyotik kaset pBR-bdp kodlanmış FLP Rekombinaz tarafından eksize edilmesi gerekir) </td><td> BioRad </td><td> 165-2100 </td><td> Veya karşılaştırılabilir electroporators </td></tr></tbody></table>

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

Çeşitli yöntemler bakteri kromozomu 1-3 işlemek için kullanılabilir. Bu protokollerin çoğu (örneğin, pir-bağımlı veya ısıya duyarlı Replikon 1,2 yataklık) şartlı replikatif plazmid ekleme güveniyor. Bu plazmid homoloji-aracılı rekombinasyon bazlı bakteri kromozomu içine entegre edilmiştir. Bu tür mutantlar insersiyonal çoğu kez direkt olarak deney ortamlarda kullanılır. Alternatif olarak, onun kaybı, ardından plazmid çıkarılması için seçim sık sık SACB gen tarafından kodlanan 4 karşı tarafından seçilebilir levan sukraz enzimi dayanır Gram-negatif bakteriler için, hangi gerçekleştirilebilir. Eksizyonu ya da ön-yerleştirme genotip ya da kromozom ve modifiye geninin plazmid ile kodlanmış kopya arasında bir değişim ile sonuçlanır geri olabilir. Bu yöntemin bir dezavantajı, zaman alıcı olmasıdır. Plazmid ilk klonlanmış gerekmektedir; bunu V. içine yatay transferi gerektirir cholerae (en fazla n<html> otably E ile çiftleştirilerek coli suşu verici) ya da ikinci bir yapay dönüştürülmesi; ve plazmid eksizyonu ve rasgele ya da ilk genotip geri ya da herhangi bir pozitif seçim sarf edilir ise, istenen değişikliği oluşturabilir. Burada, V. hızlı manipülasyon için bir yöntem mevcut cholerae kromozom (lar) 5 (Şekil 1). Bu TransFLP yöntem, V. gibi cins Vibrio bu organizmanın 6 ve diğer temsilci doğal yetkinlik yeni keşfedilen kitin-aracılı indüksiyon dayanmaktadır fischeri 7. Doğal yetkinlik PCR tarafından oluşturulan DNA parçaları dahil olmak üzere ücretsiz DNA alımını sağlar. Bir kez alınır, DNA homolog bölgenin 8 sınırdaş 250-500 bp asgari varlığını verilen kromozomu ile yeniden birleştirilmesi kastedilmektedir. Bu komşu bölgeleri arasındaki bir seçim işaretleyici İçeren sık görülen transformantlar kolay algılanmasını sağlar. T &quot;&gt; Bu yöntem, V. cholerae, farklı genetik manipülasyonu için kullanılabilir ve potansiyel olarak da diğer doğal olarak yeterli bakteri olabilir. bizim daha önce yayınlanmış bir gen silme üzerinde çalışma ve ek olarak, bu yöntem tarafından gerçekleştirilebilir ne üç yeni örnekler sağlamak benzerlik etiketlenmesi sekansları 5 ek. kitin kaynaklı doğal dönüşüm 6 başlangıç ​​protokolünü ilgili birçok iyileştirme adımlar bu TransFLP protokol içinde bu buluşa katılmıştır. Bunlar, diğerlerinin arasında ticari olarak mevcut kitin pul 8, PCR tarafından yardım yengeç kabuk parçalarından replasman, madde 9, ve FLP-rekombinasyon hedef sitesi (FRT) 5 transforme ilave olarak türetilmiş DNA. FRT sitesi FLP rekombinaz 10 tarafından aracılık seçim işaretleyicinin her bölge-yönlendirmeli eksizyon izin verir.

Üç örnek Örnek 6 için sonuçlar Şekil 4&#39;te gösterilmiştir. İlk yaklaşım komşu genlerin CTxA ve ctxB silinmesi hedeflenmiştir. Birlikte V. önemli virulans faktörü kodlayan cholerae kolera toksin. Yukarıda tarif edilen ve Şekil 3&#39;e uygun olarak biz TransFLP yöntem için oligonükleotidler tasarlanmıştır. Ebeveyn zorlanma, ctxAB orijinal DNA lokusu ve ctxAB için silinir ve direnç kasedi k...

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

Arasında genomunu değiştirmeye hızlı bir yöntem<em&gt; V. cholerae</em&gt; Tarif edilmektedir. Bu değişiklikler tek genlerin, gen kümeleri ve genomik adaları gibi kısa dizileri (örn. promotor elemanlarına veya benzeşim-etiket dizileri) ve entegrasyon silme içerir. Yöntem, doğal transformasyon ve FLP-rekombinasyon dayanmaktadır.

TransFLP - Genetiği değiştirmek için bir yöntem<em&gt; Vibrio cholerae</em&gt; Doğal Dönüşüm ve FLP-rekombinasyon sonucu

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

14.6K Görüntüleme.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Arasında genomunu değiştirmeye hızlı bir yöntem V. cholerae Tarif edilmektedir. Bu değişiklikler tek genlerin, gen kümeleri ve genomik adaları gibi kısa dizileri (örn. promotor elemanlarına veya benzeşim-etiket dizileri) ve entegrasyon silme içerir. Yöntem, doğal transformasyon ve FLP-rekombinasyon dayanmaktadır.

Video: TransFLP — 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.

Kolonizasyon Euprymna scolopes Squid tarafından Vibrio fischeri</em

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

İmmünoloji ve Enfeksiyon

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.

Hücre içi Karşı Antileishmanial Tarama için Parasite Kurtarma ve Dönüşüm Testi<em&gt; Leishmania donovani</emTHP1 İnsan Akut Monositik Lösemi Hücre Hattı&gt; Amastigotes

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.

Klinik Uygulama<em&gt; Sleeping Beauty</emGenetiği Periferik ve Göbek Kordon Kanı alınan T hücreleri Değiştir Sunan Hücreler&gt; ve Yapay Antijen

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.

Bir Cam destekli Planar çift katlı lipid Doğal Öldürücü Hücre İmmünoloji Synapse Süper çözünürlüklü Görüntüleme

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.

Bir Geçirgen Membran Ekle tabanlı Enfeksiyon Sisteminin Uygulanması memeli konakçı hücreleri üzerinde salgılanan Bakteriyel Toksinler Etkilerinin Eğitim için

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.

Tespiti için daha duyarlı ve özgül Chromogenic Agar Orta Gelişimi<em&gt; Vibrio parahaemolyticus</em&gt; Ve Diğer<em&gt; Vibrio</em&gt; Türler

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

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.

Floresans tabanlı Nöraminidaz İnhibisyonu Tahlili antivirallerin nöraminidaz inhibitörü Class Nezle Virüslerinden eğilimi değerlendirilebilir

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.

Biyotiplerin Bakımında ve Türevinde Kullanılan Laboratuvar Teknikleri<emVibrio cholerae</em&gt; Klinik ve Çevresel İzolatlar

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

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

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.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

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 ...

TransFLP - Genetiği değiştirmek için bir yöntem

Özet

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Bu videodaki bölümler

Kolonizasyon Euprymna scolopes Squid tarafından Vibrio fischeri

Hücre içi Karşı Antileishmanial Tarama için Parasite Kurtarma ve Dönüşüm Testi

Klinik Uygulama

Bir Cam destekli Planar çift katlı lipid Doğal Öldürücü Hücre İmmünoloji Synapse Süper çözünürlüklü Görüntüleme

Bir Geçirgen Membran Ekle tabanlı Enfeksiyon Sisteminin Uygulanması memeli konakçı hücreleri üzerinde salgılanan Bakteriyel Toksinler Etkilerinin Eğitim için

Tespiti için daha duyarlı ve özgül Chromogenic Agar Orta Gelişimi

Floresans tabanlı Nöraminidaz İnhibisyonu Tahlili antivirallerin nöraminidaz inhibitörü Class Nezle Virüslerinden eğilimi değerlendirilebilir

Biyotiplerin Bakımında ve Türevinde Kullanılan Laboratuvar Teknikleri

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için