وأود أن أنوه أولغا سيلفا دي سوزا للحصول على المساعدة الفنية. وأيد هذا العمل من قبل المؤسسة الوطنية للعلوم السويسري (SNSF) منحة 31003A_127029.

ويتمثل الأسلوب TransFLP (الشكل 1) من ثلاثة مناهج مختلفة: I) حذف اثنين من الجينات المجاورة ترميز المحددات الفوعة من V. الكوليرا (على سبيل المثال، ΔctxAB)؛ II) إزالة جزيرة المرضية (مثل ΔVPI-1)، وIII) إدماج بوليميراز RNA T7 التي تعتمد على تسلسل المروج المنبع من الجينات المصلحة من الذي يسمح له لاحقا اصطناعية التعبير في الخلفية سلالة منها (على سبيل المثال، T7-tfoX) (الشكل 2). 1. إعداد رقائق كيتين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الوزن 50-80 ملغ من رقائق كيتين في مستوى 1،5 مل أنبوب بلاستيكي. يمكن إعداد هذه بكميات كبيرة. كيتين رقائق متاحة تجاريا من سيغما (الفئة رقم C9213). </li><li style=";text-align:right;direction:rtl"> الأوتوكلاف رقائق الحفاظ على أغطية من الأنابيب مفتوحة. </li><li style=";text-align:right;direction:rtl"> إغلاق الأغطية على الفور بعد الأوتوكلاف وتبريده. </li><li style=";text-align:right;direction:rtl"> متجر تعقيمها كيتين رقائقفي درجة حرارة الغرفة. </li></ol> 2. اختياري: تحويل الاصطناعي V. الكوليرا مع FLP Recombinase ترميز البلازميد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد electrocompetent V. الكوليرا الخلايا باستخدام الطرق القياسية 11. تخزين مأخوذة من الخلايا المختصة في -80 ° C. </li><li style=";text-align:right;direction:rtl"> إضافة 1-2 ميكرولتر من إعداد مصغرة العادية للبلازميد PBR-FLP 5 إلى electrocompetent V. الخلايا الكوليرا ونقل الخليط إلى كوفيت الصعق الكهربائي (0.2 عرض الفجوة سم). </li><li style=";text-align:right;direction:rtl"> تطبيق النبض عند 1.6 كيلو فولت. </li><li style=";text-align:right;direction:rtl"> إضافة 0.9 مل SOC المتوسطة، ونقل بلطف الخلية إلى أنبوب القياسية مل 14. غير احتضان الحركة ل2،5 حتي 3 ساعة عند 30 ° C. </li><li style=";text-align:right;direction:rtl"> لوحة 100 ميكرولتر و 300 ميكرولتر على لوحات تحتوي على 100 ميكروغرام LB / مل الأمبيسلين واحتضان في 30 ° C بين عشية وضحاها. </li><li style=";text-align:right;direction:rtl"> تنقية استنساخ واحدة ومخزن والأسهم الجلسرين للتجارب TransFLP المستقبل. </li></ol> 3. قليل النوكليوتيد تصميم وتفاعل البلمرة سلسلة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مطلوب لا يقل عن ستة [أليغنوكليوتيد] لتضخيم المنطقة DNA (ق) من الفائدة وكذلك FRT-يحيط كاسيت المضادات الحيوية عن طريق PCR (الشكل 3). فمن المستحسن أن تدرج أيضا زوج من خارج فتيلة أليغنوكليوتيد] جزء PCR المدرجة. هذا يسمح التحقق من التكامل والختان الصحيح للكاسيت-FRT يحيط المقاومة للمضادات الحيوية (مثل صورت على أنها &quot;CHK إلى أعلى &#39;والاشعال&quot; CHK إلى أسفل &quot;في أرقام 3 إلى 5). </li><li style=";text-align:right;direction:rtl"> تصميم أليغنوكليوتيد] # 2، # 3، # 4، # 5 وبعناية. ينبغي أن تكون قادرة على يصلب بما فيه الكفاية (على سبيل المثال ~ 28 بي بي) إلى DNA القالب. الحمض النووي القالب الحمض النووي الجيني (gDNA) لالاشعال رقم 2 ورقم 5 و FRT-اساسه-FRT التي تحتوي على الحمض النووي البلازميد مثل pROD17 12 و PBR-FRT-KAN FRT-(هذه الدراسة) لالاشعال رقم 3 ورقم 4 ( الرقم 3A). </li><li style=";text-align:right;direction:rtl"> تصميم الاشعال رقم 2 لرقم 5 واسعة تسمح قاعدة الاقتران في نهاية 5&#39;ما بين رقم 2 / رقم 3 ورقم 4 / # 5 على التوالي (الشكل 3A </stronز&gt; الشكل). جولتين من PCR متابعة. </li><li style=";text-align:right;direction:rtl"> أداء ثلاث تفاعلات PCR مستقلة والجولة 1 القديس PCR. وأول واحد تضخيم المنطقة المنبع للجين / DNA المنطقة في المصالح مع المعونة من [أليغنوكليوتيد] رقم 1 ورقم 2. ينبغي أن يؤدي إلى PCR جزء من شركة بريتيش بتروليوم لا يقل عن 500 في الطول للسماح إعادة التركيب مثلي داخل الخلايا. يمكن أقصر شظايا (~ 250 BP) من 8 كافية ولكن لا ينصح. </li><li style=";text-align:right;direction:rtl"> بالتوازي أداء PCR الثانية، التي تزيد من الكاسيت FRT-يحيط المضادات الحيوية. لتقديم أمثلة ونحن هنا تستخدم أساسا APH (اساسه R). [أليغنوكليوتيد] استخدام رقم 3 ورقم 4 لهذا التفاعل (الشكل 3A). </li><li style=";text-align:right;direction:rtl"> وفي نفس الوقت، تضخيم المنطقة DNA بواسطة PCR المصب (عينة الثالثة). يجب أن يكون جزء PCR أيضا لا يقل عن 500 شركة بريتيش بتروليوم في الطول. أداء PCR مع gDNA كقالب وأليغنوكليوتيد] # 5 و # 6 (الشكل 3A). </li><li style=";text-align:right;direction:rtl"> بعد تنقية جميع PCR 3شظايا، نفذ الجولة الثانية 2 من PCR. استخدام خليط متساو من ثلاثة اجزاء على جميع الحصول عليها في الجولة الأولى كقالب. يتم تحفيز التضخيم من قبل أليغنوكليوتيد] رقم 1 ورقم 6. جزء PCR الناتجة (الشكل 3B) بمثابة تحويل DNA في التجربة التحول الطبيعي (الشكل 1). </li></ol> 4. كيتين الناجم عن التحول الطبيعي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو V. الكوليرا في الخلايا الهوائية المتوسطة الغنية في 30 ° C حتى تصل إلى الكثافة البصرية في 600 نانومتر تقريبا 0.5. </li><li style=";text-align:right;direction:rtl"> حصاد البكتيريا عن طريق الطرد المركزي. غسل بيليه مرة واحدة في المتوسط ​​اصطناعية محددة (DASW 6) قبل resuspending الخلايا في 2 مجلدات من DASW. إضافة 1 مل من ثقافة إلى 50-80 ملغ من رقائق كيتين العقيمة (تحت أوضح نقطة 1). إذا البكتيريا الميناء البلازميد PBR-FLP (اختياري نقطة 2)، إضافة الأمبيسلين (50 ميكروغرام / مل) إلى الثقافة. احتضان في C ° 30 ساعة ل16-24 دون حركة. </li><li style=";text-align:right;direction:rtl"> إضافة &amp; Gه و 200 نانوغرام من جزء الثانية على مدار PCR المستمدة 2 (شرح تحت النقطة 3). مزيج بعناية دون فصل على نطاق واسع من البكتيريا على أسطح كيتين. احتضان في C ° 30 لمدة 24 ساعة دون حركة. </li><li style=";text-align:right;direction:rtl"> دوامة الثقافة على نطاق واسع ل≥ 30 ثانية. نشر 100-300 ميكرولتر على انتقائية لوحات متوسطة LB (مثل لوحات أو الكاناميسين LB التي تحتوي على جنتاميسين للأمثلة المقدمة هنا). استخدام لوحات مزدوجة انتقائية إذا كان المرفأ البكتيريا البلازميد PBR-FLP بإضافة الأمبيسلين بصورة متزامنة إلى المضادات الحيوية الأخرى. احتضان لوحات في C ° 30 ساعة ل16-24 أو حتى المستعمرات مرئية. </li><li style=";text-align:right;direction:rtl"> عزل واحدة من لوحات transformants انتقائية. وحلت هذه transformants موضع الكروموسومات الأصلي من جزء PCR نتيجة لحدث كروس مزدوجة. ويمكن استخدام هذه السلالات مباشرة لمزيد من التجارب في حالة إزالة كاسيت المضادات الحيوية ليست ضرورية. </li></ol> 5. إزالة راديو كاسيت الانتقائية (ق) من FLPإعادة التركيب، <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحويل مصطنع transformants الخاص بك مع البلازميد PBR-FLP كما هو موضح في النقطة 2 إذا لم تفعل قبل الفحص التحول الطبيعي. </li><li style=";text-align:right;direction:rtl"> تنمو هذه البكتيريا على لوحات أجار LB C تحتوي على أمبيسلين في ل16-24 ° 37 ساعة. خيارات: تغيير درجة الحرارة في ما بين ساعة لمدة 2-3 إلى 40 ° C والتعبير عن FLP من البلازميد FLP-PBR ودي في قمع 5،10 ارتفاع درجات الحرارة؛ البكتيريا نقل لوحات جديدة بعد تقريبا. 8 ساعات من الحضانة. </li><li style=";text-align:right;direction:rtl"> اختبار الحساسية للمضادات الحيوية، (أظهر هنا لالكاناميسين؛ الشكل 1) من restreaking استنساخ بالتوازي على لوحات أجار المضادات الحيوية التي تحتوي على المضادات الحيوية والحرة. عزل المستعمرات الحساسة واحد. تجميد الأمبيسلين مقاومة للاستنساخ كما الجلسرين الأسهم إذا كنت تنوي تعديل المزيد من سلالة أخرى مع حذف / الإدراج باستخدام TransFLP. </li></ol> 6. بلازميد علاج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو بين عشية وضحاها الثقافة في الظروف الجوية وفي 30 ° C. استخدام المتوسطة الغنية دون إضافة أي مضاد حيوي. </li><li style=";text-align:right;direction:rtl"> اختياري: نمو ثقافة جديدة للموارد البشرية 3-6 من تمييع 1:100 الثقافة بين عشية وضحاها في وسط المضادات الحيوية خالية الطازجة. </li><li style=";text-align:right;direction:rtl"> لوحة خط أو التخفيف (ق) للثقافة في سهل LB وحة أجار (ق) واحتضان في 30 درجة مئوية لمدة 8-16 ساعة (حتى المستعمرات مرئية). </li><li style=";text-align:right;direction:rtl"> اختبار لالأمبيسلين الحساسية من الحيوانات المستنسخة من restreaking استنساخ بالتوازي على لوحات أجار المضادات الحيوية التي تحتوي على المضادات الحيوية وخالية من (الشكل 1). تجميد الأمبيسلين سلالة حساسة والأسهم الجلسرين. </li></ol>

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الإعلان عن أي تضارب في المصالح.

وصف طريقة TransFLP أعلاه وغيرها وقد تم على نطاق واسع 5 المستخدمة في مختبرنا. هذا النوع من التلاعب الجيني التي هي ممكنة، ضمن جملة أمور: حذف الجينات واحد ومجموعات الجينات، حذف الجزر الجيني (على سبيل المثال، VPI-1)، إدراج تسلسل المنبع من الجين في المصالح (على سبي...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> كيتين رقائق </td><td> سيجما </td><td> C9213 </td><td> التعقيم المطلوبة </td></tr><tr><td> OPTIONAL: Micropulser (في حال وينبغي رفعه من قبل المضادات الحيوية كاسيت recombinase FLP المشفرة على PBR-FLP) </td><td> Biorad </td><td> 165-2100 </td><td> أو قابلة للمقارنة electroporators </td></tr></tbody></table>

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

تتوفر العديد من الطرق لمعالجة الكروموسومات البكتيرية 1-3. معظم هذه البروتوكولات تعتمد على إدخال البلازميدات تنسخي مشروط (مثل إيواء replicons البير التي تعتمد على درجة الحرارة أو تراعي 1،2). يتم دمج هذه البلازميدات البكتيرية في الكروموسومات تقوم على التماثل بوساطة إعادة التركيب. وغالبا ما تكون مثل هذه المسوخ غرزي استخدامها مباشرة في إعدادات التجريبية. وبدلا من ذلك، يمكن إجراء الختان لاختيار البلازميد تليها خسارتها، والتي لالجراثيم سلبية الغرام تعتمد في أغلب الأحيان على انزيم مكافحة اختيار سكراز ليفان المشفرة بواسطة الجينات هيئة تنسيق المعونة 4. يمكن للالختان إما استعادة النمط الجيني ما قبل الإدراج أو تؤدي إلى تبادل بين كروموسوم ونسخة بلازميد ترميز الجين المعدلة. عيوب هذا الأسلوب هو أنه يستغرق وقتا طويلا. البلازميد لابد من استنساخ الأولى؛ أنه يتطلب نقل الأفقي إلى V. الكوليرا (ن معظم<html> otably من التزاوج مع E. سلالة الإشريكية المانحة) أو التحول الاصطناعي لهذه الأخيرة؛ واستئصال البلازميد هو عشوائي ويمكن إما استعادة النمط الجيني الأولي أو إنشاء التعديل المطلوب إذا لم يمارس اختيار إيجابية. هنا، نقدم طريقة للتلاعب السريع للV. الكوليرا كروموسوم (ق) 5 (الشكل 1). ويستند هذا الأسلوب على تحريض TransFLP كيتين بوساطة اكتشفت مؤخرا من الكفاءة الطبيعية في هذا الحي ممثل 6 و أخرى من جنس الضمة مثل V. fischeri 7. الكفاءة الطبيعية يسمح للامتصاص الحمض النووي DNA مجانا بما في ذلك شظايا ولدت PCR. تؤخذ مرة واحدة حتى، ويعيد توحيد DNA مع كروموسوم معين وجود ما لا يقل عن 250-500 من بي بي المرافقة مثلي المنطقة 8. منها علامة الاختيار في الفترات الفاصلة بين هذه المناطق المحيطة يسمح الكشف سهولة transformants التي تحدث في كثير من الأحيان. ر &quot;&gt; ويمكن استخدام هذه الطريقة لمعالجات وراثية مختلفة من الكوليرا V. وربما غيرها أيضا البكتيريا المختصة بشكل طبيعي. نحن نقدم أمثلة ثلاثة على رواية ما يمكن تحقيقه باستخدام هذا الأسلوب بالإضافة إلى دراستنا المنشورة سابقا على الحذف جين واحد وعلى إضافة علامة تقارب-متواليات 5. يتم دمج عدة خطوات التحسين بشأن بروتوكول الأولي من الكيتين التي يسببها التحول الطبيعي 6 في هذا البروتوكول TransFLP. وتشمل هذه وغيرها استبدال شظايا قذيفة السلطعون من رقائق كيتين المتاحة تجاريا 8، على التبرع PCR المستمدة من الحمض النووي وتحويل المواد 9، وإضافة المواقع المستهدفة FLP-إعادة التركيب (FRT) 5. مواقع FRT يسمح الختان الموقع موجه للعلامة الاختيار بوساطة recombinase FLP 10.

وتظهر نتائج ممثل في الأمثلة الثلاثة أرقام 4 إلى 6. النهج الأول يهدف إلى حذف الجينات المجاورة ctxA وctxB. معا ترميز عامل الفوعة الرئيسية للV. الكوليرا، بكتيريا الكوليرا. قمنا بتصميم [أليغنوكليوتيد] للأسلوب TransFLP كما هو موضح أعلاه وفقا للالشكل 3. سل...

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

وهناك طريقة سريعة لتعديل جينوم<em&gt; V. الكوليرا</emيوصف&gt;. وتشمل هذه التعديلات حذف الجينات واحد، مجموعات الجينات والجزر الجيني وكذلك التكامل من متواليات قصيرة (على سبيل المثال عناصر المروج أو تسلسل النسب، علامة). وتستند هذه الطريقة على التحول الطبيعي وإعادة التركيب FLP.

TransFLP - طريقة لتعديل وراثيا<em&gt; ضمة الكوليرا</em&gt; بناء على التحول الطبيعي وإعادة التركيب، FLP

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 Views.  Ecole Polytechnique Fédérale de Lausanne (EPFL). وهناك طريقة سريعة لتعديل جينوم V. الكوليرا. وتشمل هذه التعديلات حذف الجينات واحد، مجموعات الجينات والجزر الجيني وكذلك التكامل من متواليات قصيرة (على سبيل المثال عناصر المروج أو تسلسل النسب، علامة). وتستند هذه الطريقة على التحول الطبيعي وإعادة الترك?...

Video: 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.

استعمار Euprymna scolopes الحبار بواسطة الضمة fischeri</em

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

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 الفحص الإنقاذ والتحول لفحص مضاد الليشمانيات الطفيلي داخل الخلايا ضد<em&gt; الليشمانيا الدونوفانية</em&gt; Amastigotes في THP1 الحادة اللوكيميا الإنسان الوحيدات الخط الخليوي

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.

السريرية تطبيق<em&gt; الجمال النائم</em&gt; ومستضد تقديم الخلايا الاصطناعية لتعديل وراثيا خلايا T من دم الحبل السري والمحيطية

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.

فائقة الدقة التصوير من الخلايا القاتلة الطبيعية المناعية المشبك على دعم الزجاج مستو الدهن طبقة ثنائية

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.

تنفيذ نظام العدوى أساس إدراج-نافذ غشاء لدراسة آثار يفرز البكتيرية السموم على خلايا المضيف الثدييات

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.

تطوير أكثر حساسية وخصوصية اللونية آجار المتوسطة للكشف عن<em&gt; الضمة نظيرة الحالة للدم</em&gt; وغيره<em&gt; الضمة</em&gt; الأنواع

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.

ومنصة للفحوصات مكافحة بيوفيلم مناسبة لاستكشاف المكتبات مركب طبيعي

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.

مضان القائم على النورامينيداز تثبيط الفحص لتقييم مدى حساسية فيروسات الأنفلونزا إلى والنورامينيداز المانع فئة من الأدوية المضادة للفيروسات

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.

تقنيات المختبر تستخدم للحفاظ على والتمايز بيوتيبس<em&gt; ضمة الكوليرا</em&gt; العوازل السريرية والبيئية

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.

نظام تغذية خالية رواية للإنتاج الضخم للخلايا القاتلة الطبيعية مورين في المختبر

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 ...