وأيد هذا العمل من خلال منحة من جديد مبتكر برنامج جائزة مدير المعاهد الوطنية للصحة في (ED). وأيد AA في جزء من الجوائز البكالوريوس من المعهد الوطني للسرطان ومؤسسة العلوم الوطنية.

1. جعل النواقل الفيروسية تسلسل الترميز لهذا الجين من الفائدة، فضلا عن الضوابط الإيجابية والسلبية، تندس في pBABEpuro بالطرق التقليدية الاستنساخ (PCR التضخيم، والهضم تقييد وربط). هناك أربعة مواقع قيود على ناقلات حيث يمكن إدخال DNA: إنزيم BamHI، SnaBI، EcoRI وسالي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد ترنسفكأيشن الصف بلازميد DNA من خلايا المختصة باستخدام QIAGEN بلازميد ميدي عدة. </li><li style=";text-align:right;direction:rtl"> قياس تركيز الحمض النووي باستخدام معمل NanoDrop2000. </li></ol> 2. الارتجاعي الإنتاج سيتم استخدام خط الخلية التعبئة والتغليف بلات-E لإنتاج الفيروس الارتجاعي ecotropic التي سيلقي [كدنا] التي تهم خلايا NIH 3T3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء الثقافات بلات-E من خلايا مجمدة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الجليد بسرعة خلايا بلات-E في 37 ° C حمام الماء ونقل إلى 15 مل أنبوب مخروطي الشكل. إضافة ببطء 9 مل من بلات-E المتوسطة (دوlbecco في التعديل النسر متوسطة (DMEM) + 10٪ مصل بقري جنيني (FBS) + البنسلين والستربتوميسين). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الأنبوب في 180 × ز لمدة 5 دقائق وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> اعادة تعليق الخلايا مع 10 مل من بلات-E متوسطة ونقل إلى صحن ثقافة 10 سم. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا في 37 درجة مئوية، 5٪ CO 2 الحاضنة حتى تكون 80-90٪ متموجة (حوالي 2 أيام). </li></ol></li><li style=";text-align:right;direction:rtl"> تقسيم الخلايا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نضح المتوسطة ويغسل خلايا مرة واحدة مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> إضافة 2 مل من 0.05٪ التربسين-EDTA واحتضان لمدة 1 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> فصل الخلايا عن طريق الإصبع والتنصت، وإضافة 10 مل من بلات-E المتوسطة، ونقل تعليق خلية إلى أنبوب 15 مل. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الأنبوب في 180 × ز لمدة 5 دقائق وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا مع 10 مل من بلات-E المتوسطة والبذور منها في أطباق جديدة في 1: 4-1: 6 التخفيفات. </li></ol></li><li style=";text-align:right;direction:rtl"> بلات-E البذر وترنسفكأيشن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البذور 2 × 10 6خلايا لكل 10 سم صحن الثقافة باستخدام بلات-E وسيط وبدون أي المضادات الحيوية. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا O / N في 37 ° C، 5٪ CO 2 الحاضنة. </li><li style=";text-align:right;direction:rtl"> اليوم التالي، ونقل 300 ميكرولتر من غروب-MEM إلى 1.5 مل microfuge أنبوب. </li><li style=";text-align:right;direction:rtl"> إضافة 27 ميكرولتر من polyethylenimine (PEI)، وكاشف ترنسفكأيشن فعالة من حيث التكلفة 12، إلى أنبوب استعداد مع غروب-MEM. المزيج بلطف بواسطة الإصبع التنصت واحتضان لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> إضافة 9 ميكروغرام من DNA البلازميد ترنسفكأيشن الصف في أنبوب غروب-MEM / PEI، مزيج من قبل vortexing بلطف، واحتضان لمدة 15 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> إضافة DNA / PEI قطرة من الحكمة معقدة في طبق بلات-E، واحتضان O / N عند 37 درجة مئوية، و 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> اليوم التالي، نضح في المتوسط ​​تحتوي على كاشف ترنسفكأيشن وإضافة 10 مل من جديد بلات-E متوسطة (بدون المضادات الحيوية). </li><li style=";text-align:right;direction:rtl"> عودة إلى الخلايا الحاضنة. </li></ol></li></ol> 3. خلايا NIH 3T3 والعدوى <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء المعاهد الوطنية للصحة3T3 الثقافات والبذر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الجليد بسرعة خلايا NIH 3T3 في 37 ° C حمام الماء ونقل إلى أنبوب 15 مل. إضافة ببطء 9 مل من NIH 3T3 المتوسطة (DMEM + 10٪ FBS). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي الأنبوب في 180 × ز لمدة 5 دقائق وتجاهل طاف. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا مع 10 مل من NIH 3T3 المتوسطة ونقل إلى صحن ثقافة 10 سم. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا في 37 درجة مئوية، 5٪ CO 2 الحاضنة. من المهم جدا أن الخلايا NIH 3T3 يتم الاحتفاظ دائما underconfluent (50-60٪)، كما تردد التحول التلقائي (وبالتالي مستويات القاعدية من تشكيل بؤر) يزيد مرة واحدة تصل إلى ثقافة confluency. </li><li style=";text-align:right;direction:rtl"> البذور 3 × 10 5 خلايا لكل 10 سم طبق واحتضان O / N للعدوى في اليوم التالي. </li></ol></li><li style=";text-align:right;direction:rtl"> عدوى <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حصاد طاف فيروسات من الطبق بلات-E باستخدام 10 مل حقنة واحدة، وتصفية عليه من خلال 0.45 ميكرون حجم المسام النايلون مرشح غشاء، وتحويلها إلى 15 ملأنبوب. </li><li style=";text-align:right;direction:rtl"> إضافة 10 مل من جديد بلات-E المتوسطة للخلايا (بدون المضادات الحيوية)، وإعادتها إلى الحاضنة. </li><li style=";text-align:right;direction:rtl"> نضح المتوسطة من الطبق خلية NIH 3T3 للإصابة، وإضافة 5 مل من العادية المتوسطة NIH 3T3 و 5 مل من التي تحتوي على فيروس تصفيتها طاف. </li><li style=";text-align:right;direction:rtl"> إضافة polybrene إلى الطبق بتركيز 6 ميكروغرام / مل. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا O / N عند 37 درجة مئوية، و 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> اليوم التالي، كرر الخطوات من 3.2.1 - 3.2.5 لجولة ثانية من العدوى. </li><li style=";text-align:right;direction:rtl"> استبدال المتوسطة التي تحتوي على الفيروس مع منتظم المتوسطة NIH 3T3. </li><li style=";text-align:right;direction:rtl"> السماح للخلايا NIH 3T3 لتنمو لمدة 2-3 أسابيع، لتحل محل المتوسطة حسب الضرورة. تحقق من التعبير عن بروتين الاهتمام من جانب التقليدي الكهربائي البروتين وطخة مناعية على لست] خلية كاملة من لوحات طبق الأصل. </li></ol></li></ol> 4. تلطيخ والكمي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الكريستال البنفسجي تلطيخ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نضح المتوسطة NIH 3T3 ووضع الأطباقعلى الجليد. </li><li style=";text-align:right;direction:rtl"> غسل الأطباق مرتين مع PBS الجليد الباردة. </li><li style=";text-align:right;direction:rtl"> إصلاح الخلايا مع الميثانول الجليد الباردة لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة أطباق من الجليد، ونضح الميثانول، وإضافة 3 مل من 0.5٪ محلول الكريستال البنفسجي المحرز في 25٪ الميثانول (RT). </li><li style=";text-align:right;direction:rtl"> احتضان الأطباق لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> نضح الحل الكريستال البنفسجي وشطف بعناية الطبق مع ميلي-Q H 2 O حتى يأتي لا لون ثماره في الشطف. </li><li style=";text-align:right;direction:rtl"> السماح للأطباق لتجف O / N على الفوق (كشف). </li></ol></li><li style=";text-align:right;direction:rtl"> بؤر الكمي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مرة واحدة الأطباق جافة، استخدام مسطرة لقياس مجموعات الملونة على نحو مظلم من الخلايا على طبق من ذهب. الاعتماد فقط تلك التي هي أكبر من 5 ملم في القطر بأنها &quot;بؤر&quot;. سجل عدد من البؤر وحساب المتوسطات وأهمية مقارنة مع الضوابط. </li></ol></li></ol>

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يوفر FFA طريقة سريعة وسهلة لتقييم التحول الخبيث في المختبر. فمن قابلة للفحص عدد كبير نسبيا من الجينات المرشحة، والمتطلبات التقنية المتواضعة جعلها فعالة من حيث التكلفة. وعلاوة على ذلك، اثنين أو أكثر من الجينات يمكن coexpressed (يشار إليها أحيانا باسم &quot;التعاون&quot; فحص)...

الخلايا السرطانية يمكن تمييزها عن نظيراتها العادية من قبل مجموعة واسعة من التعديلات، من أنماط التعبير الجيني لepigenomics إلى تغيرات شكلية والتكاثري. ومن بين هذه الأخيرة، وخفض الاعتماد على المصل، وفقدان الاتصال (الكثافة) تثبيط، واقتناء الانتشار مرسى مستقل، وفي نهاية المطاف، القدرة على تكوين الأورام عند حقنه في الحيوانات، مؤشرات قابلة للقياس مفيدة التحول الخبيث 3. عدة في المختبر والمقايسات فيفو تم وضعها من أجل التحول الخلوي. في المختبر فحوصات تهدف إلى تحديد وقياس التغيرات في التشكل الثقافة (التركيز تشكيل فحص)، وديناميات الثقافة (معدل النمو والكثافة التشبع)، وعامل النمو (النمو في انخفاض المصل) أو مرسى (النمو في أجار لينة) المتطلبات. لا يزال المعيار الذهبي لتحديد طبيعة الخبيثة من نوع الخلية تشكيل الورم (xenografts) في حيوانات التجارب. ومع ذلك، رانه يكلف وطول من الدراسات في الجسم الحي لا تجعل لهم دائما مبررا كخطوة أولى التحقق من صحة أو فحص الجينات المسرطنة مرشح. على الرغم من عدم الفحص في المختبر يوفر تقييم واضح للإمكانات أنكجنيك من الجين، فإنها توفر نظرة ثاقبة إمكانات أنكجنيك التي قد تضييق المستقبل في الدراسات المجراة. واحد من أكثر الأنظمة المستخدمة على نطاق واسع لتقييم إمكانات أنكجنيك في المختبر هو التركيز تشكيل الفحص 2. ويعتمد هذا الأسلوب على استخدام NIH 3T3 الخلايا الليفية الماوس، خط خلية غير حولت الذي يظهر تثبيط اتصال قوية. overexpression من على النتائج الجين الورمي في فقدان النمو التي تعتمد على كثافة. الخلايا تحولت يمكن بعد ذلك تنمو في طبقات متعددة، وتشكيل &quot;بؤر&quot;، تصور بسهولة ضد أحادي الطبقة الخلفية للخلايا غير متحول. تشكيل التركيز الفحص، ثم، يقيس قدرة الجين الورمي مرشح للحث على التحول الخبيث، كما يتضح من فقدان الاتصال inhibition باعتباره النمط الظاهري للقياس. وقد استخدم FFA لتقييم التحول من overexpression من تحركات البروتينات (على سبيل المثال، SRC 4، BRAF 5)، عوامل النسخ (على سبيل المثال، N-MYC 6) مستقبلات، إلى جانب G-البروتين (على سبيل المثال، P2RY8 7) وGTPases (على سبيل المثال ورأس 1)، وغيرها. السهولة النسبية لهذا الاختبار يجعلها خيارا جيدا من شأنها أن توفر إجابة واضحة وسريعة بصريا ما إذا كان overexpression من الجينات يكفي لتحويل NIH 3T3 خلايا فأر الليفية في المختبر. وFFA الموصوفة في هذا البروتوكول يستخدم بلات-E خط الخلية التعبئة والتغليف 8، والذي يوفر البروتينات الفيروسية التعبئة والتغليف، وفيروسات ناقلات pBABEpuro 9 (Addgene بلازميد 1764) لإنتاج الفيروس الارتجاعي. بعد ترنسفكأيشن مع بناء pBABEpuro التي تحتوي على الجينات في المصالح، فإن خط الخلية بلات-E تنتج الارتجاعي ecotropic التي يمكن استخدامها لتصيب خلايا NIH 3T3. تيطريقة الفيروسي له من توصيل الجينات هو أكثر فعالية من الأساليب ترنسفكأيشن الكيميائية التقليدية، وأنه يوفر وسيلة للتعبير عن هذا الجين على نحو مستدام 10. مرة واحدة دمجها في جينوم الخلايا NIH 3T3، والدافع overexpression من هذا الجين من الفائدة من جانب يكرر محطة الطويلة الفيروسية (LTR) المروج 11. ويمكن استخدام هذا التعبير المستمر لتحديد ما إذا كانت الجينات في المصالح والنشاط أنكجنيك، مقاسا تشكيل بؤر، على خلايا NIH 3T3.

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			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
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			<td style="width:127px;">Sigma-Aldrich</td>
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			<td>cell line for focus formation assay</td>
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			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
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			<td>viral production reagent</td>
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			<td>viral production reagent</td>
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			<td>cell transfection reagent</td>
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			<td>cell stain reagent</td>
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			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
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			<td>cell culture supplies</td>
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			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
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			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
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			<td>cell culture supplies</td>
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			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
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			<td>cell culture media</td>
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			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td>cell culture media</td>
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focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 هو عامل النسخ الأساسي سحاب الحلزون حلقة حلزون ليسين (bHLHZ) الذي هو عضو من MYC / MAX / شبكة MAD. وهي عضو غير نمطية للأسرة MAD 13-15، وأفيد أن تشارك في التسرطن 16،17. بالمقارنة مع pBABEpuro (المراقبة السلبية) وMYC (مراقبة إيجابية)، كانت أطباق NIH 3T3 حيث تم overexpressed MXD3 أقل بكثير البؤ...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

توفر الفحص التركيز تشكيل طريقة واضحة لتقييم إمكانات تحويل من الجين الورمي مرشح.

التركيز تشكيل: A الفحص القائم على خلية لتحديد إمكانية أنكجنيك لجين

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.7K Views.  University of California, Davis.  توفر الفحص التركيز تشكيل طريقة واضحة لتقييم إمكانات تحويل من الجين الورمي مرشح. 

Video: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

المؤشرات الحيوية في نموذج حيواني لكشف الاقتران العصبية، الدموية، والسلوكية للPTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

وسيلة جديدة لإدارة الجراحية داخل الرغامى وكلاء الحيويه في نموذج الفأر الجنينية

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

الإنتاجية العالية التدفق الخلوي القائم على الفحص لكشف خلية أضداد N-ميثيل مد اسبارتاتي مستقبلات الدوبامين-2 أو مستقبلات في مصل الإنسان

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

والتعديل<em&gt; في المختبر</em&gt; الغزو الفحص لتحديد الدور المحتمل للالهرمونات، السيتوكينات و / أو عوامل النمو في التوسط السرطان غزو الخلايا

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

رواية<em&gt; في الجسم الحي</em&gt; تقنيات نقل الجينات و<em&gt; في المختبر</em&gt; فحوصات وبناء خلية لدراسة فقدان العظام في اضطرابات العضلات والعظام

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

بناء غرف بخار يستخدم لفضح الفئران إلى الكحول خلال ما يعادلها من جميع الأثلاث الثلاثة للتنمية البشرية

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

ثلاثي الأبعاد (3D) ورم الجسم الشبه الكروي الغزو الفحص

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

الاستفادة من الفحص لينة آجار مستعمرة تشكيل لالتعرف مثبطات Tumorigenicity في خلايا سرطان الثدي

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

يصح جين إسكات: عرض لHeptamer من نوع RNA دليل مكتبة صغيرة للسرطان العلاجية وكلاء المحتملة

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

حقن خلايا الفئران مسانج الميلانوما لتحديد ما إمكاناتهم الإنتقالية في الرئتين

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...