Special thanks to Dr. Peter Bradley for the antibody to detect the T. gondii mitochondria. The work was supported by National Institute of Health Grants AI072028 and AI107431 to D.G.M and a generous donation to New York Medical College for the study of tropical medicine.

The authors have no competing financial interests.

وينص البروتوكول وصف نهج غير منحاز يستخدم تفعيل الضامة الفئران المستمدة من نخاع العظام وعلم الوراثة إلى الأمام لعزل T. المسوخ الغوندية مع وجود خلل في قدرتها على البقاء على قيد الحياة تفعيل الضامة المصابة. النمط الظاهري من المسوخ تنشيط البلاعم التالية وعادة ?...

الغوندية التوكسوبلازما (T. الغوندية) هو داخل الخلايا تلزم، الممرض طفيلي. هذا هو العامل المسبب لداء المقوسات، يشكل خطرا على الصحة في الأفراد المناعة. بل هو أيضا في النظام النموذجي لمسببات الأمراض apicomplexan الأخرى التي تصيب البشر بما في ذلك الكريبتوسبوريديوم وCyclospora. ويكتسب داء المقوسات الأكثر شيوعا من خلال تناول طعام أو ماء ملوث المتباطئة أو البيوض المتكيسة مرحلة من مراحل الطفيلي. على ابتلاع، هذه المراحل تحويل إلى مرحلة tachyzoite للطفيل أن يعيد داخل الخلايا المضيفة وتنشر بشكل منتظم. خلايا T، IFN-γ، وإلى حد أقل، وأكسيد النيتريك 1-4، تعتبر مهمة للسيطرة على العدوى ولكنها ليست قادرة على القضاء على المرض، كنسبة من tachyzoites تحويل إلى مرحلة المتباطئة التي يتم حمايتها ضمن الخراجات الأنسجة مما أدى إلى عدوى مزمنة عمرا طويلا. في الواقع، لا توجد علاجات فعالة ضد الكيس الصورة المزمنالمئوية للمرض. داء المقوسات الحاد هو في معظم الأحيان بسبب إعادة تنشيط العدوى المستمرة، مع المرحلة المتباطئة للطفيل تحويل الى تكرار بسرعة tachyzoite مرحلة مميزة من العدوى الأولية والحاد. البقاء على قيد الحياة في وقت مبكر في مواجهة الاستجابة المناعية الفطرية مهم للسماح الطفيلي للوصول إلى أعداد الطفيل كافية، وكذلك للوصول إلى المواقع البعيدة، لتمكين المؤسسة من عدوى مزمنة. T. وقد تطورت الغوندية استراتيجيات لمواجهة آليات دفاع المضيف التي تساهم المرجح أن قدرته على تكرار ونشر في وقت مبكر من العدوى. أولا، T. الغوندية يشكل PV فريدة من نوعها خلال الغزو الطفيلي الذي هو الفصل بين الجنسين إلى حد كبير من عمليات التقامي وexocytic من الخلية المضيفة مقارنة لمسببات الأمراض داخل الخلايا الأخرى 5-9. أيضا، مثل كل ناجح داخل الخلايا مسببات الأمراض T. الغوندية يعدل الخلية المضيفة لخلق بيئة متساهلة وأو النمو. ويشمل هذا المضيف إعادة برمجة الخلايا التعبير الجيني عن طريق تغيير عوامل النسخ الخلية المضيفة بما في ذلك تلك الأهمية لتنظيم تنشيط الخلايا 10-15. ROP16 16-19، GRA15 20، GRA16 21 و 22 GRA24 كلها قد ثبت أن تكون مهمة في تنظيم استجابة النسخي وخلية يشير شلالات من الخلايا المضيفة المصابين T. الغوندية. وكانت الدراسات التي أجريت مؤخرا باستخدام الصلبان الوراثية بين سلالات طفيلي مع الظواهر متميزة مثمرة للغاية في تحديد الجينات الطفيليات التي تكمن وراء الصفات التي تعتمد على التركيب الوراثي الطفيليات بما في ذلك التهرب من GTPases الحصانة ذات الصلة (IRGs) 16،19،23-26. في الفئران، GTPases الحصانة ذات الصلة (IRGs) هي الحاسمة من أجل السيطرة على النوع الثاني والثالث التراكيب الوراثية للطفيل في حين تطورت خبيث جدا من النوع الأول المورثات آليات للتهرب من IRGs الفئران. ومع ذلك، فمن الواضح أيضا أن الطفيليات قد تطورت آليات للتهرب من وسائل الإعلام المضادة للجراثيميمكن الحفاظ الاختصاصات بالإضافة إلى IRGs وأن بعض هذه الآليات عبر المورثات الطفيلي 27،28. وبالإضافة إلى ذلك، فإن القليل جدا هو المعروف عن الوسطاء الحرجة من خلايا مناعة ذاتية الحكم ضد T. الغوندية خلال داء المقوسات البشري. الجينات الطفيليات مهمة لمقاومة وسطاء من خلية مناعة ذاتية الحكم قد يكون من المهم أيضا للبقاء على قيد الحياة خلال tachyzoite إلى المتباطئة التحويل والتي يمكن أيضا أن يكون سببها المضيف الاستجابات المناعية. على سبيل المثال، يمكن أن أكسيد النيتريك عند مستويات مرتفعة قمع تكرار الطفيلي في الضامة المصابة ولكن يمكن أيضا تحفيز tachyzoite لتحويل المتباطئة مما أدى إلى إنتاج الكيس 30-32. ToxoDB هي قاعدة بيانات الجينوم وظيفية للT. الغوندية الذي يعمل كمورد حاسم للحقل من حيث توفير المعلومات عن تسلسل جينوم طفيلي والوصول إلى المنشورة وغير المنشورة البيانات على نطاق والجيني بما في ذلك الشروح المجتمع، إكسب الجين<html> ression والبروتيوميات البيانات 33. وعلى غرار العديد من مسببات الأمراض الطفيلية، والغالبية العظمى من الجينوم يتكون من جينات افتراضية مع أية معلومات متاحة على أساس التماثل الجيني لتوفير نظرة ثاقبة وظائفها المحتملة. وهكذا، وعلم الوراثة إلى الأمام هو أداة قوية لتحديد الجينات الطفيليات جديدة مهمة للتهرب من المناعة، وتحويل الكيس وغيرها من المهام الحرجة للطفيلي المرضية فضلا عن التحويل بين مراحل نمو متميزة. وقوة إضافية من علم الوراثة إلى الأمام هو أنه يمكن استخدامها كنهج نسبيا غير منحازة لاستجواب الطفيلي لالجينات التي تعتبر مهمة لأداء مهام محددة في المرضية، بما في ذلك التهرب المناعة، وتشكيل الكيس. جعلت من طريقة الاختيار لتحديد الجينات المسؤولة عن الطفيليات علم الوراثة الدراسات إلى الأمام باستخدام الطفرات سواء الكيميائية وإقحامي 34-37 التحسينات الأخيرة في الجيل القادم التسلسل لالتنميط التحولي. ntent &quot;&gt; ومن المهم تحديد نقاط الضعف في T. الغوندية التي يمكن استغلالها لتعزيز فعالية الخلايا آليات مناعية ذاتية الحكم ضد الطفيليات وخاصة تلك التي قد تكون أيضا فعال ضد مرحلة الكيس مقاومة. وتحقيقا لهذا الهدف، وهو في المختبر الفئران وقد وضعت العدوى البلاعم ونموذج التشغيل لتحديد الطفرات في الطفيليات التي تضر تحديدا T. الغوندية اللياقة البدنية بعد تفعيل الضامة المصابة ولكن ليس في الضامة ساذجة. تم استخدام هذه الشاشة البلاعم لاستجواب مكتبة من T. الغوندية المسوخ إقحامي من أجل نهاية المطاف تحديد الجينات T. الغوندية مهمة لمقاومة أكسيد النيتريك 27،28. وعزلة لجنة من المسوخ T. الغوندية مع المقاومة ضعف لتفعيل الضامة المصابة، وخاصة حساسية ملحوظة إلى أكسيد النيتريك، وثبت فائدة الشاشة لتحديد الجينات الطفيليات هامة للمقاومةالى وسطاء من خلية مناعة ذاتية الحكم أخرى من آليات المقاومة وصفه لIRGs الفئران 28. الطفرات إقحامي لها مزايا أكثر من الطفرات الكيميائية من حيث توليد عدد محدود من الطفرات العشوائية في كل استنساخ طفيلي و، من الناحية النظرية، وتحديد أسهل للموقع من الطفرة. ومع ذلك، وتحديد الموقع الجيني البلازميد الإدراج في T. المسوخ إقحامي الغوندية، في الممارسة العملية، كان من الصعب المستغرب في كثير من الحالات 37. إدخال البلازميد إلى الجين ربما سيؤدي الى تعطيل وظيفة الجين على النقيض من الطفرات الكيميائية التي تنتج عادة في التغيرات النوكليوتيدات واحدة أيضا. ومع ذلك، الطفرات الكيميائية إما N-إيثيل-N-نتروزويوريا (ENU) أو سلفونات ethylmethane (EMS) قد تقدم زيادة القدرة على تحليل جزء أكبر من الجينوم طفيلي، بالمقارنة مع الطفرات إقحامي، كما أنه يخلق متعددة الأشكال النوكليوتيدات واحدة ( تقدر 10 -100) في متحولة 34(38 عاما). وعلاوة على ذلك، جعلت التطورات الحديثة في التنميط الجينوم كله من الممكن استخدام الجيل القادم التسلسل للتعرف على الجينات المرشحة الأكثر احتمالا المسؤولة عن النمط الظاهري المحددة للطفيلي تحور 34،38. بغض النظر عن النهج الطفرات، تأكيدا لدور هذا الجين الطفيلي في المقاومة لتفعيل بلعم يتطلب في نهاية المطاف حذف الجينات وتكامل للوفاء المسلمات الجزيئي كوخ. القدرة على تشريح وظيفة الجين عن طريق التلاعب الجيني لكل من الطفيليات والبلاعم لا يقل أهمية عن العديد من الجينات التي تم تحديدها عن طريق الوراثة قدما في T. الغوندية، فضلا عن مسببات الأمراض الأخرى، لا يزال يوصف الجينات افتراضية مع قليل من دون التماثل تسلسل لبروتينات أخرى ذات وظائف معروفة. وتحدد الورقة الحالية نهجا عاما يمكن استخدامه لتحديد ما إذا كانت الجينات تعطلت في متحولة مهم لمقاومة معروفة أووسيط غير معروف من خلية المناعة الذاتية الحكم. أجريت التحاليل الأولي من المضيف العوامل المضادة للجراثيم من خلال تقييم بقاء النوع البري والطفيليات متحولة في الضامة من نوع الفئران البرية مقابل تلك مع الحذف الجينات المحددة في محرض أكسيد النيتريك سينسيز (iNOS)، GP-91 phox (NADPH أوكسيديز)، و GTPases الحصانة المرتبطة محددة (IRGs). وهذا تحديد ما إذا كانت الجينات الطفيليات التي تم تحديدها هي مهمة لمقاومة أكسيد النيتريك، وسيطة الاكسجين التفاعلية أو حصانة المتعلقة GTPases 28 على التوالي أو إذا آلية مناعية غير معروفة هي المعنية. تفعيل الضامة المصابة مع كل من IFN-γ وLPS، وصفت في البروتوكول الحالي، النتائج في المقام الأول في عزل جينات الطفيلي مهمة لمقاومة أكسيد النيتريك 28. استخدام وكلاء الدوائية التي تحفز على أكسيد النيتريك في غياب تفعيل البلاعم (المانحين أكسيد النيتريك) وأكد أن غالبية الجينات التي جرى تحديدها هامة لإعادةاملساعدات إلى أكسيد النيتريك بدلا من أكسيد النيتريك في الحفل مع وسطاء الإضافية المرتبطة تنشيط البلاعم 28. خطوة واحدة واثنين من وصف شاشة الوراثة إلى الأمام تهدف إلى عزل المسوخ طفيلي مع وجود خلل اللياقة البدنية بعد تفعيل الضامة المشتقة من نخاع العظم المصاب في المختبر. خطوة واحدة تصف تحليل جرعة المعايرة لتحديد تجريبيا جرعة من γ IFN- وLPS لاستخدامها لتنشيط البلاعم التي تقلل من تكرار الطفيلي ولكن لا تمنع تماما تكرار نوع T. البرية سلالة الأبوية الغوندية التي يتم استخدامها لإنشاء مكتبة من المسوخ طفيلي. تصف الخطوة الثانية الشاشة الوراثية إلى الأمام من الحيوانات المستنسخة طافرة في الضامة في لوحات 96-جيدا. وتحدد الخطوة الثالثة نهجا لتأكيد النمط الظاهري من كل متحولة المحددة في الشاشة لوحات جيدا 96 وتقييم ما إذا كان الخلل في كل متحولة يؤثر بقاء الطفيلي، والنسخ،أو إنتاج الكيس ردا على تنشيط البلاعم. الخطوة الرابعة تصف استخدام الضامة المشتقة من نخاع العظام من الفئران مع الحذف في مسارات المضادة للميكروبات معينة للتعرف على وسطاء المناعية التي متحولة الطفيلي هو عرضة على وجه التحديد. وتحدد الخطوة الخامسة نهجا لتحديد ما إذا كان خطر المسخ الطفيلي أيضا لفي الجسم الحي المرضية حسب تقييم إنتاج الكيس في أدمغة الفئران المصابة.

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			<td colspan="2" height="21" style="height:21px;">Ted Pella EM grade formaldehyde</td>
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			<td align="right">18505</td>
			<td>http://www.tedpella.com/chemical_html/chem3.htm#anchor267712</td>
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			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Fisher</td>
			<td>BP151</td>
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			<td height="21" style="height:21px;">Alexa 488 - protein conjugation kit</td>
			<td>Life Technologies</td>
			<td>A20181</td>
			<td>http://www.lifetechnologies.com/order/catalog/product/A10235</td>
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			<td height="21" style="height:21px;">goat serum</td>
			<td>MP Biomedicals</td>
			<td>ICN19135680</td>
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			<td height="21" style="height:21px;">Vectashield mounting media</td>
			<td>Vector Labs</td>
			<td>H1200</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=428</td>
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			<td height="21" style="height:21px;">FITC-conjugated dolichos</td>
			<td>Vector Labs</td>
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			<td>https://www.vectorlabs.com/catalog.aspx?prodID=188</td>
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			<td height="21" style="height:21px;">Antibody to LAMP1</td>
			<td colspan="2">Developmental Studies Hybridoma Bank</td>
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			<td height="21" style="height:21px;">LysoTracker</td>
			<td>Life Technologies</td>
			<td>L-7526</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/L7526?ICID=search-product</td>
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			<td height="21" style="height:21px;">C57BL6 mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">664</td>
			<td>http://jaxmice.jax.org/strain/000664.html</td>
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			<td height="21" style="height:21px;">gp91 phox knock out mice</td>
			<td>Jackson Labaoratories</td>
			<td align="right">2365</td>
			<td>http://jaxmice.jax.org/strain/002365.html</td>
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			<td height="21" style="height:21px;">iNOS knock out mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">2609</td>
			<td>http://jaxmice.jax.org/strain/002609.html</td>
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			<td height="21" style="height:21px;">sodium nitroprusside</td>
			<td>ACROS Organics</td>
			<td>AC21164-0250&#160;</td>
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			<td height="21" style="height:21px;">DETA NONOate</td>
			<td>ACROS Organics</td>
			<td>AC32865-0250</td>
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			<td colspan="2" height="21" style="height:21px;">Monoclonal mouse anti-Toxoplasma gondii Ab</td>
			<td></td>
			<td>10T19A</td>
			<td>http://1degreebio.org/reagents/product/1069274/?qid=652947</td>
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forward genetics screens using macrophages to identify toxoplasma gondii genes important for resistance to ifn-&#947;-dependent cell autonomous immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

التوكسوبلازما يعيد بحرية في الضامة ساذجة ولديه الوقت تضاعف بين 6-12 ساعة تبعا لسلالة من الطفيلي الشكل 1 يظهر الطفيليات تمثيلية في السذاجة مقابل تنشيط العظم الضامة المشتقة من نخاع الشكل 2 يوضح التشكل العام للطفيليات في HFF الخلايا المضيفة في 2 و 4 و ...

Watch this Scientific Journal Video about Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-Î³-Dependent Cell Autonomous Immunity at JoVE

الشاشات الوراثية المتقدمة باستخدام البلاعم لتحديد جينات التوكسوبلازما جوندي المهمة لمقاومة المناعة الذاتية للخلايا المعتمدة على IFN-γ | علم المناعة والعدوى | فيديو

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

الى الامام علم الوراثة شاشات طريق الضامة لتحديد<em&gt; التوكسوبلازما</em&gt; الجينات هامة الحصانة الذاتية للمقاومة إلى الإنترفيرون γ التي تعتمد على خلية

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

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Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Unit 2

Population Genetics

Analyzing Gene Expression and Function

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11.0K Views.  New York Medical College. Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion. 

Video: Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

الحصول على عالي النقاء المقوسة الغوندية البيضات من قبل التدرج كلوريد السيزيوم متقطع

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

التوكسوبلازما جدار الكيس الغوندية تشكيل المنشط تقوم البلاعم في نخاع العظام والمستمدة من الشروط المتباطئة

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear الفئران مع الخصوصيات المحددة مسبقا ضد مستقبلات الخلايا T المقوسة الغوندية الحصول عليها عن طريق SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

اختيار المتصورة المنجلية الطفيليات لCytoadhesion لخلايا المخ البشري غشائي

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

شاشة الوراثية لعزل التوكسوبلازما جوندياي المسوخ الخروج المضيف الخلية

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

التلاعب الجيني في<em&gt; Δku80</em&gt; سلالات لتحليل الجينوم الوظيفي لل<em&gt; التوكسوبلازما جوندياي</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

النهج الوراثية إلى الأمام في<em&gt; الكلاميديا ​​المتدثرة</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

باستخدام الحمض النووي الريبي التدخل للتحقيق في الاستجابة المناعية الفطرية في الفأر الضامة

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.