وأيد هذا العمل من المنح المقدمة من المعاهد الوطنية للصحة لDJB (AI041930، AI073142، AI075931، AI091461 و).

<ol>
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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

ونحن هنا نقدم بروتوكول لجينة فعالة تستهدف في Δ ku80 سلالات طفيلي لتمكين استرداد فعالة من ذرية التلاعب بها وراثيا التي تمتلك الحذف استهداف الجينات، واستبدال الجين، و / أو الجينات الموسومة. تنفيذ متسلسلة من هذه الأساليب يوفر نهجا موثوق بها لعزل المسوخ طفيلي التي تحتوي على التلاعب الجيني المستهدفة واحد أو عدة 1-3. في حين أن الجيل من حذف استهدفت يعتمد على عوامل متعددة، استخدام Δ ku80 سلالة مع الاستراتيجية المقدمة وبروتوكول يزيد بشكل كبير من كفاءة وسهولة صنع سلالات متحولة محددة بدقة من T. المقوسة للدراسات الجينية وظيفية. جينوم T. وفرداني المقوسة خلال مراحل اللاجنسي. وبالتالي النظر إلى جعل عند التخطيط لحذف الجين هو أن هذا الجين لا يجب أن يكون أساسيا في المختبر. ومع ذلك، فإن كفاءة targeتينغ بالضربة القاضية جين في Δ ku80 سلالات عالية جدا، وهذا يتيح للعزل سلالات متحولة مع معدلات تكرار ضعاف بشكل ملحوظ. إذا كان الجين المستهدف هو ضروري، والطفيليات غالبا ما تتوقف لتكرار أثناء الاختيار. آخر عاملا حاسما في التلاعب الجيني المستهدف هو أن التماثل الكمال مع ما لا يقل عن 620 شركة بريتيش بتروليوم من الجهة استهداف الجيني هو مطلوب للحصول على التكاملات كشف استهدفت 2. المناطق أطول من التماثل إنتاج كفاءات أعلى من الاستهداف وباستخدام استهداف الأجنحة الحمض النووي من حوالي 1،000 سنة مضت هو نهج موثوقة وفعالة 1-4. لهذا السبب من المهم أن يتم إنشاء الأجنحة استهداف الجيني من نفس السلالة من T. المقوسة (RH، PRU) لان السلالة التي يتم التلاعب بها وراثيا. عدم تناظر كافية يمكن أن يؤدي في كثير من الأحيان في التكامل استهدفت واحدة من الجينوم الجهة الاستهداف، ولكن ليس غيرها. التكامل ناقصة أو عدم الحصول على ماجستير خروج المغلوبY يكون أيضا بسبب استمرار episomal. مباشرة بعد ترنسفكأيشن، يتم nonintegrated كل من الجزيئات التي تستهدف episomes، وأحيانا تستهدف الجزيئات يمكن أن تستمر كما episomes لأجيال عديدة. باستخدام الأجنحة DNA الهدف من ~ 1 KBP والاستمرار في تمرير الطفيليات تحت الاختيار قبل إعادة subcloning كثيرا ما يحل المشاكل المرتبطة استمرار episomal. بمجرد التحقق من صحة بالضربة القاضية ويتم إزالة علامة HXGPRT، ويمكن تكرار هذه الاستراتيجية تستهدف الجين على الحكومة العراقية الثانية لتوليد متعددة الحذف الجينات في سلالة الطفيلي واحدة 1-3. ويمكن أيضا توليد الحذف، واستبدال، أو الجينات الموسومة إنجازه باستخدام علامات اختيار مختلفة (بليوميسين، CAT، بيريميثامين مقاومة DHFR، الخ). على الرغم من أن علامة اختيار CAT موجود في النوع الثاني Δ ku80 PRU الجينوم 1 حاليا، يمكن إزالة هذه العلامة باستخدام HXGPRT </EM&gt; بروتوكول اختيار صفها هنا. في أيدينا، HXGPRT الاختيار هو الأكثر أمانا والأكثر كفاءة مع علامة أعلى كفاءة الاستهداف حيث A-نسخة واحدة الإدراج المستهدف هو كاف لاختيار القوي في MPA + X المتوسطة. HXGPRT يوفر أيضا النهج الوحيد المفيد حاليا للحذف المستهدفة من علامة اختيار (الشكل 3) باستخدام 6 thioxanthine (6TX) اختيار 2،3. وهكذا يوفر هذا البروتوكول النهج الحالي فقط لتطوير سلالات متحولة تعريف مع العديد من التلاعب الجيني المستهدفة. يوفر هذا البروتوكول طريقة قيمة وفعالة للتلاعب الجيني المستهدفة في Δ ku80 سلالات من المقوسة الغوندية وينطبق على تحليل جين واحد، عائلة من الجينات، أو على نطاق الجينوم دراسات الجينية وظيفية. قبل توافر Δ ku80 سلالات 1،2، وكانت هذه الطرق غير مجدية على نطاق واسع بسبب المعهد الوطني للإحصاءfficiency من هذه الأساليب. وبالتالي، فإن هذا البروتوكول ينطبق على نطاق واسع للتلاعب الجيني المستهدفة من المقوسة الغوندية ومتاح لجميع المحققين مهتمون في معالجة مجموعة من الأسئلة على الطفيليات علم الأحياء، استجابة المضيف، وعلم الجينوم وظيفية.

التوكسوبلازما جوندياي هو مشترك تلزم طفيلي داخل الخلايا التي في كثير من الأحيان ويصيب بشكل مزمن مجموعة واسعة من الحيوانات والبشر 5. وتشير التقديرات إلى أن أكثر من 1 مليار البشر حاليا وبالعدوى بشكل مزمن من قبل هذا الممرض. بالإضافة إلى أهمية الأمراض التي تسببها T. عدوى المقوسة، وزيادة توفر أدوات تجريبية، والموارد الجينوميات قوي 6، وسهولة في النمو المختبر ونماذج الماوس ممتازة جعلت T. المقوسة نظام نموذجا رائدا لدراسة أوسع نطاقا من مسببات الأمراض بين الخلايا حقيقية النواة وغيرها من الطفيليات apicomplexan كبيرة التي تسبب الأمراض المدمرة مثل الملاريا (المتصورة س.) وآريبتوسبوريديوم (الكريبتوسبوريديوم) 5،7. لقد كان وجود قيود كبيرة من المقوسة الغوندية باعتباره النموذج الحي الانتعاش غير الكفء للذرية التي تحمل تستهدف التلاعب الجيني. هذه مشاكلم في استهداف الجين ويرجع ذلك إلى التردد المنخفض من إعادة التركيب مثلي نسبة إلى تردد عال جدا من إعادة التركيب امتماثلان في السلالات البرية من نوع T. المقوسة حتى عندما يتم توفير التماثل DNA واسعة في الجزيئات المستهدفة الحمض النووي المستخدمة في الدراسات الوراثية 2. ونحن في الآونة الأخيرة منعت وراثيا المسار الرئيسي لإعادة التركيب امتماثلان في النوع الأول والنوع الثاني من سلالات المقوسة التوكسوبلازما عن طريق حذف الجينات التي تشفر بروتين 1،2 KU80. النوع الناتج الأول والنوع الثاني Δ ku80 سلالات تظهر معدلات النمو الطبيعي، وحجم والسلوك على حد سواء في المختبر والمجراة خلال tachyzoite ومراحل المتباطئة في المختبر والمجراة، ومع ذلك، هذه السلالات يحمل أساسا على تردد 100٪ من إعادة التركيب مثلي وهذا النمط الظاهري يزيد من احتمال عزل بسرعة ذرية المطلوب من التلاعب الجيني المستهدفة من قبل عدة مئات إلى عدة ثوusand أضعاف 1،2،4. استخدام الأخيرة على نطاق المجتمع المحلي من النوع الأول والنوع الثاني Δ سلالات ku80 ورفعت بشكل ملحوظ على وتيرة ومتنوعة، فضلا عن نسبة نجاح النهج الوراثية المستهدفة في T. المقوسة 1-4،8-13. نحن هنا وصف بروتوكول شامل للحذف استهداف الجينات، واستبدال الجينات، ووضع علامات الجينات في Δ ku80 سلالات من T. المقوسة. ونحن لشرح كيفية تصميم واستهداف موثوق التلاعب الجيني لجينات معينة أو مواضع الوراثية في Δ ku80 سلالات المقوسة التوكسوبلازما.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Overlap Primers</td>
			<td>Integrated DNA Technologies</td>
			<td>&nbsp;</td>
			<td>4 nmole Ultramer DNA oligos</td>
		</tr>
		<tr>
			<td>Validation Primers</td>
			<td>Integrated DNA Technologies</td>
			<td>&nbsp;</td>
			<td>100 nmole DNA Oligo</td>
		</tr>
		<tr>
			<td>Yeast Strain #90845</td>
			<td>ATCC</td>
			<td>&nbsp;</td>
			<td>Designation FY834</td>
		</tr>
		<tr>
			<td>Shuttle Vector pRS416</td>
			<td>ATCC</td>
			<td>87521</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DH10B E. coli</td>
			<td>Invitrogen</td>
			<td>12033-015</td>
			<td>SOC broth in kit with E. coli</td>
		</tr>
		<tr>
			<td>Resuspension Buffer</td>
			<td>Qiagen</td>
			<td>&nbsp;</td>
			<td>Buffer P1 in QIAprep Spin Miniprep Kit</td>
		</tr>
		<tr>
			<td>Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>QIAprep Spin Miniprep Kit</td>
		</tr>
		<tr>
			<td>Glass Beads</td>
			<td>Scientific Industries</td>
			<td>SI-BG05</td>
			<td>0.5 mm acid-washed</td>
		</tr>
		<tr>
			<td>Qiacube Automated Robotic Work Station</td>
			<td>Qiagen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporation Cuvette</td>
			<td>USA Scientific</td>
			<td>9104-5050</td>
			<td>2 mm-gap</td>
		</tr>
		<tr>
			<td>BTX600 electroporator</td>
			<td>BTX</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12662</td>
			<td>QIAprep Spin Maxiprep Kit</td>
		</tr>
		<tr>
			<td>25 cm2 Canted neck plug seal flask</td>
			<td>Corning</td>
			<td>430168</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>150 cm2 Canted Neck plug seal flask</td>
			<td>Corning</td>
			<td>430823</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human Foreskin Fibroblasts (HFF) cells</td>
			<td>ATCC</td>
			<td>SCRC1041</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Swin-lok Filter Holder</td>
			<td>Whatman</td>
			<td>420200</td>
			<td>25-mm-diameter</td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td>Whatman</td>
			<td>110612</td>
			<td>Nucleopore Track-etched Membrane 3 mm pore size, 25-mm-diameter</td>
		</tr>
		<tr>
			<td>Mycophenolic acid (MPA)</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xanthine (X)</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-well Tissue Culture Tray</td>
			<td>Corning Costar</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>24-well Tissue Culture Tray</td>
			<td>Corning Costar</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM Eagle Media</td>
			<td>Lonza Biowhittaker</td>
			<td>12-611F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140-111</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (Anti-Anti)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spin Column</td>
			<td>Primm Labs</td>
			<td>PAE-100</td>
			<td>Easy Clean DNA Extraction Spin Kit</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6-thioxanthine</td>
			<td>Acros Organics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue DNA Minikit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>QIAamp DNA Blood Mini Kit</td>
		</tr>
		<tr>
			<td>Cell Culture Freeze/Recovery Media</td>
			<td>Gibco</td>
			<td>126-48-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td>minus calcium, minus magensium</td>
		</tr>
	</tbody>
</table>

genetic manipulation in &delta;ku80 strains for functional genomic analysis of toxoplasma gondii

التلاعب الجيني المستهدفة باستخدام إعادة التركيب مثلي هو الأسلوب المفضل لتحليل الجينوم وظيفية للحصول على عرض تفصيلي لوظيفة الجين والنمط الظاهري (ق). تطوير سلالات متحولة مع الحذف المستهدفة الجين، والطفرات المستهدفة، وتكمل وظيفة الجين، و / أو الجينات الموسومة يوفر استراتيجيات قوية لمعالجة وظيفة الجين، لا سيما إذا كانت هذه التلاعب الجيني يمكن أن تستهدف بكفاءة إلى موضع الجينات ذات الاهتمام باستخدام بوساطة التكامل بالنقر المزدوج عبر أكثر من مثلي إعادة التركيب. وقد يرجع ذلك إلى معدلات عالية جدا من امتماثلان إعادة التركيب، والتحليل الجيني وظيفية من المقوسة الغوندية تقتصر في السابق بسبب عدم وجود وسائل فعالة لاستهداف الجينات الحذف والاستبدال الجيني لمواضع جينية محددة. مؤخرا، ونحن ألغى مسارا رئيسيا من إعادة التركيب امتماثلان في النوع الأول والنوع الثاني من سلالات T. المقوسة عن طريق حذف encodin الجينز 1،2 بروتين KU80. سلالات Δku80 تتصرف بشكل طبيعي خلال tachyzoite (الحادة) والمتباطئة مراحل (المزمنة) في التجارب المختبرية والحية ويحمل في جوهره تردد 100٪ من إعادة التركيب مثلي. سلالات ku80 Δ جعل دراسات الجينية وظيفية مجدية على جين واحد، وكذلك على مقياس الجينوم 1-4. هنا، ونحن التقرير أساليب لاستخدام النوع الأول والنوع الثاني سلالات Δku80Δhxgprt لتعزيز نهج استهداف الجينات في T. المقوسة. ونحن الخطوط العريضة وسائل فعالة لتوليد الحذف الجينات، واستبدال الجينات، والجينات الموسومة من قبل الإدراج أو الحذف المستهدفة من هيبوزانتين-الزانثين-جوانين ناقلة الفسفوريبوزيل (HXGPRT) علامة اختيار. الجين هو موضح استهداف بروتوكول يمكن استخدامها في مجموعة متنوعة من الطرق في Δku80 سلالات للنهوض تحليل وظيفي للجينوم الطفيل وتطوير سلالات واحدة التي تحملمتعددة تستهدف التلاعب الجيني. إن تطبيق هذه الطريقة الوراثية وفحوصات المظهري لاحقة تكشف عن جوانب أساسية وفريدة من نوعها لبيولوجيا T. المقوسة وما يتصل بها من مسببات الأمراض البشرية الهامة التي تسبب الملاريا (المتصورة س.) وداء خفيات الأبواغ (الكريبتوسبوريديوم).

Watch this Scientific Journal Video about Genetic Manipulation in Î”ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii at JoVE.com

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;

نحن هنا التقرير طريقة لاستخدام النوع الأول والنوع الثاني<em&gt; Δku80</em&gt; سلالات<em&gt; التوكسوبلازما جوندياي</em&gt; لتوليد بكفاءة الحذف الجينات المستهدفة وبدائل الجينات لتحليل الجينوم وظيفية.

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 ...

genetic-manipulation-ku80-strains-for-functional-genomic-analysis

Research

JoVE Journal

Immunology and Infection

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

16.9K Views.  The Geisel School of Medicine at Dartmouth. نحن هنا التقرير طريقة لاستخدام النوع الأول والنوع الثاني Δku80 سلالات التوكسوبلازما جوندياي لتوليد بكفاءة الحذف الجينات المستهدفة وبدائل الجينات لتحليل الجينوم وظيفية.

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

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 ...

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 ...

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 ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

التالية في الوقت الحقيقي تأثير العوامل الالتهاب الرئوي الحاد الفوعة في نموذج الفأر الالتهاب الرئوي عن طريق البكتيريا للإضاءة الحيوية

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

التصوير في الوقت الحقيقي من خلايا الدم النخاعي في ديناميات<em&gt; APC<sup&gt; مين / +</sup</em&gt; الأورام المعوية التي كتبها غزل القرص متحد البؤر المجهر

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

كشف الجليكوجين في خلايا الدم المحيطي وحيدات النوى مع الدوري حمض شيف تلطيخ

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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.

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 ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

والرزن التصاق الديناميكي للتحليل الوظيفي من العلاجات المضادة الالتصاق في مرض التهاب الأمعاء

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...