We thank Cindy Voisine and Yoko Shibata for helpful discussion and critical comments on the manuscript. We acknowledge the High Throughput Analysis Laboratory (HTAL) and the Biological Imaging Facility (BIF) at Northwestern University for their assistance. This work was funded by grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, and the Daniel F. and Ada L. Rice Foundation (to R.I.M.). C.I.N.-K. was supported by the Deutsche Forschungsgemeinschaft (KR 3726/1-1).

The authors declare no competing financial interests.

الأساليب المذكورة هنا تساعد على توضيح نشر والخلية معقدة المستقلة وغير المستقلة سمية الخلايا من البروتينات مثل بريون. اكتشفنا مؤخرا أن يؤخذ على عصاري خلوي مجال بريون المعرضة للتجميع ما يصل إلى الحويصلات بغشاء في عملية الالتهام الذاتي ذات الصلة. مجموعة فرعية معينة من...

وترتبط العديد من الأمراض العصبية، بما في ذلك مرض الزهايمر (AD)، ومرض باركنسون (PD)، الضموري العضلي التصلب الجانبي (ALS)، والاعتلال الإسفنجي المعدية (TSEs)، مع البروتينات المعرضة للتجميع، وبالتالي تعرف باسم البروتين misfolding اضطرابات (تطوير إدارة الأداء ). TSEs أو أمراض بريون تشكل فئة فريدة من تطوير إدارة الأداء في أنها يمكن أن تكون معدية في كل من البشر والحيوانات 1. على المستوى الجزيئي، والبريونات تكرار من خلال تجنيد وتحويل أحادى α-الحلزون الغنية المشفرة المضيف بي ار بي الخلوي (بي ار بي C) في المرضية ورقة β الغنية بي ار بي الشوري التشكل 2،3. وقد تم تحديد المجاميع البروتين نشر النفس أيضا في الفطريات، التي تشترك في الخصائص المهمة مع البريونات الثدييات 4،5. بالإضافة إلى ذلك، والبريونات الثدييات قادرة على الانتقال من خلية الى خلية وتصيب خلايا ساذجة 6،7. في حين تطوير إدارة الأداء الغيرإيه من TSEs ليست معدية، إلا أنهما يشتركان مبدأ المسببة للأمراض مشتركة مع الأمراض بريون 8،9. على الرغم من أن البروتينات ترتبط كل من تطوير إدارة الأداء لا ترتبط في هيكل أو وظيفة، وأنهم جميعا المجاميع شكل من خلال عملية تشبه بلورة تسمى منوى أو المصنفة البلمرة. وعلاوة على ذلك البذور البروتينية تنمو من خلال تجنيد الإسوية القابلة للذوبان على 2،10،11. كفاءة لنشر الذاتي تختلف في الجسم الحي، وهذا يتوقف على الخصائص الجوهرية للبروتين، والتي جنبا إلى جنب مع العوامل الخلوية إضافية مثل المحرمين الجزيئي تحدد في نهاية المطاف معدلات التنوي الكلي، البذر، والتجزؤ ونشر 12-15. وبالتالي، يجب ان يكون هناك توازن دقيق بين هذه العوامل التي تسمح نشر كفاءة من البروتين التجميع. قد يفسر هذا أيضا لماذا فقط بعض المجاميع amyloidogenic تؤوي خصائص بريون، وبالتالي ليس كل تطوير إدارة الأداء والمعدية. ويبدو أن البريونات لتمثيل س &#39;أعلى فناني الأداءاتحاد كرة القدم طيف واسع من المجاميع البروتينية الذاتي تكرار، مما يجعلها أداة قوية لدراسة تطوير إدارة الأداء 8،13. يثير الاهتمام والفضول، وسمية المرتبطة المجاميع المرتبطة المرض غالبا ما يكون غير خلية عنصر مستقل 16،17. وهذا يعني أنها تؤثر على الخلايا المجاورة التي لا تعبر عن الجين المقابل، على النقيض من تأثير مستقل الخلية بدقة، مما يعني أن فقط الخلايا معربا عن المعرض الجين النمط الظاهري محدد. وقد تجلى ذلك بشكل مقنع عن طريق التعبير الأنسجة محددة أو هدم البروتينات منها في نماذج عديدة من أمراض الاعصاب 18-26. وقد اقترحت آليات مختلفة كأساس لهذه الخلايا غير سمية الحكم الذاتي في تطوير إدارة الأداء، بما في ذلك إمدادات تقلص المواد الغذائية، وخلل في الإشارات العصبية، الغلوتامات التحفيز الزائدة، وneuroinflammation 16،27،28. وبالإضافة إلى ذلك، migh حركة تشبه بريون من المجاميع المرتبطة المرض بين الخلايار المساهمة في هذا الجانب 29،30. وتشير أدلة متزايدة على أن الادراج البروتين أخرى من البريونات يمكن أن تنقل من خلية الى خلية، وهو ما قد يفسر نشر سمة من الأمراض التي لوحظت في العديد تطوير إدارة الأداء 30-36. ومع ذلك، لم يتم بعد تحديد ما إذا كانت هناك علاقة سببية واضحة بين الحركة بين الخلايا البروتينات المرض وتأثير سام على الخلايا المجاورة. ولذلك، فهم أفضل للمسارات الخلوية التي تكمن وراء انتقال خلية الى خلية خلية وغير سمية الحكم الذاتي أمر ضروري وأساسي لتطوير علاجات جديدة. ومع ذلك، لا يفهم كثير من جوانب بريون مثل نشر والخلوية العوامل التي تؤثر على انتقال الخلية الى خلية من البروتينات تتجمع في metazoans جيدا، ولا سيما على مستوى العضوي. الديدان الخيطية ايليجانس انواع معينة لديها العديد من المزايا التي توفر إمكانات ل اكتشاف جوانب جديدة من تشبه بريون SPREADIنانوغرام في metazoans 17. انها شفافة، والسماح لفي الجسم الحي تتبع البروتينات من الموسومة fluorescently في الكائن الحي. وعلاوة على ذلك، يتم الحفاظ العديد من العمليات الخلوية والفسيولوجية المتضررين من المرض من الديدان إلى الإنسان، وC. ايليجانس هي أيضا قابلة للطائفة واسعة من التلاعب الجيني والتحليلات الجزيئية والكيمياء الحيوية 37-39. بالضبط 959 الخلايا الجسدية تشكل خنثى الكبار مع خطة الهيئة البسيطة التي لا يزال لديها العديد من أنواع الأنسجة متميزة، بما في ذلك العضلات، والخلايا العصبية والأمعاء. لوضع نموذج جديد في بريون C. ايليجانس، اخترنا للتعبير عن خارجيا وتتميز كذلك الجلوتامين / الأسباراجين (Q / N) الغنية بريون NM المجال من عصاري خلوي بروتين بريون Sup35 الخميرة، حيث لا توجد بروتينات بريون الذاتية المعروفة في الديدان 4،40. وقد البريونات الخميرة لا تقدر بثمن في توضيح الآليات الأساسية لتكرار بريون 41-44. وعلاوة على ذلك، NM هو التنوبالحادي وعصاري خلوي البروتين مثل بريون التي قد تظهر لتلخيص دورة الحياة الكاملة للبريون في الثقافة خلايا الثدييات 45،46. وبالمثل، عندما أعرب في C. ايليجانس، المجال بريون Sup35 اعتمدت بشكل جيد لمتطلبات مختلفة للنشر في الخلايا metazoan بالمقارنة مع خلايا الخميرة والملامح الرئيسية عرضت من بريون البيولوجيا ارتبط 40. NM التجميع مع النمط الظاهري السامة العميق، بما في ذلك تعطيل سلامة الميتوكوندريا ومظهر من مختلف الحويصلات الالتهام الذاتي ذات الصلة على المستوى الخلوي، فضلا عن اعتقال الجنينية واليرقات، تأخر في النمو، واضطراب واسع النطاق للبيئة طي البروتين على المستوى العضوي. لافت للنظر، يسلك المجال بريون خلية مستقلة وغير خلية سمية مستقلة، مما يؤثر على الأنسجة المجاورة التي لم أعرب التحوير. وعلاوة على ذلك، يتم مراقبة النقل حويصلي في مجال بريون داخل وبين خلايا في الوقت الحقيقي &lt;em&gt; في الجسم الحي 40. نحن هنا وصف كيفية فحص نشر مثل بريون في C. ايليجانس. سوف نشرح كيفية مراقبة النقل داخل الأقاليم وفيما بين الخلايا من الحويصلات التي تحتوي على المجال بريون باستخدام الوقت الفاصل بين مضان المجهري. سوف نؤكد على أن استخدام أجهزة الاستشعار الأنسجة محددة قابلة للطي وأعرب بتواجد مطلق للصحفيين الإجهاد لتقييم آثار خلية خلية مستقلة تتمتع بالحكم الذاتي وغير على اللياقة البدنية الخلوية. وأخيرا، فإننا سوف تصف الإجراء من الجينوم واسعة تدخل الحمض النووي الريبي (رني) شاشة أجريت مؤخرا لتحديد معدلات جديدة من سمية التي يسببها بريون. في الجمع، ويمكن لهذه الأساليب تساعد على ندف بصرف النظر مسارات الجينية التي تساهم في الحركة بين الخلايا البروتينات وغير خلية سميتها مستقلة.

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
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			<td height="21" style="height:21px;width:211px;">Reagent</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Nanosphere size standards 100 nm</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:92px;">3100A</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Levamisole</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:92px;">L-9756</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">IPTG</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:92px;">15502-10G</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Ahringer RNAi library</td>
			<td style="width:107px;">Source BioScience LifeSciences&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.lifesciences.sourcebioscience 
			.com/clone-products/non-mammalian/c-elegans/c-elegans-rnai-library/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Equipment</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Sorvall Legend XTR Refrigerated Centrifuge, 120VAC</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:92px;">75004521</td>
			<td style="width:255px;">http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_ 
			XTR_Refrigerated_Centrifuge_120 
			VAC/EW-17707-60</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">96 pin replicator&#160;</td>
			<td style="width:107px;">Scionomix&#160;&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.scinomix.com/all-products/96-pin-replicator/</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">HiGro high-capacity, incubating shaker&#160;</td>
			<td style="width:107px;">Digilab</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.digilabglobal.com/higro</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Multidrop Combi Reagent Dispenser&#160;</td>
			<td style="width:107px;">Titertrek</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://groups.molbiosci.northwestern.edu/hta/titertek.htm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Biomek FX AP96 Automated Workstation&#160;</td>
			<td style="width:107px;">Beckman Coulter</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:211px;">Innova44 shaker</td>
			<td style="width:107px;">New Brunswick</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.eppendorf.com/int///index.php?sitemap=2.3&#38;pb=d78efbc05310ec 
			04&#38;action=products&#38;contentid=1&#38; 
			catalognode=83389</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:211px;">M205 FA&#160;</td>
			<td style="width:107px;">Leica</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">ORCA-R2 C10600-10BDigital CCD camera</td>
			<td style="width:107px;">Hamamatsu</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:211px;">Spinning Disc AF Confocal Microscope&#160;</td>
			<td style="width:107px;">Leica</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">Falcon 4M60 camera&#160;</td>
			<td style="width:107px;">Teledyne Dalsa&#160;</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Software</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td style="width:107px;">Molecular Devices</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">Hamamatsu SimplePCI Image Analysis Software</td>
			<td style="width:107px;">Meyer Instruments</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://meyerinst.com/imaging-software/hamamatsu/index.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">ImageJ</td>
			<td style="width:107px;">NIH</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">wrMTrck plugin for ImageJ</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.phage.dk/plugins/wrmtrck.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">C. elegans strains</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:211px;">N2 (WT)</td>
			<td style="width:107px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">http://www.cgc.cbs.umn.edu/strain.php?id=10570</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">AM815&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; rmIs323[myo-3p::sup35(r2e2)::rfp]</td>
			<td style="width:107px;">Morimoto lab</td>
			<td style="width:92px;"></td>
			<td style="width:255px;">available from our laboratory&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:211px;">See table 1 for a source for folding sensor and stress reporter strains</td>
			<td style="width:107px;"></td>
			<td style="width:92px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

investigating the spreading and toxicity of prion-like proteins using the metazoan model organism c. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

مراقبة بيخلوي نشر من البروتينات مثل بريون من قبل في الجسم الحي الوقت الفاصل بين التصوير المعدلة وراثيا C. خطوط ايليجانس معربا عن المجال بريون بشكل خاص مناسبة تماما لتحليل جوانب معينة من مثل بريون البروتين?...

Watch this Scientific Journal Video about Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans at JoVE.com

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

التحقيق في نشر وسمية البروتينات مثل بريون باستخدام Metazoan نموذج حي<em&gt; C. ايليجانس</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

investigating-spreading-toxicity-prion-like-proteins-using-metazoan

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Biology

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

15.7K Views.  Northwestern University. Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Video: Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

توليد مستقرة ايليجانس جيم المعدلة وراثيا عن طريق Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

تشريح والتسجيل من مفرق العضلية جيم ايليجانس

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

لإنتاج C. ايليجانس الجينات المحورة عبر Recombineering مع galK إختيار ماركر

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

خلق بيئات الغازية محددة لدراسة تأثير نقص الأكسجة في<em&gt; C. ايليجانس</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

باستخدام الحمض النووي الريبي التي تتوسط استراتيجية التغذية التدخل للكشف عن الجينات المسؤولة عن تنظيم حجم الجسم في نيماتودا<em&gt; C. ايليجانس</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

تلوين الأجسام المضادة في<em&gt; C. ايليجانس</em&gt; عن طريق &quot;تجميد تكسير&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

قياس آثار البكتيريا على<em&gt; C. ايليجانس</em&gt; السلوك باستخدام مقايسة الاحتفاظ البيض

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

نموذج نقص الأكسجين المجاعة لنقص التروية / ضخه في<em&gt; C. ايليجانس</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.