وقد تم تمويل هذا العمل في جزء من المعاهد الوطنية للصحة (K08 - DK073545 معاهد الصحة القومية وبرعاية مركز أبحاث الإعاقة الفكرية الجديدة والتنموي جائزة الباحث) ، ومؤسسة فيلادلفيا ، وجامعة ولاية بنسلفانيا جائزة مكابي (MJF) ، فضلا عن أن تريستان صندوق مولين (MJF وبلدي). المحتوى هو فقط من مسؤولية الكتاب ولا تمثل بالضرورة وجهة النظر الرسمية للمعاهد الوطنية للصحة.

<ol>
	<li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans: A practical approach. , (1999).</li><li>Dingley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+dysfunction+variably+increases+oxidant+stress+in+Caenorhabditis+elegans.">Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans.</a> Mitochondrion. 10, 125-136 (2010).</li><li>Jones, B. N., Gilligan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=o-Phthaldialdehyde+precolumn+derivatization+and+reversed-phase+high-performance+liquid+chromatography+of+polypeptide+hydrolysates+and+physiological+fluids.">o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids.</a> Journal of chromatography. 266, 471-482 (1983).</li><li>Falk, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+pathway+profiling+of+mitochondrial+respiratory+chain+mutants+in+C.+elegans.">Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans.</a> Molecular genetics and metabolism. 93, 388-397 (2008).</li><li>Yudkoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+in+vivo+ureagenesis+with+stable+isotopes.">Measuring in vivo ureagenesis with stable isotopes.</a> Molecular genetics and metabolism. 100, 37-41 (2010).</li><li>Tsang, W. Y., Sayles, L. C., Grad, L. I., Pilgrim, D. B., Lemire, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+deficiency+in+Caenorhabditis+elegans+results+in+developmental+arrest+and+increased+life+span.">Mitochondrial respiratory chain deficiency in Caenorhabditis elegans results in developmental arrest and increased life span.</a> J Biol Chem. 276, 32240-32246 (2001).</li></ol>

الإعلان عن أي تضارب في المصالح.

تطبيق مطياف الكتلة لقياس وفرة النظائر في الأيضات سيط يوفر صورة تفصيلية رائعة من التغيرات الحيوية الهامة 5. هنا ، لقد قدمنا ​​بروتوكولات وإجراءات تفصيلية لاستغلال هذه المنهجية حساسة للغاية ومحددة لتقييم تدفق سيط الأيض في نموذج حيواني تنوعا وراثيا ، C. ايليجانس. في الواقع ، وتغذية الحيوانات التي تعيش مع النظائر مستقرة المسمى السلائف الأيضية (مثل C - 13 الجلوكوز) تصاريح البصيرة الرواية إلى التمويه وسيط المسار الأيضي عند قياس تخصيب النظائر في الأيضات المصب. قمنا بتطوير منهجية موثوقة للحصول على تحليل دقيق للتدفق من خلال تحلل ، والتمثيل الغذائي بيروفات ، ودورة حمض الكربوكسيليك. ويمكن تحقيق ذلك في كل من الحيوانات التي تتغذى من النظائر مستقرة المراحل المبكرة التنموية اليرقات أو يبدأ في فترة ما بعد الإنقسامية الكبار. ويمكن دراسة تخصيب النظائر في الأنواع المختلفة المستقلب بالتزامن مع دودة كاملة مجانا التنميط الأحماض الأمينية التي كتبها عالية الأداء اللوني السائل ، كما هو موضح سابقا 4 ، لتحديد تخصيب النظائر المطلقة في أنواع مختلفة من ألانين خال من الديدان ، اسبارتاتي ، الغلوتامات ، والجلوتامين. في الواقع ، ويتم الحصول على أنماط ثابتة لتخصيب النظائر عند قياس الأحماض الأمينية والأحماض العضوية في analytes aliquots من 1000 إلى 2000 الديدان الصغيرة متزامن الكبار. هذه المنهجية يمكن أن تميز بحساسية تدفق غير طبيعي وسيط في سلالات الميتوكوندريا النووي القائم على الجينات الطافرة المتعلقة البرية من نوع الديدان. هذا الأسلوب يحمل قيمة للتحقيق تغييرات في مسارات تدفق البيوكيميائية محددة ذات صلة الأخطاء الشائعة في استقلاب خلقي. على وجه الخصوص ، والاستفادة من الجلوكوز المسمى مع النظائر مستقرة يبلغ تدفق الكربون من خلال المسارات الأيضية المركزية ذات الصلة لأمراض الميتوكوندريا. في الواقع ، تشير البيانات المتوفرة لدينا تطبيق هذه المنهجية يمكن أن تميز بحساسية تدفق غير طبيعي وسيط في جين المستندة الميتوكوندريا النووية سلالة الثالث معقدة متحولة الوحيدات (ISP - 1 (qm150)) نسبة إلى البرية من نوع الديدان. من المهم أن ندرك أنه منذ اكتمال التعرض النظائر على مدى فترة من واحد إلى عدة أيام ، وتخصيب النظائر كميا يمثل &quot;حالة ثابتة&quot; قيمة التخصيب بدلا من قياس تدفق على مدى فترة محددة من الزمن كما قد يتم تحديدها في الخلية المستندة إلى النماذج تتعرض لالنظير لمدة دقيقة إلى ساعة. آخر المحتملة الحد من تدفق مسار التحقيق على مدى تطور النيماتودا يتعلق اختلاف أطوال نمو اليرقات التي تحدث في سلالات متحولة معينة. على سبيل المثال ، لا يعرف العديد من الطفرات الميتوكوندريا شديدة لسبب الاعتقال في المرحلة الثالثة اليرقات (L3) 6. وبالتالي ، قد دمج تحليل النظائر فقط في الحيوانات التي تعيش إلى سن البلوغ لا تكون ممثلة للسكان متحولة بأكمله. ومع ذلك ، يمكن تكييف هذه المنهجية لتقييم إدماج النظائر في الأيضات الوسيط في مرحلة اليرقات محددة (مثل L2) بدلا من انتظار الحيوانات للوصول الى مرحلة البلوغ. وبالمثل ، قد غيرت الحيوانات التي تدخل في مرحلة اليرقات البديلة المعروفة باسم dauer المرجح بدرجة كبيرة (على الأرجح أقل من ذلك) الأيضية التدفق النسبي للحيوانات الذين المتابعة مباشرة من خلال مراحل اليرقات الأربعة. ويمكن أيضا أن يتم تنفيذ دراسة في المستقبل لتقييم التأسيس مباشرة النظائر في الحيوانات مرحلة dauer من خلفيات وراثية مختلفة. تعريض الحيوانات لنظائر مستقرة لفترة زمنية محددة (هنا ، و 24 إلى 48 ساعة) بداية الحيوانات مرة واحدة وصلت إلى مرحلة الشباب البالغين (B كما هو مفصل في البروتوكول) يزيل أثر التنموية فترات متفاوتة. في حين أن 13 C - الجلوكوز باستجواب فقط تحلل ، والتمثيل الغذائي البيروفات وتدفق دورة حمض الكربوكسيليك ، يمكن استشفاف يحتمل النظائر الأخرى المقررة لاستجواب البيوكيميائية تدفق المسار من خلال مسارات أخرى ذات أهمية في الديدان الخيطية. على سبيل المثال ، قد تكون استخدمت 15 N - جليكاين لتقييم دوران حامض أميني في الديدان الخيطية. آخر قيود محتملة لهذا النهج هو مستوى حساسية الكشف المستقلب. تجربتنا تشير مطلوبة بحد أدنى من 500 الديدان الخيطية البالغين الشباب لتحديد بنجاح إدماج النظائر بالطرق HPLC و GC / MS يعملون هنا. استخدام الأدوات مع قدر أكبر من الحساسية ، مثل فائقة الأداء اللوني السائل (UPLC) ، قد تتيح المزيد من خفض عدد الحيوانات التي تمت دراستها ، على الرغم من أننا نتوقع هذا من غير المحتمل أن تسمح الكميات موثوقة من الأيضات ودمج النظائر في المستقلب الأنواع من الديدان واحد. درسنا السكان دودة من 1000 حيوان في التجربة ، والتي وجدنا لكشف موثوق الأيضات فرة منخفضة في كل سلالة. ومع ذلك ، لا يمكن إلا بعض الأيضات ، مثل GABA ، يمكن قياسها كميا ، يعتمد عليه في أعداد أكبر بكثير من الديدان الخيطية ، بناء على أمر من1x10 6 الحيوانات 4. باختصار ، النظائر المستقرة يوفر التنميط غير الغازية وآمنة (غير المشعة) النهج الذي لطالما استخدمت لدراسة أخطاء في استقلاب خلقي. تطبيق هذه المنهجية إلى C. ايليجانس يوفر قدرة الرواية على التحقيق في الجسم الحي ، في الوقت الحقيقي ، والتعديلات الأيضية التي تحدث على مستوى كل الحيوانات ، والتي قد تنجم عن الاضطرابات الجينية الفردية و / أو التعرض للعوامل الدوائية.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>S. basal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1 - page 59</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Cholesterol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-8503</td>
<td>Protocol B</td>
</tr>
<tr>
<td>OP50 E. coli</td>
<td>&nbsp;</td>
<td>Caenorhabditis Genetics Center</td>
<td>&nbsp;</td>
<td>Protocols A and C</td>
</tr>
<tr>
<td>K12 E. coli</td>
<td>&nbsp;</td>
<td>Caenorhabditis Genetics Center</td>
<td>&nbsp;</td>
<td>Protocol B</td>
</tr>
<tr>
<td>NGM agar</td>
<td>&nbsp;</td>
<td>Research Products INT</td>
<td>N81800-1000.0</td>
<td>Protocols A, B, and C</td>
</tr>
<tr>
<td>13C glucose (universally labeled)</td>
<td>&nbsp;</td>
<td>Cambridge Isotope labs</td>
<td>CLM-1396</td>
<td>Protocol A and C</td>
</tr>
<tr>
<td>13C glucose (1,6 labeled)</td>
<td>&nbsp;</td>
<td>Cambridge Isotope labs</td>
<td>CLM-2717</td>
<td>Protocol B</td>
</tr>
<tr>
<td>60% Perchloric acid (PCA)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>MK2766500</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>&epsilon;-aminocaproic acid (Internal Standard)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-2504</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>MTBSTFA</td>
<td>&nbsp;</td>
<td>Regis</td>
<td>270243</td>
<td>Protocol E</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Regis</td>
<td>270010</td>
<td>Protocol E</td>
</tr>
<tr>
<td>AG 1-X8, 100-200, Chloride form resin</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>140-1441</td>
<td>Protocols A and B (organic acid extraction)</td>
</tr>
<tr>
<td>AG 50W-X8, 100-200, Hydrogen form resin</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>142-1441</td>
<td>Protocols A and B (amino acid extraction)</td>
</tr>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-8875</td>
<td>Protocol C</td>
</tr>
<tr>
<td>NaOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8045</td>
<td>Protocol C</td>
</tr>
<tr>
<td>3N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol D</td>
</tr>
<tr>
<td>1N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol A</td>
</tr>
<tr>
<td>0.1 N HCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>320331</td>
<td>Protocol D</td>
</tr>
<tr>
<td>4N NH4OH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>30501</td>
<td>Protocol D</td>
</tr>
<tr>
<td>4N KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>484016</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Deionized water</td>
<td>&nbsp;</td>
<td>Milli Q Biocel</td>
<td>ZMQA60F01</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Helium</td>
<td>&nbsp;</td>
<td>Airgas</td>
<td>24001364</td>
<td>Protocol C2</td>
</tr>
<tr>
<td>SMZ-800 Zoom Stereo-Microscope</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>NI MNA41000</td>
<td>Protocols A, B, and C</td>
</tr>
<tr>
<td>pH meter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S68167</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Table top centrifuge - 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>05-400-93</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Incubated platform shaker</td>
<td>&nbsp;</td>
<td>New Brunswick</td>
<td>M1324-0004</td>
<td>Protocol B</td>
</tr>
<tr>
<td>Mini LabRoller Rotator</td>
<td>&nbsp;</td>
<td>Labnet</td>
<td>H-5500</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Pestles</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>K749520-0090</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Drill</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>K749540-0000</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>1 L glass beaker</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-539P</td>
<td>&nbsp;</td>
<tr>
<td>1 L Erlenmeyer Flasks</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-368</td>
<td>&nbsp;</td>
<tr>
<td>25 mL Erlenmeyer Flasks</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-356</td>
<td>Protocol B</td>
</tr>
<tr>
<td>7ml round-bottom glass tubes + rubber stopper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VT6431</td>
<td>Protocols A, B, and C1,C2</td>
</tr>
<tr>
<td>10 ml round-bottom glass tubes + rubber stopper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VT6430</td>
<td>Protocol C2</td>
</tr>
<tr>
<td>Blue top tubes</td>
<td>&nbsp;</td>
<td>Labco</td>
<td>438B</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical plastic tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>14-959-49A</td>
<td>All protocols</td>
</tr>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-681-320</td>
<td>Protocols A and B</td>
</tr>
<tr>
<td>Syringes (1 mL, 10 mL, 20 mL)</td>
<td>&nbsp;</td>
<td>BD Medical</td>
<td>301025, 301029, 301031</td>
<td>Protocols C1 and C2</td>
</tr>
<tr>
<td>25 gauge needles</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-253-131</td>
<td>Protocols C1 and C2</td>
</tr>
<tr>
<td>Poly-Prep Chromatography Columns</td>
<td>&nbsp;</td>
<td>BIO-RAD</td>
<td>731-1550</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14673-043</td>
<td>Protocol D</td>
</tr>
<tr>
<td>60mm Petri dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25373-085</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Glass chamber (cut bottom of 1 L flask) fitted with grease-sealed 3-way stopcock</td>
<td>&nbsp;</td>
<td>Custom Made in Glass Shop</td>
<td>&nbsp;</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Optically transparent glass plate</td>
<td>&nbsp;</td>
<td>Custom Made in Glass Shop</td>
<td>&nbsp;</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Reacti-Vap III Evaporator</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>18826</td>
<td>Protocol D</td>
</tr>
<tr>
<td>Cotton</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14224-516</td>
<td>Protocol D</td>
</tr>
<tr>
<td>High Vacuum Grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>Protocol C1</td>
</tr>
<tr>
<td>Aluminum Foil</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>01-213-18</td>
<td>Protocol B</td>
</tr>
<tr>
<td>Timer</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>T-2504-5</td>
<td>Protocol C</td>
</tr>
<tr>
<td>Gas-Ratio Mass Spectrometer</td>
<td>&nbsp;</td>
<td>Thermophinigan</td>
<td>&nbsp;</td>
<td>Protocol D</td>
</tr>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>HP</td>
<td>5980/5971</td>
<td>Protocol D</td>
</tr>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>6980N/5973N</td>
<td>Protocol D</td>
</tr>
<tr>
<td>HPLC</td>
<td>&nbsp;</td>
<td>Varian</td>
<td>9010</td>
<td>Protocol D</td>
</tr>		</tbody>
		</table>
		</div>

stable isotopic profiling of intermediary metabolic flux in developing and adult stage caenorhabditis elegans

وقد سمحت التنميط النظائر مستقرة طويلة التحقيقات الحساسة من عواقب الطفرات الوراثية الاستقلابية و / أو علاجات دوائية في النماذج الخلوية والثدييات. هنا ، نحن تصف الأساليب التفصيلية لأداء التنميط النظائر المستقرة لعملية التمثيل الغذائي وتدفق وسيط الأيض في الخيطية ، ايليجانس انواع معينة. يتم وصف أساليب التنميط دودة كاملة مجانا الأحماض الأمينية ، وصفت ثاني أكسيد الكربون ، الأحماض العضوية المسمى ، والأحماض الأمينية المسمى في الحيوانات المعرضة لنظائر مستقرة سواء من التنمية في وقت مبكر على لوحات نمو الديدان الخيطية آغار وسائل الإعلام أو بداية وصغار البالغين في حين تتعرض لعلاجات دوائية مختلفة في الثقافة السائل. كميا والأحماض الأمينية الحرة بحلول عالية الأداء اللوني السائل (HPLC) في aliquots دودة كاملة في استخراج حمض البيركلوريك 4 ٪. المسمى C - 13 عالميا الجلوكوز أو 1،6 -- 13 C - 2 يستخدم الجلوكوز والسلائف والنظائر المستقرة التي تسمى الكربون التي تتبعها مطياف الكتلة في ثاني أكسيد الكربون (سواء في الغلاف الجوي والمنحلة) وكذلك في الأيضات يدل على تدفق من خلال تحلل ، والتمثيل الغذائي بيروفات ، ودورة حمض الكربوكسيليك. وشملت النتائج ممثل لإظهار آثار التعرض الوقت النظير ، ومختلف بروتوكولات تبادل المعلومات البكتيرية ، والطرق البديلة لانقطاع في الدودة الخيطية من النوع البري ، فضلا عن مدى قريب التأسيس النظائر في الميتوكوندريا معقدة الديدان الطافرة الثالث (ISP - 1 (qm150) ) نسبة إلى البرية من نوع الديدان. تطبيق التنميط النظائر مستقرة في الديدان الخيطية المعيشة يوفر قدرة الرواية على التحقيق على مستوى كل الحيوانات تعديلات الأيض في الوقت الحقيقي التي هي سبب الاضطرابات الجينية الفردية و / أو علاجات دوائية.

Watch this Scientific Journal Video about Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans at JoVE.com

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

يوصف التنميط النظائر مستقرة التحليل اللوني للغاز الكتلة الطيفي لتدفق الاستقلابية المتوسطة في الديدان الخيطية<em&gt; انواع معينة ايليجانس</em&gt;. وترد تفاصيل أساليب تقييم تخصيب النظائر في ثاني أكسيد الكربون والأحماض العضوية ، والأحماض الأمينية بعد التعرض النظير إما أثناء تطوير أغار على لوحات أو أثناء مرحلة البلوغ في الثقافة السائل.

النظائر مستقرة من التنميط التمويه الاستقلابية المتوسطة في مرحلة تطوير والكبار انواع معينة ايليجانس</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

stable-isotopic-profiling-intermediary-metabolic-flux-developing

Research

JoVE Journal

Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

13.4K Views.  The Children's Hospital of Philadelphia. يوصف التنميط النظائر مستقرة التحليل اللوني للغاز الكتلة الطيفي لتدفق الاستقلابية المتوسطة في الديدان الخيطية انواع معينة ايليجانس. وترد تفاصيل أساليب تقييم تخصيب النظائر في ثاني أكسيد الكربون والأحماض العضوية ، والأحماض الأمينية بعد التعرض النظير إم...

Video: Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis 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 ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

والعزلة في المختبر تفعيل انواع معينة ايليجانس الحيوانات المنوية

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

الوقت الفاصل بين مرحلة التطور الجنيني المبكر من الميكروسكوب في انواع معينة ايليجانس

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

الأساسية<em&gt; Caenorhabditis ايليجانس</em&gt; طرق: التزامن والمراقبة

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

استخراج البروستاجلاندين والتحليل في<em&gt; انواع معينة ايليجانس</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

زراعة<em&gt; انواع معينة ايليجانس</em&gt; في ثلاثة أبعاد في المختبر

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

الجمع بين النوكليوتيدات والاستخراج البروتين في ايليجانس كاينورهابديتيس

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

قياس معدلات استهلاك الأوكسجين في سليمة ايليجانس كاينورهابديتيس

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

تنقية وتحليل Caenorhabditis elegans خارج الخلية الحويصلات

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

النمذجة العمر المرتبطة الأمراض العصبية في Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...