وأيد هذا العمل من قبل ويلكوم ترست وجلاكسو سميث كلاين (GSK) والمعهد الاسكتلندي للتشوير الخليوي (وهي الآن جزء من الفسفرة البروتين MRC وحدة Ubiquitylation).

<ol>
	<li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+ubiquitin-like+proteins.">Origin and function of ubiquitin-like proteins.</a> Nature. 458, 422-429 (2009).</li><li>Schulman, B. A., Harper, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-like+protein+activation+by+E1+enzymes:+the+apex+for+downstream+signalling+pathways.">Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways.</a> Nat Rev Mol Cell Biol. 10, 319-331 (2009).</li><li>Siomi, H., Siomi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+road+to+reading+the+RNA-interference+code.">On the road to reading the RNA-interference code.</a> Nature. 457, 396-404 (2009).</li><li>Kaelin, W. G., Ratcliffe, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensing+by+metazoans:+the+central+role+of+the+HIF+hydroxylase+pathway.">Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.</a> Mol Cell. 30, 393-402 (2008).</li><li>Melvin, A., Mudie, S., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromatin+remodeler+ISWI+regulates+the+cellular+response+to+hypoxia:+role+of+FIH.">The chromatin remodeler ISWI regulates the cellular response to hypoxia: role of FIH.</a> Mol Biol Cell. 22, 4171-4181 (2011).</li><li>Bhinder, B., Djaballah, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+analyzing+actives+in+random+RNAi+screens:+introducing+the+&amp;#34;H+Score&amp;#34;+for+hit+nomination+&amp;+gene+prioritization.">A simple method for analyzing actives in random RNAi screens: introducing the &amp;#34;H Score&amp;#34; for hit nomination &amp; gene prioritization.</a> Comb Chem High Throughput Screen. 15, 686-704 (2012).</li><li>Bett, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P-body+component+USP52/PAN2+is+a+novel+regulator+of+HIF1A+mRNA+stability.">The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability.</a> Biochem J. 451, 185-194 (2013).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Birmingham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat Methods. 6, 569-575 (2009).</li><li>Stagg, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TRC8+E3+ligase+ubiquitinates+MHC+class+I+molecules+before+dislocation+from+the+ER.">The TRC8 E3 ligase ubiquitinates MHC class I molecules before dislocation from the ER.</a> J Cell Biol. 186, 685-692 (2009).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF146+is+a+poly(ADP-ribose)-directed+E3+ligase+that+regulates+axin+degradation+and+Wnt+signalling.">RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling.</a> Nat Cell Biol. 13, 623-629 (2011).</li><li>Jackson, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profiling+reveals+off-target+gene+regulation+by+RNAi.">Expression profiling reveals off-target gene regulation by RNAi.</a> Nat Biotechnol. 21, 635-637 (2003).</li><li>Birmingham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3'+UTR+seed+matches,+but+not+overall+identity,+are+associated+with+RNAi+off-targets.">3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.</a> Nat Methods. 3, 199-204 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whither+RNAi.">Whither RNAi.</a> Nat Cell Biol. 5, 489-490 (2003).</li></ol>

والكتاب ليس لديهم ما يكشف.

وقد أثبت استخدام شاشات سيرنا على نطاق الجينوم في خلايا الثدييات أن تكون قيمة للغاية في تحديد المنظمين رواية من المسارات البيولوجية متميزة. هنا، ونحن قد وصفت استخدام شاشة ubiquitome سيرنا المستهدفة لتحديد المنظمين للاستجابة بوساطة HIF1A الخلوية لنقص الأكسجة. شاشات المستهد?...

تعديل البروتينات مع اليوبيكويتين وتشبه جزيئات اليوبيكويتين (UBLs) يمثل نظام البيوكيميائية توسعية الذي ينظم مسارات بيولوجية متنوعة واستجابات التوتر. المرفق التساهمية من UBLs إلى البروتينات المستهدفة يمكن أن يكون لها نتائج مختلفة تنظم الاستقرار والتوطين، وظيفة أو interactome الركيزة 1. أنشئت الخطوات الأنزيمية الكامنة التعديل الأول لأسامة بن لادن اليوبيكويتين، ويخدم الآن كنموذج للتعديل مع معظم UBLs، بما في ذلك السومو، NEDD8، ISG15 وFAT10. لتعديل لتحدث، ويتم تنشيط مجموعة الكربوكسيل من diglycine عزر أسامة بن لادن أول من انزيم تفعيل E1 لتشكيل ثيول عالية الطاقة التي يتم نقلها إلى السيستين موقع نشط من انزيم التصريف E2. ثم يتفاعل مع E2-E3 يغاز الركيزة ملزمة للتوسط نقل أسامة بن لادن على (عادة) بقايا يسين الهدف خلق سلسلة متفرعة (isopeptide) الربط 2. يمكن أن يحدث جولات متعاقبة من التعديل لبناء سلاسل isopeptide على الركيزة، والتي لاليوبيكويتين يمكن أن يحدث من خلال أي من سبعة lysines لها، أو من خلال-N محطة ميثيونين لخلق سلاسل اليوبيكويتين الخطية. هذه التعديلات تشكل طبولوجيا منفصلة مع أغراض متنوعة مثل إنشاء الزخارف التفاعل جديدة واستهداف البروتينات للتدهور قبل إزالة أسامة بن لادن بواسطة البروتياز المتخصصة. في حالة اليوبيكويتين هناك اثنين من الانزيمات E1، E2 30-40 الانزيمات التصريف، 600 E3 على الأقل ونحو 100 ligases الانزيمات deubiquitylating (يصف). في حين أن مسارات هي أقل توسعية لأخرى 10 أو نحو ذلك UBLs، وتعقد ubiquitome الشاملة يتيح التنوع الهائل في النتيجة البيولوجية للتعديل خاصة أسامة بن لادن. ومع ذلك، في حين تم إحراز تقدم كبير في علم الأحياء أسامة بن لادن، لا تزال الأدوار الخلوية الدقيقة للأغلبية هذه المكونات ubiquitome غير معروف. استخدام الباحث قصيرةوقد برزت erfering حمض النووي الريبي (الرناوات siRNAs) كأداة قوية في مجال علم الوراثة العكسية ويرجع ذلك إلى قدرة الرناوات siRNAs خصيصا لاستهداف mRNAs والخلوية للتدمير، والسماح للدور الجينات الفردية لفحصها في سياقات البيولوجية المختلفة 3. وقد استخدمت شاشات الجينوم لتحديد والتحقق من صحة المنظمين جديدة من العديد من العمليات الخلوية، وخلقت ثروة من البيانات المفيدة في متناول المجتمع العلمي على نطاق أوسع. ومع ذلك، في حين أثبتت شاشات الجينوم مفيدة للغاية، وشاشات استهدفت تزداد شعبية لأنها أرخص وأسرع وأقل تنطوي على إدارة البيانات والتقرير فقط على أعضاء الجينوم التي المحقق هو الأكثر اهتماما. لذلك، من أجل فهم أفضل التي تشارك العمليات الخلوية أسامة بن لادن مكونات الأسرة في، قمنا بتجميع مكتبة سيرنا الإنسان تستهدف جميع المعروفة ومكونات ubiquitome توقع. ويشمل هذا UBLs، E1 الانزيمات تفعيل، E2 التصريفالإنزيمات، ligases E3، مجال ملزم اليوبيكويتين (UBD) التي تحتوي على البروتينات ويصف. هذه المكتبة يمكن استخدامها لشاشة ضد طائفة واسعة من خطوط الخلايا مراسل من المشاكل البيولوجية متميزة، مما يتيح تحديد موضوعية حول مكونات أسامة بن لادن الرواية التي تحكم هذه الممرات. يصف بروتوكول التالية كيفية إجراء صارم سيرنا المستهدفة ubiquitome الشاشة لتحديد المنظمين رواية من الاستجابة تعتمد على HIF1A لنقص الأكسجة. تحت التوتر الأكسجين العادي، HIF1A يخضع لprolyl الهيدروكسيل أن يؤدي إلى الاعتراف بها والمستهدفة للتدهور من قبل فون هيبل لينداو (VHL) E3 يغاز معقدة 4. نقص الأكسجة يمنع الهيدروكسيل prolyl مما يؤدي إلى استقرار HIF1A واللاحقة ملزمة لعناصر استجابة نقص الأكسجة (HRES) لدفع التعبير الجيني. هنا، نحن تصف الشاشة باستخدام خلايا عظمية U20S معربا عن ستابلي يراعة luciferase تحت سيطرة ثلاث نسخ جنبا إلى جنب من هايpoxia استجابة عنصر (الخلايا U20S-تعليم حقوق الإنسان) 5. هذا البروتوكول يمكن تكييفها لأي مسار بيولوجي إذا قوي للقراءة من النشاط الصحفي هو تحقيق ويمكن أن يقترن مع الضوابط الإيجابية والسلبية المناسبة.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:288px;">Automated Liquid Dispenser</td>
			<td style="width:147px;">Fluid-X</td>
			<td style="width:200px;">XPP-721</td>
			<td style="width:156px;">http://www.fluidx.eu/BIOTRACK/xpp-721-liquid-handling-system.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Automated cell counter</td>
			<td>Nexcelom Bioscience</td>
			<td>Cellometer Auto T4</td>
			<td>http://www.nexcelom.com/Cellometer-Auto-T4/index.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hypoxia Workstation</td>
			<td>Ruskin</td>
			<td>In vivo2 300</td>
			<td>http://www.ruskinn.com/products/invivo2300</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Automated Cell Dispenser</td>
			<td>Thermo Scientific</td>
			<td>Matrix Wellmate</td>
			<td>http://www.matrixtechcorp.com/automated/pipetting.aspx?id=11</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Plate Shaker</td>
			<td>Heidolph</td>
			<td>Titramax 1000</td>
			<td>http://www.heidolph-instruments.co.uk/products/shakers-mixers/platform-shakers/vibrating/titramax/titramax-1000/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Luminometer</td>
			<td>Perkin Elmer</td>
			<td>Envision 2104 Multilabel Reader</td>
			<td>http://www.perkinelmer.co.uk/Catalog/Family/ID/EnVision%20Multilabel%20Plate%20Readers</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">White Walled Assay Plate</td>
			<td>Greiner Bio One</td>
			<td>655083</td>
			<td>http://www.greinerbioone.com/en/row/articles/catalogue/article/37_11/13221/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Clear Plate Film</td>
			<td>Perkin Elmer</td>
			<td>1450-461</td>
			<td>http://www.perkinelmer.co.uk/Catalog/Product/ID/1450-461</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">siRNA library</td>
			<td>Thermo Scientific</td>
			<td>On-Target Plus</td>
			<td>http://www.thermoscientificbio.com/rnai-and-custom-rna-synthesis/sirna/on-targetplus-sirna/search-gene/</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Transfection reagent</td>
			<td>Invitrogen</td>
			<td>Lipofectamine RNAimax</td>
			<td>http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Transfection-Selection/lipofectamine-rnaimx.html</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reduced Serum Medium</td>
			<td>Invitrogen</td>
			<td>Optimem</td>
			<td>http://products.invitrogen.com/ivgn/product/31985062?ICID=search-product</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
			<td>http://products.invitrogen.com/ivgn/product/41965039#</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>https://products.invitrogen.com/ivgn/product/16000044?ICID=search-product#</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tryspin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product#</td>
		</tr>
	</tbody>
</table>

sirna screening to identify ubiquitin and ubiquitin-like system regulators of biological pathways in cultured mammalian cells

تعديل آخر متعدية من البروتينات مع اليوبيكويتين وتشبه جزيئات اليوبيكويتين (UBLs) والناشئة باعتبارها شبكة الإشارات الخلوية الحيوية التي تنظم مسارات بيولوجية متنوعة بما في ذلك الاستجابة نقص الأكسجة، proteostasis، استجابة الحمض النووي من التلف والنسخ. لفهم أفضل لكيفية UBLs تنظيم مسارات ذات الصلة إلى الأمراض التي تصيب البشر، قمنا بتجميع سيرنا الإنسان &quot;ubiquitome&quot; مكتبة تتكون من 1،186 سيرنا حمامات دوبلكس تستهدف جميع المعروفة ومكونات نظام مسارات أسامة بن لادن توقع. هذه المكتبة يمكن فحص ضد مجموعة من خطوط الخلايا معربا عن صحفيين من المسارات البيولوجية المتنوعة لتحديد المكونات التي تعمل أسامة بن لادن كما المنظمين إيجابية أو سلبية للمسار في السؤال. هنا، نحن تصف بروتوكول الاستفادة من هذه المكتبة لتحديد ubiquitome المنظمين للاستجابة بوساطة HIF1A الخلوية لنقص الأكسجين باستخدام مراسل luciferase القائم على النسخ. مرحلة التطوير الأولي مقايسةيتم تنفيذها لوضع معايير الفرز مناسبة من خط الخلية قبل تنفيذ الشاشة في ثلاث مراحل: الابتدائي والثانوي والجامعي الفحص / deconvolution. استخدام استهدفت أكثر من مكتبات الجينوم سيرنا كله تزداد شعبية لأنها تتيح ميزة الإبلاغ فقط على أعضاء المسار الذي المحققين هي الأكثر اهتماما. على الرغم من القيود المتأصلة في سيرنا الفرز، على وجه الخصوص، ايجابيات كاذبة الناجمة عن سيرنا خارج الهدف آثار، وتحديد المنظمين رواية حقيقية من المسارات في مسألة تفوق أوجه القصور هذه، والتي يمكن التغلب عليها عن طريق إجراء سلسلة من التجارب التي تضطلع التحكم بعناية.

قبل الفحص، يتم تأسيس نقص الأكسجة، استجابة الخلايا U20S-تعليم حقوق الإنسان. الخلايا U20S-تعليم حقوق الإنسان تعبر عن بناء مراسل تتكون من يراعة luciferase تنصهر المصب من ثلاث نسخ جنبا إلى جنب من العنصر استجابة نقص الأكسجة، والتي لا بد من مغاير HIF1A/HIF1B عند التعرض لنقص الأكسجة (...

Watch this Scientific Journal Video about siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells at JoVE.com

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

هنا، نحن تصف منهجية لإجراء سيرنا المستهدفة &quot;ubiquitome&quot; الشاشة لتحديد اليوبيكويتين الرواية والمنظمين مثل اليوبيكويتين الاستجابة بوساطة HIF1A الخلوية لنقص الأكسجة. وهذا يمكن أن تتكيف مع أي مسار بيولوجي حيث قراءة قوية من النشاط الصحفي هو متاح.

سيرنا فحص لتحديد اليوبيكويتين واليوبيكويتين مثل المنظمين نظام مقررات تمهيدية البيولوجية في خلايا الثدييات مثقف

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

sirna-screening-to-identify-ubiquitin-ubiquitin-like-system

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Biology

11.3K Views.  University of Dundee.  هنا، نحن تصف منهجية لإجراء سيرنا المستهدفة "ubiquitome" الشاشة لتحديد اليوبيكويتين الرواية والمنظمين مثل اليوبيكويتين الاستجابة بوساطة HIF1A الخلوية لنقص الأكسجة. وهذا يمكن أن تتكيف مع أي مسار بيولوجي حيث قراءة قوية من النشاط الصحفي هو متا?...

Video: siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

متجانسة الكشف السريع عن طريق فحوصات البيولوجي المغناطيسي نظام Biosensing التحوير

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

esiRNA بعثة لفحص رني في خلايا الثدييات

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

LentiPlex بعثة مجمع shRNA الفرز مكتبة في خلايا الثدييات

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

[رني الفرز لتحديد الظواهر تال للمرحلة الجنينية في C. ايليجانس</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

عزلة البروتينية ريبوسوم الوليدة منضم<em&gt; في المختبر</em&gt; لتحديد المواقع وقفة بالحركة على طول مرنا

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

A الكمي الفحص لدراسة البروتين: تفاعلات الحمض النووي، اكتشاف المنظمين الترانسكربتي التعبير الجيني، وتحديد رواية وكلاء المضادة للورم

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

عكس الخميرة نظام هجين اثنين من التعرف على الثدييات مستقبلات النووية التي تتفاعل مع بقايا تقوم الليجندات و / أو الخصوم

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

فحوصات مراسل المتوازية نطاق واسع في خلايا الثدييات مثقف

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

التصوير المحلية الكالسيوم<sup&gt; 2+</sup&gt; الإشارات في خلايا الثدييات مثقف

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...