עבודה זו נתמכה על ידי קרן Wellcome, GlaxoSmithKline (GSK) ואת המכון הסקוטי לאיתות סלולרי (כיום חלק של זירחון חלבון MRC ויחידת 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>

יש המחברים אין לחשוף.

השימוש במסכי siRNA הגנום בתאי יונקים הוכיחו להיות יקר מאוד בזיהוי רגולטורים רומן של מסלולים ביולוגיים מובהקים. הנה, יש לנו תיארתי את השימוש במסך siRNA ubiquitome ממוקד לזהות רגולטורים של התגובה התאית בתיווך HIF1A להיפוקסיה. מסכי ממוקדים הופכים אטרקטיביים יותר ויותר כמו שהם בדרך...

שינוי של חלבונים עם מולקולות יוביקוויטין וכמו היוביקוויטין (UBLs) מייצג מערכת ביוכימית רחבת ידיים המווסתת את המסלולים ביולוגיים מגוונים ותגובות לחץ. הקובץ המצורף קוולנטיים של UBLs לחלבוני היעד שלהם יכול להיות תוצאות שונות המסדירות את היציבות, לוקליזציה, פונקציה או interactome של המצע 1. הצעדים האנזימטית שבבסיס שינוי UBL הוקמו ראשון ליוביקוויטין, וכעת משמשים כמופת לשינוי עם רוב UBLs, כולל SUMO, NEDD8, ISG15 וFAT10. ללהתרחש שינוי, קבוצת carboxylate של מוטיב diglycine UBL מופעלת לראשונה על ידי אנזים הפעלת E1 כדי ליצור תיאול עתירה אנרגיה שמועברת לציסטאין הפעיל באתר של אנזים conjugating E2. E2 אז אינטראקציה עם האנזים E3 בכריכת מצע לתווך העברת UBL על (בדרך כלל) שאריות ליזין יעד יצירת מסועפת שרשרת (isopeptide) הצמדה 2. סיבובים רצופים של שינוי יכולים להתרחש לבנות רשתות isopeptide על גבי המצע, אשר להיוביקוויטין יכול להתרחש דרך כל השבעה lysines, או באמצעות מתיונין N-המסוף שלה כדי ליצור שרשרות יוביקוויטין ליניארי. שינויים אלה יוצרים טופולוגיות דיסקרטיות עם מטרות מגוונות כגון יצירת מוטיבים אינטראקציה חדשות ומיקוד חלבונים להשפלה לפני הסרת UBL ידי פרוטאזות מומחה. במקרה של היוביקוויטין יש שני E1 אנזימים, 30-40 אנזימי conjugating E2, לפחות 600 E3 ligases וכ 100 אנזימים deubiquitylating (ממכנים). בעוד שהמסלולים הם פחות מרחיב לאחר 10 או כך UBLs, מורכבות ubiquitome הכוללות מאפשרת גיוון עצום בתוצאה הביולוגית של שינוי UBL מסוים. עם זאת, בעוד התקדמות גדולה בביולוגיה UBL נעשה, התפקידים הסלולריים המדויקים של רוב רכיבי ubiquitome אלה אינם ידועים. השימוש בint הקצרחומצת erfering ריבונוקלאית (siRNAs) התפתחה ככלי רב עוצמה בגנטיקה הפוכה בשל היכולת של siRNAs למקד במיוחד mRNAs הסלולרי להשמדה, מה שמאפשר את תפקידם של גנים בודדים כדי להיבחן בהקשרים ביולוגיים שונים 3. מסכי הגנום כולו נעשו שימוש כדי לזהות ולאמת רגולטורים חדשים של תהליכים תאיים רבים, ויצרו שפע של מידע שימושי לנגיש לקהילה המדעית הרחבה יותר. עם זאת, בעוד מסכי הגנום כולו הוכיחו מאוד שימושיים, מסכי ממוקדים הופכים פופולריים יותר ויותר כמו שהם זולים יותר, מהירים יותר, כרוכים בפחות ניהול נתונים ודיווח רק על חברים של הגנום שבו החוקר הוא מעוניין ביותר. לכן, כדי להבין טוב יותר אילו רכיבי משפחת התהליכים תאיים UBL מעורבים, יש לנו מלוקט ספרייה אנושית siRNA מיקוד כל ידוע וחזויי מרכיבי ubiquitome. זה כולל את UBLs, אנזימי הפעלת E1, E2 conjugatingאנזימים, ligases E3, מחייב רישום היוביקוויטין (UBD) המכיל חלבונים וממכנים. ספרייה זו יכולה לשמש למסך נגד מגוון רחב של שורות תאי כתב של בעיות ביולוגיות שונות, ובכך מאפשרת זיהוי משוחד של רכיבי UBL רומן המסדירים מסלולים אלה. הפרוטוקול הבא מתאר כיצד לבצע siRNA ממוקד קפדני ubiquitome מסך לזהות רגולטורים רומן של תגובת HIF1A תלויה להיפוקסיה. תחת מתח חמצן רגיל, HIF1A כפוף להידרוקסילציה prolyl שגורם לו להיות מוכרים וממוקדות לשפלה על ידי פון היפל לינדאו (VHL) מורכב האנזים E3 4. היפוקסיה מעכבת הידרוקסילציה prolyl המובילה להתייצבות HIF1A ובה לאחר מכן מחייב אלמנטי תגובת היפוקסיה (HREs) לנהוג ביטוי גנים. כאן, אנו מתארים מסך באמצעות תאי אוסטאוסרקומה U20S יציבות להביע גחלילית בלוציפראז תחת שליטתו של שלושה עותקי טנדם של Hypoxia תגובת אלמנט (תאי U20S-HRE) 5. פרוטוקול זה יכול להיות מותאם לכל מסלול ביולוגי אם קריאה מתוך חזק של פעילות העיתונאית הוא בר השגה והוא יכול להיות בשילוב עם בקרות חיוביות ושליליות מתאימות.

<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

שינוי שלאחר translational של חלבונים עם מולקולות יוביקוויטין וכמו היוביקוויטין (UBLs) מתגלה כרשת איתות תאית דינמית המווסתת את המסלולים ביולוגיים מגוונים כולל תגובת היפוקסיה, proteostasis, תגובת הנזק לדנ&quot;א ושעתוק. כדי להבין טוב יותר כיצד UBLs להסדיר מסלולים רלוונטיים למחלות בבני אדם, יש לנו מלוקט siRNA אנושי &quot;ubiquitome&quot; ספרייה בהיקף של 1,186 בריכות דופלקס siRNA מיקוד כל ידוע וחזויי רכיבים של מסלולי מערכת UBL. ספרייה זו יכולה להיות מוקרנת נגד מגוון רחב של שורות תאים להביע כתבים של מסלולים ביולוגיים מגוונים כדי לקבוע אילו רכיבי UBL לפעול כרגולטורים חיוביים או שליליים של המסלול בשאלה. כאן אנו מתארים פרוטוקול ניצול בספרייה זו כדי לזהות ubiquitome-רגולטורים של התגובה התאית בתיווך HIF1A היפוקסיה באמצעות כתב בלוציפראז מבוסס שעתוק. שלב פיתוח assay ראשונימבוצע כדי לקבוע פרמטרים הקרנה מתאימים של הקו הסלולרי לפני ביצוע המסך בשלושה שלבים: הקרנה / deconvolution ראשונית, שניונית ושלישוניים. השימוש ממוקד על ספריות siRNA הגנום כולו הופך לפופולרי יותר ויותר כפי שהוא מציע את היתרון של דיווח רק על חברים של המסלול שבו החוקרים מעוניינים ביותר. למרות מגבלות מובנות של הקרנת siRNA, בפרט שווא חיובי שנגרם על ידי siRNA השפעות מחוץ היעד, זיהוי של רגולטורים רומן אמיתיים של המסלולים בשאלה עולה ליקויים אלה, אשר ניתן להתגבר על ידי ביצוע סדרה של ניסויי שליטה התחייבו בזהירות.

לפני ההקרנה, היפוקסיה, יכולת התגובה של תאי U20S-HRE היא הוקמה. תאי U20S-HRE להביע לבנות כתב בהיקף של גחלילית בלוציפראז התמזגו במורד הזרם של שלושה עותקי טנדם של אלמנט תגובת היפוקסיה, שהוא מחויב heterodimer HIF1A/HIF1B בחשיפה להיפוקסיה (איור 1 א). תאים ממוקמים בתחנת עבודה היפוקסי?...

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

כאן, אנו מתארים מתודולוגיה לבצע siRNA ממוקד מסך &quot;ubiquitome&quot; לזהות היוביקוויטין רומן ורגולטורים כמו היוביקוויטין של התגובה התאית בתיווך HIF1A להיפוקסיה. זה יכול להיות מותאם לכל מסלול ביולוגי שבו חזקה לקריאה מהפעילות העיתונאית היא זמינה.

הקרנת siRNA לזיהוי יוביקוויטין ורגולטורים מערכת דמויות יוביקוויטין של מסלולים ביולוגיים בתאי יונקים בתרבית

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

Research

JoVE Journal

Biology

11.3K Views.  University of Dundee.  כאן, אנו מתארים מתודולוגיה לבצע siRNA ממוקד מסך "ubiquitome" לזהות היוביקוויטין רומן ורגולטורים כמו היוביקוויטין של התגובה התאית בתיווך HIF1A להיפוקסיה. זה יכול להיות מותאם לכל מסלול ביולוגי שבו חזקה לקריאה מהפעילות העיתונאית היא זמינה. 

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.

איתור מהיר של מבחני אחידים ביולוגית באמצעות מערכת מגנטית biosensing אפנון

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.

המשימה esiRNA עבור הקרנת RNAi בתאי יונקים

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.

Mutagenesis וניתוח פונקציונלי של תעלות יונים הביעו Heterologously בתאי יונקים

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 המשימה משולב הקרנת shRNA ספריית בתאי יונקים

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.

RNAi הקרנת לזהות פנוטיפים Postembryonic ב ג elegans</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.

הבידוד של הריבוזום פוליפפטידים המתהווה כרוכים<em&gt; במבחנה</em&gt; כדי לזהות אתרים השהה של תרגום לאורך ה-mRNA

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.

כמותי Assay לחקר חלבון: אינטראקציות-DNA, רגולטורים גלו תעתיק של ביטוי גנים, ולזהות חומרים אנטי סרטניים רומן

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.

הפוך מערכת דו היברידית שמרים לזהות יונקים הגרעיני קולטן שאריות כי אינטראקציה עם ligands ו / או יריבים

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.

מבחני כתב מקבילים מסיבי בתאי יונקים בתרבית

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.

ההדמיה המקומית Ca<sup&gt; 2 +</sup&gt; אותות בתאי יונקים בתרבית

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