אנו מודים המדע כולו Sigma החיים המשימה יעד מזהה ספריית פיתוח צוות, במיוחד קווין Gutshall למציאת טכנולוגיה משא ומתן תנאי הרשיון, והת&#39;ר Holemon על התמיכה שלה והשתתפות בדיונים לפתרון בעיות פוריות. כמו כן, אנו מודים לד&quot;ר יופ Gäken של לונדון בקינגס קולג&#39; בחופשיות על שיתוף שלא פורסמו התוצאות והצעות ו Qazi חמיד של RxBiosciences להכנת המנה הגדולה של cDNA וההתמדה שלו מקבל את זה משובטים. ברצוננו להודות נאן לין סקוט באהר מ SAFC לביצוע מערכי cDNA וניתוח נתונים. המשימה הוא סימן מסחרי רשום של Sigma-Aldrich ביוטכנולוגיה LP

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המחברים מצהירים יחסים כלכליים עם גורמים מסחריים בעלי עניין בעבודה שהוגש.

מיקרו RNA הם 20-24 RNAs נוקלאוטיד המווסתים ביטוי גנים לאחר תעתיק של התרגום מעכב mRNA, ולעתים קרובות, ערעור היציבות mRNA ממוקד (בדיקה ב [6]). מירנה אחד יכול לווסת כמה מאות mRNAs לשלוט התגובה של התא אותות התפתחותיים וסביבתיים. זיהוי ואימות mRNAs יעד הכרחי בקביעת התפקיד של מירנה ותפקוד במסלולים אלה. עם זאת, זיהוי המטרה היא לא פשוטה, כי אצל בעלי חיים, miRNAs ואתרי היעד שלהם הם לא משלים באופן מלא. &quot;הזרע&quot; באזור, מבסס 2 עד 7 מסוף 5&#39;-של מירנה, הוא בדרך כלל משלים מטרותיו. עם זאת, יש הרבה יוצאים מן הכלל זרע, במורד בסיס ההתאמה יכול לפצות על התאמה מושלמת זרע. מספר אלגוריתמים פותחו לחזות מטרות מירנה על בסיס התאמה זרע ופיצוי במורד הזרם, מבנה היעדעמדת D, שימור הרצף, ופרמטרים נוספים שונים נצפו על מטרות תוקף הניסוי (בדיקה ב [7]). בעוד סיליקו תחזיות נוח ולעשות רבות לזהות מטרות מירנה חוקיים, רובם חזו גנים להיכשל בדיקות אימות ניסיוני, ויעדים בפועל רבים אינם חזה. יתר על כן, מאז אלגוריתמים מבוססים על תכונות היעד שנקבעו קודם לכן, הם אינם מאפשרים גילוי של מטרות החורגות ממה שכבר ידוע. מספר מערכות ניסיוניות שימשו בהצלחה לזהות או לגלות מטרות תפקודיות מירנה בתאים חיים. אלה שיטות ההקרנה גלובליים כוללים microarrays ו-RNA רצף, RNA שיתוף immunoprecipitation (RIP), וסימון איזוטופ יציב עם חומצות אמינו תרבית תאים (SILAC), שיטה proteomic (בדיקה ב [7]). לכל שיטה יש יתרונות וחסרונות. מאז מירנה מיקוד לעתים קרובות מערערת mRNA מוביל לזילות שלו, אובדן oרווח R ברמת mRNA לאחר החדרת מירנה לחקות או מעכב, בהתאמה, יכול לזהות מטרות מירנה. הפסד זה או רווח של mRNA מזוהה בקלות על ידי microarray או רצף עמוק. תוך גילוי שיטות mRNA הם פשוט רגישים יותר מאשר שיטות זיהוי חלבונים, זיהוי ה-mRNA יחמיץ שום מטרות מירנה שאינם מפורקים. דיווחים אחרונים של המעבדה Bartell השוואת יעד תוצאות מירנה מ איתור RNA עם אלה SILAC [8] או פרופיל הריבוזום [9] מצביעים על כך miRNAs יונקים בעיקר לפעול על ידי הפחתת רמות ה-mRNA המטרה. עם זאת, מעבדות רבים אחרים העובדים עם מטרות מירנה בודדים לא זוהה שינוי בחלבון אך לא חל שינוי ברמת ה-mRNA. דיווחים על מה שנראה תקנה translational ללא אובדן mRNA לגילוי כוללים: miR-10b על HOXD10 [10], miR221/222 על p27kip1 [11], miR-21 על Pdcd4 [12], miR-126 על p85β [13] , miR-34 על SIRT1 [14], miR-21 על PTEN [15], miR-302d על Arid4b [16], miR-200C על JAG1 [17], ו miR-299, 297, 567,nd 609 על VEGFA [18]. יתר על כן, קלנסי ואח&#39; [19] לאחרונה דווח כי תקנה translational ידי בואו-7 התגלתה רק כאשר isoforms mRNA בודדים המכילים את האתר בואו-7 היעד התגלו באופן סלקטיבי. זה האחרון מצביע על כך, לפחות בחלק מהמקרים, רגולציה translational ניתן להתעלם בפרופיל מרוכבים - כלומר, כאשר כל isoforms mRNA מזוהים כמו mRNA 1 - כפי שנתקבל עם microarray רבים וניתוחים ברצף. שיתוף immunoprecipitation של מירנה-mRNA באמצעות מתחמי argonaute (בדרך כלל ago2) או חלבון אחר הקשור (RNA immunoprecipitation, או RIP) יהיה לבודד את מטרות מירנה ללא קשר מנגנון ויסות. בנוסף, RIP מזהה אנדוגני, ומן הסתם רלוונטי מבחינה ביולוגית, אינטראקציות. עם זאת, לא ידוע אם כל מירנה-mRNA אינטראקציות הם פונקציונליים, ולקרוע יחמיץ שום מטרות mRNA כי מקשרים רק זמני או מפורקים במהירות. יתר על כן, ספציפיים מירנה-mRNA שותפים יש להסיק bioinformatiקאלי משום שכל מירנה-mRNA זוגות שיתוף זירז יחד. לבסוף, SILAC ישירות מזהה את המוצר הסופי של התקנה מירנה, החלבון עצמו, אבל הוא לא רגיש ועל כן מתגעגע חלבונים נדירים ושינויים לקפל קטנים רמות החלבון. לאור המגבלות הנוכחיות עם מירנה היעד שיטות הזיהוי, הרגשנו assay גלובלית נוספת כדי לזהות מטרות מירנה תפקודית על ידי מנגנון חלופי היה צורך. כדי למלא את הצורך הזה, אנחנו רישיון טכנולוגיה שהומצאה על ידי יופ Gaken ו עזים Mohamedali של לונדון בקינגס קולג&#39;. ההמצאה שלהם הוא חלבון כפול היתוך הבחירה, באופן ספציפי, שילוב kinase-zeocin תימידין, מוסדר על ידי ספריית cDNA של פוטנציאל היעד רצפים מירנה ב 3&#39;UTR שלה. תאים transfected ביציבות עם ולבטא TKzeo-cDNA בונה ניתן לבחור עם zeocin, כפי שמודגם באיור 2. לאחר הבעת עניין מירנה, מטרות של מירנה ניתן לבחור אינטרנטganciclovir ה, כפי שמודגם באיור 3. Ganciclovir הורג תאים להביע תימידין קינאז, כלומר, כל התאים לבטא TKzeo-cDNA חסר מטרה מירנה של עניין. ממוקד cDNA יכול להיות מבודד על ידי הגברה PCR של ה-DNA של תאים ששורדים primers באמצעות ganciclovir כי האגף מוצרים cDNA ו PCR יכול להיות רצף לזהות מטרות. פיתחנו טכנולוגיה במכללת המלך לתוך כלי חדש לזיהוי הגילוי העולמי של מטרות תפקודיות מירנה אדם - יעד המשימה ספריית מזהה. עם הספרייה מזהה מטרה, משתמשים יכולים לבודד את מטרות מירנה על ידי שורה של transfection התרבות יונקים התא ובחירת צעדים סמים. הספרייה היא מקיפה, והוא מכיל 66-79% מכלל הגנים האנושיים. תוצאות ראשוניות מצביעות על כך גם גילה בעבר, כמו גם מטרות חדשות יכול להיות מבודד ספריית יעד המשימה מזהה. אף על פי הפרוטוקול הוא באורך כלשהו,שימוש בספריית מזהה יעד מחייב תקן טכניקות מעבדה מולקולרית - תרבית תאים transfection יונקים,, בחירת התרופה, PCR, וסדר - ולכן, צריך להיות גם במסגרת האמצעים של רוב הביולוגים. אנו מציעים תשומת לב מספקת ישולם עיצוב ניסיוני נכונה, אופטימיזציה של תנאי התרבות, והכי חשוב, לפני בדיקה של כל סוג תא חדש כדי לקבוע רמות התרופה האופטימלי עבור צעדים הבחירה (kill של עקומות) כדי להבטיח הצלחה עם ספריית מזהה מטרה. כמו בכל יעד מירנה שיטות הזיהוי, מומלץ מאוד כי מטרות שזוהו על ידי הקרנת ספריית מזהה מטרה להיות מאומת עם השיטה השנייה, כגון microarray, qRT-PCR, assay הכתב (כלומר, בלוציפראז), או ניתוח המערבי.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> התחלה מהירה REDTaq רדימיקס </td><td> Sigma-Aldrich </td><td> P0982 </td><td></td></tr><tr><td> Ganciclovir </td><td> Sigma-Aldrich </td><td> G2536 </td><td></td></tr><tr><td> G418 </td><td> Sigma-Aldrich </td><td> G8168 </td><td></td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P9620 </td><td></td></tr><tr><td> Topo ת&quot;א </td><td> Invitrogen </td><td> KNM4500-01 </td><td></td></tr><tr><td> GenElute היונקים הדנ&quot;א הגנומי Miniprep Kit </td><td> Sigma-Aldrich </td><td> G1N10 </td><td></td></tr><tr><td> Nucleofection ספציפי תא kזה </td><td> Lonza </td><td></td><td></td></tr><tr><td> Nucleofector </td><td> Lonza </td><td> AAD-1001 </td><td></td></tr><tr><td> Zeocin </td><td> Invitrogen </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=R25001">R250-01</a> </td><td></td></tr><tr><td height="1"> היעד מזהה הספרייה </td><td> Sigma-Aldrich </td><td> MREH01 </td><td></td></tr></tbody></table>

genome-wide screen for mirna targets using the mission target id library

ספריית מזהה יעד נועד לסייע מטרות גילוי וזיהוי של microRNA (מירנה). ספריית מזהה היעד הוא פלסמיד מבוסס, הגנום כולו ספריית cDNA משובטים לתוך הזרם 3&#39;UTR של חלבון מבחר כפול היתוך, תימידין קינאז-zeocin (TKzeo). הסיבוב הראשון של הברירה היא transformants יציבות, ואחריו עם הקדמה של מירנה עניין, ובסופו של דבר, בחירה עבור cDNAs המכילים היעד של מירנה. CDNAs נבחרים מזוהים על ידי רצף (ראה איור 1-3 עבור זרימת עבודה מזהה ספריית היעד לפרטים נוספים). כדי להבטיח כיסוי רחב של transcriptome האנושי, יעד מזהה ספריית cDNAs נוצרו באמצעות תחול oligo-DT באמצעות מאגר של RNA הכל מוכן מרקמות אדם מספר רב של שורות תאים. וכתוצאה מכך מגוון cDNA מ 0.5 עד 4 קילו, עם גודל ממוצע של 1.2 קילו, והיו משובטים לתוך כפול מבחר p3TKzeo פלסמיד (ראה איור 4 על מפת הפלסמיד). הגן מטרות המיוצגות library ניתן למצוא באתר האינטרנט Sigma-Aldrich. תוצאות Illumina רצף (לוח 3), עולה כי הספרייה כולל 16,922 של 21,518 גנים ייחודיים UCSC RefGene (79%), או 14,000 גנים עם 10 או יותר קורא (66%).

Watch this Scientific Journal Video about Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library at JoVE.com

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

ספריית מזהה היעד הוא פלסמיד מבוסס, הגנום כולו אוסף של cDNA משובטים לשמש לזיהוי מטרות מירנה. כאן אנו מדגימים את השימוש והיישום.

הגנום כולו מסך עבור מטרות מירנה שימוש יעד המשימה ספריית מזהה

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

genome-wide-screen-for-mirna-targets-using-mission-target-id

Research

JoVE Journal

Biology

17.5K Views.  Sigma-Aldrich. ספריית מזהה היעד הוא פלסמיד מבוסס, הגנום כולו אוסף של cDNA משובטים לשמש לזיהוי מטרות מירנה. כאן אנו מדגימים את השימוש והיישום.

Video: Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

הגנום כולו ניתוח (טעינה) רמות Aminoacylation של tRNA שימוש microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

הגנום כולו ניתוח באמצעות שבב זיהוי איזופורם ספציפי מטרות ג&#39;ין

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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 ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

כמותיים אוטומטי תפוקה גבוהה הגנום כולו RNAi המסכים ג elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

אלגוריתם רומן ייס שינוי נקודות לניתוח הגנום רחב, של סוגי נתוני ChIPseq מגוונים

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

מחיקות Gene הגנום ב<em&gt; סטרפטוקוקוס sanguinis</em&gt; על ידי תפוקה הגבוהה PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

זיהוי של מטרות מירנה בתפוקה גבוהה באמצעות assay 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

מיפוי הגנום של תרופות-DNA בתאי אינטראקציות עם COSMIC (Crosslinking של מולקולות קטנות לבודד הכרומטין)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

טיהור הגנום כולו של DNA המעגל extrachromosomal מתאי איקריוטיים

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

זרימת עבודה מקיפה עבור הביטוי מטא אנליזה ATL E3 אוביקוויטין ליגאז ג'ין משפחת גפן וזיהוי הגנום כולו

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...