עבודה זו נתמכה על ידי מענק מטעם המרכז Packard עבור ALS מחקר באוניברסיטת ג&#39;ונס הופקינס (ADG), פרס ממציא ניו מנהל NIH 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), אלן ריטה פרס קרן Scholar. ADG הוא חוקר Pew במדעי ביו, נתמך על ידי Pew Charitable Trusts.

1. ההכנות לשינוי שמרים פרוטוקול זה מיועד עשר צלחות 96-טוב אבל יכול להיות scaled למעלה או למטה בהתאם. מצאנו כי פרוטוקול זה לא עובד טוב יותר מעשרים 96-היטב צלחות בכל סבב של טרנספורמציה. הליך השינוי כולו (משלב I.3) ייקח בערך שמונה שעות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Aliquot 5-10μL של DNA פלסמיד (100 ng / μl) מן הספרייה שמרים ORF FLEXGene לתוך הבאר התחתון של כל עיגול 96-גם צלחת עם המטפל RapidPlate Biorobot נוזלי. המקום חשפו צלחות 96-היטב מכסה המנוע ספסל נקי קטר לילה יבש. מצאנו יעילות טרנספורמציה דומה עם CEN ו פלסמידים 2μ שמרים. </li><li style=";text-align:right;direction:rtl"> לחסן 200 מ&quot;ל של שמרים peptone דקסטרוז (YPD) מדיה (10g / L תמצית שמרים, 20 גרם / L peptone, 20 גרם / גלוקוז L) עם זן שאילתה בקבוק Erlenmeyer 500ml מבולבל. דגירה לילה בשעה 30 ° C עם רועד (200 סל&quot;ד). במידת הצורך, מדיה סלקטיבי יכול לשמש גם עבור אלו תרבויות המתנע. </li><li style=";text-align:right;direction:rtl"> למחרת בבוקר, לדלל את המתח שאילתה OD 600 = 0.1 ב 2L של YPD. Shake עבור 4-6 שעות בקצב של 30 ° C (200 סל&quot;ד) עד התרבות מגיע OD 600 = 0.8-1.0. לצמיחה אופטימלית, לגדול שתי התרבויות 1 ליטר ב 2.8 צלוחיות נפרדות L מבולבל. במידת הצורך, מדיה סלקטיבי יכול לשמש גם במקום YPD. </li></ol> 2. שמרים שינוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קציר תאים על ידי צנטריפוגה. מלאו ארבעה צינורות הפנויה חרוטי 225mL עם התרבות שמרים ספין על 3000rpm (~ 1800 XG) במשך 5 דקות בצנטריפוגה השולחן (Eppendorf 5810R). יוצקים את supernatant וחזור עד שכל התאים נקצרים. </li><li style=";text-align:right;direction:rtl"> שטפו תאים 200 מ&quot;ל של autoclaved מזוקקים H 2 O. Resuspend תא גלולה ידי ניעור או vortexing. שלב התאים הגליל האחד חרוט 225mL. ספין על 3000rpm במשך 5 דקות. הסר supernatant. </li><li style=";text-align:right;direction:rtl"> הכן פתרון 0.1MLiOAc/1XTE (0.1M LiOAc, 10mm טריס, 1mm EDTA, pH 8 במים מזוקקים autoclaved). שטפו תאים 0.1MLiOAc/1XTE 100 מ&quot;ל. ספין על 3000rpm במשך 5 דקות. הסר supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend תא גלולה ב 70mL 0.1M LiOAc/1xTE. Shake ו / או מערבולת כדי להבטיח גלולה הוא resuspended לחלוטין. </li><li style=";text-align:right;direction:rtl"> דגירה עם רועד ב 30 מעלות צלזיוס במשך 30 דקות (200 סל&quot;ד). </li><li style=";text-align:right;direction:rtl"> הוסף β-mercaptoethanol ל 0.1M. דגירה עם רועד ב 30 מעלות צלזיוס במשך 30 דקות (200 סל&quot;ד). הערה: שלב זה ניתן להשמיט אם באמצעות זני שמרים הרגישים mercaptoethanol-β. מצאנו כי צעד זה אינו הכרחי לשינוי מוצלח, אולם הוא עושה לשפר את יעילות הטרנספורמציה. </li><li style=";text-align:right;direction:rtl"> מרתיחים 2ml של ה-DNA זרע sonicated סלמון (10mg/mL) למשך 15 דקות ולאחר מכן לצנן על הקרח. </li><li style=";text-align:right;direction:rtl"> הוסף 2ml של ה-DNA סלמון מבושל זרע לתערובת התא. זהירות: המשקע עלול להיווצר על תוספת של ה-DNA סלמון זרע לתערובת התא. שימוש pipetman ו -200 μl טיפ, להסיר בזהירות כל המשקע שנוצר, כי זה עלול להפריע pipetting במורד הזרם. </li><li style=";text-align:right;direction:rtl"> 50μL Aliquot תערובת של התא היטב בכל צלחת 96-היטב באמצעות Rapidplate BioRobot (ניתן גם להשתמש מרובת ערוצים פיפטה). אל תערבב. </li><li style=";text-align:right;direction:rtl"> דגירה בטמפרטורת החדר למשך 30 דקות בלי לרעוד. </li><li style=";text-align:right;direction:rtl"> הכן 200 מ&quot;ל של 40% PEG-3350/10% DMSO/0.1M LiOAc (160mL של 50% PEG-3350, DMSO 20mL, LiOAc 20mL 1M). הכן פתרון מיד לפני השימוש. </li><li style=";text-align:right;direction:rtl"> הוסף 125μL הפתרון PEG / LioAc / DMSO היטב כל אחד. מערבבים על ידי aspirating מחלק ההשעיה תא שמונה פעמים עם RapidPlate. </li><li style=";text-align:right;direction:rtl"> דגירה בטמפרטורת החדר למשך 30 דקות בלי לרעוד. </li><li style=";text-align:right;direction:rtl"> הלם מחממים את התאים על 42 מעלות צלזיוס במשך 15 דקות בתוך אינקובטור יבש. אל ערימת הצלחות. הערה: מצאנו כי צעד זה הלם חום אינו חיוני לשינוי מוצלח. עבור זני שמרים מסוימים (למשל, מוטציות רגישות טמפרטורה), הלם החום מזיקות. צמצמנו בעבר הזמן הלם החום דקה אחת הצליחו להפוך בהצלחה זן שמרים מחסה מוטציה ypt1 טמפרטורה רגיש בפרוטוקול זה. </li><li style=";text-align:right;direction:rtl"> ספין צלחות על 2500rpm (~ 1100 XG) במשך 5 דקות, באמצעות מתאמים צנטריפוגות כדי להכיל 96-גם הצלחות. הסר הפתרון PEG על ידי צלחות היפוך מעל דלי פסולת. כתם צלחת הפוכה על מגבת נייר כדי להסיר את נוזל שיורי (תאים יישארו בתחתית בארות). </li><li style=";text-align:right;direction:rtl"> יש לשטוף את התאים על ידי הוספת 200μL התקשורת מינימלי (למשל SD /-עורה) היטב עם כל RapidPlate. </li><li style=";text-align:right;direction:rtl"> ספין צלחות ב 2500rpm במשך 5 דקות ולהסיר supernatant ידי דלי היפוך פסולת שוב על מגבות נייר סופג. </li><li style=";text-align:right;direction:rtl"> הוסף 200μL התקשורת מינימלי (למשל SD /-עורה) היטב עם כל RapidPlate. </li><li style=";text-align:right;direction:rtl"> לדגור על 30 מעלות צלזיוס למשך 2 ימים בלי לרעוד. אחרי 2 ימים, מושבות קטנות של תאים צריך להתחיל לגבש בתחתית כל טרנספורמציה בהצלחה היטב. </li></ol> 3. אכון assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לוותר 200μL התקשורת raffinose מינימלי מבוסס (כגון השרף /-עורה) לתוך הבאר אחדחדש שטוח 96-התחתונה גם צלחת. </li><li style=";text-align:right;direction:rtl"> מערבבים היטב את כל הצלחת השינוי על ידי aspirating מחלק ההשעיה תא שמונה פעמים עם Rapidplate. </li><li style=";text-align:right;direction:rtl"> לחסן צלחות השרף עם 5μL תערובת תא מהצלחת שינוי. </li><li style=";text-align:right;direction:rtl"> לדגור על 30 מעלות צלזיוס למשך יום אחד. מושבות קטנים צריכים להיות נוכחים בחלק התחתון של כל טוב אחרי יום אחד. </li><li style=";text-align:right;direction:rtl"> ספוט מושבות על SD /-עורה ואת SGal /-עורה צלחות מכסה המנוע. לעקר 96-הבריח משכפל (Frogger) על ידי בוערים. מערבבים עם מושבות Frogger לפני תצפית על צלחות התקשורת סלקטיבית. לאחר תצפית, לאפשר צלחות לייבוש מכסה המנוע למשך 5-10 דקות. </li><li style=";text-align:right;direction:rtl"> לדגור על 30 מעלות צלזיוס במשך 2-3 ימים. </li></ol> צלחות צילום עם מצלמה דיגיטלית, ו חזותית להשוות צמיחה של מושבות על SGal /-עורה צלחות למצוא מושבות שבהן רעילות זן שאילתה משופרים (צמיחה איטית / מושבות פחות צפוף) או מודחקים (צמיחה מהיר / במושבות צפופות יותר). 4. נציג התוצאות: <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="איור 1"/> באיור 1. Overexpression פלסמיד שמרים המסך כדי לזהות מדכאי ו משפרי של רעילות TDP-43. TDP-43 הוא חלבון אנושי כי היה מעורב בפתוגנזה של טרשת לרוחב amyotrophic (מחלת לו גריג). אגרגטים cytoplasmic של TDP-43 להצטבר במוח הנוירונים בחוט השדרה של חולי ALS 17. הבעת TDP-43 תוצאות בתאי שמרים צבירה cytotoxicity 18. השתמשנו במערכת זו מודל להגדיר מנגנוני הרעילות TDP-43 19,20. מוצגות דוגמאות של צלחות repesentative מן השמרים שלנו TDP-43 משנה מסך רעילות. לוחות אלה מציגים מושבות עם משולבת גלקטוז ועין מתנהלת-TDP-43 פלסמיד והפך גם עם פלסמידים מהספריה ביטוי FLEXGene ORF. צלחת בצד שמאל מכיל גלוקוז, אשר מדכא ביטוי TDP-43 או פלסמידים FLEXGene. צלחת בצד ימין מכיל גלקטוז, אשר מעוררת את הביטוי של TDP-43 ואת ORFs של פלסמידים FLEXGene. החץ הירוק מצביע על המושבה הפכה עם פלסמיד זה מדכא את הרעילות של TDP-43. החץ האדום מצביע על המושבה הפכה עם פלסמיד זה משפר את הרעילות של TDP-43.

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אין ניגודי אינטרסים הכריז.

כאן אנו מציגים פרוטוקול לבצע המסך תפוקה גבוהה overexpression פלסמיד בשמרים. גישה זו מאפשרת להקרנה מהירה משוחדת עבור מכפילי הגנטי של פנוטיפים רבים סלולריים שונים. שימוש בגישה זו, חוקר יכול המסך חלק ניכר של הגנום שמרים ב עניין של שבועות. גישה זו משוחדת גם מאפשר זיהוי של מכפילי...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 הבריח משכפל (Frogger) </td><td> V &amp; P מדעי </td><td> VP404 </td></tr><tr><td> FLEXGene ORF הספריה </td><td> המכון Proteomics, מבית הספר לרפואה הארוורד </td><td></td></tr><tr><td> Tabletop צנטריפוגות </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> בקבוק מבולבל 500ml </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L משולשת מבולבל Fernbach צפחת </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate פיפטה טיפים </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate פיפטה טיפים </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

נבגי שמרים, cerevisiae Saccharomyces, היא מערכת מודל עוצמה להגדרת המנגנונים הבסיסיים של תהליכים תאיים רבים חשובים, כולל אלה עם שייכות ישירה מחלות אנושיות. בגלל הזמן הקצר של הדור שלה היטב מאופיין הגנום, יתרון הניסוי העיקריים של מערכת מודל שמרים היא היכולת לבצע מסכי גנטית כדי לזהות גנים המסלולים המעורבים בתהליך נתון. במשך שלושים השנים האחרונות מסכי גנטי כזה כבר בשימוש על מנת להבהיר את מחזור התא, מסלול הפרשה, והיבטים רבים יותר שמור ביותר של ביולוגיה של התא אוקריוטים 1-5. בשנים האחרונות, ספריות genomewide מספר זני שמרים פלסמידים נוצרו 6-10. אוספים אלה מאפשרים כעת לחקירה שיטתית של תפקוד הגן באמצעות רווח, והפסד של פונקציה גישות 11-16. כאן אנו מספקים פרוטוקול מפורט לשימוש פרוטוקול תפוקה גבוהה טרנספורמציה שמרים עם רובוט טיפול הנוזל לבצע מסך overexpression פלסמיד, באמצעות ספריה ערוכים של 5500 פלסמידים שמרים. אנחנו משתמשים במסכים אלה כדי לזהות מכפילי הגנטי של רעילות הקשורות הצטברות של צבירה נוטה חלבונים אנושיים מחלות ניווניות. השיטות שהוצגו כאן הם להתאמה בקלות ללימוד פנוטיפים סלולריים אחרים בעלי עניין.

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

כאן אנו מתארים מסך overexpression פלסמיד ב<em&gt; Saccharomyces cerevisiae</em&gt;, באמצעות פלסמיד ספריה ערוכים ו תפוקה גבוהה טרנספורמציה פרוטוקול שמרים עם רובוט טיפול נוזלי.

תפוקה גבוהה Overexpression פלסמיד שמרים מסך

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

high-throughput-yeast-plasmid-overexpression-screen

Research

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Biology

16.3K Views.  University of Pennsylvania School of Medicine. כאן אנו מתארים מסך overexpression פלסמיד ב Saccharomyces cerevisiae, באמצעות פלסמיד ספריה ערוכים ו תפוקה גבוהה טרנספורמציה פרוטוקול שמרים עם רובוט טיפול נוזלי.

Video: High-throughput Yeast Plasmid Overexpression Screen

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

מסך התפוקה גבוהה עבור Biomining פעילות cellulase מספריות Metagenomic

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

תפוקה גבוהה Saccharification Assay חומרים Lignocellulosic

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

טיהור תפוקה גבוהה של חלבונים רקומביננטי זיקה מתויגת-

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

חיזוי מבנה המשני RNA באמצעות SHAPE תפוקה גבוהה

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

התפוקה microinjections גבוה של zygotes קיפודי הים

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

כותרות תפוקה גבוהה של וירוסי רקומביננטי לוציפראז- להביע

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

מסך ידני מולקולה קטנה מתקרב תפוקה גבוהה באמצעות עוברי דג הזברה

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

שחזור של לבבות למבוגרים דג הזברה עבור יישומי תפוקה גבוהה

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

הקרנת תפוקה גבוהה עבור כימית מאפננים של גני Post-תעתיק מוסדר

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

תפוקה גבוהה<em&gt; Danio rerio</em&gt; Assay הוצאה אנרגיה

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...