אנו מודים לורי לי לקריאה ביקורתית של כתב היד. TWC נתמך על ידי predoctoral מלגת איגוד לב אמריקאי (12PRE6590007). MRB נתמך על ידי מענק לאומי לסרטן מכון אימון (CA119925 T32). SSH נתמך על ידי המכון הלאומי לבריאות (R01DK078640). EL נתמך על ידי המכון הלאומי לבריאות (R01GM081635 וR01GM103926).

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

תמצית ביצי Xenopus היא מערכת ביוכימית חזקה לחקירת מחזור β-catenin. הריכוז של β-catenin בתמצית ביצי Xenopus הוא ~ 25 ננומטר 2. בתנאים אופטימליים, תמצית הביצה היא מסוגלת משפיל β-catenin בשיעור של 50-100 ננומטר / שעה וחצי מקסימאלי ב200 ננומטר 24. ישנם מספר צעדים קריטיים לכינו?...

A correction was made to <a href="http://www.jove.com/video/51425">Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract</a>. At the time of publication there were some instances where an incorrect volume notation was used. These instances were corrected from:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 ml extract for each time point.
3.2.3. At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 ml equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated &#946;-catenin-luciferase fusion).
to:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 &#181;l pelleted beads to 200 &#181;l extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 &#181;l extract for each time point.
3.2.3. At the designated time point, remove 1-5 &#181;l of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 &#181;l equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/&#181;l of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 &#181;l of the in vitro-translated &#946;-catenin-luciferase fusion).

Erratum: Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

תמצית ביצת laevis Xenopus כבר נעשה שימוש נרחב כדי ללמוד תהליכים בתא ביולוגיים רבים כולל דינמיקת cytoskeletal, הרכבה ויבוא גרעיניים, אפופטוזיס, חילוף חומרים של היוביקוויטין, התקדמות מחזור התא, העברת אותות, ומחזור חלבון 1-17. מערכת תמצית ביצי Xenopus ניתן לניתוח ביוכימי של לגיון של תהליכים תאיים, כי תמצית ביצה מייצגת בעצם ציטופלסמה חי המכילה את כל המרכיבים החיוניים cytoplasmic דרוש לביצוע תהליכים אלו ולאפשר חקירה. ניתן להכין כמויות גדולות של תמצית ביצים בפעם אחת למניפולציות ביוכימיות הדורשות כמויות גדולות של חומר (למשל, טיהור חלבון או הקרנת תפוקה גבוהה) 18-20. יתרון נוסף הוא שהריכוז של חלבונים ספציפיים בתמצית ביצי Xenopus יכול להיות מותאם בדיוק על ידי תוספת של חלבון ו / או immunodepletion של endog רקומביננטיחלבונים בוגדניים בניגוד לtransfection של DNA פלסמיד שבו ביטוי של החלבון של עניין הוא קשה לשליטה. בנוסף, המחסור בחלבונים רקומביננטיים זמינים ניתן להתגבר על ידי התוספת של תמלילים המקודדים את החלבון של עניין, תוך ניצול של הקיבולת הגבוהה של תמצית ביצי Xenopus מוכנה הטרי לתרגם mRNA הוסיף אקסוגני. הרגולציה של פירוק חלבונים היא קריטית לשליטה רבים מסלולי הסלולר ומעבדת 21. תמצית ביצי Xenopus כבר נעשה שימוש נרחב ללמוד פירוק חלבונים, שכן המערכת מאפשרת מספר דרכים לעקוב אחר מחזור חלבון ללא השפעות מבלבלות של שעתוק ותרגום. מסלול איתות Wnt הוא מסלול איתות שמור ביותר שמשחק תפקידים קריטיים בהתפתחות ומחלה. מחזור של β-catenin, מפעיל העיקרי של מסלול Wnt, מוסדר מאוד, ואני במצב יציב מוגבראיבל של β-catenin הוא קריטי להפעלה של גנים מטרת Wnt. החשיבות של השפלה β-catenin מודגשת על ידי העובדה שמוטציות במסלול Wnt המעכבות השפלה β-catenin מצאו ב~ 90% מכל מקרים ספורדיים של סרטן מעי גס 22. השפלה β-catenin על ידי רכיבים של מסלול Wnt יכולה להיות סכמה בנאמנות בתמצית ביצי Xenopus ללמוד את המנגנון של המחזור שלה, כמו גם לזהות מאפנני מולקולה קטנים רומן של השפלה שלה 2, 19, 20, 23-29. שיטות להכנה של תמצית ביצי Xenopus לחקר מחזור התא תוארו בפרסומי יופיטר הקודמים 30-32. הפרוטוקול הנוכחי מתאר שינוי של שיטות אלה, והוא מותאם לשפלה של [35 S]-radiolabeled β-catenin ומתויג לוציפראז β-catenin בתמצית ביצי Xenopus. Assay השפלה radiolabeled מאפשר הדמיה ישירה של רמות חלבון באמצעות autoradiography. מתיונין [35 S] הוא שולב החלבון של עניין באמצעות תגובה בתרגום חוץ גופית שלאחר מכן ניתן להוסיף ישירות לתגובת השפלה. בנוסף, assay מחזור חלבון radiolabeled אינו דורש נוגדן נגד החלבון של עניין או תג אפיטופ, שיכול להשפיע על יציבות חלבון. כי אפילו שינויים קטנים ברמות חלבון, כפי שבאו לידי ביטוי בשינויים בעוצמתה של להקת חלבון רדיואקטיבי, הם דמיינו בקלות על ידי autoradiography, [S 35]-radiolabeled assay השפלה מייצג שיטה שימושית מאוד להדמיה של חלבון מחזור 2. שילוב של β-catenin לגחלילית בלוציפראז (להלן בפשטות &quot;לוציפראז&quot;) מאפשר למדידות כמותיות מדויקות וקלילות של רמות חלבון, כדיכדי לקבוע את מאפייני הקינטית של מחזור β-catenin 19, 20. יתרון עיקרי של assay בלוציפראז הוא שהיא מספקת מערכת כמותיים חזקה הלשנותם בקלות. הפרוטוקול הבא מספק שיטות פשוטות למנסה לאמוד השפלה β-catenin ושיטה חזקה, יעילה ואפקטיבית להקרנת תפוקה גבוהה של מאפנני β-catenin רומן.

<table><tbody><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>ProSpec</td><td>hor-272-A</td><td>Reconstituted with distilled water before use.&amp;nbsp;</td></tr><tr><td>Human chorionic gonadotropin</td><td>Sigma</td><td>CG10-10VL</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>BP366-1</td><td></td></tr><tr><td>Sodium chloride</td><td>Research Products International</td><td>S23020-5000.0</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Calcium chloride</td><td>Acros Organics</td><td>AC42352-5000</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-1</td><td></td></tr><tr><td>Cysteine</td><td>Acros Organics</td><td>AC17360-1000</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884-10MG</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153-10MG</td><td></td></tr><tr><td>Pepstatin</td><td>Sigma</td><td>P4265-5MG</td><td></td></tr><tr><td>Cytochalasin B</td><td>Sigma</td><td>C8273-10MG</td><td></td></tr><tr><td>3 ml syringe: Luer Lock tip</td><td>Becton Dickinson</td><td>309657</td><td></td></tr><tr><td>27 G needle</td><td>Becton Dickinson</td><td>305109</td><td></td></tr><tr><td>96 well solid white polystyrene microplate, round bottomed</td><td>Corning</td><td>3605</td><td></td></tr><tr><td>Steady-Glo luciferase assay system</td><td>Promega</td><td>E2520</td><td>Store long-term at -80 &amp;deg;C, can store for up to 1 month at -20 &amp;deg;C</td></tr><tr><td>TNT Sp6 coupled reticulocyte lysate system</td><td>Promega</td><td>L4600</td><td></td></tr><tr><td>TNT Sp6 high-yield wheat germ protein expression system</td><td>Promega</td><td>L3260</td><td>Generally higher yield than reticulocyte lysate&amp;nbsp;</td></tr><tr><td>EasyTag Express protein labeling mix [S35]</td><td>Perkin Elmer</td><td>NEG772007MC</td><td></td></tr><tr><td>Creatine phosphate</td><td>Sigma</td><td>27920-5G</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A2383-5G</td><td></td></tr><tr><td>Creatine phosphokinase</td><td>Sigma</td><td>C3755-35KU</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D8418-50ML</td><td></td></tr><tr><td>Dual-Glo luciferase assay system</td><td>Promega</td><td>E2920</td><td>Same storage conditions as Steady-Glo</td></tr><tr><td>50 ml Centrifuge tubes</td><td>Fisher Scientific</td><td>0556214D</td><td></td></tr><tr><td>Sorvall SS-34 fixed angle rotor</td><td>Thermo Scientific</td><td>28020</td><td></td></tr><tr><td>115 V 50/60 Hz Minicentrifuge</td><td>Fisher Scientific</td><td>05-090-128</td><td></td></tr><tr><td>mMessage mMachine Sp6 kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>Anti-firefly luciferase antibody</td><td>Abcam</td><td>ab16466</td><td></td></tr><tr><td>Anti-GSK3 antibody</td><td>BD Transduction Laboratories</td><td>610201</td><td></td></tr><tr><td>FLUOStar Optima</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>Sorvall RC-6 Plus centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>16 &amp;deg;C Incubator</td><td>Percival Scientific</td><td></td><td></td></tr></tbody></table>

reconstitution of &#946;-catenin degradation in xenopus egg extract

תמצית ביצת laevis Xenopus היא מערכת מאופיינת היטב, חזקה ללימוד הביוכימיה של תהליכים תאיים שונים. תמצית ביצי Xenopus נעשתה שימוש כדי ללמוד מחזור חלבון בהקשרים רבים תאיים, כולל את מחזור התא ומסלולים הולכים אותות 1-3. בזאת, שיטה מתוארת לבידוד תמצית ביצי Xenopus שעבר אופטימיזציה כדי לקדם את ההשפלה של המרכיב הקריטי Wnt המסלול, β-catenin. שתי שיטות שונות מתוארות להעריך פירוק חלבוני β-catenin בתמצית ביצי Xenopus. שיטה אחת היא מבחינה ויזואלית אינפורמטיבי ([S 35]-radiolabeled חלבונים), ואילו השני הוא לשנותם בקלות רבה יותר למבחני תפוקה גבוהה (גחלילית חלבוני היתוך מתויג לוציפראז). הטכניקות המתוארות יכולות לשמש, אך אינם מוגבלות, להערכת מחזור חלבון β-catenin ולזהות רכיבים מולקולריים תרמו למחזור שלה. בנוסף, ability לטהר כמויות גדולות של תמצית ביצים הומוגנית Xenopus בשילוב עם קריאת הנתונים הכמותיים וקלילות של חלבונים מתויגים לוציפראז מאפשר מערכת זו כדי להתאים בקלות להקרנת תפוקה גבוהה למאפננים של השפלה β-catenin.

סכמטי של השפלה β-catenin בתמצית ביצי Xenopus מוצג באיור 2 א. 35 β-catenin כותרת-S הודגר בתמצית ביצי Xenopus, aliquots (1 מיליליטר לחלץ שווה ערך) הוסרו במועדים המתאימים, ודגימות היו נתונים SDS-PAGE ואחרי autoradiography. השפלה β-catenin על ידי רכיבים של מסלול Wnt מתווכת על ידי מערכת ה...

Watch this Scientific Journal Video about Reconstitution Of Î²-catenin Degradation In Xenopus Egg Extract at JoVE.com

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

שיטה מתוארת לניתוח פירוק חלבונים באמצעות רדיואקטיבי וחלבונים לוציפראז-Fusion בתמצית ביצי Xenopus והתאמת להקרנת תפוקה גבוהה למאפנני מולקולה קטנים של פירוק חלבונים.

הכינון מחדש של השפלה β-catenin ב<em&gt; Xenopus</em&gt; חלץ ביצה

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

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Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Unit 1

Translation: RNA to Protein

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12.0K Views.  Vanderbilt University Medical Center.  שיטה מתוארת לניתוח פירוק חלבונים באמצעות רדיואקטיבי וחלבונים לוציפראז-Fusion בתמצית ביצי Xenopus והתאמת להקרנת תפוקה גבוהה למאפנני מולקולה קטנים של פירוק חלבונים. 

Video: Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

שיחזור התנודות מחזור התא ב- Microemulsions של תמציות ביצה ללא תא צפרדע רפואית

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Biochemistry

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

הכנה ההיפוך חלוקה של Xenopus laevis ביצה תמציות

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

הפקה של טראנסגנטי Xenopus laevis על ידי שילוב אנזימים מתווכת הגבלת והשתלות גרעיני

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity.
This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or &#946;-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Signaling levels are known to regulate cell fate, indicating that regulation of Wnt signaling constitutes an interesting therapeutic target. Here, we describe flow cytometry and confocal microscopy analysis methods for a robust murine canonical Wnt signaling reporter model that measures distinct Wnt signaling levels.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt ...

Developmental Biology

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

טרנסלוקציה מקוטב של חלבוני ניאון ב Xenopus האאקטודרם בתגובה איתות Wnt

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

חקר מחסום הניזק לדנ&quot;א באמצעות<em&gt; Xenopus</em&gt; תמציות ביצה

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

ייצור של<em&gt; Xenopus tropicalis</em&gt; ביצה מחלץ לזהות RNAs microtubule הקשורים

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis כמודל לזיהוי תרגום ירידת ערך

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

תא Assay חינם לחקר הכרומטין Decondensation בסוף מיטוזה

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Assay חינם-נייד באמצעות<em&gt; Laevis Xenopus</em&gt; עוברי מחלץ לחקור מנגנונים של תקנת גודל גרעינית

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

הכינון מחדש של השפלה β-catenin ב

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