כספים סטארט-אפ לא דיקינסון מVCU תמכו בעבודה זו. המחברים מבקשים להודות דן Nacu לכשרון האמנותי שלו ביצירת האיור סכמטי.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+anatomy+of+the+face+in+Down's+syndrome:+anthropometric+proportion+indices+in+the+craniofacial+regions.">Surface anatomy of the face in Down's syndrome: anthropometric proportion indices in the craniofacial regions.</a> J Craniofac Surg. 12, 519-524 (2001).</li><li>Scheuer, H. A., Holtje, W. J., Hasund, A., Pfeifer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognosis+of+facial+growth+in+patients+with+unilateral+complete+clefts+of+the+lip,+alveolus+and+palate.">Prognosis of facial growth in patients with unilateral complete clefts of the lip, alveolus and palate.</a> J Craniomaxillofac Surg. 29, 198-204 (2001).</li><li>Parsons, K. J., Andreeva, V., James Cooper, W., Yelick, P. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bentho-pelagic+divergence+of+cichlid+feeding+architecture+was+prodigious+and+consistent+during+multiple+adaptive+radiations+within+African+rift-lakes.">Bentho-pelagic divergence of cichlid feeding architecture was prodigious and consistent during multiple adaptive radiations within African rift-lakes.</a> PLoS One. 5, (2010).</li><li>Klingenberg, C. 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יש לי המחברים אין לחשוף.

laevis Xenopus הפך לכלי שימושי עבור לנתח מנגנוני התפתחות פיתוח orofacial בסיס; עם זאת, אין כיום פרוטוקולים מתארים גודל ובצורה של שינויים באזור זה בצפרדעים. השיטה המתוארת כאן תתרום באופן משמעותי לתחום של פיתוח orofacial כך שהוא מאפשר לכימות קפדני יותר של פנוטיפים orofacial בצפרדע?...

בין הסוגים הנפוצים וההרסניים ביותר של מומים מולדים אנושיים כאלה המשפיעים על הפה ופנים, הם כמו בקיעי orofacial 1. ילדים עם מבני orofacial פגומים לעבור ניתוחים רבים במהלך חייהם ואת המאבק עם מומי פנים, דיבור, שמיעה ובעיות אכילה. לכן, קידום מחקר חדש בפיתוח cranio- וorofacial הוא בעל חשיבות עליונה למניעה וטיפול בסוגים אלה של מומים מולדים בבני אדם. Laevis Xenopus התפתח ככלי חדש ללנתח את המנגנונים לפיקוח על בניית craniofacial (כמה דוגמאות כוללות את 2,3,4 -11). לכן, שיטה כמותית לניתוח שינויים בגודל ובצורה במהלך פיתוח של הראש והפנים של מין זה יכול להיות מאוד חזק 3. כאן, אנו מציגים שיטה כאמור; שילוב מדידות גודל מסורתיים עם morphometrics הגיאומטרי מותאם ממחקר צפרדעים 12 </sup&gt; ושפע של מחקרי ניתוח מבנה פנים אנושיים 13-15. מטרת פרוטוקול זה היא לאפשר לחוקרים לכמת גודל פנים וצורות כדי להבחין בין פנוטיפים orofacial השונים במהלך התפתחות נורמלית ולא נורמלית. ניתוח זה יאפשר לבידול טוב יותר בין פגמי craniofacial העדינים כגון אלה הנובעים מהשפעות סינרגיסטי של גנים ו / או גורמים סביבתיים. בנוסף, שיטת כימות זה עשויה גם לחשוף אפילו שיפור או הצלה קל של פגם orofacial. זה כן הופך אותו למדריך שימושי בניתוח הרפוי פוטנציאלי. השילוב של מדידות פנים וmorphometrics הגיאומטרי שאנו מציגים כאן מאפשר ניתוח סטטיסטי מקיף יותר של שניהם בגודל ובצורה של אזור orofacial מאשר פרוטוקולים הנוכחיים אשר במידה רבה לנצל רק אחת או 15-18 האחר. יתר על כן, אנו מציגים דרך פשוטה להעריך הן את המדיאלי ומטוסים לרוחב שלהפנים ללא צורך בציוד הדמיה תלת-ממדי מתוחכם המשמש במחקרים הנוכחיים 13,19. אנו מדגימים פרוטוקול זה על צפרדעי laevis עוברים שטופלו במעכב קולטני חומצה רטינואית שגורם פיתוח orofacial חריג ו2,3 חיך שסוע חציון. כימות של הממדים וצורה של אזור orofacial בעוברים אלה חשפה שינויים בmidface שדומה לבני אדם עם בקיעי חיך דומים ומודלים של עכברי 20,21. עם זאת, פרוטוקול זה יכול להיות מנוצל כדי להעריך את ההשפעות של תרכובות אחרות בפיתוח orofacial כגון חומרים טבעיים, קוטלי עשבים, או חלבונים כגון גורמי גדילה. יתר על כן, שינויי גודל וצורת orofacial הנובעים מהפרעות של ביטוי גנים באמצעות הפסד או רווח של ניסויי פונקציה (באמצעות morpholinos antisense או מצננים / Talens) יכול להיות גם לכמת באמצעות פרוטוקול זה. לבסוף, פיתחנו specifica שיטה זוlly להעריך מורפולוגיה צפרדעים; עם זאת, הוא שונה בקלות לניתוח של כל חוליות. יישומים אחרים יכולים לכלול גם שימוש בפרוטוקול זה להשוואה בין מינים קשורים באופן הדוק למחקרים אבולוציוניים או אקולוגיים. בעוד הדוגמא שאנו מספקים כאן מנצלת פרוטוקול זה כדי לתאר את הניתוח באזור orofacial, זה יכול בקלות להיות שונה לניתוח של אזורים אחרים, איברים, או מבנים. פרוטוקול כימות orofacial זה יהפוך למשאב יקר ערך עבור קהילת המחקר, כמו גם כלי ללימוד מצוין לסטודנטים לתואר ראשון כהפגנת וידאו.

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Name of the reagent</td>
			<td style="width:143px;">Company</td>
			<td style="width:136px;">Catalogue number</td>
			<td style="width:236px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>fitted with AxioCamICC1 camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Inox forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, disposable scalpel</td>
			<td>Sklar</td>
			<td>06-2015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">24-well plate</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:136px;">087721</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Standard Disposable transfer pipettes</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:136px;">13-711-7M</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">150 mm X 15 mm Petri dishes</td>
			<td style="width:143px;">Falcon</td>
			<td style="width:136px;">351058</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Incubators</td>
			<td style="width:143px;">Ectotherm</td>
			<td style="width:136px;"></td>
			<td style="width:236px;">set to 15C or 20C&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:304px;">Modeling Clay</td>
			<td style="width:143px;">Premo, or other non-toxic modeling clay</td>
			<td style="width:136px;"></td>
			<td style="width:236px;">in black or white</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Straight teasing needle</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:136px;">19010</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:304px;">Capillary Tubing (for needles)</td>
			<td>FHC</td>
			<td>30-30-1</td>
			<td style="width:236px;">Borosil 1.0mm OD x 0.5mm ID/Fiber, 100 mm each</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:304px;">Needle Puller, Model P-97&#160;</td>
			<td style="width:143px;">Sutter Instrument Co,&#160;</td>
			<td style="width:136px;">Needle Puller: P-97 Flaming/ Bown micropipette puller Filament: FB300B</td>
			<td style="width:236px;">For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:304px;">Pipettemen</td>
			<td style="width:143px;">Gilson</td>
			<td style="width:136px;">F144802, F123600, F123602</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">BMS-453</td>
			<td style="width:143px;">Tocris</td>
			<td style="width:136px;">3409</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:304px;">DMSO</td>
			<td style="width:143px;">American Bioanalytical</td>
			<td style="width:136px;">AB00435-01000</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Cysteine</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:136px;">52-90-4</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Paraformaldehyde powder</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:136px;">158127</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:304px;">Petri dishes</td>
			<td style="width:143px;">Falcom</td>
			<td style="width:136px;">353003, 351058</td>
			<td style="width:236px;">100 mm diameter and 150 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">100% Ethanol</td>
			<td style="width:143px;">VWR</td>
			<td>89125-170</td>
			<td style="width:236px;"></td>
		</tr>
	</tbody>
</table>

quantification of orofacial phenotypes in xenopus

צפרדעים הפכו לכלי חשוב עבור לנתח את המנגנונים לפיקוח על בנייה ופגמי craniofacial. שיטה לכמת פיתוח orofacial תאפשר לניתוח קפדני יותר של פנוטיפים orofacial על ביטול עם חומרים שיכולים מבחינה גנטית או מולקולרי לתפעל ביטוי גנים או תפקוד חלבון. שימוש בשתי תמונות ממדיות של הראשים העובריים, ממדים-כגון גודל מסורתיים כרוחב orofacial, גובה וarea- נמדדים. בנוסף, מדד עגלגלות של פתיחת הפה העוברית משמש לתאר את הצורה של הפה. morphometrics הגיאומטרי של שתי תמונות ממדיות אלה מבוצעים גם כדי לספק מבט מתוחכם יותר של שינויים בצורתו של אזור orofacial. ציוני דרך מוקצות לנקודות ספציפיות באזור orofacial וקואורדינטות נוצרות. ניתוח מרכיבי עיקרון משמש להפחתת קואורדינטות ציון דרך לרכיבי עיקרון שאז להפלות הטיפולקבוצות. תוצאות אלו מוצגות כעלילת פיזור שבו אנשים עם צורות orofacial דומות להתקבץ יחדיו. זה גם שימושי לביצוע ניתוח פונקצית מבחין, שסטטיסטי משווה את עמדותיהם של ציוני הדרך בין שתי קבוצות טיפול. ניתוח זה מוצג ברשת שינוי בהם שינויים בעמדת ציון דרך נתפסים כוקטורים. רשת היא על גבי וקטורים אלה, כך שדפוס השתאה מוצג כדי להראות היכן עמדות ציון דרך משמעותיות השתנו. שינויי צורה בניתוח פונקצית המבחין מבוססים על מדד סטטיסטי, ולכן יכולים להיות מוערך על ידי p-value. ניתוח זה הוא פשוט ונגיש, הדורש רק stereoscope ותוכנה חופשית, ובכך יהיה משאב מחקר והוראה רב-ערך.

כאן, ניתוח כמותי של גודל orofacial וצורה הודגם להשוות עוברים שטופלו במעכב חומצה רטינואית קולט (מעכב RAR) לבקרה שלא טופלה. טופלו עוברים עם ריכוז 1 מיקרומטר של מעכב כימי זה משלב 24-30 (26-35 hpf), נשטף החוצה, וקבוע בשלב 42 (82 hpf). לאחר מכן הם עובדו ונותחו כמתואר בפרוטוקול. תוצאות הן הנתוני...

Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

Quantification of Orofacial Phenotypes in Xenopus

שיטה לכמת את גודלו וצורת orofacial של עוברי laevis Xenopus פותחה. בפרוטוקול זה, מדידות גודל מסורתיות בשילוב עם morphometrics הגיאומטרי כדי לאפשר ניתוחים מתוחכמים יותר של התפתחות וליקויי orofacial.

כימות Orofacial פנוטיפים ב<em&gt; צפרדעים</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

quantification-of-orofacial-phenotypes-in-xenopus

Research

JoVE Journal

Biology

Quantification of Orofacial Phenotypes in Xenopus

9.7K Views.  Virginia Commonwealth University.  שיטה לכמת את גודלו וצורת orofacial של עוברי laevis Xenopus פותחה. בפרוטוקול זה, מדידות גודל מסורתיות בשילוב עם morphometrics הגיאומטרי כדי לאפשר ניתוחים מתוחכמים יותר של התפתחות וליקויי orofacial. 

Video: Quantification of Orofacial Phenotypes in Xenopus

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

בשנת העצרת חוץ גופית גרעיני מופרדים באמצעות תמציות Xenopus ביצה

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection של ביציות Xenopus Laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

הדמיה ב-RNA לוקליזציה Xenopus ביציות

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

כימות γH2AX מוקדים בתגובה קרינה Ionising

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

RNAi הקרנת לזהות פנוטיפים Postembryonic ב ג elegans</em

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

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

השתלת פנים ב<em&gt; Laevis Xenopus</em&gt; עוברים

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

<em&gt; Xenopus</em&gt; טכניקת ביצית Cut-פתוח וזלין גאפ מתח מהדק עם Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

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

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

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 ...

Spinal Cord Transection In Xenopus laevis Tadpoles

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.

Spinal Cord Transection In Xenopus laevis Tadpoles

Xenopus laevis tadpole spinal cord transection is a relevant injury method to study spinal cord injury and regeneration by making a transverse cut that completely severs the spinal cord at the thoracic level.

העברת חוט השדרה בקסנופוס לאביס ראשנים

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis ...

זילוח דם בצפרדעים אפריקאיות בעלות טפרים לתמלול ופרוטאומיקה

Effective Rapid Blood Perfusion in Xenopus

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

מחבר זרקור: זילוח דם מהיר יעיל ב Xenopus  

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

זילוח דם מהיר יעיל בקסנופוס

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...