אנו מודים לראדק Sindelka על עזרתו, וקאש Bresilla לסיוע עם בעלי צפרדע והכנת עובר. עבודה זו מומנה על ידי NIH באמצעות R01DE021109 המענק לHLS לורה Jacox מומן על ידי הרשל סמית בוגר המלגה באוניברסיטת הרווארד ומענק מלגה בודדת F30 F30DE022989-01 דרך NIDCR.

<ol>
	<li>Gorlin, R. J., Cohen, M., Levin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Syndromes of the head and neck. , (1990).</li><li>Trainor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniofacial+birth+defects:+The+role+of+neural+crest+cells+in+the+etiology+and+pathogenesis+of+Treacher+Collins+syndrome+and+the+potential+for+prevention.">Craniofacial birth defects: The role of neural crest cells in the etiology and pathogenesis of Treacher Collins syndrome and the potential for prevention.</a> Am. J. Med. Gen. A. 152, 2984-2994 (2010).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+primary+mouth+in+Xenopus+laevis.">Development of the primary mouth in Xenopus laevis.</a> Dev. Bio. 295, 700-713 (2006).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positioning+the+extreme+anterior+in+Xenopus:+cement+gland,+primary+mouth+and+anterior+pituitary.">Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary.</a> Sem. Cell Dev. Bio. 18, 525-533 (2007).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+antagonists+Frzb-1+and+Crescent+locally+regulate+basement+membrane+dissolution+in+the+developing+primary+mouth.">The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.</a> Dev. 136, 1071-1081 (2009).</li><li>Gilbert, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Developmental Biology. , (2010).</li><li>Borchers, A., Epperlein, H. H., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+to+study+migratory+behavior+of+cranial+neural+crest+cells+in+Xenopus.">An assay system to study migratory behavior of cranial neural crest cells in Xenopus.</a> Dev. Genes Evol. 210, 217-222 (2000).</li><li>Grunz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homoiogenetic+neural+inducing+activity+of+the+presumptive+neural+plate+of+Xenopus+laevis.">Homoiogenetic neural inducing activity of the presumptive neural plate of Xenopus laevis.</a> Dev. Growth Differ. 32, 583-589 (1990).</li><li>Servetnick, M., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+neural+and+lens+competence+in+Xenopus+ectoderm:+evidence+for+an+autonomous+developmental+timer.">Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.</a> Dev. Bio. 112, 177-188 (1991).</li><li>Servetnick, M., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeogenetic+neural+induction+in+Xenopus.">Homeogenetic neural induction in Xenopus.</a> Dev. Bio. 147, 73-82 (1991).</li><li>Couly, G., Coltey, P., Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triple+origin+of+skull+in+higher+vertebrates:+a+study+in+quail-chick+chimeras.">The triple origin of skull in higher vertebrates: a study in quail-chick chimeras.</a> Dev. 117, 409-429 (1993).</li><li>Couly, G. F., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+early+neural+primordium+in+quail-chick+chimeras+:+I.">Mapping of the early neural primordium in quail-chick chimeras : I.</a> Developmental relationships between placodes, facial ectoderm. 110, 422-439 (1985).</li><li>Couly, G. F., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+early+neural+primordium+in+quail-chick+chimeras+II.+The+prosencephalic+neural+plate+and+neural+folds:+Implications+for+the+genesis+of+cephalic+human+congenital+abnormalities.">Mapping of the early neural primordium in quail-chick chimeras II. The prosencephalic neural plate and neural folds: Implications for the genesis of cephalic human congenital abnormalities.</a> Dev. Bio. 120, 198-214 (1987).</li><li>Lievre, A. L., Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+development+of+cranial+sensory+ganglia+and+the+potentialities+of+their+component+cells+studied+in+quail-chick+chimeras.">The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras.</a> Dev. Bio. 94, 291-310 (1982).</li><li>Kennedy, A., Dickinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Median+facial+clefts+in+Xenopus+laevis:+roles+of+retinoic+acid+signaling+and+homeobox+genes.">Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes.</a> Dev. Bio. 365, 229-240 (2012).</li><li>Trainor, P., Tam, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm+and+neural+crest+of+the+mouse+embryo-+codistribution+in+the+craniofacial+mesenchyme+but+distinct+segregation+in+the+branchial+arches.">Cranial paraxial mesoderm and neural crest of the mouse embryo- codistribution in the craniofacial mesenchyme but distinct segregation in the branchial arches.</a> Dev. 121, 2569-2582 (1995).</li><li>Sive, H. L., Grainger, R. M., Harland, R. 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יש המחברים אין לחשוף.

צעדים ומגבלות קריטיים: הליך השתלת הפנים EAD הוא זמן ועבודה אינטנסיבי. זה דורש תרגול, ידיים יציבים, ומיומנות מושלמת. פרוטוקול השתלת פנים מסתמך על יכולתו של החוקר כדי להסיר ביעילות וברקמות להשתלה. אם אחד לוקח יותר מדי זמן להכניס את ההשתלה בפרצופו של המארח, את פני המאר?...

כדי להבין את המנגנונים העומדים בבסיס ליקויי craniofacial לידת 1-2, רקמות חשובות ותרומות האיתות שלהם במהלך התפתחות craniofacial חייב להיות מזוהה. בצפרדע Xenopus laevis, חלק מהפנים, כוללים את הפה וצורת נחיריים מ&quot; אקסטרים Anterior דומיין &quot;(EAD), שבו האאקטודרם והאנדודרם ישירות לצד זה 3-4. EAD פועל גם כמרכז איתות להשפיע על רקמות הסובבות, כוללים הרכס העצבי גולגולתי, המהווה את הלסתות ואזורי פנים אחרים 5. כדי לזהות את הגנים שתורמים לתפקוד EAD, טכניקה &#39;השתלת הפנים&#39; פותחה, שבו רקמה מושתלת מתורם לעובר מארח, לאחר הסרת אזור המארח המתאים. בעקבות ההשתלה, וכתוצאה מפיתוח פנים הוא העריך. לכן, ההשפעות של אובדן התפקוד (LOF) או רווח של פונקציה (GOF) לגן מסוים בEAD מנותחים באופן מקומי, שבו שאר שעותEAD והגוף מורכב מרקמות מסוג בר. השתלת הגומלין יכולה להתבצע, שבו רקמה מסוג בר שהושתלה עוברים עם LOF הגלובלי או GOF בגנים ספציפיים. השתלה כבר בשימוש תכוף בXenopus וחומוס לומד 6. לדוגמא, השתלת Xenopus התייחסה אינדוקציה homogenetic עצבית, עדשה ויכולת עצבית, והגירה רכס עצבית 7-10. השתלת chimeric שליו חומוס נתחה פיתוח של הצלחת הקדמית העצבית, רכס עצבי קדמי, רכס עצבי, ועצמות גולגולת 11-14. זוהי טכניקת ההשתלה הראשונה למחקר של התפתחות craniofacial Xenopus. טכניקה זו הוכיחה תפקיד רומן למעכבי Wnt Frzb1 וסהר בויסות היווצרות קרום במרתף בפה המשוער 5. Laevis Xenopus הוא מודל אידיאלי למחקר של התפתחות craniofacial כעוברים גדולים, לפתח כלפי חוץ,nd הפנים גלויים ונגיש, המאפשרים המיקרומניפולציה והדמיה של פיתוח. מופיעים מנגנוני פיתוח פנים נשמרו, מה שמעיד כי ממצאים שנעשו בצפרדע לספק תובנות לגבי ההתפתחות אנושית 4,15-16.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pasteur pipette</td>
			<td rowspan="3" style="width:101px;">VWR</td>
			<td rowspan="3" style="width:136px;">14672-400</td>
			<td style="width:128px;">Lime Glass&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Size 5 3/4&#8217;&#8217;</td>
			<td style="width:128px;">Cotton Plugged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:128px;">Disposable</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:195px;">Graduated Transfer Pipette</td>
			<td rowspan="2" style="width:101px;">VWR</td>
			<td rowspan="2" style="width:136px;">16001-180</td>
			<td style="width:128px;">Disposable&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Polyethylene</td>
		</tr>
		<tr height="80">
			<td height="100" rowspan="2" style="height:100px;width:195px;">#5/45 forceps</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools by Dupont medical</td>
			<td rowspan="2" style="width:136px;">11251-35</td>
			<td rowspan="2" style="width:128px;">Angled 45 degrees</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="40">
			<td height="81" rowspan="3" style="height:81px;width:195px;">Standard Pattern Forceps</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">11000-20</td>
			<td style="width:128px;">Straight; serrated tip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Stainless Steel;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">20cm long</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Capillary Tubing (for needles)</td>
			<td rowspan="2" style="width:101px;">FHC</td>
			<td rowspan="2" style="width:136px;">30-30-1</td>
			<td style="width:128px;">Borosil 1.0mm OD x 0.5mm ID/Fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">100mm each</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cover slip&#160;</td>
			<td rowspan="4" style="width:101px;">VWR</td>
			<td style="width:136px;">48393 252&#160;</td>
			<td style="width:128px;">24x60mm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">micro cover glass</td>
			<td style="width:136px;">or&#160;</td>
			<td style="width:128px;">or&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">(for glass bridges)</td>
			<td style="width:136px;">48393 230</td>
			<td style="width:128px;">24x40mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">No.1.5</td>
			<td style="width:136px;"></td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Ficoll 400</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">F9378</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Needle Puller&#160;</td>
			<td style="width:101px;">Sutter Instrument Co</td>
			<td rowspan="3" style="width:136px;">Needle Puller: discontinued Filament: FB300B</td>
			<td rowspan="3" style="width:128px;">The most similar, currently available&#160; needle puller is the P-97. For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Model P-80</td>
			<td style="width:101px;">Flaming / Brown micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">(discontinued)</td>
			<td style="width:101px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Stereomicroscope</td>
			<td rowspan="2" style="width:101px;">Zeiss</td>
			<td rowspan="2" style="width:136px;"></td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Zeiss Stemi 1000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:195px;">Stereomicroscope Lighting by Fostec</td>
			<td style="width:101px;">Fostec</td>
			<td style="width:136px;"></td>
			<td style="width:128px;">Use a light box with 2 fiberoptic arms.&#160;&#160;</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Nickel Plated Pin Holder</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">26018-17</td>
			<td style="width:128px;">Jaw Opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Length: 17cm</td>
		</tr>
		<tr height="60">
			<td height="101" rowspan="3" style="height:101px;width:195px;">Moria Nickel Plated Pin Holder</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">26016-12</td>
			<td style="width:128px;">Jaw opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Length: 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;"></td>
		</tr>
		<tr height="40">
			<td height="60" rowspan="2" style="height:60px;width:195px;">Tungsten Needles</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">10130-05</td>
			<td rowspan="2" style="width:128px;">0.125mm Rod diameter</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Van Aken Plastalina</td>
			<td rowspan="2" style="width:101px;">Blick&#160;</td>
			<td rowspan="2" style="width:136px;">#33268-2981</td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Modeling Clay- white, red or yellow</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">mMessage mMashine SP6 or T7 Kit</td>
			<td style="width:101px;">Ambion</td>
			<td style="width:136px;">AM1340</td>
			<td style="width:128px;"></td>
		</tr>
	</tbody>
</table>

facial transplants in xenopus laevis embryos

מומים מולדים Craniofacial להתרחש ב1 מתוך כל 700 לידה חיות, אבל אטיולוגיה ידועה לעתים רחוקות בגלל הבנה מוגבלת של פיתוח craniofacial. כדי לזהות היכן מסלולים ורקמות איתות לפעול בדפוסים של הפנים פיתוח, טכניקה &#39;השתלת הפנים&#39; פותחה בעוברים של צפרדע Xenopus laevis. אזור של רקמות פנים משוערות (&quot;אקסטרים Anterior דומיין&quot; (EAD)) מוסר מעובר תורם בשלב tailbud, ולהשתיל לעובר מארח של אותו השלב, שממנו האזור שווה הערך הוסר. זה יכול לשמש כדי ליצור פנים chimeric בי הרקמה המארחת או תורם יש הפסד או רווח של תפקוד בגן, ו / או כוללת תווית שושלת. לאחר ריפוי, התוצאה של פיתוח היא בפיקוח, ומצביעה על תפקידים של מסלול האיתות תוך התורם או רקמות מארח שמסביב. Xenopus הוא מודל יקר ערך לפיתוח פנים, כמו אזור הפנים הוא גדול וקלותccessible להמיקרומניפולציה. ניתן assayed עוברים רבים, על פני פרק זמן קצר מאז פיתוח מתרחש במהירות. ממצאים בצפרדע רלוונטיים להתפתחות אנושית, שכן תהליכי craniofacial מופיעים נשמרים בין Xenopus ויונקים.

רקמות המושתלות צריכים להיות מוכנסות במלואו לתוך ראש המארח לאחר ההשתלה כפי שמוצגות באיור 3 א, ויש לי גשר זכוכית להציב כראוי על פניו של העובר, כפי שמוצגים באיור 2Bc. הרקמות המושתלות התורמות חייבים להיות בגודל נכון לפתיחה המארחת, להשתלה כדי להצליח. רקמת E...

Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Facial Transplants in Xenopus laevis Embryos

טכניקה להשתלה &quot;אקסטרים Anterior דומיין&quot; רקמות פנים בין Xenopus laevis עובר כבר מפותחת. רקמה ניתן להעביר מהרקע ביטוי גנים אחת לאחרת, ומאפשרת הלימוד של דרישות מקומיות לפיתוח craniofacial ולאיתות אינטראקציות בין אזורי פנים.

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. ...

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Facial Transplants in Xenopus laevis Embryos

10.1K Views.  Harvard University.  טכניקה להשתלה "אקסטרים Anterior דומיין" רקמות פנים בין Xenopus laevis עובר כבר מפותחת. רקמה ניתן להעביר מהרקע ביטוי גנים אחת לאחרת, ומאפשרת הלימוד של דרישות מקומיות לפיתוח craniofacial ולאיתות אינטראקציות בין אזורי פנים. 

Video: Facial Transplants in Xenopus laevis Embryos

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Dissection של ארגונית הקוטב Explants בעלי חיים מעוברים Xenopus laevis והרכבה של Assay הידבקות התא

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

פלסטיק הטבעת ואת חתך של עוברים Xenopus laevis

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

קבלת ביצים Xenopus laevis נקבות

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

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 ...

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 ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

רקמות קביעת שווי שימוש בבעלי חיים להשתלות (ACT) Assay ב Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-תא הדמיה וניתוח כמותי של תאים אפיתל עובריים ב Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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 ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

Dissection של<em&gt; Laevis Xenopus</em&gt; העצבי קרסט עבור<em&gt; במבחנה</em&gt; Explant תרבות או<em&gt; In vivo</em&gt; השתלות

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

באמצעות plusTipTracker תוכנה כדי למדוד microtubule Dynamics ב<em&gt; Laevis Xenopus</em&gt; קונוסים צמיחה

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...