Name: functional cloning using a xenopus oocyte expression system
Uploaded: 2016-01-30T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

Alle experimentellen Verfahren wurden von der University of Virginia Institutional Animal Care und Verwenden Committee genehmigt. ANMERKUNG: Abbildung 1 eine schematische Übersicht über die experimentellen Verfahren. 1. Herstellung von Oozyten <ol><li> Pre-prime X. laevis Weibchen mit 150 U Pregnant Mare Serum Gonadotropin (PMSG) etwa eine Woche im Voraus über Eizelle Isolation. Injizieren Sie 1 ml 150 U / ml PMSG in Rücken Lymphsack mit 1 cc sterile Spritze mit 29 G-Nadel. </li><li> Bereiten Sie Lösungen für Oocyteninjektion und Oozyten-Tierkappe Assay. <ol><li> Vorbereitung Ca ++ / Mg ++ -freier OR2 (OR2-): 82 mM NaCl, 2,5 mM KCl, 1,5 mM Na 2 HPO 4, 50 mM HEPES pH 7,2. </li><li> Bereiten OR2: OR2- plus 1 mM CaCl 2 und 1 mM MgCl 2. </li><li> Vorbereitung OCM: 60% Leibovitz L15, 0,4 mg / ml BSA, 100 ug / ml Gentamycin, pH 7,8. </li><li> Prepare 1x MBS: 88 mM NaCl, 1 mM KCl, 0,7 mM CaCl 2, 1 mM MgSO 4, 5 mM HEPES (pH 7,8), 2,5 mM NaHCO 3. </li><li> Bereiten Sie 3% Ficoll in 1x MBS. </li><li> 1fach NAM: 110 mM NaCl, 2 mM KCl, 1 mM Ca (NO 3) 2, 1 mM MgSO 4, 0,1 mM EDTA, 1 mM NaHCO 3, 2 mM Na 3 PO 4, pH 7,4. </li></ol></li><li> Betäuben die Frau in einer 0,03% igen Lösung von Ethyl-3-aminobenzoat Methansulfonatsalz (MS222) in 0,1 MBS (erste aufzulösen 0,3 g MS222 in 10 ml 95% Ethanol, dann in 1 L 0,1 x MBS verdünnen), indem Frosch in Lösung 10-15 Minuten oder bis nicht mehr reagiert. <ol><li> Prüfen Sie, ob der Anästhesie abgeschlossen durch Drehen narkotisierten Frosch auf den Rücken, um sicherzustellen, es reagiert nicht (vollständig betäubten Froschschenkel zu bewegen oder drehen Körper über) ist. </li></ol></li><li> Chirurgisch zu isolieren Eierstockfragmente, indem sie ein Bauchschnitt durch die Haut und Körper Wand mit Skalpell, Isolieren Eierstockgewebe mit einer Pinzetteund Schere. Zeigen Eierstock Fragmente in OR2-. Schließen Sie die Körperwand mit einem 3-0 Seidennaht auf einem 24 mm gebogene Nadel und schließen Sie die Haut separat mit den gleichen Nähten. Eine Wiederherstellung der weiblichen in 1 g / l Aquarium Salz in Wasser. </li><li> Verwenden feinen Pinzette, reißEierStockGewebe in kleine (10 - 20 Eizellen) Stücke und Transfer zum frischen OR2-. </li><li> Defolliculate ovarian Fragmente in 2,0 mg / ml Collagenase A in OR2- durch Rühren vorsichtig auf einem Schüttler für 1 Stunde, der Übertragung auf frische Kollagenase und Rühren für eine weitere Stunde. </li><li> Wasch Oozyten 10mal OR2 [Ca ++ / Mg ++ enthält], Verwerfen kleiner Oozyten. </li><li> Waschen Sie Oozyten in OCM zweimal und übertragen an die frische OCM. Isolieren Stufe VI Oozyten nach Größe bei Sichtprüfung und entsorgen unreifen (kleinere) Oozyten: Stufe VI Oozyten sind größer als die unreifen Eizellen mit noch Pigmentierung im Tier Hemisphäre und etwa 1,2 bis 1,4 mm Durchmesser. </li><li> Pflegen Oozyten bei 18 - 20 ° C in OCM vor der Injektion. Hinweis: Agarose-beschichteten Petrischalen verwendet werden, um ein Anhaften von Eizellen zu minimieren, austeilen wird. </li></ol> 2. Injektion Bibliothek Transcripts <ol><li> Vorbereitung einer direktionalen cDNA-Bank 15 unter Verwendung von RNA in einem geeigneten Stadium der Entwicklung (beispielsweise neuronale Platte Stufe 14 10) extrahiert. Erwerben Sie eine cDNA-Bibliothek oder konstruieren eine Verwendung eines kommerziellen Kits pro Übersicht in den vier Schritten unten. <ol><li> Isolieren Sie etwa 5 ug mRNA mit einem Produkt, für Poly (A) + RNA (Siehe Tabelle der Materialien) zu bereichern und nach den Anweisungen des Herstellers. Anmerkung: Die Transkripte verwendet werden, um die cDNA-Bibliothek kann auf ein induzierendes Gewebe beschränkt sein zu erzeugen (wie beispielsweise das neuronale Platte nach Mikrodissektion des gewünschten Gewebes) oder kann aus ganzen Embryonen in einem bestimmten Stadium können. </li><li> Produzieren cDNA mit einem kommerziellen Kit nach Herstelleranweisungen. </li><li> Ligieren ungefähr 20 ng cDNA in einen vector, das mit dem kommerziellen Kit oder einem anderen geeigneten Vektor. Einen geeigneten Plasmidvektor enthält Sequenzen, die für mRNA-Stabilität, wie beispielsweise 5&#39;-β-Globin-3 Poly (A) -Sequenzen (pCS2 +, pTnT, pCS105). </li><li> Transform-Ligation in kompetente Bakterienzellen mit Lieferanten empfohlenen Protokoll für Hitzeschock-Transformation. Hinweis: Alternativ ist eine Sammlung von offenen Leserahmen (ORFeome) Klone Handel erhältlich und können verwendet werden, um Transkripte für die Injektion zu erzeugen. </li></ol></li><li> Herstellung von 10 Pools von Plasmiden von 10 3 bis 10 4 Komplexität (10. April - 10. MAI Gesamtkomplexität). Dies entspricht 10 Platten mit 1.000 - je 10.000 Kolonien. <ol><li> Plattenbibliothekskultur auf 10 15-cm-LB-Ampicillin-Platten, wachsen 12 bis 18 h bei 37 ° C, und sammeln Sie Kolonien von jeder durch sanften Druck mit einem Glas Treuer in 7 ml LB. </li><li> Bereiten Sie eine Glycerin-Stamm von 0,5 ml (0,2 ml sterilem Glycerin und Speicher hinzuzufügen bei -20 &amp;# 730; C), und verwenden Sie die verbleibenden 6,5 ml, um DNA mit einem Standard-Vorbereitung im Handel erhältlichen DNA Miniprep Kit folgenden Herstellers. </li></ol></li><li> Linearisieren gepoolte Plasmid-DNA (1,0 bis 2,0 &amp; mgr; g) mit geeigneten Restriktionsenzymverdau 16 bei 37 ° C für 1 - 1,5 Stunden. Isolieren Sie die linearisierte DNA mit Phenol / Chloroform-Extraktion, gefolgt von Ethanolfällung und Resuspension in Wasser nach den Vorgaben der RNA-Polymerase-Kit verwendet wird. Synthetisieren Sinn-RNA mit einer RNA-Polymerase kommerziellen Kits nach Herstelleranweisungen. </li><li> Vorbereitung Nadeln für die Mikroinjektion (Verwendung einer Nadel Abzieher mit Glaskapillarröhren) von etwa 20 um Durchmesser; messen Nadelspitzen auf einem zusammengesetzten Mikroskop mit einem kalibrierten Okularmikrometer. Hinweis: Nadelzieher Einstellungen müssen empirisch bestimmt, um eine Nadel mit einer feinen Spitze, so dass, wenn sie mit einer feinen Pinzette gebrochen ergibt die gewünschte Düsengröße zu erzeugen. </li><li> Prepare (Push Ton ingleichmäßige Schicht auf der Unterseite der Schale) Lehm ausgekleideten 35 x 10 mm Petrischalen mit parallelen Rillen an Eizellen in während Mikroinjektion zu halten; produzieren Nuten mit einem Einkaufszentrum Sonde oder Pasteur-Pipette an der Spitze in Flammen fusioniert. Als Alternative zu Ton, vorzubereiten Agarose Gerichte machen Vertiefungen mit einem Elastomer Form 17. Übertragungs Oozyten mit weiter Bohrung Pipette bis 3% Ficoll in 1X MBS (ca. 2 ml) in Reihen in den Lehm ausgekleidet Gerichte. </li><li> Verwendung Mikroinjektor, füllen Nadel mit 1 ng / nl RNA und stellen Balance leichten Überdruck zu erzeugen (Ausarbeitung Eizelle Zytoplasma zu verhindern). </li><li> Injizieren Oozyten, die mit etwa 20 nl RNA in der Äquatorregion. Lassen Sie 1 Stunde für die injizierten Oozyten in Ficoll-MBS bleiben, dann übertragen Sie vorsichtig, um 1x MBS. Inkubation für 8 bis 24 Stunden bei 20 ° C vor der animal cap Assay. </li></ol> 3. Tier Cap Assay <ol><li> Bereiten Sie 3 / 4x NAM und erhalten feinen Pinzette, Haarschleife, gekrümmte Deckglas-Fragmente, Ton ausgekleideten dishes mit becherförmigen Vertiefungen, um Eizellen zu halten. Machen Sie gebogene Deckglas-Fragmente durch Auseinanderbrechen Deckgläser in kleine Fragmente (ungefähr 1-2 mm x 2 - 4 mm) und durch Flammen, bis Kanten polieren und hängen, wodurch ein Kurvenstück. </li><li> Düngung X. laevis Eiern 18 durch in vitro oder natürliche Paarung und Kultur Stufen Gastrula (10 - 11 Stunden nach der Befruchtung bei RT) in 0,1 x MBS vor der Sortierung nach Stufe; Sammeln Mitte der Gastrula (Stufe 11 bis 11,5) 10 Embryonen. </li><li> Transfer-Embryonen in eine Petrischale etwa zur Hälfte mit 3/4-fach NAM. Die Verwendung von zwei Paaren von feinen Pinzette, entfernen Fertilisation (Dotter) Membran von Gastrulen. </li><li> Transfer Oozyten bis 3 / 4x NAM (etwa 2 ml) in Ton ausgekleideten Gerichte und unbeweglich in einzelnen Eindrücke im Ton, Erzeugung Eindrücke auf einzelne Embryonen unterzubringen als Nuten wurden oben beschrieben. </li><li> Transfer-Embryonen, um den Ton ausgekleideten Gerichte und schneiden animal caps off gastrulae mit zwei Paaren von feinen Pinzette. Achten Sie darauf, Tierkappe Ektoderm nur und nicht die Äquatorial Gewebe 18 zu isolieren. </li><li> Platzieren eines Tieres Kappe auf dem Tierhemisphäre jede Oozyte mit der Innenfläche des Tierkappe Kontaktieren der Eizelle. Halten Rekombinanten gemeinsam durch Anlegen gekrümmten Deckglas-Fragment und nach unten gedrückt, um das Glas, Abflachen des Ektoderms wie die Deckkontakte des Tons (animal caps kann mit der Innenschicht auf die Oberfläche der Eizelle für 6 ausgesetzt bleiben offen - 8 h oder länger ). Hinweis: Sie können auch den Tierkappe in einem Ton-Einzug mit seiner Innenfläche nach oben und legen Sie eine Eizelle an der Kappe; sichern die rekombinante mit kleinen Erweiterungen von Ton. </li><li> Kultur bei 20 ° C, bis Steuer Embryonen reach gewünschte Bühne für Assay. </li><li> Separate rekombinanten durch Entfernen Deckglas / Ton und Isolieren Ektoderm mit einer Pinzette und einer Haarschleife. </li><li> Fix ektodermalen Fragmente für 1 Stunde in MEMFA (3,8%Formaldehyd in MEM [0,1 M MOPS, pH 7,4, 2 mM EGTA und 1 mM MgSO 4]). </li><li> Transfer-Fragmente (und Kontrolle Embryonen) aus MEMFA zu Ethanol und bei -20 ˚C. </li></ol> 4. Analyse der Antwort in Ektoderm durch in situ Hybridisierung (ISH) <ol><li> Bereiten Sie Lösungen für die ISH. <ol><li> Vorbereitung 1x PBS: 0,01 M phosphatgepufferter Kochsalzlösung, 0,138 M NaCl, pH 7,4 </li><li> Vorbereitung PBS-Tween (PTW): 1x PBS, 0,1% Tween-20 </li><li> Vorbereitung 100x Denhart-Lösung: 2% BSA, 2% Polyvinylpyrrolidon, 2% Ficoll </li><li> Vorbereitung Hybridisierungspuffer: 50% Formamid, 5 × SSC, 1 mg / ml Hefe-RNA torula, 1 ug / ml Heparin, 1x Denhart-Lösung, 0,1% Tween-20, 0,1% CHAPS, 10 mM EDTA, DEPC-H 2 O </li><li> Vorbereitung Maleinsäure-Puffer (MAB): 100 mM Maleinsäure, 150 mM NaCl, pH 7,5 </li><li> Bereiten MAB + Block: MAB, 2% Blockierungsreagenz (Wärme bis 60 ° C zu lösen) </li><li> Vorbereitung alkalische Phosphatase (AP) Puffer: 100 mM TrispH 9,5, 50 mM MgCl 2, 100 mM NaCl, 0,1% Tween, dH 2 O </li></ol></li><li> Bereiten Sie die RNA-Sonde. <ol><li> Unter Verwendung eines kommerziellen RNA-Polymerase-Kit und graben-NTP-Mix, fügen Sie in ein 1,5-ml-Röhrchen (50 &amp; mgr; l-Reaktion): 25,5 &amp; mgr; l DEPC-H 2 O, 10 ul 5X Transcription Buffer, 2,5 ul 10x dig-NTP-Mix, 5 ul 100 mM DTT, 2 ul RNAsin, 2 ul linearisierte DNA-Templat (~ 1 ug / ul), 3 ul RNA Polymerase und Inkubation 37 ° C für 90 min. </li><li> In 2 ul RNA Polymerase und Inkubation 37 ° C für 60 min. </li><li> Prüfen 2 ul Reaktions auf 1% Agarose-Gel. </li><li> 1 ul RQ1 RNase-Free DNase und Inkubation 37 C für 20 min. </li><li> Fällt die Sonde durch Zugabe von 50 &amp; mgr; l DEPC-H 2 O, 25 ul 10 M Ammoniumacetat und 313 ul Ethanol. Lagerung bei -20 C über Nacht (O / N), dann erholen RNA durch Zentrifugation bei 13.800 × g für 20 min. Mit 500 ul 75% Ethanol waschen, kurz drehen, entfernenEthanol und ermöglichen Pellet an der Luft trocknen. In 50 ul DEPC-H 2 O. </li><li> Hinzufügen Hybridisierungspuffer bis zu einer Endkonzentration von ~ 0,5 ug / ul. </li></ol></li><li> Bereiten Tissue für die Hybridisierung. Wenn nicht anders angegeben, füllen Fläschchen an die Spitze mit jeder Lösung Änderung beschrieben (ca. 4 ml). <ol><li> Entfernen von Ethanol aus Fläschchen und Transfer-Embryonen in 75% Ethanol / PTW, dann 50% Ethanol / PTW für jede 10 min, horizontal auf Wippe. </li><li> Dreimal Waschen in PTW für jeden auf Wippe 5 min. </li><li> Transfer zum 10 ug / ul Proteinase K-Behandlung in PTW; Rockröhren vertikal 15 min. </li><li> Spülen Sie zweimal je 10 min in 0,1 M Triethanolamin pH-Wert 7,8 - Rockröhren vertikal. </li><li> In 12,5 ul Essigsäureanhydrid, um Rohre und Rock vertikal 5 min. Wiederholen Sie mit zusätzlichen 12,5 ul Essigsäureanhydrid für 5 min. </li><li> Waschen Sie in PTW 5 min vertikal auf Wippe. </li><li> REFIX in 4% Paraformaldehyd 20 min auf Wippe. Wärmehybridisierungspufferbis 60 ° C. </li><li> Dreimal Waschen in PTW für jeden auf Wippe 5 min. </li><li> Entfernen Sie alle, aber ~ 1 ml PTW aus jedem Röhrchen und fügen Sie 250 ul Hyb-Puffer; leicht schwenken Rohre zu mischen. Rockröhren vertikal 5 min. </li><li> Ersetzen Sie mit 60 ˚C Hyb-Puffer (0,5 ml) und leicht rühren bei 60 ° C 10 min. Ersetzen mit frischem Hyb Puffer und rühren bei 60 ° C zwei bis 4 Stunden. </li><li> Wärmesonde (1 ml bei 0,5 ug / ul in Hyb-Puffer) bis 60 ° C (3 min). Entfernen Hyb Puffer und Sonde in Rohren. Leicht bewegen O / N bei 60 ° C. </li></ol></li><li> Bereiten Tissue für Antikörper. <ol><li> Warm Hyb Puffer und 2x SSC + 0,1% Tween-20-Lösungen bis 60 ° C. </li><li> Ersetzen Sondenlösung mit Hyb Buffer (Speichern Sonden bei -20 ° C für 2 - 3x Wiederverwendung). Für 10 Minuten bei 60 ° C waschen. </li><li> Dreimal bei 60 ° C in 2 × SSC-Tween (20 min je unter Rühren) zu waschen. </li><li> Dreimal bei 60 ° C in 0,2 × SSC-Tween (20 min je unter Rühren) zu waschen. </li><li> Zweimal für jeweils 15 min horizontal auf Wippe waschen in MAB (RT). </li><li> 1 ml MAB + Block. Waschen Sie 2 Stunden vertikal auf Wippe. </li><li> Transfer zum 1 ml MAB + Block mit einer 1 / 2.000 Anti-Digoxygenin-AP. Rock vertikal bei 4 CO / N. </li></ol></li><li> Bereiten Tissue für Farbreagenz. <ol><li> Ersetzen Antikörperlösung mit MAB; waschen dreimal MAB 5 min jeweils horizontal auf Wippe. </li><li> Drei Mal in MAB 1 Stunde jeweils horizontal auf Wippe waschen. </li><li> Zweimal in AP-Puffer jeweils 10 Minuten horizontal auf Wippe waschen. </li><li> Bringen Gewebe Mehr auch Kunststoffwanne, entfernen AP-Puffer und fügen BM Lila Reagenz (~ 1 ml). Erlauben Reaktion auf in der Dunkelheit 5 Minuten bis O / N fortfahren. </li><li> Wenn entsprechende Färbung erreicht ist, zu übertragen, um Gewebedurchstechflaschen mit PTW. Zwei Mal jeweils 10 Minuten auf Wippe waschen in PTW. </li><li> Fix in Bouin-Lösung bei 4 CO / N auf Wippe. </li><li> Waschen Sie Bouin-mit drei 70% Ethanol / 30% PTW Wäschen bei RT. Gehen to Aufnahme von Bildern mit Auflicht und ein Mikroskop montierten Kamera, Bleichen wenn nötig 18. </li></ol></li></ol> 5. Sib Auswahl und Klonierung <ol><li> Wählen Sie Pool mit der höchsten Aktivität (größte Resonanz in ektodermalen Gewebe). </li><li> Man titriert (verdünnte mit LB, geeignete Dichte) das Glycerin Lager für den Pool mit der höchsten Aktivität und Platte aus zehn neuen Platten mit etwa einem Zehntel der Kolonien aus dem vorherigen Schritt (unter Verwendung von Abschnitt 2 in Protokoll oben). </li><li> Poolgröße zu reduzieren, bis Aktivität auf einzelne Kolonie zurückverfolgt. </li><li> DNA-Sequenz zu erhalten, unter Verwendung von Standard-Primer im Vektor. </li></ol>

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	<li>Yergeau, D., Kelley, C. ., Zhu, H., Kuliyev, E., Mead, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+Genetic+Screens+in+Xenopus.+Using+Transposon-Mediated+Insertional+Mutagenesis.">Forward Genetic Screens in Xenopus. Using Transposon-Mediated Insertional Mutagenesis.</a> Methods in Molecular Biology. 917, 111-127 (2012).</li><li>Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis.+as+a+Model+Organism+for+Genetics+and+Genomics:+Past,+Present+and+Future.">Xenopus tropicalis. as a Model Organism for Genetics and Genomics: Past, Present and Future.</a> Methods in Molecular Biology. 917, 3-15 (2012).</li><li>Nakayama, T., Fish, M., Fisher, M., Oomen-Hajagos, J., Thomsen, G., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+Efficient+CRISPR/Cas9-Mediated+Targeted+Mutagenesis+in+Xenopus+tropicalis.">Simple and Efficient CRISPR/Cas9-Mediated Targeted Mutagenesis in Xenopus tropicalis.</a> Genesis. 51, 835-843 (2013).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis.">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis.</a> Biology Open. 1 (12), 1273-1276 (2012).</li><li>Zhao, Y., Ishibashi, S., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetic+Studies+Using+Antisense+Morpholino+Oligonucleotides.">Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides.</a> Methods in Molecular Biology. 917, 143-154 (2012).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injected+Xwnt-8.+RNA+acts+early+in+Xenopus.+embryos+to+promote+formation+of+a+vegetal+dorsalizing+center.">Injected Xwnt-8. RNA acts early in Xenopus. embryos to promote formation of a vegetal dorsalizing center.</a> Cell. 67, 753-765 (1991).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+noggin.,+a+new+dorsalizing+factor+localized+in+the+Spemann+organizer+in+Xenopus+embryos.">Expression cloning of noggin., a new dorsalizing factor localized in the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li><li>Plautz, C., Zirkle, B., Deshote, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+of+induction+of+anterior+head+ectodermal+properties+in+Xenopus.+embryos+are+mediated+by+transcriptional+cofactor+ldb1.">Early stages of induction of anterior head ectodermal properties in Xenopus. embryos are mediated by transcriptional cofactor ldb1.</a> Developmental Dynamics. 243 (12), 1606-1618 (2014).</li><li>Servetnick, M., Grainger, R. <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> Development. 112, 177-188 (1991).</li><li>Nieuwkoop, P., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li><li>Lemaire, P., Garrett, N., Gurdon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+Siamois.,+a+Xenopus.+homeobox+gene+expressed+in+dorsal-vegetal+cells+of+blastulae+and+able+to+induce+a+complete+secondary+axis.">Expression cloning of Siamois., a Xenopus. homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.</a> Cell. 81, 85-94 (1995).</li><li>Lustig, K., Kroll, K., Sun, E., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+a+Xenopus+T.-related+gene+(Xombi.)+involved+in+mesodermal+patterning+and+blastopore+lip+formation.">Expression cloning of a Xenopus T.-related gene (Xombi.) involved in mesodermal patterning and blastopore lip formation.</a> Development. 122, 4001-4012 (1996).</li><li>Hsu, D., Economides, A., Wang, X., Eimon, P., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus.+dorsalizing+factor+Gremlin+identifies+a+novel+family+of+secreted+proteins+that+antagonize+BMP+activities.">The Xenopus. dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.</a> Molecular Cell. 1 (5), 673-683 (1998).</li><li>Lustig, K., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+oocyte+expression+assay+to+reconstitute+inductive+signaling.">Use of an oocyte expression assay to reconstitute inductive signaling.</a> Proc. Natl. Acad. Sci. USA. 92, 6234-6238 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE Science Education Database. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Restriction+Enzyme+Digests.">Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests.</a> JoVE Science Education Database. , (2015).</li><li>Viczian, A., Zuber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Determination+Using+the+Animal+Cap+Transplant+(ACT)+Assay+in+Xenopus+laevis.">Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis.</a> J. Vis. Exp. (39), e1932 (2010).</li><li>Sive, H., Grainger, R., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of.Xenopus laevis.: A laboratory manual. , (2000).</li><li>Zygar, C., Cook, T., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activation+during+early+stages+of+lens+induction+in+Xenopus.">Gene activation during early stages of lens induction in Xenopus.</a> Development. 125, 3509-3519 (1998).</li><li>Medina-Martinez, O., Jamrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxe+view+of+lens+development+and+disease.">Foxe view of lens development and disease.</a> Development. 134, 1455-1463 (2007).</li><li>Amin, N., Tandon, P., Osborne, N. E., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+in+the+tetraploid+Xenopus.laevis.+enables+genome-wide+insight+in+a+classic+developmental+biology+model+organism.">RNA-seq in the tetraploid Xenopus.laevis. enables genome-wide insight in a classic developmental biology model organism.</a> Methods. 66, 398-409 (2014).</li><li>Gilchrist, M., Zorn, A., Voigt, J., Smith, J., Papalopulu, N., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+large+set+of+full-length+clones+from+a+Xenopus+tropicalis+EST+project.">Defining a large set of full-length clones from a Xenopus tropicalis EST project.</a> Developmental Biology. 271, 498-516 (2004).</li><li>Karpinka, 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=Xenbase,+the+Xenopus.+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus. model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Research. 43, D756-D763 (2015).</li><li>Zhang, S., Li, J., Lea, R., Amaya, E., Dorey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Genome-Wide+In+Vivo+Screen+Identifies+New+Regulators+of+Signaling+Pathways+during+Early+Xenopus+Embryogenesis.">A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signaling Pathways during Early Xenopus Embryogenesis.</a> PLoS ONE. , e79469 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Die für die funktionale Klonierung von Genen in der Lage, eine Antwort in kompetente Ektoderm Induzieren hier beschriebene Verfahren kann verwendet werden, um eine Vielzahl von Genprodukten zu identifizieren. Diese Methode baut auf früheren Arbeiten durch die Kombination von Gewebe-induzierende Assays mit Ausdruck Klonierungstechniken. Wir nutzen die Stoffwechselwege des Xenopus-Oozyten als Quelle der Erzeugung von induzierende Faktoren, die direkt oder indirekt folgende RNA-Injektion. Dies in Kombination mit...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

<table><tbody><tr><td>12/101 Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>12/101</td><td>Monoclonal antibody for detection of muscle tissue</td></tr><tr><td>20x SSC Buffer</td><td>Sigma</td><td>S6639</td><td>for ISH</td></tr><tr><td>Acetic anhydride</td><td>Sigma</td><td>A6404</td><td>for ISH</td></tr><tr><td>Anti-Dig-AP</td><td>Roche</td><td>11093274910</td><td>for ISH</td></tr><tr><td>Aurum Plasmid Mini Kit</td><td>Bio-Rad</td><td>732-6400</td><td>Plasmid DNA purification</td></tr><tr><td>Blocking Reagent</td><td>Roche</td><td>11096176001</td><td>for ISH</td></tr><tr><td>BM Purple</td><td>Roche</td><td>11442074001</td><td>for ISH</td></tr><tr><td>Boekel Hybridization Oven</td><td>Fisher Scientific</td><td>13-245-121</td><td>for ISH</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma</td><td>HT10132</td><td>for ISH</td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td>for OCM</td></tr><tr><td>CHAPS</td><td>Sigma</td><td>C3023</td><td>for ISH</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>Defolliculation of oocytes</td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C121800</td><td>Dejelly embryos</td></tr><tr><td>DEPC-H2O</td><td>Fisher Scientific</td><td>BP5611</td><td>for ISH</td></tr><tr><td>Dig-RNA Labeling Mix</td><td>Roche</td><td>11277073910</td><td>for ISH probes</td></tr><tr><td>Dumont #5 forceps</td><td>World Precision Instruments</td><td>500233</td><td>for Vitelline envelope removal</td></tr><tr><td>Ethyl 3-aminobenzoate</td><td>Sigma</td><td>A5040</td><td>MS222 anesthetic</td></tr><tr><td>Ficoll PM 400</td><td>Sigma</td><td>F4375</td><td>for Injection media</td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td>for ISH</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma</td><td>G1914</td><td>for OCM</td></tr><tr><td>Glass capillaries</td><td>World Precision Instruments</td><td>4878</td><td>3.5&quot; long, I,D, 0.530 mm</td></tr><tr><td>Glass sample vials</td><td>Fisher Scientific</td><td>06-408B</td><td>for ISH</td></tr><tr><td>Hair loop</td><td></td><td></td><td>Hair affixed in pasteur pipette for tissue manipulation</td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H4784</td><td>for ISH</td></tr><tr><td>Injector Nanoliter 2010</td><td>World Precision Instruments</td><td>Nanoliter 2010</td><td>Microprocessor-controlled microinjector</td></tr><tr><td>Instant Ocean</td><td>Carolina</td><td>972433</td><td>Aquarium Salt for frog recovery</td></tr><tr><td>IRBG XGC Xenopus verified full-length cam cDNA</td><td>Source Bioscience</td><td>989_IRBG</td><td>cDNA library&amp;nbsp;</td></tr><tr><td>LB Agar plates with 100 &amp;micro;g/ml Ampicillin</td><td>Teknova</td><td>L5004</td><td>150 mm pre-poured LB-Amp plates for sib selection</td></tr><tr><td>LB Luria Broth</td><td>Teknova</td><td>L8650</td><td>LB for collecting colonies in sib selection from plates and dilution of cultures</td></tr><tr><td>Magnetic mRNA Isolation Kit</td><td>New England BioLabs</td><td>S1550S</td><td>for isolation of poly(A)-enriched RNA</td></tr><tr><td>Maleic Acid</td><td>Sigma</td><td>M0375</td><td>for ISH</td></tr><tr><td>Manual Microfil Micromanipulator</td><td>World Precision Instruments</td><td>M3310R</td><td>Manual micromanipulator</td></tr><tr><td>Nutating Mixer</td><td>Fisher Scientific</td><td>22-363-152</td><td>Rocker for ISH</td></tr><tr><td>Permoplast</td><td>Nasco</td><td>SB33495M</td><td>Clay for injection and dissection dishes</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>P5368</td><td>for ISH</td></tr><tr><td>PMSG</td><td>Sigma</td><td>G4877</td><td>to stimulate oocyte development</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>PVP40</td><td>for ISH</td></tr><tr><td>Programmable Puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>Micropipette needle puller</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td>for ISH</td></tr><tr><td>pTnT Vector</td><td>Promega</td><td>L5610</td><td>cDNA library construction</td></tr><tr><td>Riboprobe Combination System</td><td>Promega</td><td>P1450</td><td>in vitro transcription</td></tr><tr><td>Superscript Full Length cDNA Library Construction Kit</td><td>Life Technologies</td><td>18248013</td><td>kit for cDNA library construction</td></tr><tr><td>Sutures, 3-0 silk</td><td>Fisher Scientific</td><td>19-037-516</td><td>Suture thread and needle for post-oocyte removal</td></tr><tr><td>Torula RNA</td><td>Sigma</td><td>R3629</td><td>for ISH</td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>T1502</td><td>for ISH</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td>for ISH</td></tr><tr><td>Universal RiboClone cDNA Synthesis System</td><td>Promega</td><td>C4360</td><td>alternative kit for cDNA library construction</td></tr><tr><td>Xenopus Full ORF Entry Clones - ORFeome Collaboration</td><td>Source Bioscience</td><td>5055_XenORFeome</td><td>ORFeome Clones</td></tr><tr><td>XL2-Blue Ultracompetent Cells</td><td>Agilent Technologies</td><td>200150</td><td>cells for transformation of cDNA library</td></tr></tbody></table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Als Reaktion auf die Expression von mRNA in Oozyten injiziert und reagiert animal cap-Gewebe wurde für die Expression von OTX2 durch in situ-Hybridisierung (Figur 2 und Tabelle 1) untersucht; OTX2 wird in der mutmaßlichen Linsen Ektoderm (PLE) aus Neuralrohrschlusses durch Linsenplakode exprimiert Verdickung 19. Da jedoch OTX2 ist auch in der vorderen neuronalen Ektoderm sowie nichtneuralen Kopf Ektoderm außerhalb des PLE...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

Functional Cloning Mit ein<em&gt; Xenopus</em&gt; Eizellen Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

functional-cloning-using-a-xenopus-oocyte-expression-system

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K Aufrufe.  Shepherd University. We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Serie: Functional Cloning Using a Xenopus Oocyte Expression System

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation und<em&gt; In-vitro-</em&gt; Ausbau der<em&gt; Xenopus laevis</em&gt; Eizellen, von Intrazytoplasmatische Spermieninjektion gefolgt, um embryonale Entwicklungsstudie

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Forschung

Entwicklungsbiologie

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Mittels konfokaler Analyse<em&gt; Xenopus laevis</em&gt; Um Modulatoren der Wnt und Shh Morphogen Gradienten Untersuchen

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

Keim Urzelle Transplantation für CRISPR/Cas9-basierte in Xenopus überspringen

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Genetik

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<em&gt; In-vivo-</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

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.

Mikroinjektion von Xenopus laevis Oozyten

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

Biologie

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Mikroinjektion von DNA in Augenknospen in Xenopus laevis Embryonen und Bildgebung von GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Die Befruchtung der Xenopus Oozyten Über den Host-Transfer-Methode

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

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-Cell-Imaging und Quantitative Analyse der embryonalen Epithelzellen in 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.

Herstellung transgener Xenopus laevis Durch Restriktionsenzym-vermittelte Integration und Reaktorsicherheit 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 ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

Anwendung von Maus parthenogenetischen Haploid embryonalen Stammzellen als Ersatz für Sperma

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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