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>

<ol>
	<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 border="0" cellpadding="0" cellspacing="0" width="766">
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			<td height="42" style="height:42px;width:207px;">12/101 Antibody</td>
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			<td style="width:233px;">Monoclonal antibody for detection of muscle tissue</td>
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			<td height="21" style="height:21px;width:207px;">20X SSC Buffer</td>
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			<td style="width:233px;">for ISH</td>
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			<td height="21" style="height:21px;width:207px;">Acetic anhydride</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A6404</td>
			<td style="width:233px;">for ISH</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Anti-Dig-AP</td>
			<td style="width:191px;">Roche</td>
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			<td height="21" style="height:21px;width:207px;">Aurum Plasmid Mini Kit</td>
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			<td style="width:233px;">Plasmid DNA purification</td>
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			<td height="21" style="height:21px;width:207px;">BM Purple</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11442074001</td>
			<td style="width:233px;">for ISH</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Boekel Hybridization Oven</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">13-245-121</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Bouin&#39;s Solution</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">HT10132</td>
			<td style="width:233px;">for ISH</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">BSA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A9647</td>
			<td style="width:233px;">for OCM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">CHAPS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C3023</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Collagenase A</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">10103578001</td>
			<td style="width:233px;">Defolliculation of oocytes</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cysteine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C121800</td>
			<td style="width:233px;">Dejelly embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">DEPC-H2O</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">BP5611</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Dig-RNA Labeling Mix</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11277073910</td>
			<td style="width:233px;">for ISH probes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Dumont #5 forceps</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">500233</td>
			<td style="width:233px;">for Vitelline envelope removal</td>
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			<td height="21" style="height:21px;width:207px;">Ethyl 3-aminobenzoate</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A5040</td>
			<td style="width:233px;">MS222 anesthetic</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ficoll PM 400</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F4375</td>
			<td style="width:233px;">for Injection media</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Formamide</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F9037</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Gentamicin sulfate</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G1914</td>
			<td style="width:233px;">for OCM</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Glass capillaries</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">4878</td>
			<td style="width:233px;">3.5&#34; long, I,D, 0.530mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glass sample vials</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">06-408B</td>
			<td style="width:233px;">for ISH</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Hair loop</td>
			<td style="width:191px;"></td>
			<td style="width:135px;"></td>
			<td style="width:233px;">Hair affixed in pasteur pipette for tissue manipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Heparin sodium salt</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">H4784</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Injector Nanoliter 2010</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">Nanoliter 2010</td>
			<td style="width:233px;">Microprocessor-controlled microinjector</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Instant Ocean</td>
			<td style="width:191px;">Carolina</td>
			<td style="width:135px;">972433</td>
			<td style="width:233px;">Aquarium Salt for frog recovery</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">IRBG XGC Xenopus verified full-length cam cDNA</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">989_IRBG</td>
			<td style="width:233px;">cDNA library&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">LB Agar plates with 100 &#181;g/mL Ampicillin</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L5004</td>
			<td style="width:233px;">150mm pre-poured LB-Amp plates for sib selection</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:207px;">LB Luria Broth</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L8650</td>
			<td style="width:233px;">LB for collecting colonies in sib selection from plates and dilution of cultures</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Magnetic mRNA Isolation Kit</td>
			<td style="width:191px;">New England BioLabs</td>
			<td style="width:135px;">S1550S</td>
			<td style="width:233px;">for isolation of poly(A)-enriched RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Maleic Acid</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">M0375</td>
			<td style="width:233px;">for ISH</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Manual Microfil Micromanipulator</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">M3310R</td>
			<td style="width:233px;">Manual micromanipulator</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Nutating Mixer</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">22-363-152</td>
			<td style="width:233px;">Rocker for ISH</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Permoplast</td>
			<td style="width:191px;">Nasco</td>
			<td style="width:135px;">SB33495M</td>
			<td style="width:233px;">Clay for injection and dissection dishes</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate Buffered Saline</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P5368</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PMSG</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G4877</td>
			<td style="width:233px;">to stimulate oocyte development</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Polyvinylpyrrolidone</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">PVP40</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Programmable Puller</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">PUL-1000</td>
			<td style="width:233px;">Micropipette needle puller</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Proteinase K</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P6556</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">pTnT Vector</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">L5610</td>
			<td style="width:233px;">cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Riboprobe Combination System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">P1450</td>
			<td style="width:233px;">in vitro transcription</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Superscript Full Length cDNA Library Construction Kit</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:135px;">18248013</td>
			<td style="width:233px;">kit for cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Sutures, 3-0 silk</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">19-037-516</td>
			<td style="width:233px;">Suture thread and needle for post-oocyte removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Torula RNA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">R3629</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Triethanolamine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">T1502</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Tween 20</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P9416</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Universal RiboClone cDNA Synthesis System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">C4360</td>
			<td style="width:233px;">alternative kit for cDNA library construction</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">Xenopus Full ORF Entry Clones - ORFeome Collaboration</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">5055_XenORFeome</td>
			<td style="width:233px;">ORFeome Clones</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">XL2-Blue Ultracompetent Cells</td>
			<td style="width:191px;">Agilent Technologies</td>
			<td style="width:135px;">200150</td>
			<td style="width:233px;">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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Das Verständnis frühen Organogenese das vereinfachte<em&gt; In-situ-</em&gt; Hybridisierung Protokoll<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Forschung

Entwicklungsbiologie

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Live-Imaging von angeborenen Immun und Präkanzeröse Zell-Interaktionen unter Verwendung eines induzierbaren Gal4 / UAS Expression System in Zebrafisch-Larven Haut

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Alginat Encapsulation pluripotenter Stammzellen Mit einer Co-Axial Düsen

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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 ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Tracking-Zellen in GFP-transgenen Zebrafisch Mit dem Photokonvertierbare PSmOrange-System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technik auf Zielmikroinjektions an die Entwicklungs<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Zeitliche Reihenfolge der dynamischen Ausdruck von Daten aus Detaillierte Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Funktionelle Manipulation der mütterlichen Gene Produkte verwenden<em&gt; In Vitro</em&gt; Oozytenreifung in Zebrabärbling

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Fetale kardiovaskuläre Bildgebung mit einem Hochfrequenz-Ultraschall (30 / 45MHZ) Maus

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Dreidimensionales Angiogenese-Assay-System mit Co-Culture-Spheroiden, die von endotheliaalen Koloniebildenden Zellen und mesenchymalen Stammzellen gebildet werden

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...