We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

<ol>
	<li>Miyamoto, K., Gurdon, J. B., Trounson, A., Gosden, R., Eichenlaub-Ritter, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+amphibian+egg+to+understand+the+mammalian+oocyte.">Insights into the amphibian egg to understand the mammalian oocyte.</a> Biology and Pathology of the Oocyte: Role in Fertility, Medicine, and Nuclear Reprogramming. , 1-11 (2013).</li><li>Heasman, J., Kofron, M., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-catenin+signaling+activity+dissected+in+the+early+Xenopus+embryo:+a+novel+antisense+approach.">Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach.</a> Developmental biology. 222 (1), 124-134 (2000).</li><li>Oelgeschlager, M., Kuroda, H., Reversade, B., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordin+is+required+for+the+Spemann+organizer+transplantation+phenomenon+in+Xenopus+embryos.">Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.</a> Developmental cell. 4 (2), 219-230 (2003).</li><li>Szenker, E., Lacoste, N., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+requirement+for+HIRA-dependent+H3.3+deposition+revealed+at+gastrulation+in+Xenopus.">A developmental requirement for HIRA-dependent H3.3 deposition revealed at gastrulation in Xenopus.</a> Cell reports. 1 (6), 730-740 (2012).</li><li>Heasman, J., Holwill, S., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> Methods in cell biology. 36, 213-230 (1991).</li><li>Heasman, 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=Overexpression+of+cadherins+and+underexpression+of+beta-catenin+inhibit+dorsal+mesoderm+induction+in+early+Xenopus+embryos.">Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos.</a> Cell. 79 (5), 791-803 (1994).</li><li>Hulstrand, A. M., Schneider, P. N., Houston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+antisense+oligonucleotides+in+Xenopus+oocytes.">The use of antisense oligonucleotides in Xenopus oocytes.</a> Methods. 51 (1), 75-81 (2010).</li><li>Miyamoto, K., 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=Nuclear+Wave1+is+required+for+reprogramming+transcription+in+oocytes+and+for+normal+development.">Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development.</a> Science. 341 (6149), 1002-1005 (2013).</li><li>Halley-Stott, R. P., 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=Mammalian+nuclear+transplantation+to+Germinal+Vesicle+stage+Xenopus+oocytes+-+a+method+for+quantitative+transcriptional+reprogramming.">Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.</a> Methods. 51 (1), 56-65 (2010).</li><li>Astrand, C., Belikov, S., Wrange, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+characterizes+chromatin+presetting+by+NF1+and+Oct1+and+enhances+glucocorticoid+receptor+binding+to+the+MMTV+promoter.">Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.</a> Experimental cell research. 315 (15), 2604-2615 (2009).</li><li>Amaya, E., Kroll, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+generating+transgenic+frog+embryos.">A method for generating transgenic frog embryos.</a> Methods in molecular biology. 97, 393-414 (1999).</li><li>Kroll, K. L., 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=Transgenic+Xenopus+embryos+from+sperm+nuclear+transplantations+reveal+FGF+signaling+requirements+during+gastrulation.">Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.</a> Development. 122 (10), 3173-3183 (1996).</li><li>Smith, S. J., Fairclough, L., Latinkic, B. V., Sparrow, D. B., Mohun, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+transgenesis+by+sperm+nuclear+injection.">Xenopus laevis transgenesis by sperm nuclear injection.</a> Nature protocols. 1 (5), 2195-2203 (2006).</li><li>Drury, K. C., Schorderet-Slatkine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cycloheximide+on+the+'autocatalytic'+nature+of+the+maturation+promoting+factor+(MPF)+in+oocytes+of+Xenopus+laevis.">Effects of cycloheximide on the 'autocatalytic' nature of the maturation promoting factor (MPF) in oocytes of Xenopus laevis.</a> Cell. 4 (3), 269-274 (1975).</li><li>Smith, L. D., Ecker, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+oocyte+nucleus+in+physiological+maturation+in+Rana+pipiens.">Role of the oocyte nucleus in physiological maturation in Rana pipiens.</a> Developmental biology. 19 (3), 281-309 (1969).</li><li>Miyamoto, K., Pasque, V., Jullien, J., Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+actin+polymerization+is+required+for+transcriptional+reprogramming+of+Oct4+by+oocytes.">Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes.</a> Genes &amp; development. 25 (9), 946-958 (2011).</li><li>Schneider, P. N., Hulstrand, A. M., Houston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+Xenopus+oocytes+using+the+host+transfer+method.">Fertilization of Xenopus oocytes using the host transfer method.</a> J. Vis. Exp. (45), (2010).</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>Sakane, Y., 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=Targeted+mutagenesis+of+multiple+and+paralogous+genes+in+Xenopus+laevis+using+two+pairs+of+transcription+activator-like+effector+nucleases.">Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.</a> Development, growth &amp; differentiation. 56 (1), 108-114 (2014).</li><li>Nakayama, T., 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=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 (12), 835-843 (2013).</li><li>Guo, X., 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=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li></ol>

The authors have nothing to disclose.

Abbiamo qui i dettagli di un nuovo metodo per esaurire i fattori materni o iperespressione fattori esogeni prima della fecondazione. Questo sistema richiede due microiniezioni, ma salta la chirurgia di rane, utilizzato nel metodo di trasferimento ospitante 7,17. E &#39;ottimale per rimuovere il passo chirurgia rana in termini di cura degli animali. Inoltre, non dobbiamo prendere in considerazione per la qualità delle rane femminili accoglienza per gli esperimenti di trasferimento, il che significa che possiamo sbarazzarci di un fattore biologico che influenza il successo degli esperimenti. Pertanto in questo sistema la qualità degli ovociti ottenuti da rane PMSG-innescato è un importante fattore biologico che è fondamentale per gli esperimenti di successo. La qualità degli ovociti può essere giudicato dopo la raccolta di ovaio o dopo defolliculation. Se qualsiasi anomalia si osserva in queste fasi, è ottimale per raccogliere un&#39;altra ovaio da una nuova rana. Un altro punto in cui è possibile controllare la qualità degli ovociti è dopo la maturazione degli ovociti. Se meno di 80% di progesteronee-trattati ovociti mostrano segni di maturazione, non si può essere in grado di ottenere il numero sufficiente di embrioni per ulteriori analisi. L&#39;uso di defolliculation enzimatica anziché defolliculation manuale è anche un altro vantaggio di questo sistema per ridurre il lavoro necessario per defolliculation. Tuttavia, è ancora possibile che defolliculation manuale può dare uno sviluppo migliore del trattamento enzimatico. Come mostrato in Figura 3, quasi il 10% del maturato in vitro ovociti può raggiungere la fase di nuoto girino. Questi dati sono raccolti da 13 esperimenti indipendenti, compresi quelli riportati in 8 e nuovi esperimenti di iniezione. Metodo di trasferimento Host bisogno 75-150 ovociti in ogni trattamento per ottenere dati significativi dal 30-60% degli ovociti trasferiti può raggiungere la fase neurula 17. Il nostro metodo di solito inizia con 200-300 ovociti in ciascun gruppo sperimentale dal momento che circa il 40-60% degli embrioni spaccati può raggiungere ilfase risposta muscolare. Questi dati suggeriscono che entrambi i metodi supportano una velocità di sviluppo ragionevole. Finora abbiamo ottenuto 10 rane vive di controllo mRNA-iniezione e controllo antisenso ovociti oligo-iniettato con questa tecnica, che indica che questo approccio supporta lo sviluppo attraverso la metamorfosi. Il nostro metodo di manipolazione ovocita-ICSI offre l&#39;opportunità di verificare il ruolo dei fattori materni durante molto precoce dello sviluppo embrionale, subito dopo la fecondazione. Inoltre, la sovraespressione di fattori che modificano la cromatina prima della fecondazione può rendere possibile rimodellare la cromatina materna prima della fecondazione per comprendere gli stati cromatina materni necessari per lo sviluppo. Questa strategia può anche lavorare bene con gli attuali sistemi di editing gene come la trascrizione attivatore-like effettrici nucleasi (TALEN) 18,19 e CRISPR / CAS9 20,21 dal momento che questi può essere espresso anche prima della fecondazione. Pertanto, il nostro nuovo approccio ha un Potential da utilizzare per molte applicazioni in futuro.

Xenopus laevis è un diffuso, potente organismo modello per studiare lo sviluppo 1. Questo perché le uova di Xenopus sono insolitamente grande (circa 1,2-1,4 mm, in confronto alle controparti di mammifero essendo circa 0,1 mm) e abbondante. Le uova contengono componenti maternamente sintetizzati e conservati, che sono sufficienti per guidare lo sviluppo embrionale fino transizione a metà blastula (MBT, che si verificano nella fase 8-8.5 con 4.000-8.000 cellule). MBT è accompagnata da attivazione del genoma zygotic, che quindi produce prodotti genici embrionali che indirizzano un ulteriore sviluppo. Numerosi studi volti a identificare i fattori materni importanti per lo sviluppo. Molti studi si basano sulla iniezione di oligonucleotidi antisenso (oligonucleotidi) compresi oligonucleotidi morfolino in embrioni fecondati, nel qual caso la degradazione di proteine ​​materni può essere osservato nella fase gastrula 2-4. In alternativa, mRNA sono iniettati a em fecondatobryos a disturbare le funzioni del gene o di tracciare sorti di proteine ​​sovraespresse. Tuttavia, l&#39;iniezione in embrioni stadio unicellulare normalmente non influenza i livelli di espressione delle proteine ​​materne alle primissime fasi embrionali prima MBT. Heasman e Wylie stabilito il metodo di trasferimento dell&#39;host per superare questo problema 5. In loro metodo, gli ovociti defolliculated manualmente vengono iniettati con oligonucleotidi antisenso e trasferiti in femmine di accoglienza 6. Le proteine ​​sono inibiti prima della fecondazione in modo che i ruoli delle proteine ​​ha diminuito in sviluppo embrionale precoce possono essere esaminate. Questo metodo esaurimento materno ha portato all&#39;identificazione di numerosi ruoli di sviluppo uniche di proteine ​​materne, come rivisto in 7. In questo rapporto, abbiamo dettaglio il nostro metodo recentemente sviluppato, in cui è raggiunto l&#39;esaurimento materno o sovraespressione di mRNA prima della fecondazione, senza defolliculation manuale e trasferimento di accoglienza8. Defolliculation manuale richiede un sacco di tempo e di trasferimento di accoglienza spesso ha bisogno di tecniche abili e la licenza specifica per la chirurgia degli animali, ostacolando così l&#39;uso frequente del metodo di esaurimento materno. Defolliculation e trasferimento di accoglienza sono sostituite da defolliculation enzimatica 9,10 e iniezione intracitoplasmatica di spermatozoi (ICSI) 11-13, rispettivamente. ICSI alle uova è stato originariamente utilizzato per la produzione di rane transgeniche 12. Sperma ed embrionali nuclei sono stati trapiantati in in vitro ovociti maturati anfibi 11,14,15. Qui, dimostriamo il nostro metodo step-by-step per iniettare sperma al vitro ovociti maturati che sono stati pre-iniettato con oligonucleotidi antisenso o mRNA.

<table><tbody><tr><td>Pregnant Mare Serum Gonadotropin (PMSG)&amp;nbsp;</td><td>MSD Animal Health</td><td>Vm 01708/4309</td><td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td><td>Sigma</td><td>A5040</td><td>For anesthesia</td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>For oocyte defolliculation. Store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Drummond Nanoject</td><td>Drummond Scientific Company</td><td>3-000-205/206</td><td>For microinjection</td></tr><tr><td>glass capillary&amp;nbsp;</td><td>Alpha laboratories</td><td>7&amp;rdquo; Drummond #3-000-203-G/XL</td><td>For microinjection</td></tr><tr><td>Micropipette Puller&amp;nbsp;</td><td>SUTTER Instrument</td><td>Model D-97</td><td>For microinjection</td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-500</td><td>For &lt;em&gt;in vitro&lt;/em&gt; maturation and ICSI</td></tr><tr><td>14 ml ultra-clear centrifuge tube&amp;nbsp;</td><td>Beckman Coulter United Kingdom Ltd</td><td>344060</td><td>For sperm purification</td></tr><tr><td>OptiPrep Density Gradient Medium (Iodixanol)</td><td>Sigma</td><td>D1556</td><td>For sperm purification</td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Cell permeabilization reagent</td></tr><tr><td>Shaker</td><td>Hybaid&amp;nbsp;</td><td>HB-SHK-1</td><td>For oocyte defolliculation</td></tr><tr><td>Dissecting microscope (Stereo zoom microscope)</td><td>ZEISS</td><td>Semi SV6</td><td>For oocyte collection and microinjection</td></tr><tr><td>50 &amp;mu;m pore filter</td><td>CellTrics</td><td>04-0042-2317</td><td>For sperm purification</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-100XP</td><td>For sperm purification</td></tr><tr><td>50 ml centrifuge tube</td><td>Cellstar Greiner Bio-One</td><td>227261 or 210261</td><td>Oocyte collection and defolliculation reaction</td></tr><tr><td>15 ml centrifuge tube</td><td>FALCON</td><td>352097</td><td>For sperm purification</td></tr><tr><td>90 mm Petri dish&amp;nbsp;</td><td>Thermo Scientific</td><td>101VR20/C</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 60&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 35&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>351008</td><td></td></tr></tbody></table>

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.

Lo sviluppo embrionale di embrioni ICSI con ovociti in vitro maturazione è stata esaminata (Figura 3A). Tassi maturazione degli ovociti GV alla fase MII sono variabili e dipende in gran parte la qualità degli ovociti. In buone esperimenti, quasi il 100% degli ovociti GV rispondere alle progesterone e mostrano segni di maturazione degli ovociti, diventando infine le uova in fase MII. Tutti ovociti maturati sono stati sottoposti a ICSI e circa il 25% di uova iniettate spaccati (Figura 3A, n = 13 per il controllo scrambled ovociti oligo-iniettato e n = 7 per non ovociti oligo-iniezione). Circa il 60% o 80% degli embrioni spaccati, prodotti a partire da ovociti oligo-iniettato controllo o ovocita non per iniezione, rispettivamente, hanno raggiunto la fase blastula / gastrula. Tra gli embrioni blastula / gastrula, circa la metà degli embrioni in campioni oligo-iniettato erano di buona qualità (quasi segni di scissione anormale e apoptosi), mentre l&#39;82% degli embrioni blastula / gastrula erano di buona qualità in ncampioni on-iniettati (Figura 3A). Infine, il 41% e l&#39;11% degli embrioni spaccati in campioni oligo-iniettato raggiunsero la risposta muscolare e le fasi di nuoto girino, rispettivamente, mentre il 60% e il 29% degli embrioni spaccati in campioni non iniettato fatto (Figura 3A). Questi embrioni ICSI sono la miscela di embrioni normali e patologici (Figura 3B). Alcuni di loro subiscono metamorfosi e si sviluppano a maturare rane 8. Questi risultati suggeriscono che l&#39;iniezione di oligonucleotidi antisenso in ovociti GV, seguiti da IVM e ICSI, permette efficiente sviluppo embrionale precoce. Anche se l&#39;iniezione di oligonucleotidi antisenso si diminuisce lo sviluppo embrionale, siamo ancora in grado di ottenere un numero sufficiente di embrioni per molti scopi sperimentali come il controllo i tassi di sviluppo, RT-PCR, Western Blot e così via. <img alt="Figura 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> Fico<html> ure 1: Esempi tipici di Xenopus laevis ovociti di buona qualità o di cattiva qualità per esperimenti di maturazione in vitro. (A) Un esempio di ovociti di qualità cattiva. Ad esempio, gli ovociti mostrano pigmentazione frammentarie non vengono utilizzate per gli esperimenti successivi. Ogni Xenopus laevis ovocita è di circa 1,2-1,4 mm di diametro. (B) Un ovocita parzialmente defolliculated. La metà destra del ovocita è coperta da cellule follicolari, che possono essere distinti dalla presenza di vasi sanguigni. Una freccia indica una zona priva di cellule del follicolo e in cui l&#39;ago di iniezione viene iniettato. (C) Un esempio di ovociti di buona qualità, che sono altrettanto dimensioni e mostrano emisferi animali uniformemente pigmentate con netto contrasto tra l&#39;emisfero animale e l&#39;emisfero vegetale. 2496 / 52496fig2highres.jpg &quot;/&gt; Figura 2:. Xenopus laevis ovociti prima e dopo la maturazione degli ovociti dopo la maturazione, macchie bianche chiare appaiono nella parte superiore degli emisferi animali e cellule follicolari sono staccata. Ogni Xenopus laevis ovocita è di circa 1 mm di diametro. <img alt="Figura 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Figura 3: Sviluppo di embrioni ICSI, prodotti da ovociti in vitro maturato. (A) Sviluppo di ICSI embrioni per ogni fase è riassunta. Gli oociti sono stati iniettati con controllo strapazzate oligonucleotidi antisenso (iniezione oligo di controllo) o senza iniezione (non iniezione), seguiti da IVM e ICSI. Il corrispondente numero di embrioni in ciascuna fase è indicata sopra le barre. Media ± SEM è mostrato. N = 4-13 esperimenti indipendenti / diverso females. (B) Esempi di sopravvivere embrioni ICSI. Questi embrioni tailbud fase sono stati prodotti in un esperimento, in cui circa 200 ovociti sono stati usati come materiale di partenza.

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

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.

Manipolazione e<em&gt; In Vitro</em&gt; Maturazione di<em&gt; Xenopus laevis</em&gt; ovociti, seguito da Iniezione intracitoplasmatica di spermatozoi, per studiare lo sviluppo embrionale

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

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

22.4K Views.  University of Cambridge. 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.

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

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.

Utilizzando Analisi confocale di<em&gt; Xenopus laevis</em&gt; Per Indagare modulatori di Wnt e Shh morphogen gradienti

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

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Biologia dello sviluppo

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.

Cellule staminali simil-<em&gt; Xenopus</em&gt; Embrionali espianti di studiare Caratteristiche precoce dello sviluppo neurale<em&gt; In Vitro</em&gt; E<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, ...

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.

Functional Cloning Using a Xenopus Oocyte 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.

Clonazione funzionale utilizzando un<em&gt; Xenopus</em&gt; Ovociti 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 ...

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.

Tecnica a Target microiniezione per lo sviluppo<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 ...

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.

Manipolazione funzionale delle materne genica Utilizzando<em&gt; In Vitro</em&gt; La maturazione degli ovociti in Zebrafish

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

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualizzazione e analisi quantitativa di angiogenesi embrionale in<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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.

Microiniezione degli ovociti di Xenopus Laevis

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

Biologia

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.

Fertilizzazione Ovociti di Xenopus Utilizzando il metodo di trasferimento Host

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 e analisi quantitativa della embrionali in cellule epiteliali 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.

Produzione di transgenici Xenopus laevis Integrazione mediata da enzima di restrizione e trapianto nucleare

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 ...

Manipolazione e

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Capitoli in questo video

Utilizzando Analisi confocale di

Cellule staminali simil-

Clonazione funzionale utilizzando un

Tecnica a Target microiniezione per lo sviluppo

Manipolazione funzionale delle materne genica Utilizzando

Visualizzazione e analisi quantitativa di angiogenesi embrionale in

Microiniezione degli ovociti di Xenopus Laevis

Fertilizzazione Ovociti di Xenopus Utilizzando il metodo di trasferimento Host

Live-cell imaging e analisi quantitativa della embrionali in cellule epiteliali Xenopus laevis

Produzione di transgenici Xenopus laevis Integrazione mediata da enzima di restrizione e trapianto nucleare