We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

HINWEIS: Tierversuche wurden in Übereinstimmung mit den National Institutes of Health Guide für die Pflege und Verwendung von Labortieren durchgeführt. Der Ausschuss für Tierversuche der Tohoku University School of Medicine hat den hier beschriebenen experimentellen Verfahren. 1. Anästhesie und Laparotomie <ol style="margin-left: 40px;"><li> Für die Blutentnahme, verwenden Sie spezifischen Pathogen-freien männlichen Sprague-Dawley Ratten. Schnell die Ratten für mindestens 18 Stunden, während die Bereitstellung von Wasser. Verwenden Sie im Ruhestand männlichen Ratten Züchter bei 6-8 Monaten (550-650 g) zum Sammeln von Blut. </li><li> Durchfluss von 2,5 bis 4,0% Isofluran, die ein Inhalationsnarkosemittel ist, in eine Box zu einem Narkosegerät angeschlossen, um Anästhesie bei 2,0 bis 3,5 l / min für 10 Minuten vorstellen. Übertragen Ratten in der Box auf die Anästhesie einzuführen. <ol><li> Betäubung zu bestätigen, indem der Verlust der Reaktion auf die Stimulation an den Pfoten mit einer Pinzette. Decken Sie die Nase des Ratten with eine Maske auf ein Narkosegerät, um während der Blutabnahme tief betäuben die Ratten verbunden. Die Konzentration und Strömungs werden bei 2,5-4,0% gehalten und 2,0-3,5 L / min. HINWEIS: Halothan sollte nicht zur Inhalationsanästhesie verwendet, weil Embryonen im Serum, die von Ratten, die mit diesem Reagens weisen eine Verzögerung in der embryonalen Entwicklung im Vergleich zu Embryonen in der von Ratten mit Isofluran betäubt 22 erhaltene Serum kultiviert betäubt gesammelt, kultiviert werden. </li></ol></li><li> Um eine Kontamination des Fells zu vermeiden, die Haut desinfiziert durch Eingießen 70% Ethanol auf den Bauch der narkotisierten Ratte. Pick-up die Haut desinfiziert und Bauchwand am unteren Bereich mit einer Pinzette und gleichzeitig geschnitten, diese Schichten in Richtung der Thorax-Bereich mit einem Paar große Schere, um die inneren Organe aus. Reflect den Darm außerhalb der Bauchhöhle mit einer Pinzette. </li><li> Schneiden Sie die Haut und Bauchwand in Richtung der Hinterbeine mit einerPaar große Schere, um die hinteren Bauchbereich weiter aus. Reflektieren Sie das Extra-Fett außerhalb der Bauchhöhle mit einer Pinzette. </li><li> Nehmen Sie das viszerale Fett an der Mittellinie mit zwei Pinzetten und teilen das Fett vorsichtig in einer Richtung parallel zu der Bauchschlagader freizulegen. Wiederholen Sie diesen Vorgang, um das viszerale Fett in Längsrichtung geteilt, um leicht die Gabelung der Bauchaorta. Zusätzlich ist die Aorta pulsatilen und kann von der benachbarten Hohlvene mit diesen charakteristischen unterscheiden. </li><li> Nehmen Sie das Fett um die Bauchaorta und die Bifurkation vorsichtig mit zwei Pinzetten und trennen Sie das Fett auf der Bauchaorta. </li></ol> 2. Blutentnahme <ol style="margin-left: 40px;"><li> Vorsichtig die Spitze einer 21 G Nadel in eine 20 ml Spritze unmittelbar kranial der Bifurkation der Aorta, mit dem Kegel nach unten verbunden. Halten Sie die Spritze mit einemHand, und ziehen Sie die Spritze langsam zu 15 ml Blut aus einer männlichen Ratte zu sammeln. </li><li> Entfernen Sie die Nadel aus der Spritze, und gießen Sie das Blut in zwei eiskalte 10-ml sterilen Röhrchen (Abbildung 1A). Halten Sie die Röhrchen auf Eis, bis Zentrifugation, um die Bildung der Blutgerinnsel verzögern. Reduzieren Sie die Zeit Zentrifugation durch Zentrifugieren mehrere Rohre auf einmal. </li><li> Euthanize die narkotisierten Ratten durch Thorakotomie und Schneiden des Herzens oder Enthauptung. </li></ol> 3. Rat Serum Vorbereitung <ol style="margin-left: 40px;"><li> Zentrifugieren des gesammelten Blutes für 5 min bei 1.200 × g und Raumtemperatur (RT), um das Blut in einen oberen und unteren Lagen (1C) zu trennen. </li><li> Halten Sie die Röhrchen bei 4 ° C für 1,5-2 Stunden, um die Fibringerinnsel beheben. </li><li> Holen das Fibringerinnsel in der oberen Schicht unter Verwendung eines Paars von gebogenen Pinzette und quetschen das Fibringerinnsel, das Serum zu trennen. Drücken Sie dann die Fibringerinnsel mit den gebogenen Pinzette gegen downwards nahe der Grenzfläche zwischen den beiden Schichten (Fig. 1D). </li><li> Wiederholen Sie Schritt 3.3 weiter lösen das Fibringerinnsel. </li><li> Zentrifugieren Sie die Röhrchen für 5 min bei 1.200 xg und RT (1E). </li><li> Das Serum vorsichtig in einem 15 ml sterilen Röhrchen übertragen mit einer sterilen Pipette in einer Laminar-Air-Flow-Gehäuse. </li><li> Zentrifugieren des gesammelten Rattenserum für 5 min bei 1.200 × g und Raumtemperatur, um jegliche verbleibenden roten Blutkörperchen zu entfernen. </li><li> Pool Serum vorsichtig in ein neues 15 ml sterilisierten Röhrchen unter Verwendung einer sterilen Pipette, um eine Kontamination durch restliche Erythrozyten am Boden des Rohres (1F) zu vermeiden. </li><li> Inkubieren des Serums in einem Wasserbad bei 56 ° C für 30 min, um das Komplementsystem zu inaktivieren. </li><li> Kühlen Sie die Rohre aus Rattenserum auf RT im laminaren Luftstrom Schrank. Das Serum sollte auf 10 ml, einzeln steril Röhrchen aliquotiert werden. Die Röhrchen bei -20 ° C bis zur Verwendung (1H). </li></ol>

<ol>
	<li>New, D. A. T., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Mammalian Postimplantation Embryos. A Practical Approach. , 1-14 (1990).</li><li>Eto, K., Takakubo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+development+of+rat+embryos+in+culture+during+the+period+of+craniofacial+morphogenesis.">Improved development of rat embryos in culture during the period of craniofacial morphogenesis.</a> J Craniofac Genet Dev Biol. 5, 351-355 (1985).</li><li>Takahashi, M., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+method+of+rodent+whole+embryo+culture+using+the+rotator-type+bottle+culture+system.">The method of rodent whole embryo culture using the rotator-type bottle culture system.</a> J Vis Exp. (42), e2170 (2010).</li><li>New, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+embryo+culture,+teratogenesis,+and+the+estimation+of+teratologic+risk.">Whole embryo culture, teratogenesis, and the estimation of teratologic risk.</a> Teratology. 42, 635-642 (1990).</li><li>Matsuo, 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=A+mutation+in+the+Pax-6+gene+in+rat+small+eye+is+associated+with+impaired+migration+of+midbrain+crest+cells.">A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells.</a> Nat Genet. 3, 299-304 (1993).</li><li>Nomura, T., Holmberg, J., Frisen, J., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6-dependent+boundary+defines+alignment+of+migrating+olfactory+cortex+neurons+via+the+repulsive+activity+of+ephrin+A5.">Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5.</a> Development. 133, 1335-1345 (2006).</li><li>Yamamoto, M., 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=Nodal+antagonists+regulate+formation+of+the+anteroposterior+axis+of+the+mouse+embryo.">Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo.</a> Nature. 428, 387-392 (2004).</li><li>Inoue, 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=Role+of+cadherins+in+maintaining+the+compartment+boundary+between+the+cortex+and+striatum+during+development.">Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.</a> Development. 128, 561-569 (2001).</li><li>Takahashi, M., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+regulates+specification+of+ventral+neurone+subtypes+in+the+hindbrain+by+establishing+progenitor+domains.">Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains.</a> Development. 129, 1327-1338 (2002).</li><li>Calegari, F., Haubensak, W., Yang, D., Huttner, W. B., Buchholz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+postimplantation+mouse+embryos+with+endoribonuclease-prepared+short+interfering+RNA.">Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.</a> Proc Natl Acad Sci U S A. 99, 14236-14240 (2002).</li><li>Takahashi, M., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferring+genes+into+cultured+mammalian+embryos+by+electroporation.">Transferring genes into cultured mammalian embryos by electroporation.</a> Dev Growth Differ. 50, 485-497 (2008).</li><li>Pryor, S. E., Massa, V., Savery, D., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension+analysis+in+mouse+whole+embryo+culture.">Convergent extension analysis in mouse whole embryo culture.</a> Methods Mol Biol. 839, 133-146 (2012).</li><li>Kikkawa, 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=Dmrta1+regulates+proneural+gene+expression+downstream+of+Pax6+in+the+mammalian+telencephalon.">Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon.</a> Genes Cells. 18, 636-649 (2013).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240, 237-246 (2001).</li><li>Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+backscatter+microscopy+of+mouse+embryos.">Ultrasound backscatter microscopy of mouse embryos.</a> Methods Mol Biol. 135, 235-243 (2000).</li><li>Pierfelice, T. J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+microinjection+into+the+mouse+forebrain+in+utero+at+E9.5.">Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.</a> J Vis Exp. (45), e2047 (2010).</li><li>Quinlan, G. A., Khoo, P. L., Wong, N., Trainor, P. A., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+grafting+and+labeling+in+postimplantation+mouse+embryos.">Cell grafting and labeling in postimplantation mouse embryos.</a> Methods Mol Biol. 461, 47-70 (2008).</li><li>Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R., Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roller+culture+of+postimplantation+embryos.">Roller culture of postimplantation embryos.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , 237-241 (2003).</li><li>Garcia, M. D., Udan, R. S., Hadjantonakis, A. K., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+for+culturing+mouse+embryos.">Preparation of rat serum for culturing mouse embryos.</a> Cold Spring Harb Protoc. 2011, 391-393 (2011).</li><li>Brown-Woodman, P. D., Ritchie, H. E., Korabelnikoff, A., Emmanuel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+ether+with+alternate+volatile+anesthetics+for+collection+of+rat+serum+used+in+embryo+culture.">Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.</a> Toxicol In Vitro. 18, 719-724 (2004).</li><li>Gray, J., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tube+closure+in+mouse+whole+embryo+culture.">Neural tube closure in mouse whole embryo culture.</a> J Vis Exp. (56), e3132 (2011).</li><li>Machado, C. B., 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=Reconstruction+of+phrenic+neuron+identity+in+embryonic+stem+cell-derived+motor+neurons.">Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.</a> Development. 141, 784-794 (2014).</li><li>Glanville-Jones, H. C., Woo, N., Arkell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+whole+embryo+culture+with+commercially+available+reagents.">Successful whole embryo culture with commercially available reagents.</a> Int J Dev Biol. 57, 61-67 (2013).</li><li>Ornoy, A., Yacobi, S., Yaffee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+culture+of+11.5-day-old+rat+embryos+in+DMEM/F12+and+20%+fetal+bovine+serum.">A simple method of culture of 11.5-day-old rat embryos in DMEM/F12 and 20% fetal bovine serum.</a> J Anat. 203, 419-423 (2003).</li><li>Moore-Scott, B. A., Gordon, J., Blackburn, C. C., Condie, B. G., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+serum-free+in+vitro+culture+technique+for+midgestation+mouse+embryos.">New serum-free in vitro culture technique for midgestation mouse embryos.</a> Genesis. 35, 164-168 (2003).</li><li>Kalaskar, V. K., Lauderdale, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Embryonic+Development+in+a+Serum-free+Whole+Embryo+Culture+System.">Mouse Embryonic Development in a Serum-free Whole Embryo Culture System.</a> J Vis Exp. (85), e50308 (2014).</li></ol>

No conflicts of interest are declared.

Reproduzierbare Ergebnisse in WEC Experimente sind abhängig von der Verwendung von hochwertigen IC Rattenserum und genaue Embryo Dissektionstechnik 3. Den Zeitraum des Fastens vor Blutentnahme nicht kürzen, weil das Fasten ist notwendig, um Glucose-Konzentrationen im Blut zu standardisieren. Weibliche Ratten, nicht zur Herstellung von Serum verwendet werden, da Hormonspiegel ändern bei weiblichen Tieren nach dem Östrus-Zyklus, und diese Änderungen in Hormone Östradiol für Embryokulturen geeignet sind. ...

Eine Vielzahl von Modelltiere sind in der Entwicklungsbiologie zur Entwicklungsmechanismen auf molekularer und zellulärer Ebene zu untersuchen. Zum Beispiel Amphibien-und Vogelspezies wurden weithin als klassische Modell Tiere, die für die direkte Behandlung der Embryonen sind, weil diese Embryonen entwickeln sich außerhalb der Mutter eingesetzt. Im Gegensatz zu diesen Tieren wachsen Säugetier-Embryonen in die Gebärmutter der Mutter, und das Wachstum in späteren Phasen hängt entscheidend von der Funktion der Gebärmutter. Daher ist es in der Regel schwierig, Säugetierembryos, wie sie aus der Maus und Ratte in einem frühen Stadium direkt zu manipulieren. In den 1960er Jahren wurde eine neue Denis ganze Säugetierembryokultur (WEC)-Technik mit WEC Geräte mit einer kontinuierlichen Sauerstoffzufuhr und Wärme ein. WEC können Maus-und Rattenembryos zu wachsen ex vivo (dh außerhalb des Uterus). Obwohl die WEC-Technik wurde oft in Teratologie durch Zugabe verschiedener chemic verwendetAl-Verbindungen in dem Kulturmedium wurde diese Technik auch in verschiedenen Entwicklungsbiologie Studien verwendet worden, um einzigartige Entwicklungsmechanismen in Säugetieren 2-4 zu prüfen. Zum Beispiel wird WEC mit anderen Techniken, wie z. B. Zellmarkierung, in Wildtyp-und Mutanten-Embryos unter Verwendung fluoreszierender Farbstoff 5, Zelltransplantation 6 und Gen-Einführung über 7 Lipofektion und Elektroporation 8-13 kombiniert. Kürzlich wurde in utero Manipulation verwendet wurde, um Entwicklungsprozesse in Nagetierembryonen in späteren Stadien zu analysieren und mit Elektroporationsverfahren 14-16 zusammengefasst. Jedoch sind diese Techniken nicht für die Handhabung von Postimplantationsembryonen und midgestation aufgrund von Schwierigkeiten Erzielung einer genauen lokalen Injektion der DNA-Lösung in Embryonen im Frühstadium. Obwohl Ultraschall-geführte Zelltransplantation und Injektion von viralen Vektoren in frühen Embryonen (dh, E8.5-E-9.5 in der Maus) in utero wurde berichtet, zuvor 17,18, sind sehr gute Kenntnisse erforderlich, um diese Experimente mit einer hohen Erfolgsquote durchzuführen. Daher WEC mit hoher Zugänglichkeit ex vivo hat Vorteile in Bezug auf die Manipulation der Maus-und Rattenembryos. Sofort zentrifugiert (IC) Rattenserum von männlichen Ratten vorbereitet wird oft für WEC Medium verwendet. Wenn Embryonen sind in der Postimplantationsstufe (dh früher als E8.0 im E10.0 Maus oder der Ratte) kultiviert wird eine Mischung aus synthetischen Mediums und IC Rattenserum häufig als Medium für WEC 19 verwendet. Doch die Kultur Embryonen in der Mitte der Schwangerschaft (dh., E8.0-12.5 in Maus-Embryonen oder E10.0-E14.5 in Rattenembryonen), 100% Serum sollte als Medium verwendet werden, da es derzeit keine alternative Medien ermöglicht Embryonen in vitro normalerweise länger als 2 Tage wachsen. Die Herstellung von hochwertigen RattenserumEin entscheidender Schritt zur Erreichung Reproduzierbarkeit WEC Experimenten. Verglichen mit verzögerter Zentrifugation hat den IC Vorteil der Reduzierung der Hämolyse, wenn das Serum wird durch Zusammendrücken des Fibringerinnsels gesammelt, da die meisten der roten Blutzellen bereits aus der Fibrin-Gerinnsel getrennt. Als hämolytischer Rattenserum nicht auf das normale Wachstum der Ratten-und Maus-Embryonen zu unterstützen, ist die Herstellung von Serum mit sofortiger Zentrifugation bevorzugt Zubereitung mit verzögerter Zentrifugation. Unser Protokoll enthält zwei zusätzliche Schritte im Vergleich zu anderen Protokollen 18-20 (dh., Speichern des gesammelten Bluts auf Eis vor der ersten Zentrifugation und Halten der gesammelten Blutproben 2 h bei 4 º C nach der ersten Zentrifugation). Die ersteren Stufe die Bildung des Blutgerinnsels zu verzögern, und der letztere Schritt fördert die Verfestigung des Fibringerinnsels für einfache Quetschen. Daher kann unser Protokoll von Anfängern verwendet werden. Allerdings reproduzieren Blutentnahme und SerumExtraktion genau durch einfaches Protokoll Bücher bezieht, ist extrem schwierig, 19-21. In diesem Video-Beitrag zeigen wir, unsere Standard-Protokoll für die Herstellung der optimalen IC Rattenserum, die Blutentnahme aus der Bauchaorta von männlichen Ratten und Gewinnung von Serum durch Zentrifugation enthält.

<table><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>For rat anesthesia.</td></tr><tr><td>Large scissors</td><td>Napox</td><td>B-7H</td><td>Stelized scissors for cutting the skin and muscle of rats.&amp;nbsp;</td></tr><tr><td>Curved forceps</td><td>Napox</td><td>A-3-2</td><td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Charles Rivers Laboratories</td><td></td><td>Retired male rats from colony in the lab or purchased retired male rats.</td></tr><tr><td>Syringe (20 ml)</td><td>TERUMO</td><td>SS-29ESZ</td><td></td></tr><tr><td>Needle (21 G x 5/8&quot;)</td><td>TERUMO</td><td>NN-2116R</td><td></td></tr><tr><td>Sterile test (spitz) tube (10 ml)</td><td>ASIAKIZAI</td><td>1101C000B-10</td><td>For collection of boold</td></tr><tr><td>Sterile disposable pipette</td><td>Eiken Chemical</td><td>CD2000 No.4</td><td></td></tr><tr><td>Sterilie disposable tube (15 ml)&amp;nbsp;</td><td>Falcon</td><td>2196</td><td></td></tr></tbody></table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Abbildung 1 zeigt repräsentative Ergebnisse von den beschriebenen Verfahren zur Abtrennung von Blutzellen und Serum. Wir erhalten in der Regel 15 ml Blut aus einem pensionierten männlichen Ratte (Abbildung 1A). Durch Zentrifugieren, kann das gesammelte Blut in eine obere Schicht, die das Serum und das Fibringerinnsel, die mit einem Pfeil angedeutet ist, und eine untere Schicht, die die Blutzellen (Fig. 1C) getrennt werden. Wichtiger ist, hämolytische Blutproben nicht...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Herstellung von Ratten Serum Geeignet für Säugetier Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

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Research

JoVE Journal

Biology

13.8K Aufrufe.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Serie: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Ein optogenetische Ansatz zur Abschätzung Bildung neuronaler Verbindungen in einer Co-Kultursystem

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forschung

Entwicklungsbiologie

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Neuronale Differenzierung von embryonalen Stammzellen der Maus in Serum-freiem Monolayer-Kultur

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Methoden für die genau zu lokalisieren Übertragung von Zellen oder DNA in frühen Postimplantations Maus Embryonen

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Herstellung von Ratten Oligodendrozyten Vorläuferkulturen und Quantifizierung von Oligodendrogenesis Mit Doppel-Infrarot-Fluoreszenz-Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolierung von murinen embryonalen Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolierung und Kultivierung von embryonalen Ratten-Nervenzellen: ein Quick-Protokoll

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neurowissenschaften

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Herstellung von Ratten-Gehirn-Aggregate Kulturen für Neuron und Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Biologie

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Kultur von Mausembryonen von der Prägastrulation bis zur fortgeschrittenen Organogenese

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Die Methode der Nager Whole Embryo Culture mit der Rotator-type Bottle Kultur-System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Maus embryonalen Entwicklung in einem Serum-freien Whole Embryo Culture-System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...