We thank Mr. Hajime Ichijo for video-recording and helpful advices for editing the video. We also thank Drs.Yuji Tsunekawa and Kaichi Yoshizaki for kind assistant for video-recording. This work is supported by KAKENHI on Young Scientist B and on Priority Areas- Molecular Brain Science from MEXT of Japan. We acknowledge the support of Global COE Program "Basic and Translational Research Center for Global Brain Science" from MEXT of Japan and The Core Research for Evolutional Science and Technology (CREST) from Japanese Science and Technology Corporation from Japanese Science and Technology Agency (JST).

1. Setting up the WEC system
<ol>
	<li>Joint a 0.22 &mu;m membrane filter to the inlet silicon tube within WEC system a).</li>
	<li>Open the valve of a gas cylinder containing O2 (95%) and CO2 (5%). Flow the gas mixture into the drum via the bubbling bottle containing autoclaved water.</li>
	<li>Adjust the flow volume of gas mixture to 50 cc.</li>
	<li>Insert the out let silicon tube into a water bottle and check out the flow of gas mixture.</li>
	<li>Rotate the drum at the speed ...

<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=Intoroduction.+In+mammalian+postimplantation+embryos.">Intoroduction. In mammalian postimplantation embryos.</a> In Mammalian Postimplantation Embryos A Practical Approach. , 1-14 (1990).</li><li>Cockroft, D. L., 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=Dissection+and+culture+of+post+implantation+embryos.">Dissection and culture of post implantation embryos.</a> In Mammalian Postimplantation Embryos A Practical Approach. , 15-40 (1990).</li><li>Morris-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postimplantation+mammalian+embryos.">Postimplantation mammalian embryos.</a> Essential Developmental Biology A Practical Approach. , 55-66 (1993).</li><li>Fujinaga, M., Tuan, R. S., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+rodent+embryos+during+the+early+postimplantation+period.">In vitro culture of rodent embryos during the early postimplantation period.</a> Developmental Biology Protocols. , 53-76 (2000).</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>New, D. A., 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=A+rotating+bottle+culture+method+with+continuous+replacement+of+the+gas+phase.">A rotating bottle culture method with continuous replacement of the gas phase.</a> Experientia. 35, 138-140 (1979).</li><li>Osumi-Yamashita, N., Ninomiya, Y., Eto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+craniofacial+embryology+in+vitro.">Mammalian craniofacial embryology in vitro.</a> Int. J. Dev. Biol. 41, 187-194 (1997).</li><li>Inoue, T., Nakamura, S., 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=Fate+mapping+of+the+mouse+prosencephalic+neural+plate.">Fate mapping of the mouse prosencephalic neural plate.</a> Dev. Biol. 219, 373-383 (2000).</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>Inoue, Y. U., Asami, J., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+labeling+of+mouse+rhombomeres+by+Cadherin-6::EGFP-BAC+transgenesis+underscores+the+role+of+cadherins+in+hindbrain+compartmentalization.">Genetic labeling of mouse rhombomeres by Cadherin-6::EGFP-BAC transgenesis underscores the role of cadherins in hindbrain compartmentalization.</a> Neurosci. Res. 63, 2-9 (2009).</li><li>Calegari, F., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inhibition+of+cyclin-dependent+kinases+that+lengthens,+but+does+not+arrest,+neuroepithelial+cell+cycle+induces+premature+neurogenesis.">An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis.</a> J. Cell Sci. 116, 4947-4955 (2003).</li><li>Osumi, N., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+cultured+mammalian+embryos+by+electroporation.">Gene transfer into cultured mammalian embryos by electroporation.</a> Methods. 24, 35-42 (2001).</li><li>Arai, Y. <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+Fabp7,+a+downstream+gene+of+Pax6,+in+the+maintenance+of+neuroepithelial+cells+during+early+embryonic+development+of+the+rat+cortex.">Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex.</a> J. 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There are two critical steps in rodent WEC for the success. First, the dissection procedure should be accurate not to damage embryos, especially blood vessels. Second, the procedure should be as quickly as possible because oxygen and nutrient are no longer supplied via the placenta after isolation from the uterus. This is critical for older embryos. In the case of rat WEC after E12.5, we should transfer dissected embryos into culture bottles within 30 minutes.
We have analyzed migration patte...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td colspan="5">Setting up WEC </td>
 </tr>
 <tr>
 <td>WEC system (10-0310)</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-0310</td>
 <td>Another small model is also available.</td>
 </tr>
 <tr>
 <td>Silicon plug without a hole</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gas mixture cylinder</td>
 <td>Tool</td>
 <td>Nikko Sanso</td>
 <td>-</td>
 <td>Containing 95% oxygen &amp; 5% carbon dioxide. Custom order.</td>
 </tr>
 <tr>
 <td>Gas regulator</td>
 <td>Tool</td>
 <td>Ono Seisakusho</td>
 <td>WR-11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 &mu;m filter Millex GS</td>
 <td>Tool</td>
 <td>Millipore</td>
 <td>SLGS033SS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5">Anesthesia and isolation of the uterus </td>
 </tr>
 <tr>
 <td>Large scissors</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>B-7H</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>A-3-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disposal Petri dish 
 (90 mm x 15 mm) </td>
 <td>Tool</td>
 <td>Iwaki</td>
 <td>SH90-15</td>
 <td>Deep-type dish is the best for dissection.</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Reagent</td>
 <td>Abbott</td>
 <td>B506</td>
 <td>For anesthesia</td>
 </tr>
 <tr>
 <td>Pentobarbital sodium</td>
 <td>Reagent</td>
 <td>Schering-Plough Animal Health</td>
 <td>-</td>
 <td>For anesthesia</td>
 </tr>
 <tr>
 <td>Tyrode's saline</td>
 <td>Reagent</td>
 <td>-</td>
 <td>-</td>
 <td>According as the protocol in Ref. 2. Save at 4&deg;C. </td>
 </tr>
 <tr>
 <td>Timed-pregnant Sprague-Dawley rat</td>
 <td>Animal</td>
 <td>Charles Rivers Laboratories Japan</td>
 <td>-</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5">Preparation of culture medium </td>
 </tr>
 <tr>
 <td>Culture glass bottle</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-05</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disposal culture bottle</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-06</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silicon plug with hole</td>
 <td>Tool</td>
 <td>Ikemoto Rika</td>
 <td>010-032-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aluminum foil</td>
 <td>Tool</td>
 <td>Any company</td>
 <td>-</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Autoclaving bag</td>
 <td>Tool</td>
 <td>Hogy</td>
 <td>HM-26</td>
 <td>For culture bottles</td>
 </tr>
 <tr>
 <td>Autoclaving bag</td>
 <td>Tool</td>
 <td>Hogy</td>
 <td>HM-14A</td>
 <td>For silicon plugs</td>
 </tr>
 <tr>
 <td>0.45 &mu;m filter Millex HA</td>
 <td>Tool</td>
 <td>Millipore</td>
 <td>SLHA033SS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml conical tube</td>
 <td>Tool</td>
 <td>Becton Dickinson</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 ml beaker</td>
 <td>Tool</td>
 <td>Iwaki</td>
 <td>TE-32</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rat IC serum</td>
 <td>Reagent</td>
 <td>Charles Rivers Laboratories Japan</td>
 <td>-</td>
 <td>Refer to: Mr. Kunihiko Morisaki, 
 TEL: +81-(0) 45-474-9336; FAX: +81-(0) 45-474-9340</td>
 </tr>
 <tr>
 <td>D(+)-Glucose</td>
 <td>Reagent</td>
 <td>Wako</td>
 <td>041-00595</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibiotic-antimyotic liquid</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>15240</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="5">Dissection of embryos </td>
 </tr>
 <tr>
 <td>Ophthalmic straight scissors</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>MB50-7</td>
 <td>For cutting the uterine wall</td>
 </tr>
 <tr>
 <td>Ophthalmic curved scissors</td>
 <td>Tool</td>
 <td>Napox</td>
 <td>MB54-2</td>
 <td>For cutting the yolk sac</td>
 </tr>
 <tr>
 <td>Forceps #5 </td>
 <td>Tool</td>
 <td>Vigor</td>
 <td>T6715</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forceps #5F</td>
 <td>Tool</td>
 <td>Regine</td>
 <td>T6819</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass pipette</td>
 <td>Tool</td>
 <td>-</td>
 <td>-</td>
 <td>Made by hand using Pasteur pipette.</td>
 </tr>
 <tr>
 <td>Autoclaving bag</td>
 <td>Tool</td>
 <td>Hogy</td>
 <td>HM-4</td>
 <td>For glass pipettes</td>
 </tr>
 <tr>
 <td>Binocular microscope</td>
 <td>Tool</td>
 <td>Leica</td>
 <td>Mz7s</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Light</td>
 <td>Tool</td>
 <td>Leica</td>
 <td>CLS 150XD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oil stone</td>
 <td>Tool</td>
 <td>Uchida Yoko</td>
 <td>833-2000</td>
 <td>For sharpening of forceps</td>
 </tr>
 <tr>
 <td>Machine oil</td>
 <td>Tool</td>
 <td>Uchida Yoko</td>
 <td>835-0000</td>
 <td>For sharpening of forceps</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System at JoVE.com

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

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.

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

the-method-rodent-whole-embryo-culture-using-rotator-type-bottle

Research

JoVE Journal

Biology

23.5K vues.  Tohoku University Graduate School of Medicine. Whole technique de culture d'embryons de souris nous permet de culture et d'embryons de rat Ex vivo durant des périodes limitées correspondant aux stades midgestation. Dans ce protocole vidéo, nous démontrons nos procédures standard de la culture d'embryon de rat entière après E12.5 en utilisant le système des rotateurs de type flacon de culture.