この作品は、スペイン語MinisterioデEducación Y Ciencia（BIO 2006から11792）とのGeneralitat de Catalunyaの（2005 - SGR00437）によってサポートされていました。著者は、メディアを準備する彼らの技術支援のためのMarc Puigcerverとジョナタンルーカスに感謝します。 ServeiドールEstabulariからすべてのスタッフは動物の住宅、そしてこのビデオの一部を記録する上での追加の貢献のために特にフアンホセマーティンとハビエルニートのために認められている。 NCBは、 ポルトガル語Fundaçãoパラの仲間Ciência電子TecnologiaとSGはスペイン語MinisterioデEducación Y Cienciaの仲間です。

動物のケアと手順は、バルセロナ、スペインの理学部自治の動物と人間の研究に倫理委員会で承認されたガイドラインおよび規則に従って行った。 I.卵母細胞のコレクション<ol><li>厚い膣分泌物（排卵後の放電）の存在のためのゴールデンシリアの株の、雌のハムスター（8-12週の古いゴールデンハムスター）を確認して選択します。 </li><li> 60時間後にPMSG 40 IUの腹腔内注射（妊馬血清性性腺刺激ホルモン）HCG（ヒト絨毛性性腺刺激ホルモン）を40 IUが続くことにより過剰排卵するために選択された女性を誘導する。 </li><li> 16時間二酸化炭素室内のhCGの投与後の女性のハムスターを安楽死させる。 </li><li>ビデオに示すように雌から卵を隔離し、KSOM - H培地のドロップに移す。 </li><li> 37ヒアルロニダーゼのドロッ​​プで実体顕微鏡下で卵管膨大部を引き裂くことによって卵母細胞の卵丘複合体（156 U / ml）を集める℃に卵母細胞は、約5分で卵丘細胞から解放されます。 </li><li>ヒアルロニダーゼから卵母細胞をピックアップし、KSOM - H培地で二度それらを洗う。 </li></ol>マウス胚の収集のために、雌マウス（ハツカネズミ、6〜8週の古いが）後でhCGの48時間の5 IUが続くと雄マウスと交配PMSGの5 IUの腹腔内注射による過剰排卵するように誘導されています。女性は44から46または54から56時間、それぞれ、前核、2セルまたは4細胞期胚の取得を示しますためにhCGの投与後、24から26で頸椎脱臼により屠殺されています。ハムスターの卵母細胞について記載した前核胚のコレクションが正確に実行されます。 2セルと4セルの段階で胚がKSOM - H培地[6]で卵管をフラッシュすることによって収集されます。 注：このプロトコルに続いて約20〜30卵母細胞/胚は各女性から収集することができます。 II。ソリューションを凍結融解の準備<ol><li> KSOM - H（propilenglycolの1.5 M）5mlにpropilenglycolの0.57 mlを希釈することにより、I溶液を準備します。 </li><li> 0.2052グラムのショ糖（スクロースの0.3 M）をチューブにKSOM - H 2 mlを添加することにより融解溶液を調製します。 </li><li>私は0.0684グラムのショ糖（propilenglycolと0.1 Mのショ糖の1.5 M）をチューブに溶液2 mlを添加することにより凍結溶液を調製します。 </li></ol> 注：ソリューションの私とソリューションを凍結融解はちょうど彼らの使用の前に準備する必要があります。しかし、凍結と融解の溶液の調製に使用するショ糖の指定された金額は、事前に重み付けし、数ヶ月間、室温で3 ML -チューブに格納されることがあります。 III。凍結プロトコル<ol><li>卵母細胞/胚KSOM - H培地からの溶液Iの低下に移転し、室温で7月15日分間インキュベートする。 </li><li>一方、それはビデオ（各ソリューション/わらの1滴）に示すように凍結融解ソリューション90μlの滴を配置することでストローをロードする90ミリメートルのペトリ皿を準備する。 </li><li>凍結液滴に卵母細胞/胚の希望数を転送する。 注：私達は通常5から30卵母細胞または胚/ドロップ間で転送する。 </li><li>口制御吸引システムにわらを添付し、この順序に従って、それをロードする：溶液I、空気、卵母細胞と凍結ドロップ、空気は、ドロップ、空気を解凍。 </li><li>ストローがロードされると、最初のドロップは、わらとアザラシこの最後の上端にあるポリマーに達する（溶液I）を導入するまで吸引してください。プラスチック製のプラグと藁やシールのもう一方の端を熱シールします。 </li><li>プログラム可能な冷凍庫のスイッチを入れ、凍結プログラムを選択します。このプロトコルで使用されている凍結プログラムは、もともとラサールらによって発表された冷却速度に基づいています。 [7]： <ul><li> -7〜室温から2℃/分° C 0℃/ 10分間分播種の温度平衡と誘導を可能にする（下記参照） </li><li> -7〜0.3℃/ minで-30 ° CにC 0 ° Cの温度平衡化を可能にするために2分間/分</li><li> 35 ° C / minで-30℃から-130℃まで</li></ul></li><li>融解溶液の液滴とわらの部分が外側に向いていることを確認し、プログラム可能な冷凍庫のホルダーのスロットにストローをロードする。凍結プログラムを起動します。 </li><li>サンプルの温度が-7℃、冷却がhold状態にあるに達すると、手動シーディングを実行します。液体窒素に突入する鉗子は、直ちに解決策を融解の滴を含むストローの一部を公開するためにわずかに冷凍庫の所有者を引いて、と鉗子でストローのこの部分に触れてください。ホルダー内の各ホルダーと各わらでも同じ処理を繰り返し、最後まで実行する凍結プログラムをしましょう​​。 </li><li>プログラムが完了すると、液体窒素で所有者を突き刺す。 </li><li>からストローを削除します。ホルダーとラベルの付いたゴブレに転送する。 </li><li>最後に、液体窒素タンク内のゴブレを格納する。 </li></ol> IV。プロトコルを解凍<ol><li>液体窒素タンクからストローを削除します。 </li><li> 40秒間室温でわらを維持する。 </li><li> 40秒間に30℃の水浴中でわらを浸す。 </li><li>わらからプラグを外したり、熱シールドチップをカット。 </li><li> 35mmのペトリ皿の中でわらを空にし、軽く凍結と融解のソリューションを含む2滴を混ぜる。 </li><li>室温で15分間、混合物中の卵母細胞/胚のままにしておきます。 </li><li> 37℃でさらに15分間KSOM - Hの新鮮なドロップ℃に卵母細胞/胚を転送する</li><li>卵母細胞/胚は、現在、さらに操作のための準備が整いました。 </li></ol> 注：手順6と7の間は、融解した卵母細胞/胚は徐々に水分補給のために、最大膨潤ているはずです。 V.代表の結果凍結保存プロトコルを使用すると、ここにハムスターの卵母細胞のための約95％のそれぞれ&gt; 90％、前核、2セルと4セルの段階で85％とマウスB6CBAF1胚は80％、の生存率の結果を報告した。マウスの胚を解凍後KSOMにin vitroで培養すると、少なくとも70〜80％の最適な培養条件で胚盤胞期に到達する必要があります。 それはあなたがそれぞれの凍結実験で、卵母細胞/胚の残りの部分はさらなる操作のために使用される前に、このグループを解凍その卵母細胞/胚の対照群（わら）を含めることをお勧めします。対照群の生存率が予想よりも低い場合、同一ロットから別のわらを解凍。この2番目のグループの生存率も低い場合、この凍結ロットから残りのストローを破棄。

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196+and+-269+&#176;C.">Survival of mouse embryos frozen to -196 and -269 &#176;C.</a> Science. 178, 411-414 (1972).</li><li>Wilmut, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cooling+rate,+warming+rate,+cryoprotective+agent+and+stage+of+development+on+survival+of+mouse+embryos+during+freezing+and+thawing.">The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing.</a> Life Sci. 11, 1071-1079 (1972).</li><li>Parkening, T. A., Tsunoda, Y., Chang, M. C. <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+various+low+temperatures,+cryoprotective+agents+and+cooling+rates+on+the+survival,+fertilizability+and+development+of+frozen-thawed+mouse+eggs.">Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.</a> J. Exp. Zool. 197, 369-374 (1976).</li><li>Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+principles+of+embryo+freezing.">Cryobiological principles of embryo freezing.</a> J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).</li><li>Vajta, G., Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cryopreservation+systems.">Improving cryopreservation systems.</a> Theriogenology. 65, 236-244 (2006).</li><li>Duselis, A. R., Vrana, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+mouse+oocytes.">Retrieval of mouse oocytes.</a> JoVE. 3, (2007).</li><li>Lassalle, B., Testart, J., Renard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryo+features+that+influence+the+success+of+cryopreservation+with+the+use+of+1,2+propanediol.">Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.</a> Fertil. Steril. 44, 645-651 (1985).</li><li>Ib&#225;&#241;ez, E., Grossmann, M., Vidal, F., Egozcue, J., Santal&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+caprine+beta-lactoglobulin+transgenic+mouse+embryos.">Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.</a> Cryobiology. 35, 290-298 (1997).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Tateno, H., Kamiguchi, Y., Mikamo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+freezing+and+thawing+method+of+hamster+oocytes+designed+for+both+the+penetration+test+and+chromosome+assay+of+human+spermatozoa.">A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.</a> Mol. Reprod. Dev. 33, 202-209 (1992).</li><li>Biggers, J., McGuinnis, L. K., Raffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+preimplantation+development+of+the+mouse+in+the+protein-free+KSOM.">Amino acids and preimplantation development of the mouse in the protein-free KSOM.</a> Biol. Reprod. 63, 281-293 (2000).</li></ol>

このプロトコルでハムスターの卵母細胞およびマウス胚は、正常に凍結保存することができます。一度凍結、卵母細胞/胚が無期限に液体窒素タンクに貯蔵し、必要に応じて任意の時間または場所で回収することができます。これにより、必要なときに使用する準備がほぼ試料を有するという利点を提供しています。一方、このプロトコルはまた、生きた動物のコロニーの維持に経済的かつ安全な代替として...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French type mini-straw 0.25 ml</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>13407/0010</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Farma-Lepori, Spain</td>
<td>749036.4</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-3884</td>
<td>&nbsp;</td>
<tr>
<td>KSOM-H medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref. [11]</td>
</tr>
<tr>
<td>Liquid Nitrogen </td>
<td>&nbsp;</td>
<td>Air Liquide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic plugs</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>19046</td>
<td>&nbsp;</td>
<tr>
<td>PMSG</td>
<td>&nbsp;</td>
<td>Intervet, Spain</td>
<td>1.614/8447</td>
<td>&nbsp;</td>
<tr>
<td>Propilenglycol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>82280</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>107687</td>
<td>&nbsp;</td>
<tr>
<td>Surgical Scissors</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BC-060R; BG-061R</td>
<td>&nbsp;</td>
<tr>
<td>Thermo-sealer</td>
<td>&nbsp;</td>
<td>SIZ</td>
<td>220</td>
<td>&nbsp;</td>
<tr>
<td>35 and 90 mm culture dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>153066; 150350</td>
<td>&nbsp;</td>
<tr>
<td>10 ml tubes</td>
<td>&nbsp;</td>
<td>Greiner Bio-one</td>
<td>163160</td>
<td>&nbsp;</td>
<tr>
<td>Nitrogen tank</td>
<td>&nbsp;</td>
<td>MVE, Cryogenics</td>
<td>XC 43/28</td>
<td>&nbsp;</td>
<tr>
<td>Programmable Freezer</td>
<td>&nbsp;</td>
<td>Planner Products Ltd.</td>
<td>Kryo-10 Series III</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BD-331R; BD-053R; OC-020R</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

collection and cryopreservation of hamster oocytes and mouse embryos

時ウィッティンガム胚および卵母細胞が最初に成功し、30年以上前に凍結保存した<i&gt;ら。</i&gt;<sup&gt; 1</sup&gt;とウィルムット<sup&gt; 2</sup&gt;別々にパークニング、マウスの胚は° Cと、数年後に-196で凍結して保存することができることが記載<i&gt;ら</i&gt;。<sup&gt; 3</sup&gt;凍結卵母細胞の体外受精（IVF）に起因するライブ子孫の誕生を報告した。それ以来、凍結保存技術の使用は急速に、ヒトと動物の生殖補助の実施及び動物遺伝資源の保全に不可欠なコンポーネントになるために広がっている。緩慢凍結とガラス化：現在のところ、卵母細胞および胚を凍結保存するために使用される2つの主な方法があります。アプローチの多種多様な子どもたちの動物と何千もの両方の技術と数百万人が凍結胚から生まれてきた改善しようとするために使用されている。しかし、凍結保存に関連する重要な欠点は、まだ氷結晶の形成以来、克服しなければならない、ソリューションの効果と浸透圧ショック後の融解卵母細胞および胚におけるいくつかのcryoinjuriesを引き起こすようです。プログラム可能な冷凍庫で緩慢凍結は、通常、化学物質の毒性および浸透圧ショックに関連付けられている凍結防止剤、低濃度を使用する利点がありますが、低濃度での氷結晶の形成を避けるために、彼らの能力は限られている。緩慢凍結では、手動または自動シーディングを使用して避けなければならない冷却効果を誘発する<sup&gt; 4</sup&gt;。ガラス化の過程においては、凍結保護剤の高濃度は、氷結晶の形成を阻害し、水が固化されているガラスのようなガラス固化体状態の形成につながるが、展開されません。しかし、使用される濃度でcyroprotectantsの毒性のために、卵母細胞/胚は、直接液体窒素でサンプルを沈める前に、時間のと最小容積の溶液中で非常に短い期間のために凍結保護ソリューションに公開することができます<sup&gt; 5</sup&gt;。それは高価な機器（プログラマブル冷凍庫）が必要とされている非常に迅速な方法であるため、過去10年間では、ガラス化は、より一般的になってきている。しかし、緩慢凍結では、卵母細胞/胚凍結保存のための最も広く使われている方法であり続けている。このビデオ、記事では、収集し、徐々に高​​解凍後の生存率とハムスターの卵母細胞を凍結する方法を、ステップバイステップ、を示しています。同じ手順でもうまく着床前の発達の異なる段階でマウスの胚を凍結融解に適用することができます。

Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

このビデオ、記事では、高解凍後の生存率とハムスターの卵母細胞を収集し、凍結保存する方法についてステップバイステップのデモンストレーションを提示する。同じ手順でもうまく着床前の発達の異なる段階でマウスの胚を凍結融解に適用することができます。

コレクションとハムスターの卵母細胞とマウス胚の凍結保存

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

collection-and-cryopreservation-of-hamster-oocytes-and-mouse-embryos

Research

JoVE Journal

Biology

16.8K ビュー.  Universitat Autonoma de Barcelona. このビデオ、記事では、高解凍後の生存率とハムスターの卵母細胞を収集し、凍結保存する方法についてステップバイステップのデモンストレーションを提示する。同じ手順でもうまく着床前の発達の異なる段階でマウスの胚を凍結融解に適用することができます。

ビデオ: Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

マウス卵母細胞への核移転

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

生物学

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPCのマウス胚の解剖

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

マウス卵母細胞の検索

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

着床前の牛の胚、羊、ヤギの凍結保存

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

エチレングリコール系ガラス化によるマウス胚の凍結保存

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

siRNA媒介サイレンシングによるマウス卵母細胞における減数分裂紡錘体の評価

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

ヒト胚形成期における栄養芽細胞の単一細胞回収

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...