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...

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

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

16.9K 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

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 ...

生物学

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

マウス着床前胚幹細胞の誘導

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

発生生物学

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

遺伝子組み換えマウスの高効率生産のための凍結解凍胚の使用

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

遺伝学

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未熟ネコの最小体積ガラス化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデン ハムスターの生産

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

生物工学

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

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.

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 ...

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.

無血清全胚培養系で、マウス胚発生

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

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.

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

要約

さらに動画を探す

この動画の章

マウス卵母細胞の検索

マウス着床前胚幹細胞の誘導

遺伝子組み換えマウスの高効率生産のための凍結解凍胚の使用

未熟ネコの最小体積ガラス化

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデンハムスターの生産

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

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

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

無血清全胚培養系で、マウス胚発生

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

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

要約

さらに動画を探す

この動画の章

マウス卵母細胞の検索

マウス着床前胚幹細胞の誘導

遺伝子組み換えマウスの高効率生産のための凍結解凍胚の使用

未熟ネコの最小体積ガラス化

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデン ハムスターの生産

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

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

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

無血清全胚培養系で、マウス胚発生

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

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデンハムスターの生産