USDAマルチ状態の研究プロジェクトW - 2171&quot;家畜の改善のための生殖細胞と胚発生と操作が&quot;感謝されるから部分的な資金調達。この記事のビデオ部分のためのテクニカルイラストレーションを作成したライアンキャラハンの芸術的才能は、、また感謝する。

1。凍結保存1の胚の適合性を評価する<ol><li>個々のドナーの女性の生殖器官から採取した試料は実体顕微鏡を用いた評価に先立って10分間、等張胚保持媒体中に配置する必要があります。胚移植の子孫の家系の同定を可能にするために互いに独立した各ドナーの女性から胚を保つために一定である。 </li><li>低倍率（≤10倍）を使用して、個々のドナーの女性からの検体は未受精卵の卵子で構成される別々のグループに分離されるべきである、胚、または譲渡品質の胚を縮退。同期受信者の女性は、品質、グレード3の胚と約25の転送後の妊娠率のために利用可能な限り、開発の拡張胚盤胞の段階を経てコンパクトな桑実胚でのみ品質のグレード1または2の胚は凍結保存に適しており、他のすべては（破棄される必要があります-30％）は許容範囲内と考えられます。 </li><li>胚の透明帯が損なわれていないこととは素材が透明帯に付着（例えば、細胞、粘液）がないことを保証するために≥50倍の倍率で高品質のグレード1と拡張胚盤胞期の胚を介した2コンパクト桑実胚を検査する。ひび割れや欠落している透明帯付きまたは付着物質を有する透明帯を持つ任意の胚は、他の胚から分離されるべきであり、どちらか破棄するか別々に処理。 </li></ol> 2。疾病伝播2の可能性を減らすために胚を洗浄<ol><li>培地を洗浄する胚の最初のウェルに培地を保持している張胚の最小限の量で透明帯、無傷の胚（例えば、リン酸塩は生理食塩水の抗生物質とウシ血清アルブミン、新生児ウシ血清、またはポリビニルアルコールで補強バッファリング）に移動します。各ドナーの女性からの胚とは別に保管してください、と一度に10以上の胚を洗浄しないでください。連続希釈≥1:100を達成するために各連続洗浄に培地の最小限の量で胚を移動し、それぞれの連続した​​洗浄の間に変更されている無菌胚の取扱いのヒントを使用するようにしてください。 </li><li>の2.1に記載されてさらに4回洗濯しても胚を移動します。 </li><li>ウイルス性疾患の伝播が問題となる場合、最長で90秒のトリプシンに合計複合曝露時間については、0.25％トリプシン溶液に胚を2回洗浄する。 </li><li> twoトリプシン回洗浄後、2.1で説明したように胚の洗浄培地で5倍以上の胚を洗浄。 </li><li>最終的な胚の洗浄の後、等張性胚への場所の胚は、メディアを保持する。 </li></ol> 3。凍結保存3に前に胚を脱水<ol><li>胚は、例えばエチレングリコールやグリセリンなどの凍結防止剤（CPA）の高張液（1.4から1.5モル濃度）に培地を保持している張胚の最小限の量で転送する必要があります。 </li><li>彼らは浸透圧平衡に到達するまでの胚は凍結培地（高張CPA液）に座ってすることができます。エチレングリコールは、より急速にグリセロール、平衡化時間は、通常、グリセロール（10分）に比べてエチレングリコール（5分）での胚のための小さいしないより胚細胞に浸透しているため。胚の間と後にこの平衡化時間の一部を達成することができるストロー（次のステップで説明）にロードされます。 </li></ol> 4。凍結保存のために0.25mlのプラスチックストローに胚のロード<ol><li>胚のローディング装置に0.25mlのプラスチックストロー（11.5センチメートル作業長）の綿栓の端を取り付け、適切にラベル付けさわらに高張CPA液の約1.3センチメートルを吸引除去する。ラベリング手順の様々なわらシーリングプラグのどちらか、またはわらアダプタに接続された別のわらに接続されている印刷されたラベルの使用を含め、存在する。 </li><li> CPAソリューションからわらを削除し、藁の中で小さな気泡を作成するための空気の約0.15センチメートルを吸引除去する。 </li><li>わらに高張CPA液の約0.8 cmの単位で、単一の胚を吸引除去する。 </li><li> CPAソリューションからストローを取り出して、ストロー内の第小さな気泡（空気の泡が物理的に藁の中で胚を分離するのに役立つ）を作成するために空気の約0.15センチメートルを吸引除去する。 </li><li>わらの綿栓の端内に存在するポリ塩化ビニル（PVC）粉を湿らせてCPAソリューションの十分な量で描画する特定され、完全に藁を埋めるためにCPAのソリューションの約9.1センチメートルを吸引除去する。 CPAソリューションは、ゲルに塩ビの粉を発生させ、ストローの端部をシール。 </li><li> PVCの粉末、プラスチック製のストローのシーリングプラグ、またはヒートシーラーとわらのもう一方の端をシールします。 </li></ol> 5。制御された速度の胚の冷凍機への胚の配置<ol><li>メタノール浴または液体窒素蒸気の凍結機のどちらかは、着床前の胚の凍結保存のためにプログラムすることができます。 </li><li>エンブリーを含むストローをロードする<html>温度周囲温度から-6まで冷却された冷凍機へのOS℃に負荷のストローは、綿栓の終わりまで（プラスチック製のストローのシーリングプラグやわらアダプタは綿栓の端が下に置かれている場合には、使用されている場合を除く）。 </li><li>胚は、先に進む前に、少なくとも2分間この温度で​​座ることができます。 </li></ol> 6。種まき<ol><li>一度胚は、-6に冷却している° C、トングを（または綿タッチして、わらの内部にCPAの溶液中で氷の結晶形成を誘導するために液体窒素（または液体窒素に浸漬綿棒）で過冷却トングのペアを使用します。胚の上または下のどちらかのソリューションの列に先端が棒）。 </li><li> CPAの溶液中の水は、液体窒素にさらされる領域では結晶化し、氷の結晶はすぐに胚を取り巻くCPAソリューションの列に広がってしまいます。 </li><li>さらに、冷却前に10分間、温度をシードで胚を保持する。 </li></ol> 7。胚の継続的な脱水<ol><li> 0.5℃/ -34の温度にダウン分° Cの割合でクール胚この冷却速度は、胚の継続的な脱水性を確保することが重要です。 </li><li> -34で胚を保持℃で10分間液体窒素（-196℃）に胚を急落する前に。 </li><li>場所は、適切に標識されたサトウキビに接続されて適切にラベル付けさゴブレット（液体窒素で満たされた）に胚を凍結保存し、短期または長期保存用液体窒素デュワーのキャニスターに杖を置く。 </li></ol> 8。凍結保存胚の融解<ol><li>キャニスターは、以下のデュワーのフロストラインを保つように、液体窒素デュワーの首にキャニスターを引き出します。 </li><li>融解する胚を含む杖を見つけ、迅速かつ慎重にゴブレットに他のストローを暖めるにならないようにという、ゴブレットからわらを取り除く。 </li><li>追加の25〜30秒、37℃の水浴中に3〜5秒（ひび透明帯4の発生率を減少させる）ために空気中のわらを押しながら、水没。 </li><li> 、水浴からわらを削除わらを（藁で胚の識別を汚れに注意しながら）ワイプ、（藁の非綿プラグの端をチョキンと切るために藁切断装置を使用して、密封を使用して熱やPVCパウダー）、または慎重にどちらかのプラスチック製のシーリングプラグを取り外し、と。 <ol type="a"><li>同期胚の受信者に、可能な限り迅速に胚移植のデバイスとの転送に藁をロードする（ただし胚は、CPAとしてエチレングリコールを用いて凍結保存した場合のみ）、または</li><li>ショ糖の1.0モル濃度（非透過した化合物）を含む皿上のわらの開放端を保持し、わらの綿栓の端を切断するはさみのペアを使用。わらの内容は自由にショ糖溶液にフローする必要がありますが、任意の残留媒体は藁内部に存在する場合はストローで空気の小さな列をプッシュする必要がある場合があります。胚を10分間蔗糖溶液の中に残さないようにして、10分のための媒体を保持する等張胚に胚を移す。胚を解凍後、新しいわらに負荷し、適切な受信者の女性への転送を評価する。 </li></ol></li></ol> 9。代表的な結果： 様々な要因は、妊娠に影響を及ぼすと同期受信者の女性5〜凍結融解初期胚の移転に起因する出生率を生きることができます。コンパクトな桑実胚よりも高度なまたは拡張胚盤胞よりも高度な発達段階での胚はしばしば同様にコンパクトな桑実胚、初期胚盤胞、胚盤胞、および胚発生の拡張胚盤胞の段階で胚などの凍結保存プロセスを生きていけない。品質のグレード1と2収量の胚の品質グレード3の胚は（一般的に低いため解凍後の妊娠率に凍結保存されていない）よりも高い解凍後の妊娠率。胚は低下妊娠率を示す可能性のある胚の凍結またはプロシージャを解凍胚中に誤って処理。その発情周期のドナーの女性のそれと同期していない受信者の女性に転送胚は通常、低妊娠率を生成する。 （例えば、生検または二等分） をin vitroで生成されるか、いくつかの方法で操作胚は通常より低い妊娠率が得られます。最適な条件下では、 生体由来胚の凍結融解の転送後に得られる妊娠率は、牛6,7、一般的に羊8,9,10,11 in 65から75パーセント、そしてヤギの60から70パーセント60から70パーセントです。 12,13,14,15。同様に、 体外生産胚の凍結融解の転送後に得られる妊娠率は、通常、牛6,16の40から50パーセント、羊17の25から35パーセント、およびヤギ18の30〜40％で。 BLE 1&quot;/&gt; 同期受信者の女性に国内の反すう家畜種からの凍結融解胚の転送は、次の表1。予想される妊娠率。

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胚が水から主に構成されているため、それが着床前の胚は十分に脱水し、徐々に細胞内氷晶形成を回避するために本明細書に記載のプロトコルに従って冷却することが重要です。冷却プロセスが開始されると、それは、単方向の温度変化を維持するために、温度の変動を回避することが重要です。特定のCPAのソリューションのための適切な温度で氷の結晶の形成（&quot;種まき&quot;）の開始が重要です。 この徐冷/着床前の胚の凍結保存のための迅速な解凍方法に変更を加えると、一段階法（わらにロードされたメディアの最後の列には、ショ糖の代わりに、CPAのソリューションです）19と胚は、エチレングリコール中で凍結直接転送方法を（含まれています第一胚）20からのエチレングリコールを除去せずに解凍し、受信者の女性の子宮に直接転送されます。エチレングリコールの量を削減するため、直接転送（DT）受信者の女性の子宮に入金、それは6 CMは前述のように続行する前に培地を保持していると0.25mlのストローを記入することをお勧めします。それは自主的な標準がDTのために凍結保存し着床前の胚は黄色のストローで凍結し、液体窒素デュワーに黄色のゴブレットに格納するように商業的な胚移植の業界内で存在することに留意すべきである。コマーシャル業界にも従わなければならないすべての凍結保存胚のための固有のラベル表示要件があります。 この方法の1つの代替手段はガラス化21、より高濃度（6-8 M）CPAソリューションはCPAのソリューションは、氷なしで液体から&quot;ガラス&quot;状態に変化させる超急速に冷却され凍結保存の非平衡方式です。結晶形成。ガラス化は、その温度上昇の感受性のためにコンパクトな桑実胚よりも発育の高度な胚の凍結保存のために可能性が適しています。 この手法は、独自の遺伝資源の資源の保全22アプリケーションだけでなく、2008暦年に転送さ約50万ウシ初期胚の55％以上が凍結保存された国際的な商業胚移植の業界にしています。23

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> BioLifeの凍結媒体</td><td> Agtech、（株） </td><td> C18、C18A </td><td> 1.5 Mエチレングリコール</td></tr><tr><td> Emcareエチレングリコール凍結ソリューション</td><td> ICPBio生殖</td><td> IC8、IC10 </td><td> 1.5 Mエチレングリコール</td></tr><tr><td> ViGroのエチレングリコールの凍結に加え、 </td><td> Bionicheアニマルヘルス</td><td> EVM835 </td><td> 1.5 Mエチレングリコール</td></tr><tr><td> Emcare 10％グリセロール凍結ソリューション</td><td> ICPBio生殖</td><td> IC12 </td><td> 1.34 Mのグリセロール</td></tr><tr><td> Emcare 1 Mのショ糖の雪解け</td><td> ICPBio生殖</td><td> IC16 </td><td> 1.0 Mのショ糖</td></tr><tr><td> ViGroワンステップの融解</td><td> Bionicheアニマルヘルス</td><td> EVM247、EVM847 </td><td> 1.0 Mのショ糖</td></tr><tr><td> ViGro雪解け1-2-3プラス</td><td> Bionicheアニマルヘルス</td><td> EVM248、EVM848 </td><td> 5％、2.5％、0.5、0.5で0％グリセロール、0.6％ショ糖</td></tr><tr><td> Emcare CSU解凍キット</td><td> ICPBio生殖</td><td> IC20 </td><td> 6％、3％、10.3％のショ糖と0％のグリセロール</td></tr><tr><td> BioLife保持と伝達媒体</td><td> Agtech、（株） </td><td> C15、C15A </td><td> 0.4パーセントBSA（ウシ血清アルブミン）で修正されたPBS </td></tr><tr><td> Emcare保持ソリューション</td><td> ICPBio生殖</td><td> IC2、IC4、IC6 </td><td> 0.4パーセントBSA（ウシ血清アルブミン）、MOPS緩衝液（不活性両性イオン） </td></tr><tr><td> ViGro持ち株プラス</td><td> Bionicheアニマルヘルス</td><td> EVM024、EVM824 </td><td></td></tr><tr><td> SYNGROホールディング培地</td><td> Bionicheアニマルヘルス</td><td> ESM024、ESM224、ESM828 </td><td>ヒアルロン酸が含まれていない。動物の起源のない製品を</td></tr><tr><td> BioLife修正D - PBS </td><td> Agtech、（株） </td><td> C06 </td><td>改変ダルベッコりん酸緩衝生理食塩水</td></tr><tr><td> BioLife BSAフラクションV </td><td> Agtech、（株） </td><td> B09 </td><td>凍結乾燥したウシ血清アルブミン、フラクションV </td></tr><tr><td> BioLife抗生物質、抗真菌</td><td> Agtech、（株） </td><td> B01 </td><td>アンホテリシンB、ペニシリンG、硫酸ストレプトマイシン</td></tr><tr><td> BioLifeト​​リプシンキット</td><td> Agtech、（株） </td><td> C22A </td><td> 25グラム凍結乾燥トリプシン、滅菌水、滅菌D - PBS </td></tr></tbody></table><table border="1"><tbody><tr><th>機器の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> Biol -クール制御された速度の冷凍庫</td><td> FTSシステムズ</td><td> BC - IV -40 </td><td>メタノール浴のフリーザー</td></tr><tr><td>コントロール制御された速度の冷凍庫を凍結する</td><td> Cryologic </td><td> CL2200 </td><td>液体窒素冷凍庫</td></tr></tbody></table>

cryopreservation of preimplantation embryos of cattle, sheep, and goats

牛、羊、ヤギから着床前の胚は、短期または長期の保管のために凍結保存することができます。着床前の胚では、水から主に構成されており、凍結保存の過程で細胞内氷晶形成の回避は、胚の生存性を維持するために極めて重要である。細胞内脱水を促進する浸透圧勾配を作成するには、例えばエチレングリコール（EG）やグリセロールなどの凍結防止剤（CPA）（GLYC）の - 胚は高張液（1.5 M 1.4）に置かれます。胚は、CPAの溶液中で浸透圧平衡に到達した後、それらを個別に凍結用の0.25ミリリットルプラスチックストローに高張CPAのソリューションにロードされます。胚は-6℃の温度で制御された速度の冷凍庫に入れられます氷の結晶の形成は、胚を囲むCPAの溶液中で誘導され、結晶化は、さらに細胞内脱水を引き起こし、胚の外側のCPAの濃度の増加を発生させます。胚は液体窒素（-196℃）に突入する前に-34 ° Cの温度に、さらに脱水が可能、0.5℃/分の速度で冷却される。凍結胚は、受信者（代理）メスに転送する前に解凍する必要があります。胚を含むストローは5秒〜3室温の空気で開催された液体窒素デュワーから削除、25〜30秒間37℃の水浴中に配置されます。胚は、受信者（代理の）女性への転送前にCPAを除去するための10分間のショ糖の1 M溶液中に配置されているGLYCで凍結保存。 EGで凍結保存胚は、しかし、直接受信者の子宮に転送される場合があります。

Watch this Scientific Journal Video about Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats at JoVE.com

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

着床前の胚は、細胞の脱水を引き起こすことが高張凍結保護溶液中に配置した後に凍結保存することができます。平衡化後、氷の結晶形成は、胚を囲む溶液中で誘導される。胚は徐々に保管するための液体窒素中に急落する前に氷点下の気温に冷却されるとさらに脱水が発生します。

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-preimplantation-embryos-of-cattle-sheep-and-goats

Research

JoVE Journal

Biology

30.5K ビュー.  Iowa State University. 着床前の胚は、細胞の脱水を引き起こすことが高張凍結保護溶液中に配置した後に凍結保存することができます。平衡化後、氷の結晶形成は、胚を囲む溶液中で誘導される。胚は徐々に保管するための液体窒素中に急落する前に氷点下の気温に冷却されるとさらに脱水が発生します。

ビデオ: Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

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 ...

生物学

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

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 ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

の直立イメージングショウジョウバエ胚

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

キネシンの単離及び精製からショウジョウバエ胚

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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 ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ゼブラフィッシュ胚における酸化ストレスの分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

ダニ Ex Vivo 唾液腺培養物および細胞外小胞からのマイクロRNAの単離

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

<meta charset="utf8"></head><body>絶滅危惧種のサンゴからのサンゴ精子凍結保存の最適化

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

<meta charset="utf8"></head><body>著者スポットライト:全球的なサンゴ礁の修復のための効率的な凍結保存およびバイオバンキング技術の開発

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

バイオバンキングと水産養殖をサポートするためのサンゴ精子の凍結保存のための半自動ワークフロー

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around ...