この作品は、BTの動物や鳥科学省からの資金によって支えられている。

全ての動物実験は、米国農務省動物実験ガイドラインに従ってベルツヴィルエリア動物管理使用Comittees（BAACUC 11から015）によって承認された。 1。麻酔および鎮痛（外科的処置の両方に共通） <ol><li>マウスを計量し、27 Gの針を2 1ミリリットルの注射器で、次の麻酔薬や鎮痛剤をロードします。 <ol><li>ケタミン（0.1ミリグラム/グラム：0.01ミリリットル/ 10 mg / mlの溶液）およびキシラジン（0.01ミリグラム/グラム：2 mg / mlの溶液0.005ミリリットル/ g）。 </li><li>ブプレノルフィン（0.1μgの/ G：0.01 mg / mlの溶液を0.01ミリリットル）。 </li></ol></li><li>親指と人​​差し指で、できるだけ顎の近くの首筋を拾っ少しと薬指の間に尾を保持することによって、マウスを固定。 </li><li> 腹腔内にケタミン-キシラジン混合物を注入する。臓器に穿刺を回避するために、マウスを保持してわずかに腰のレベルの下に頭（ 図1A） </li><li>親指と人差し指（ 図1B）との間にネックホールドの首筋にブプレノルフィンを皮下注射する。 </li><li>暖かいステージに（きれいで、他の動物なしで）ケージにマウスを残す。 </li><li>意識不明たら、（足指のピンチによって確認された） 後足反射の不在を確認してください。目の乾燥を回避し、 眼瞼反射 （ 図1C）がないことをチェックするために眼軟膏を適用します。 </li><li>このプロトコルは、（プロトコル2および3）の下に記載の手順を実行するのに十分な30分の最小ための外科麻酔面を提供する。より長い時間が必要な場合は、1.1.1に記載の投薬量の半分でケタミン+キシラジンの追加の注射を30分後に適用することができる。より速く、不規則なものと呼吸パターンの変化が見よを示す適切な麻酔面のSS。 </li></ol> 2。精管切除<ol><li> 実績のある嵌合性能とオスを使用してください。 </li><li> 、手術器具を滅菌手術が行われる表面を洗浄し、70％エタノールで拭いてください。 </li><li>反射の消失をチェックする、（プロトコル1）以前に詳述されるように麻酔を行う。 </li><li>暖かいステージにマウスを置き、2架空の横方向の線の間の腹側領域から電気バリカンで毛を取り除く 0.5センチメートルとペニス（ 図2A）上記2.5センチメートルを置いた。 </li><li> 10％ポビドンヨードおよび70％エタノールで連続的なワイピングで剃毛面積を消毒 。 </li><li>剃毛面積を露出する穴と、滅菌タオルで外科医やカバーに向けた尻尾と仰臥位でマウスを置きます。手術領域を照らす。 </li><li> 10〜15ミリメートル縦肌を実行incisionは約1cmペニス上腹部の内側のラインで。鋸歯状の鉗子をドレッシングしてから（ 図2Aおよび2B）ハサミでカットして肌を保持する。 </li><li> 白線 5〜10mmの縦切開を行う。鋸歯状の鉗子をmicrodissectingで筋肉を持ち、ハサミ（ 図2C）で切断した。 </li><li>鋸歯状の鉗子を解剖マイクロ片側の睾丸脂肪パッドを取得し、 精巣を露出させ 、それを引っ張って、 輸精管と精巣上体 。 精管が同様の精巣壁に取り付けられていない（精巣に内側に位置しており、それが明確に区別自由チューブです血管が1辺（ 図2D）に沿って走ると精巣上体）。 </li><li>彼らは赤になるまでのマイクロ解剖鋸歯鉗子、 炎ドレッシング鉗子で輸精管を保持している （ 図2E </st（ 図2F）を一度に2点が精管を切断して焼灼するためにそれらを使用して栄&gt;）、および。カットは約5ミリメートルの部分を削除し、2明確に分離焼灼終了する（ 図2G）のままにする必要があります。 </li><li>戻って腹腔へ睾丸、精巣上体と精管を移動します。 </li><li> 他の睾丸のステップ9から進んでください 。 </li><li> 5/0吸収性縫合糸（ 図2H）で作られた1つまたは2つの水平マットレスステッチで筋肉を縫合 。 </li><li> 1または2の創傷切り（ 図2I）で皮膚を縫合 。 </li><li> vasectomised男性の識別（耳のリング、指の入れ墨を...）、 それは麻酔から回復するまでケージは暖かいステージ上に配置して観察を移動（意識し胸骨横臥を維持）。温かい生理食塩水の0.5〜1ミリリットル皮下注射は、再を改善するcovery。精管切除の転送中に発生する可能性の発生率を記録して、飲料水に抗生物質を追加します。 </li><li>創傷クリップは、10日間創傷クリッパー除去や歯ピンセットで精管切除後に除去することができる。精管切除の男性で、手術後2週間の交尾できるようになります。 </li><li>受信者を取得するためにそれを使用する前に、肥沃な雌と交配させることにより精管切除のオスの不妊をテストします 。 </li></ol> 3。子宮卵管胚移植<ol><li>マウス桑実胚または胚盤胞は2.5 DPCでの偽妊娠レシピエント雌に、この技術によって転送することができます。 </li><li> 胚操作用ガラスピペットを準備します。 <ol><li>ピペットホルダーの損傷を防ぐために、ガラスキャピラリーの先端を磨く。 </li><li>少し回転させながら、細かい炎で加熱することにより、ガラスキャピラリの中央部を柔らかく同期両手で毛細血​​管。キャピラリー部は（光赤色）柔らかく可鍛性になると、炎からすぐにそれを撤回し、130〜150程度に、その外径を狭める両端を引っ張る 。 </li><li>クールダウンしてから軽くダイヤモンドポイントの鉛筆、砥石や爪やすりで狭い部分を得点し、両側から引っ張って、それをカットするガラスのを待ちます。ブレークがきれいに垂直である必要があり</li></ol></li><li> 100〜130ミクロンの開口部を残して、非常に迅速に燃えることで先端を磨く 。ピペットは、後で使用するために保存することができる。 </li><li>暖かい胚操作メディア （CZBHまたはM2は、説明を参照してください）。 </li><li> 、手術器具を滅菌手術が行われる表面を洗浄し、70％エタノールで拭いてください。 </li><li>反射の消失をチェックする、（プロトコル1）以前に詳述されるように麻酔を行う。 </li><li> Tを維持彼温かいステージ上でマウス、膝と遠位リブ（ 図3Aと図3B）との間の背側領域から電気バリカンで毛を削除します 。 </li><li> 10％ポビドンヨードおよび70％エタノールで連続的なワイピングで剃毛面積を消毒 。 </li><li>あらかじめ温めておいた胚操作メディアにインキュベーターから胚を移動します 。 </li><li>実体顕微鏡下で暖かい舞台に受信者を移動し、（その頭を右または外科医の左側に見て）外科医に横方向に発生しやすい位置に配置します。 </li><li>剃毛面積を露出する孔を、滅菌タオルでエリアをカバーし、手術領域を照らす。 </li><li>最後の肋骨と腰と背中と腹部（ 図3Aの Aとの間のラインの背⅓の間の線の頭蓋⅓に位置してスポットにおける皮膚中の 1cmの横方向（垂直方向） の切開を行うND 3B）。鋸歯状の鉗子をドレッシングで皮膚を持ち、ハサミ（ 図3C）で切断した。 </li><li>皮膚が切断されると、卵巣（赤/オレンジ）または卵巣（白色）を囲む脂肪パッドは、体壁を介して視覚化することができる。切開は任意の大血管を切断しないスポットでの卵巣または脂肪パッド上で体壁に 0.3〜0.5センチ、横方向（垂直方向） の切開を行う。マイクロ解剖鋸歯鉗子で筋肉を持ち、ハサミ（ 図3D）で切断した。 </li><li>その頭は外科医に向いているようにマウスを移動します。 </li><li>胚操作用ピペット （ 図3E）をロードします 。 <ol><li> CZBHメディアが広い部分の約5ミリメートルまでの操作ピペットの狭い部分を通って毛細管現象によって上昇することができます。 </li><li>小さな気泡（0.2〜0.5ミリメートル）を取り上げる。 </li><li>で胚（5-10）を導入メディアの最小量（2-4 mm）である。 </li><li>別の小さな気泡（0.2〜0.5ミリメートル）とメディアの少量（0.5〜mm）を取り上げる。 </li><li>ステップ3.17の準備ができて、口の吸引器ホルダーにまたは手動デバイスに接続されているガラスピペットのままにしておきます。 </li></ol></li><li>マイクロ解剖鋸歯ピンセットで卵巣周囲の脂肪パッドを取得し、 卵巣、卵管、および腹腔のうち上部の子宮の小さい部分（ 図3Fおよび3G）を公開するために、マウスの頭の方に引き出します。 </li><li>卵管を移動し、（卵管が子宮を満たしているIE）子宮卵管接合部を露出させたマイクロ解剖鋸歯鉗子で脂肪パッドをつかむ、使用できるようになり、口の中で吸引器マウスピースを保持している。 </li><li>アクセス可能な子宮卵管接合を維持する（右利きの場合）、左手でわずかに湾曲したマイクロ解剖鉗子を取るとすぐ下に配置しますその部分の上2ミリメートル程度の卵管をつかんで子宮卵管接合。 </li><li>わずかに湾曲したマイクロ解剖鉗子で子宮卵管接合部を保持している、27のG針（ 図3H）を鉗子に近い卵管部分を穿刺 。 </li><li>子宮卵管接合（ 図3Iおよび3J）を介して子宮に針を事前に実施したオリフィスに胚操作用ピペットを挿入します 。ピペットは子宮卵管接合（ 図3K）を通過すると、簡単にスライドします。破片による子宮内膜の損傷（NO 3mm以下）とピペットブロッキングを防止するために、子宮に非常に遠く進まないでください。 </li><li>優しく（ 図3L）を吹き込んで子宮に胚を解放します 。両方の気泡が子宮を通過しなければならない。最初のバブルの上にあるメディアの中にはまた、子宮内に放出しますが、着床を妨げる可能性がある、より多くの空気を導入することを避けることができる。 </li><li>胚が子宮内に放出された直後に、ピペットを削除します 。 </li><li>脂肪パッドをつかんで戻って腹腔への卵管と卵巣を移動します。 </li><li> 5/0吸収性縫合糸（ 図3M）水平マットレスステッチで筋肉を縫合 。 </li><li>創傷クリッパー（ 図3N）で皮膚を縫合する 。 </li><li> 必要に応じて反対側にステップ10から続行する。 </li><li> 、受信者（耳のリング、指の入れ墨を...）を特定（ウォームステージ上に配置）、そのケージに移動して、麻酔から回復するまで観察 （意識し胸骨横臥を維持）。 </li><li>胚移植時に発生する可能性の発生率に注釈や飲料水に抗生物質を追加します。暖かいのSalIの0.5〜1ミリリットルを皮下注射NEのソリューションは、回復を向上させます。 </li><li>創傷クリップは、10日間創傷クリッパー除去または鉗子（ 図3O）の2つの対を有する胚移植後に除去することができる。受信者は、妊娠を評価し、仔の数を推定するために、その日に計量することができます。 15日胚移植後に受信者に寄り添う材料を提供する。 </li></ol>

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+hamster+oocytes+to+assay+the+fertilizing+capacity+of+human+spermatozoa.">Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa.</a> J. Reprod. Fertil. 66, 161-168 (1982).</li><li>Dios Hourcade, d. e., Perez-Crespo, J., Serrano, M., Gutierrez-Adan, A., A, B., Pintado, <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+and+in+vivo+development+of+mice+morulae+after+storage+in+non-frozen+conditions.">In vitro and in vivo development of mice morulae after storage in non-frozen conditions.</a> Reprod. Biol. Endocrinol. 10, 62 (2012).</li></ol>

著者らは、競合する経済的利益を宣言していません。

精管切除は大きな困難を伴わない比較的単純手術法である。ポビドンヨード、エタノールで消毒した場合は、それが腹膜を刺激する可能性がある（エタノールによる）最後の洗浄は、ポビドンヨードを削除していることを確認してください。 輸精管へのアクセスはまた、陰嚢または腹部8の横方向の切開を行うことによって達成することができる。陰嚢切開は、必要に応じて、...

胚移植は、おそらくマウスモデルで実行される最も頻繁な外科的処置である。この技術は、 インビトロでの操作技術に供胚から子孫を得ることが不可欠であり、したがって、前核注入、レンチウイルス形質導入、またはキメラ形成による遺伝的に改変されたモデルの開発のために必要なステップを構成している。しかも、この技術は、着床前の開発中に発生する多様な侮辱の発達への影響の研究を可能にする。人工生殖技術1または別の物質または代謝物2の異常な濃度への暴露の使用は、移植や胎盤形成の障害と子孫における長期的な効果をもたらした胚の発達に影響を及ぼす可能性があります。信頼性と再現性胚移植技術は、一貫性のある人間に移植すると、胎児の発育に関する実験的治療の可能性のあるマイナスの効果をテストすることが重要であるNER。 マウス着床前胚は、0.5日後に交尾の膨大部（DPC）偽妊娠レシピエント（卵管転送）3,4または2.5のDPC偽妊娠レシピエントの子宮に（子宮転送）5,6を経由して卵管にどちらレシピエント雌に転送することができます彼らの発達段階に応じて。このような胚または誘導多能性幹細胞の注入によってキメラマウスを生成するために使用されるもののような胚盤胞段階の胚は、通常、子宮転送により転送される。胚盤胞は、また0.5 DPC受信者の卵管に転送することができますが、胚は休眠を受け、注入が行われる前に発作から回復する2日間を持っているので、それは、発達かく乱にはあまり生理的テストを構成している。子宮内移植は、子宮腔への胚操作ピペットのアクセスを可能にする開口部を生成するために、狭い針で子宮壁に穴をあけることを含む。 Althoughこの技術は良い結果を得ることができ、長期の生存が（ つまり PUPに開発移植胚の割合）は、多くの場合、低および7,8予測不可能である。 子宮壁の穿刺は、いくつかの有害な副作用を伴う。まず、子宮筋層が高度に血管組織であり、その穿刺は、多くの場合、小さな出血が生じる。血液は胚移植ピペットをブロックしたり、胚の死および/または着床不全の原因となる子宮内腔に侵入​​することができる。血液細胞および破片が割球に取り付けることができるように、 透明帯のない胚は、転送されるとき、これは特に関連する。胚が転送された後第行わ開口は密封しないので、オリフィスを通って流れることができ、大きすぎる体積が子宮内に導入されたときに腹腔に排出される。ここに記載さ子宮卵管胚移植はEMBRを提供するために子宮卵管接合を利用する子宮壁を穿刺し、それによってその有害な結果9を回避する必要なく、子宮へヨーヨー。 胚移植のために使用偽妊娠レシピエント雌を精管切除した雄8と自然交配により得られる。雄性不稔で生成精液の分泌物が移植胚を受け入れるになるために子宮のために必要とされる。受信者を取得するには、生後6ヶ月〜8週間の2人の女性の最大は午後精管切除雄と配置されます。翌朝、雌を膣交尾プラグ、雄精液から凝固タンパク質の塊の存在がチェックされます。交配は通常、深夜中に発生するように、膣プラグ検出の日は0.5 DPCであると考えられている。精管切除した雄は、いくつかのベンダーから購入することができますが、ここに記載さの外科的処置は、比較的容易であり、胚移植のために必要とされるよりも、追加の機器を必要としない。

<table border="0" cellpadding="0" cellspacing="0" width="1234">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="29">
			<td height="29" style="height:29px;width:232px;">Ketamine</td>
			<td style="width:111px;">VEDCO</td>
			<td style="width:203px;">Ketaved ANADA 200-257</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:232px;">Xylazine</td>
			<td style="width:111px;">Lloyd laboratories</td>
			<td style="width:203px;">Anased NADA #139-236</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">Buprenorphine</td>
			<td style="width:111px;">Generic</td>
			<td style="width:203px;">NDC 400-42-010-01</td>
			<td style="width:691px;">To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Eye ointment</td>
			<td style="width:111px;">Novartis</td>
			<td style="width:203px;">Genteal</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:232px;">Antibiotic</td>
			<td style="width:111px;">Pfizer</td>
			<td style="width:203px;">Clavamox NADA #55-101.</td>
			<td style="width:691px;">Added to drinking water (0.3 mg/ml of amoxicillin trihydrate and 0.075 mg/ml of clavulanate potassium). Add 1.5 ml of the reconstituted 15 ml bottle to 250 ml of water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Dressing serrated forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-8120</td>
			<td style="width:691px;">Any medium size surgical-grade steel straight forceps will work.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Micro dissecting serrated forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5137</td>
			<td style="width:691px;">These ones are curved at a 90&#186; angle. Straight forceps can be used if preferred.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Slight curved micro dissection forceps</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5136</td>
			<td style="width:691px;">This model is particularly useful to hold the oviduct.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Scissors</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-5880</td>
			<td style="width:691px;">Any regular surgical grade steel small straight scissors will work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">27G needles</td>
			<td style="width:111px;">Beckton-Dickinson</td>
			<td style="width:203px;">305136</td>
			<td style="width:691px;">Smaller needles (30G) can be also used. 25G may be a bit too big.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">Clip applier</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">42763</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">9 mm Clips</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">427631</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Clip remover</td>
			<td style="width:111px;">MiKRon</td>
			<td style="width:203px;">7637</td>
			<td style="width:691px;">Two pairs of teeth forceps (ROBOZ RS-8160) can be used instead.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:232px;">Suture needle holder</td>
			<td style="width:111px;">ROBOZ</td>
			<td style="width:203px;">RS-7820</td>
			<td style="width:691px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Suture</td>
			<td style="width:111px;">Dowist Gell</td>
			<td style="width:203px;">5-0 Dexon S 7204-21</td>
			<td style="width:691px;">Can be substituted for any 4-0 to 6-0 absorbable suture with a narrow curved needle.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:232px;">Glass capillaries</td>
			<td style="width:111px;">VWR</td>
			<td style="width:203px;">100 ul calibrated pipettes 53432-921</td>
			<td style="width:691px;">It includes a mouth aspirator system that only requires to attach a 0.22 um filter in the tubing to be ready to use. More information can be obtained in ref. 8.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:232px;">Burner</td>
			<td style="width:111px;">KISAG AG</td>
			<td style="width:203px;">Typ 2002</td>
			<td style="width:691px;">Gas operated burner, can be charged with Kigas (CH-4512, from the same vendor). Alcohol burners may be also used, but gas provides a higher temperature and this burner provides a small and precise flame.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:232px;">Stereomicroscope</td>
			<td style="width:111px;">Leica</td>
			<td style="width:203px;">MZFLIII</td>
			<td style="width:691px;">This is an expensive estereomicroscope with fluorescence, that can be also used for other purposes. There are cheaper options such as Leica MZ8 or Nikon SMZ-10 or SMZ-2B, to name a few. It is better to use two stereomicroscopes, one for handling the embryos (which does not need to be a very nice one) and another one for the recipient. The one used for the recipient should display a long distance from the stage plate to the objective lense, in order to be able to focus 3-4 cm above the stage plate (where the oviduct will be placed) and still leave some room for the surgeon; most of the stereomicroscopes can do this, but some cannot.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:232px;">Fiber optics ilumination</td>
			<td style="width:111px;">Dolan Jenner</td>
			<td style="width:203px;">Fiber lite</td>
			<td style="width:691px;">To iluminate the surgical area. There are different systems available.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:232px;">Warm stages</td>
			<td style="width:111px;">American scope</td>
			<td style="width:203px;"><a href="https://exch.mail.umd.edu/owa/redir.aspx?C=1qTJKejamUSfudeYG_MlZHvTY3dsJM9Iaqr2SnwGD4ECWGzB8e8Rpg--_L7u0vJmyatXQJzGQ0k.&amp;URL=http%3a%2f%2fstore.amscope.com%2ftcs-100.html" target="_blank">http://store.amscope.com/tcs-100.html</a></td>
			<td style="width:691px;">These can be placed over the stage plate of the stereomicroscope. Some modifications (inserting a stick to level the stage) may be needed if it is too short for the stereomicroscope. A big warm stage can be used for warming the cage if it is available. If not, a regular heating pad can be used, but temperature must be checked.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">Culture dishes for embryo manipulation</td>
			<td style="width:111px;">Falcon</td>
			<td style="width:203px;">353001</td>
			<td style="width:691px;">351008 may be also used, they made narrower drops.</td>
		</tr>
	</tbody>
</table>

utero-tubal embryo transfer and vasectomy in the mouse model

サロゲートメス着床前の胚の転送は、その後の胎児の発育と成人健康上の着床前の開発中に発信し遺伝子改変マウスを製造するための、またはエピジェネティックな変化の影響を研究するために必要なステップです。効果的かつ一貫性のある胚移植技術の使用は、遺伝子改変動物の発生を増強する用語に移植率及び生存率に対する異なる処理の効果を決定するために重要である。胚盤胞期の胚は、通常、胚操作ピペットを導入するために子宮壁内の穿刺を行い、子宮転送により転送される。ピペットが取り下げられた、胚による子宮の正圧に腹腔へ流出することができた後に子宮に行わオリフィスは閉じません。パンクにも、移植性を損なう出血を生成することができ、ブロック転送ピペットおよび胚Dに影響を与える可能性が透明帯のない胚が転送され、特にevelopment、。従って、この技術は、しばしば非常に可変と全体的に低い胚の生存率をもたらす。これらの負の影響を回避、子宮卵管胚移植、胚流出を妨げる天然バリアとしての子宮卵管接合を利用して、子宮壁の穿刺を避ける。精管切除した雄は、偽妊娠の受信者を取得するために必要とされる。精管切除を行うための技術が子宮卵管胚移植を補完するものとして記載されている。

子宮卵管胚移植は、子宮胚移植2,9,10に関連する合併症のいくつかを回避子宮に胚を転送する手段を提供する。 表1に、我々は我々が説明したプロトコルに従って、CD1受信者に操作の異なる種類の対象CD1胚盤胞を転送取得しているいくつかの代表的な結果を示す。項（PUP、その結果胚の％）または（レンチウイルス曝露の場合）E15に生存への生存は受精卵の段階（IVC）から<...

Watch this Scientific Journal Video about Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model at JoVE.com

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

子宮卵管胚移植は、子宮の転送を実行するときに発生する可能性があり、胚の流出を防止するためのバリアとして子宮卵接合を使用しています。精管切除雄は、胚移植のための偽妊娠レシピエントを得るために必要とされる。両方の技術が議論されている。

マウスモデルにおける子宮卵管胚移植と精管切除

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

utero-tubal-embryo-transfer-and-vasectomy-in-the-mouse-model

Research

JoVE Journal

Biology

47.2K ビュー.  United States Department of Agriculture. 子宮卵管胚移植は、子宮の転送を実行するときに発生する可能性があり、胚の流出を防止するためのバリアとして子宮卵接合を使用しています。精管切除雄は、胚移植のための偽妊娠レシピエントを得るために必要とされる。両方の技術が議論されている。 

ビデオ: Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

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

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

鶏胚における神経誘導のためのアッセイ

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

の電気生理学的記録ショウジョウバエ胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

初期のニワトリ胚の形態形成の組織の変形を追跡

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

細胞特異的にLINC複合体を破壊するマウスモデルの検証

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

単核RNAシーケンシングのための成体マウス腎臓からの核分離

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

生殖補助医療のためのマウスにおける子宮頸部マニピュレーション誘発偽妊娠

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

著者スポットライト:非外科的胚移植または人工授精の前に精管切除された男性を必要とせずに雌マウスに偽妊娠を誘発する  

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

非外科的胚移植または人工授精の前に精管切除された雄を必要とせずに雌マウスに偽妊娠を誘導する

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant ...