ここで紹介する研究は、チャールズリバーによってサポートされていました。著者らは、動物のケアとホルモンの注射のための遺伝子組み換えモデルとサービスのグループと発生学のスタッフに最も感謝しています。

1。マウス精子の凍結保存<ol><li>凍結保存される各行は、開始前に以下を準備。 <ol><li>キットCPAの240リットルの4ウェルディッシュの1ウェル</li><li>次のように15精子の凍結保存ストロー<ol><li>注射器に最も近い綿栓で1 mLシリンジに0.25mlのストローを接続します。それぞれのわらでは、次に体外受精培地の8センチメートル（約160μl）を、空気の1cmをロードする。保持される行の名前で藁にラベルを付けます。 </li></ol></li></ol></li><li> LN 2は泡箱の奥深くに15センチメートルでなければなりません。蓋を閉じます。 </li><li>凍結保存される各行から2雄マウスを安楽死させる。マウスは、10〜60週齢の間でなければなりません。各マウスから輸精管と一緒に2つ尾副睾丸を削除します。 </li><li>事前に準備されたCPA -含む4ウェルディッシュ（ステップ1.1）のウェルに上体を置きます。 </li><li>各精巣上体5-7長手方向のカットを作成し、静かに精子を押し出すために、各輸精管を絞る。 </li><li> 3〜5分間、室温で軽く手でCPAおよび精巣上体混合物を振る。これは、精子が出て泳ぐことができるようになります。 </li><li>事前に準備したわらを使用して、精子懸濁液の5mmの（約10μl）を吸引除去し、空気の1cm。 </li><li> 15ストローの合計で最大で、このプロセスを繰り返します。最高の精子の生存のための10分以内にロードし、シールのストロー。 </li><li>慎重にわらの両側をシール加熱する。第一精子の列で凍結キャニスターにストローを置きます。 </li><li>ハンドルを延長するキャニスターにキットの一部として提供されている1 mlのピ​​ペットを取り付けます。蓋の穴から泡箱にLN 2のキャニスターを置きます。 10分間LN 2で垂直に浮いてキャニスターを許可する。 </li><li> LN2のキャニスターを浸し。精子は、現在凍結されており、長期的なLN 2ストレージに移動することができます。 </li><li>キャニスターはには使用しないでください次の行はそれまで、室温まで加温している。 </li></ol> 2。マウスの過剰排卵<ol><li>一般的に、精子のドナーと同じ背景の若いメスのマウスを使用してください。 </li><li>マウスは〜12グラム（生後3〜4週間）になります。 </li><li>妊馬血清ゴナドトロピン（PMSG）の5〜10 IUと腹腔内注射を行います。 </li><li> 46〜48時間を待つ。 </li><li>ヒト絨毛性ゴナドトロピン（hCG）の5〜10 IUと腹腔内注射を行います。 </li><li>収穫の卵母細胞後述するようにhCGを注射後14〜16時間。 </li></ol> 3。マウスの精子の回収と体外受精<ol><li>体外受精の前に、各ストロー融解するため、以下を準備し、37℃CO 2インキュベーターで1時間以上平衡化します。 <ol><li>精子の培養皿：ある35ミリメートルペトリ皿、油で覆われた精子処理媒体（STM）の90μlのドロップで。 </li><li>料理を切断：卵のコレクションには10秒の最大値からuperovulated女性、体外受精培地3mlワン35ミリメートルペトリ皿。 </li><li> IVFも：体外受精のための5過排卵女性、体外受精培地の500μlの（。。。あなたが10過排卵女性を使用している場合、たとえば、あなたは2井戸が必要となります）4ウェルディッシュの1つのウェルの各グループについて</li><li>洗濯料理：使用される4もIVFディッシュの各ウェルを洗浄するためのカリウムシンプレックス最適化された培地（KSOM）3mlのワン35ミリメートルペトリ皿</li><li>培養皿：IVFディッシュの各ウェルから胚培養のためのオイルで覆わKSOM培地50μl滴ワン35ミリメートルペトリ皿。 </li></ol></li><li> LN 2からわらを外し、2〜3分間37℃の水浴中での場所。 </li><li>水浴からストローを取り出して、余分な水分を除去するために拭きます。綿栓最も近い第1の空気の列の近くに最初のカットを行います。体外受精培地にストローをカットすることも、精子の列の前に表示される気柱が存在します。 </li><li>その後、s内のストローの端をカットecond気柱、精子の列をカットするように注意しながら。 45の角度でカット - これは、精子を吸引することが容易になります。 </li><li> 200μlのピペッターとストローの先端から精子懸濁液を吸引除去する。 </li><li> STMのドロップの中心に精子懸濁液を置き、37℃、5％CO 2インキュベーターで45〜60分間皿をインキュベートする。 </li><li>精子のインキュベーションの間に、切削皿に過剰排卵の雌の卵管を細かく分析。卵丘 - 卵子複合体（COCs）を放出、各卵管を引き裂くために30gの針やピンセットを使用してください。汚れたメディアが余分な洗浄ステップのためになるように体外受精培地の脂肪と血を避けてください。 </li><li>一度、すべての卵管が引き裂かれて、雌5から各IVF井戸にCOCsを転送する。排卵によって生成COCsの数は非常に依存株である。 </li><li> STMのドロップの周辺から精子の10〜15μlを収集し一体外受精の受精も約14-16時間はhCGのINJを投稿するection。よく使用されるそれぞれの受精を繰り返します。最終的な精子の濃度は約100万/ mlとなります。 </li><li> 37℃5％CO 2インキュベーターで4〜6時間共培養精子と卵母細胞。 </li><li> 37℃5％CO 2インキュベーター内で開発するためのKSOM降下で2倍と文化のKSOM最小で胚を洗浄してください。 </li><li>体外受精率は、一晩培養した後に使用される総卵母細胞の二つ細胞期胚外として算出した。 </li></ol> 4。代表的な結果 5チャールズリバーマウス近交系から精子を凍結させた。保護率は新鮮精子の評価値によって凍結精子の評価値で割って算出されています。精子の運動性の保護率はそれぞれ60.2パーセント、81.3パーセント、78.6パーセント、66.5パーセント、C57BL/6NCrl、129S2/SvPasCrl、FVB / NCrl、DBA/2NCrl、およびBALB / cAnNCrlのための61.0パーセントであった。急速な進歩的な運動のための保護率は47.3％、69.4パーセント、57.0パーセント、52.8パーセント、C57BL/6NCrl、129S2/SvPasの54.1％であった<html> CRL、FVB / NCrl、DBA/2NCrl、それぞれBALB / cAnNCrl、（図1）。 凍結融解精子で体外受精率はそれぞれ55.7パーセント、26.4パーセント、87.3パーセント、C57BL/6NCrl、129S2/SvPasCrl、FVB / NCrl、DBA/2NCrl、およびBALB / cAnNCrlで87.8パーセントと26.2％（図2）であった。 1.5年の期間にわたって、49 GMのマウス系統は、精子の凍結保存によってアーカイブされた。これらの行は、後に正常女性4系統の体外受精（C57BL / 6、BALB / C、DBA / 1と129Sv）（図3）で（として生まれたライブ子犬によって定義される）を回収した。 <img alt="図1。" src="/files/ftp_upload/3713/3713fig1.jpg" /> 図1マウスにおける精子の運動性と急速な進歩運動の保護比<img alt="図2。" src="/files/ftp_upload/3713/3713fig2.jpg" /> マウスにおける凍結融解精子と図2。体外受精3713fig3.jpg&quot;/&gt; 図3。GMのマウスにおける凍結融解精子による体外受精で

<ol>
	<li>Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., Ogawa, S. <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+mouse+spermatozoa+in+the+presence+of+raffinose+and+glycerol.">Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.</a> J. Reprod. Fertil. 89, 511-516 (1990).</li><li>Yokoyama, M., Akiba, H., Katsuki, M., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+normal+young+following+transfer+of+mouse+embryos+obtained+by+in+vitro+fertilization+using+cryopreserved+spermatozoa.">Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa.</a> Jikken Dobutsu. 39, 125-128 (1990).</li><li>Sztein, J. M., Farley, J. S., Young, A. F., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+cryopreserved+mouse+spermatozoa+affected+by+temperature+of+collection+and+rate+of+thawing.">Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.</a> Cryobiology. 35, 46-52 (1997).</li><li>Thornton, C. E., Brown, S. D., Glenister, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+numbers+of+mice+established+by+in+vitro+fertilization+with+cryopreserved+spermatozoa:+implications+and+applications+for+genetic+resource+banks,+mutagenesis+screens,+and+mouse+backcrosses.">Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.</a> Mamm. Genome. 10, 987-992 (1999).</li><li>Sztein, J. M., Farley, J. S., Mobraaten, L. E. <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+fertilization+with+cryopreserved+inbred+mouse+sperm.">In vitro fertilization with cryopreserved inbred mouse sperm.</a> Biol. Reprod. 63, 1774-1780 (2000).</li><li>Liu, L., Nutter, L. M., Law, N., McKerlie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+freezing+and+in+vitro+fertilization+in+three+substrains+of+C57BL/6+mice.">Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.</a> J. Am. Assoc. Lab. Anim. Sci. 48, 39-43 (2009).</li><li>Nakagata, N. <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+mouse+spermatozoa+and+in+vitro+fertilization.">Cryopreservation of mouse spermatozoa and in vitro fertilization.</a> Methods. Mol. Biol. 693, 57-73 (2011).</li><li>Mazur, P., Koshimoto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+intracellular+ice+formation+the+cause+of+death+of+mouse+sperm+frozen+at+high+cooling+rates.">Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates.</a> Biol. Reprod. 66, 1485-1490 (2002).</li><li>Jin, B., Yamasaki, C., Yamada, N., Seki, S., Valdez, D. M., Kasai, M., Edashige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+mouse+spermatozoa+are+injured+during+freezing.">The mechanism by which mouse spermatozoa are injured during freezing.</a> J. Reprod. Dev. 54, 265-269 (2008).</li><li>Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., Diekman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+signaling+pathways+involved+in+sperm+acquisition+of+fertilizing+capacity.">Novel signaling pathways involved in sperm acquisition of fertilizing capacity.</a> J. Reprod. Immunol. 53, 133-150 (2002).</li><li>Ostermeier, G. C., Wiles, M. V., Farley, J. S., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserving,+distributing+and+managing+genetically+modified+mouse+lines+by+sperm+cryopreservation.">Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.</a> PLoS ONE. 3, e2792-e2792 (2008).</li></ol>

著者らは、チャールズ川、このキットのメーカーの社員です。

1990年以来、18％ラフィノース、3％スキムミルクを使用して基本的な精子の凍結プロトコルは、マウスの多くの株で信頼性が証明されているとマウス系統の多数は、1,2,3,4,5,6,7を凍結保存されている。凍結と融解過程で精子細胞の損傷の主な原因は、氷の形成8,9および活性酸素種10に関連している。凍結培地のようなモノチオグリセロールなどのアミノ酸などのフリーラジカルスカベンジャー、および還元剤、、補充を調査し、大幅にC57BL / 6 11,6含む近交系における凍結融解精子で体外受精率を高めることが証明されました。 現在のプロトコルでは、我々は市販の抗酸化物質と氷のブロッカーとの基本的なCPAを最適化することで、精子の凍結や体外受精の手順を変更することにより、マウスの精子の凍結保存のために新しいI•クライオキットを開発しました。精子の運動性の保護率（&gt; 60％）と急速進行性の運動性は、（&gt; 45％）I•クライオキットで凍結five近交系マウスからの精子のために見られた。 5マウス近交系における凍結融解精子2細胞期の胚発生率が著しく基本的なCPA 11,6とそれに比べて改善されました。 キットを使用して、ユーザは、各行の2人の男性からの凍結精子で濃縮された精子懸濁液を作成します。唯一の凍結15ストローによってプロセスが簡単に、高速で、短期または長期の保管のための少ないストレージスペースを占有。ほとんどの時間は、マウスの線は、1または2ストローを融解​​することによって回復することができます。

<table border="1">
	<tbody>
		<tr>
			<th>
				Materials</th>
			<th>
				Supplier</th>
			<th>
				Catalogue number</th>
			<th>
				Comments</th>
		</tr>
		<tr>
			<td>CPA</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>STM</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>IVF medium</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>0.25 ml plastic straw</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Sperm freezing canister</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Cane and goblet</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>Should be embryo tested</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Falcon</td>
			<td>351008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat sealer</td>
			<td>ABTEC</td>
			<td>TISH-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-well multidish</td>
			<td>Nunc</td>
			<td>73521-424</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Gilson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C + 5% CO2 incubator</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LN2 storage system</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C water bath</td>
			<td>VWR</td>
			<td>89032-196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>Intervet</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>EMDChemicals</td>
			<td>367222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 26G 3/8 needle</td>
			<td>BD</td>
			<td>BD309625</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 gauge &frac12; needle</td>
			<td>BD</td>
			<td>305106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5135, RS-5110, RS-5005</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

mouse sperm cryopreservation and recovery using the i&middot;cryo kit

マウスの新たな遺伝子組み換え（GM）株の何千もの遺伝子導入やノックアウト技術の出現以来、作成されている。これらの貴重な動物の多くは、緊急の場合のバックアップなしプランで、生きている動物としてのみ存在します。胚の凍結保存は、このバックアップを提供するが、高価であることができる、長い手順である、と一般的には成功のための動物の多数を必要とすることができます。マウスの精子が正常に18％ラフィノース、3％スキムミルクで構成される基本的な凍結防止剤（CPA）で凍結保存することができることが発見されて以来、精子の凍結保存は、アーカイブの配布およびこれらの貴重な株の回復のために受け入れ可能でコスト効果の高い手順となっている。 ここでは、マウス精子の凍結保存のための新開発のI•クライオキットを示しています。近交系マウス五一般的に使用されている菌株からの精子は、このキットを使用して凍結し、回収した。精子の運動性の高い保護率（&gt;60％）と制御（基本CPA）に比べて急速に進行性の運動性（&gt; 45％）5近交系マウスで、このキットで凍結精子のために見られた。回収した精子で体外受精後の二細胞期胚の開発は、調査全5マウス系統で一貫して改善された。 1.5年の期間にわたって、49 GMのマウス系統は、I•クライオキットで精子の凍結保存によってアーカイブされ、後にIVFにより回収。

Watch this Scientific Journal Video about Mouse Sperm Cryopreservation and Recovery using the IÃ‚Â·Cryo Kit at JoVE.com

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

ここでは、マウス精子の凍結保存のための新開発のI•クライオキットを示しています。凍結融解精子を持つ二細胞期胚の開発は、このキットの使用で5マウス系統で一貫して改善された。 1.5年の期間に、49遺伝子改変マウス系統は、I•クライオキットで精子の凍結保存によってアーカイブされ、後に正常IVFによって回収。

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-sperm-cryopreservation-and-recovery-using-the-icryo-kit

Research

JoVE Journal

Biology

39.5K ビュー.  Charles River. ここでは、マウス精子の凍結保存のための新開発のI•クライオキットを示しています。凍結融解精子を持つ二細胞期胚の開発は、このキットの使用で5マウス系統で一貫して改善された。 1.5年の期間に、49遺伝子改変マウス系統は、I•クライオキットで精子の凍結保存によってアーカイブされ、後に正常IVFによって回収。

ビデオ: Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

収穫精子とマウスの人工授精

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

生物学

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫組織化学：ベクターLabsからベクタステインABCキットを用いたパラフィン切片

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Bio - Rad社のTotal RNA Kitを使​​用してNeuroprecursor細胞からのRNA抽出

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

シグマのエキス- N - Ampの組織PCRキットを使用してマウス組織の急速なジェノタイピング

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

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 ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes 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 ...

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 ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

魚の精子の評価ソフトウェアを使用して、冷却装置

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...