这项研究是在国家生物资源项目的合作，教育部，文化，体育，科学和技术，日本。

实验的总体方案如图 。 1。 1。试剂准备<ol><li>在100毫升玻璃瓶的基础培养基（在这里为PB1的已知）根据下面的下面的图表: <table border="1"><tbody><tr><td></td><td> 兆瓦 </td><td> mM的 </td><td> mg/100ml </td></tr><tr><td>氯化钠</td><td> 58.4 </td><td> 136.98 </td><td> 800.0 </td></tr><tr><td>氯化钾</td><td> 74.6 </td><td> 2.68 </td><td> 20.0 </td></tr><tr><td> KH 2 PO 4 </td><td> 136.1 </td><td> 1.47 </td><td> 20.0 </td></tr><tr><td> NA 2 HPO 4 .12 H 2 O </td><td> 358.14 </td><td> 8.04 </td><td> 288.1 </td></tr><tr><td>氯化镁2 · 6H 2 O </td><td> 203.3 </td><td> 0.49 </td><td> 10.0 </td></tr><tr><tD&gt;葡萄糖</td><td> 180.2 </td><td> 5.56 </td><td> 100.0 </td></tr><tr><td>钠丙酮酸</td><td> 110 </td><td> 0.33 </td><td> 3.6 </td></tr><tr><td>氯化钙2 · 2H 2 O </td><td> 147 </td><td> 0.9 </td><td> 13.2 </td></tr><tr><td>青霉素G </td><td></td><td></td><td> 6.0（约） </td></tr></tbody></table></li><li>使聚蔗糖蔗糖（FS）解决方案: <ol><li> PB1的解决方案，在50毫升管14毫升以下​​的化学品。 <table border="1"><tbody><tr><td>聚蔗糖70 </td><td> 6.0摹</td></tr><tr><td>蔗糖</td><td> 3.424克</td></tr><tr><td>牛血清白蛋白</td><td> 42.0毫克</td></tr></tbody></table></li><li>混合聚蔗糖70和蔗糖PB1的解决方案，直至完全溶解，摇匀。 </li><li>检查以上完全溶解后，加入BSA溶液的表面上，并保持它在直到BSA的摄氏4度完全溶解（&gt; 4小时或过夜）。 </li></ol></li><li>设为平衡溶液（EFS20）50毫升管和玻璃化溶液（EFS40）: <ul><li> EFS20:乙二醇20％（V / V），24％（W / V）聚蔗糖，和0.4 mol / L的蔗糖PB1与BSA </li><li> EFS40:乙二醇40％（V / V），18％（W / V）聚蔗糖，和0.3 mol / L的蔗糖PB1与BSA *对于胚胎从BALB / C或ICR株，增加蔗糖浓度为0.9 mol / L蔗糖，因为他们更敏感cryodamage 6。 </li></ul><table border="1"><tbody><tr><td></td><td> EFS20解决方案 </td><td> EFS40解决方案 </td></tr><tr><td>乙二醇</td><td> 1毫升</td><td> 2毫升</td></tr><tr><td>财政司司长解决方案</td><td> 4毫升</td><td> 3毫升</td></tr></tbody></table></li><li>使用0.45微米filte的过滤消毒的解决方案河在5毫升的聚苯乙烯管和储存，分装在4 ° C。他们可用于6个月左右。 </li><li>制备的胚胎解冻含有蔗糖（TS1）0.75米的解决方案<ol><li>在PB1的溶解7.7克的蔗糖（在步骤1.1准备），使总体积30毫升。直至蔗糖完全溶解，轻轻摇动混合。 </li><li>在表面上的解决方案中添加牛血清白蛋白90毫克，离开它的立场，直到完全溶解。 </li><li>经过滤消毒的解决方案。 </li><li>分装和储存在4 ° C他们可以用1个月左右。 </li></ol> 注:TS1也可以从市售M2的准备。 <ol start="6"><li>溶解7.7克的蔗糖在M2和带来的总体积30毫升。 </li><li>直至蔗糖完全溶解，轻轻摇动混合。 </li><li>经过滤消毒的解决方案。 </li><li>分装和储存在摄氏4度。他们可以用1个月左右。 </li></ol></li><li> TS2（解冻的解决方案含0.25 M蔗糖）: <ol><li> TS1的10毫升，20毫升PB1或M2量稀释适当。 </li><li>分装和储存在4 ° C他们可以用1个月左右。 </li></ol></li></ol> 2。 2 - 细胞小鼠胚胎玻璃化<ol><li> 2 -细胞小鼠胚胎自然交配或常规体外受精技术准备。 </li><li>分装50μL成cryotube EFS40。此后，在室温下进行玻璃化过程的其余部分。 </li><li>分装50μL的EFS20 35毫米或60毫米的塑料培养皿的底部。 </li><li>转移至30胚胎EFS20使用玻璃毛细管培养基的最低金额。启动了2分钟的计时器。 注:下降的底部放置胚胎。这使得胚胎的高效脱水。立体显微镜检查胚胎的形态。脱水胚胎萎缩形态， 如图所示。 2A。如果他们没有足够的脱水，等待1或2分钟。 </li><li>在约1.5分钟，拿起唯一的解决方案的最低金额从EFS20下降的胚胎。他们转移到EFS40在步骤2.1编写的cryotube。注:为了获得最佳效果，调整拿起使所有的胚胎可在2分钟左右转移到EFS40，胚胎的时机。 </li><li>等待1分钟。 </li><li>把cryotube直接进入液氮（第2号法律公告）。 </li></ol> 3。解冻陶瓷2 - 细胞小鼠胚胎<ol><li>解冻之前，准备一盘菜，与10μL滴恢复胚胎的胚胎培养液中（见步骤3.13）。盖在二氧化碳培养箱石油和地方菜，直到使用。注:虽然可能会在本实验中使用的常规胚胎培养任何媒体，我们建议具有较高的渗透压，如M16的介质的媒体。 </li><li>暖TS1至37 ° C。 </li><li>戴上口罩，并cryogloves。打开的LN 2吨ANK和检索cryotube含有胚胎。 </li><li>快速打开管的顶部， 并放弃2号法律公告。等待30秒，以防止冻结在下一步TS1。 </li><li>使用1000ul移液器添加到管850μLTS1（37℃），并温柔pipettings在25秒内（约10次）的混合解决方案，直到溶液均匀溶解。 </li><li>传输整个卷管60毫米的塑料培养皿（或手表玻璃）（ 图3A）。注:胚胎应在室温（22-25℃）处理，直到它们被放置在CO 2培养箱步骤3.14。 </li><li>启动了3分钟的计时器。 </li><li>在2分钟TS1中左右，轻轻摇晃的菜，直到培养基中含有胚胎是在菜的表面（ 图 3B）传播。注:这将有助于胚胎下到谷底，因为它们是浮在表面附近时转移到TS1。 </li><li> TS2的三个50μL滴摆上日E盘（ 图3C）。 </li><li> 3分钟TS1中后，检查胚胎立体显微镜的形态;他们应略有萎缩。请参阅在早期胚胎的形态（ 图2B）及更高版本（图2C）TS1中。注:如果胚胎仍然肿，保持TS1中的1至3分钟。 </li><li>拿起胚胎，并将它们传送的TS2的首次下降（ 图3D）。开始3分钟定时器</li><li> 3分钟后，转移胚胎到第二个下拉，然后到第三个下拉（ 图3E）。 </li><li>胚胎转移到步骤3.1编写的培养基。胚胎是在图所示的形状。 2D。 </li><li>将菜在二氧化碳培养箱。约10分钟后，胚胎转移到未来的培养液洗出蔗糖已经从TS2下降。 </li><li>继续在CO2培养箱中培养直至胚胎移植的文化。 请注意:传输安莉芳操作系统解冻日收件人女性输卵管。 在体外悠久的文化，可能会减少一些菌株在小鼠胚胎的可行性。 </li></ol> 4。代表性的成果: - 体外和体内的胚胎发展后的解冻是在表1和2 。本协议的优点是解冻后和其广泛的适用性，不同品系的小鼠胚胎的高生存能力。 <table border="1"><tbody><tr><td>应变</td><td>管总号</td><td>玻璃化冷冻胚胎号</td><td>回收（人数（％）） </td><td>形态正常（第（％）） </td><td>发展到囊胚（第（％）） </td></tr><tr><td>只C57BL/6J </td><td> 20 </td><td> 400 </td><td> 397（99） </td><td> 394（99） </td><td> 342（87） </td></tr><tr><td> BALB / CA </td><td> 15 </td><td> 300 </td><td> 296（99） </td><td> 282（95） </td><td> 238（84） </td></tr><tr><td> ICR </td><td> 24 </td><td> 480 </td><td> 474（99） </td><td> 443（93） </td><td> 398（90） </td></tr></tbody></table> 表1， 在体外共同小鼠品系胚胎玻璃化冷冻解冻发展<table border="1"><tbody><tr><td>胚胎的条件</td><td>收件人女性号</td><td>胚胎号转移</td><td>植入网站（人数（％）） </td><td>现场后代（人数（％）） </td></tr><tr><td>新鲜的</td><td> 12 </td><td> 180 </td><td> 141（78.3） </td><td> 110（61.1） </td></tr><tr><td>陶瓷</td><td> 16 </td><td> 242 </td><td> 202（83.5） </td><td> 125（51.7） </td></tr></tbody></table> 表2。C57BL/6J小鼠胚胎玻璃化冷冻解冻体内发展。 <img alt="图1" src="/files/ftp_upload/3155/3155fig1.jpg" /> 图1包括平衡，玻璃化冷冻，胚胎解冻的实验总体方案。 <img alt="图2" src="/files/ftp_upload/3155/3155fig2.jpg" /> 图2。胚胎解冻的每一步的形态。 <img alt="图3" src="/files/ftp_upload/3155/3155fig3.jpg" /> 图3。胚胎解冻的过程。所有的程序都在室温下进行。 （一），添加到一个cryotube TS1（37℃）850μl和解决cryotube整个卷转移到一个塑料盘。此时，胚胎看起来肿图所示。 2B。 （二），轻轻摇动的菜表面遍布的解决方案。 （三），放在塑料盘TS2的三个50μL滴。 （四），胚胎转移的TS2的首次下降。 3分钟后，胚胎看shurunken 图所示。 2C。 （五），转移他们的串行在剩下的TS2的LY下降，然后到培养液中。

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196&#176;+and+-269&#176;C.">Survival of mouse embryos frozen to -196&#176; and -269&#176;C.</a> Science. 187, 411-414 (1972).</li><li>Rall, W. F., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-free+cryopreservation+of+mouse+embryos+at+-196&#176;C+by+vitrification.">Ice-free cryopreservation of mouse embryos at -196&#176;C by vitrification.</a> Nature. 313, 573-575 (1985).</li><li>Yoshiki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+resources+at+the+RIKEN+BioResource+center.">The mouse resources at the RIKEN BioResource center.</a> Exp. Anim. 58, 85-96 (2009).</li><li>Mochida, K., Ogura, A. <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+embryos+in+laboratory+species.">Cryopreservation of embryos in laboratory species.</a> J. Mamm. Ova. Res. 27, 87-92 (2010).</li><li>Kasai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+mouse+embryos+cryopreservation+in+a+low+toxicity+vitrification+solution,+without+appreciable+loss+of+viability.">A simple method for mouse embryos cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability.</a> J. Reprod. Fert. 89, 91-97 (1990).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing+methods.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Kasai, M., Mukaida, T. <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+animal+and+human+embryos+by+vitrification.">Cryopreservation of animal and human embryos by vitrification.</a> Reprod. Biomed. Online. 9, 164-170 (2004).</li><li>Miyake, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+mouse+oocytes+and+embryos+at+various+stages+of+development+in+an+ethylene+glycol-based+solution+by+a+simple+method.">Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method.</a> Theriogenology. 40, 121-134 (1993).</li><li>Han, M. S., Niwa, K., Kasai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+rat+embryos+at+various+developmental+stages.">Vitrification of rat embryos at various developmental stages.</a> Theriogenology. 59, 1851-1863 (2003).</li><li>Jin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+vitrification+of+mouse+embryos.">Equilibrium vitrification of mouse embryos.</a> Biol. Reprod. 82, 444-450 (2010).</li></ol>

我们什么都没有透露。

以来的第一份报告于1985年2小鼠胚胎玻璃化冷冻曼仕龙和Fahy，取得了一些技术改进，以增加胚胎解冻后的生存能力。最成功的修改之一是通过使用乙二醇作为冷冻保护剂，因为它的低毒性和高膜通透性。这种优势使我们能够处理在室温下4的冷冻胚胎，其他的玻璃化冷冻方法需要胚胎稍凉移交并不总是适用于包括的 BALB / C 6小鼠的一些菌株。第一乙二醇为基础的玻璃化冷冻是由葛西等。在1990年5,7。由于原来的方法是优化作为容器的塑料吸管，我们稍微修改了协议cryotubes，这是在实验室中更容易获得和更耐物理损坏。因此，这里描述的玻璃化冷冻方法可被应用程序licable到许多实验室用小鼠作为研究模型。同样的方法也可用于小鼠胚胎的桑椹胚和囊胚阶段和大鼠胚胎9。然而，我们最近发现的cryotubes质量后，可能会影响冻融胚胎的存活率。因此，有必要检查其前vitrifying胚胎（例如，在特定的试剂和设备表）的平滑的内表面的cryotubes。 玻璃化冷冻方法比传统的慢速冷冻方法有许多优势，但他们本身有一个运输的目的方面的缺点。由于玻璃化冷冻胚胎应保持在低于-120 ° C至维持其可行性，干托运人通常是用于他们的安全运输。干托运人笨重，和他们的往返是昂贵的，特别是国际运输。我们现在目前正在制定一项新的玻璃化冷冻方法玻璃化冷冻胚胎可以保存在干冰的温度（约-80℃）至少7 天 10 。这个方法应该是下一代的玻璃化冷冻。

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>牛血清白蛋白</td><td>默克生物科学（Calbiochem） </td><td> 12657 </td><td></td></tr><tr><td>乙二醇</td><td>光纯药工业</td><td> 058-00986 </td><td></td></tr><tr><td>聚蔗糖70 </td><td> GE医疗集团</td><td> 17-0310-10 </td><td></td></tr><tr><td>血糖</td><td>光纯药工业</td><td> 041-00595 </td><td></td></tr><tr><td>氯化钠</td><td>光纯药工业</td><td> 191-01665 </td><td></td></tr><tr><td>氯化钾</td><td>光纯药工业</td><td> 163-03545 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td>光纯药工业</td><td> 169-04245 </td><td></td></tr><tr><td> NA 2 HPO 4 · 12H 2 </sUB&gt; O </td><td>光纯药工业</td><td> 500-04195 </td><td></td></tr><tr><td>氯化镁2 · 6H 2 O </td><td>光纯药工业</td><td> 135-00165 </td><td></td></tr><tr><td>氯化钙2 · 2H2O </td><td>光纯药工业</td><td> 031-00435 </td><td></td></tr><tr><td>青霉素G </td><td> Sigma - Aldrich公司</td><td> P - 4687 </td><td></td></tr><tr><td>丙酮酸钠</td><td> Sigma - Aldrich公司</td><td> P - 8574 </td><td></td></tr><tr><td>蔗糖</td><td>光纯药工业</td><td> 196-00015 </td><td></td></tr><tr><td> M16的介质</td><td> Sigma - Aldrich公司</td><td> M7292 </td><td></td></tr><tr><td> M2培养液</td><td> Sigma - Aldrich公司</td><td> M7167 </td><td></td></tr><tr><td> Cryotube </td><td>住友电木</td><td> MS - 4501 </td><td> "低温小瓶" </td></tr></table>

cryopreservation of mouse embryos by ethylene glycol-based vitrification

小鼠胚胎冷冻保存是一个技术的基础上，支持生物医学科学，因为多株小鼠已产生遗传修饰和数量一直呈逐年上升趋势。其技术的发展与慢速冷冻方法开始在20世纪70年代，1， 然后在80年代末2制定的玻璃化冷冻方法。一般来说，后者的技术优势在其快速，简便，高生存和回收胚胎。然而，冷冻保护剂中包含有剧毒，可能会影响以后的胚胎发育。因此，该技术并不适用于某些菌株的小鼠，即使解决方案冷却至4 ° C，以减轻毒性作用在胚胎处理。理化学研究所生物资源中心，超过5000个不同的遗传背景和表型的小鼠品系维持 3，所以我们优化了玻璃化冷冻TEchnique与其中，我们可以从许多不同的小鼠株冻存胚胎与胚胎存活率高的好处后，vitrifying和解冻（或液化，更精确），在环境温度4。 在这里，我们提出了一个鼠标，已成功地在我们中心的胚胎玻璃化冷冻方法。冷冻保存的解决方案包含乙二醇代替DMSO的毒性，以尽量减少胚胎5。它还含有预防反玻璃化和渗透调节的聚蔗糖和蔗糖分别。胚胎可以在室温下处理，并转移到液态氮中，5分钟之内。由于原来的方法是优化作为容器的塑料吸管，我们稍微修改了协议cryotubes，这是在实验室中更容易获得和更耐物理损坏。我们还描述了详细的玻璃化冷冻胚胎解冻的过程，因为它是一个关键的STEP为有效的恢复活体小鼠。这些方法将有助于研究人员和技术人员，他们需要在安全和成本效益的方式保存供以后使用小鼠品系。

Watch this Scientific Journal Video about Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification at JoVE.com

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

乙二醇为基础的玻璃化冷冻方法对小鼠胚胎描述。这是有利于它的简单性和胚胎毒性低的其他方法，因此可广泛适用于小鼠多株，包括自交系和基因修饰的小鼠。

基于乙二醇玻璃化冷冻小鼠胚胎的超低温保存

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

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Biology

31.0K 次观看.  RIKEN BioResource Center. 乙二醇为基础的玻璃化冷冻方法对小鼠胚胎描述。这是有利于它的简单性和胚胎毒性低的其他方法，因此可广泛适用于小鼠多株，包括自交系和基因修饰的小鼠。

视频: Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

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小鼠胚胎剖析

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

科研

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

E15小鼠胚胎在子宫内侧脑室注射和电穿孔

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

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.

收集和仓鼠卵母细胞和小鼠胚胎冷冻保存

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

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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.

活体成像和治疗卵巢癌的原位小鼠模型

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

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

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

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

植入前胚胎，牛，羊，山羊的冷冻保存

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

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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

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

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

精子冷冻保存和恢复使用I ·冷冻包

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

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

基因转移到老年人的鸡胚<em&gt;前OVO</em&gt;电穿孔

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...