Name: collection and cryopreservation of hamster oocytes and mouse embryos
Uploaded: 2009-03-27T00:00:00.000+00:00
Duration: 13 min 36 s
Description: Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

这项工作是支持西班牙Ministerio教育Ÿ Ciencia（生物2006-11792）和Generalitat加泰罗尼亚（2005 - SGR00437）。作者想感谢他们的技术援助马克Puigcerver和Jonatan卢卡斯在准备媒体。 Servei D&#39; Estabulari的所有工作人员承认动物的住房，尤其是胡安何马丁和贝尼托哈维尔为他们录制这个视频的一部分的额外贡献。 NCB是葡萄牙Fundação第一个Ciência发送Tecnologia和SG是西班牙Ministerio教育Ÿ Ciencia研究员研究员。

根据Universitat，西班牙巴塞罗那自治大学的动物和人类研究伦理委员会批准的准则和法规，动物保健和程序进行。 一，卵母细胞收集<ol><li>检查，并选择女仓鼠（Mesocricetus鲫; 8-12周的老）金厚厚的阴道放电（postovulatory放电）的存在叙利亚应变。 </li><li>诱导选定的女性，60小时后腹腔注射40 IU PMSG（孕马血清促性腺激素）40国际单位的HCG（绒毛膜促性腺激素）其次superovulate。 </li><li>安乐死女仓鼠，16小时后，HCG在二氧化碳室管理。 </li><li>隔离在视频所示的女性输卵管，并将它们传送到KSOM - H培养基下降。 </li><li>撕裂立体显微镜下输卵管壶腹部，透明质酸酶（156 U /毫升）下降收集卵母细胞卵丘复合物，在37 ° C。将被释放卵母细胞的卵丘细胞在大约5分钟。 </li><li>拿起从透明质酸酶的卵母细胞和KSOM - H培养基洗两次。 </li></ol>对于收集的小鼠胚胎，雌性小鼠（家鼠; 6-8周的老）诱导，superovulate通过腹腔注射5 IU PMSG其次是5 IU hCG的48小时内，并与雄性小鼠交配。女性在24-26颈椎脱位处死，44-46或54-56小时后，HCG为原核，2芯或4细胞阶段的胚胎，分别obtention管理。仓鼠卵母细胞原核胚胎的收集是完全执行。在2 - 细胞和4细胞阶段的胚胎收集输卵管冲洗KSOM - H培养基[6]。 注：本议定书约20至30的卵母细胞/的胚胎可以从每个女性收集 。 二。制备冷冻和解冻的解决方案<ol><li>准备的解决方案，我在KSOM - H（1.5米的propilenglycol）5毫升稀释0.57毫升propilenglycol。 </li><li>准备加入2毫升的KSOM - H与0.2052 g蔗糖（蔗糖的0.3 M）的管解冻的解决方案。 </li><li>准备加入0.0684 g蔗糖（1.5米propilenglycol和0.1 M的蔗糖）2毫升的解决方案，我管冻结的解决方案。 </li></ol> 注：我的解决方案和冷冻和解冻的解决方案必须就其使用前的准备 。 然而，蔗糖指定要编制的冷冻和解冻的解决方案中使用的金额可能会提前加权和3毫升管在室温下保存数月。 三。冻结协议<ol><li>转让卵母细胞/胚胎KSOM - H培养基的解决方案，我下降为7-15分钟，在室温下孵育。 </li><li>同时，准备了90毫米的培养皿中加载的吸管放置90μL滴冻结和解冻的解决方案，因为它是在视频（1滴每个解决方案/稻草）所示。 </li><li>转移所需的卵母细胞/胚胎数的冻结溶液滴。 注：我们通常5至30个卵母细胞或胚胎/下降之间的转移。 </li><li>将一根救命稻草，一个口控制的愿望系统，下面这个命令：解决方案，我的卵母细胞，空气，解冻下降，空气冻结下降，空气，加载它。 </li><li>一旦秸秆被加载，保持吸气，直到第一个下拉介绍（解决方案），达到了高端的秸秆和密封为此聚合物。热密封的稻草或用塑料塞密封的另一端。 </li><li>可编程的冷冻开关并选择冻结计划。冻结在本议定书中所使用的程序是基于最初由拉萨尔等发表的冷却速率。 [7]： <ul><li> 2 ° C /分钟从室温到-7 ° C 0 ° C /分钟为10分钟，让温度平衡和诱导播种（见下文） </li><li> 0.3 ° C /分钟，从-7 ° C至-30 ° C 0℃/分钟2分钟，让温度平衡</li><li> 35 ° C /分钟从-30 ° C到-130 ° C </li></ul></li><li>可编程冷冻人的插槽装入秸秆，确保秸秆解冻液下降部分是朝外。启动冻结的方案。 </li><li>当样品的温度达到-7℃，并保持冷却，进行人工播种：陷入液氮钳，稍微拉冰柜的持有人揭露的部分包含滴解冻液吸管，并立即触摸这部分镊子的吸管。重复每个持有人，持有人在每个稻草，然后让程序运行，直到最后的冻结。 </li><li>当程序完成后，投身于液氮中的持有人。 </li><li>取出的吸管持有人及他们转移到一个标记gobelet。 </li><li>最后，商店内的液氮罐gobelet。 </li></ol>四。解冻协议<ol><li>从液氮罐中取出一根稻草。 </li><li>保持秸秆在常温下为40秒。 </li><li>浸泡在30℃水浴中的稻草，在40秒。 </li><li>从秸秆中取出插头或切断热密封提示。 </li><li>空秸秆在35毫米培养皿，轻轻地混合两滴含冷冻和解冻的解决方案。 </li><li>混合物在室温下15分钟留在卵母细胞/胚胎。 </li><li>一个额外的15分钟内的卵母细胞/胚胎转移到一个新鲜的KSOM - H下降，在37 ° C。 </li><li>卵母细胞/胚胎，现在准备进一步的处理。 </li></ol> 注意：在步骤6和7，你应该看到，解冻卵母细胞/胚胎慢慢地膨胀起来由于补液。 五，代表性的成果使用的冷冻保存协议仓鼠卵母细胞中大约95％和&gt; 90％，85％和80％的鼠标B6CBAF1胚胎的原核细胞和4 - 细胞阶段，分别的成活率在这里报告的结果。当KSOM在体外培养小鼠胚胎解冻后，至少有70％至80％应达到最佳的培养条件胚泡阶段。 建议你包括在每个冻结的实验和对照组（秸秆）的卵母细胞/胚胎解冻本组之前，其余的卵母细胞/胚胎用于进一步操作。如果对照组的生存率低于预期，解冻同一地段的另一稻草。如果这个第二组的存活率也低，放弃从这个冻结大量剩余的秸秆。

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196+and+-269+&#176;C.">Survival of mouse embryos frozen to -196 and -269 &#176;C.</a> Science. 178, 411-414 (1972).</li><li>Wilmut, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cooling+rate,+warming+rate,+cryoprotective+agent+and+stage+of+development+on+survival+of+mouse+embryos+during+freezing+and+thawing.">The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing.</a> Life Sci. 11, 1071-1079 (1972).</li><li>Parkening, T. A., Tsunoda, Y., Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+various+low+temperatures,+cryoprotective+agents+and+cooling+rates+on+the+survival,+fertilizability+and+development+of+frozen-thawed+mouse+eggs.">Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.</a> J. Exp. Zool. 197, 369-374 (1976).</li><li>Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+principles+of+embryo+freezing.">Cryobiological principles of embryo freezing.</a> J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).</li><li>Vajta, G., Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cryopreservation+systems.">Improving cryopreservation systems.</a> Theriogenology. 65, 236-244 (2006).</li><li>Duselis, A. R., Vrana, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+mouse+oocytes.">Retrieval of mouse oocytes.</a> JoVE. 3, (2007).</li><li>Lassalle, B., Testart, J., Renard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryo+features+that+influence+the+success+of+cryopreservation+with+the+use+of+1,2+propanediol.">Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.</a> Fertil. Steril. 44, 645-651 (1985).</li><li>Ib&#225;&#241;ez, E., Grossmann, M., Vidal, F., Egozcue, J., Santal&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+caprine+beta-lactoglobulin+transgenic+mouse+embryos.">Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.</a> Cryobiology. 35, 290-298 (1997).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Tateno, H., Kamiguchi, Y., Mikamo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+freezing+and+thawing+method+of+hamster+oocytes+designed+for+both+the+penetration+test+and+chromosome+assay+of+human+spermatozoa.">A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.</a> Mol. Reprod. Dev. 33, 202-209 (1992).</li><li>Biggers, J., McGuinnis, L. K., Raffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+preimplantation+development+of+the+mouse+in+the+protein-free+KSOM.">Amino acids and preimplantation development of the mouse in the protein-free KSOM.</a> Biol. Reprod. 63, 281-293 (2000).</li></ol>

与本议定书​​仓鼠卵母细胞和小鼠胚胎，可以成功地冷冻保存。一旦冷冻，卵母细胞/胚胎可以无限期地储存在液氮罐，并期望在任何时间或地点恢复。这提供了样品几乎准备在需要使用时的优势。另一方面，这个协议也可以用于生成有价值的小鼠品系（如转基因株系），作为经济和更安全的替代维护活体动物殖民地，胚胎cryobanks。重要的是要注意，这个协议是优化冻存仓鼠卵母细胞和混合B6CBAF1小鼠胚胎。之?...

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

collection and cryopreservation of hamster oocytes and mouse embryos

首次成功地冻存胚胎和卵母细胞超过30年前，当惠廷厄姆<i&gt;等。</i&gt;<sup&gt; 1</sup&gt;和维尔穆特<sup&gt; 2</sup&gt;分别描述小鼠胚胎可以冷冻和储存在-196 ° C，几年后，Parkening<i&gt;等</i&gt;。<sup&gt; 3</sup&gt;报道冻存卵母细胞体外受精（试管婴儿）的生活造成后代的诞生。从那时起，冷冻保存技术的使用，已迅速蔓延，成为在人类和动物的辅助生殖的实践和在动物遗传资源的保护的重要组成部分。目前，有两种主要方法用于冻存卵母细胞和胚胎慢速冷冻和玻璃化冷冻。一个多种方法已被用来尝试改进技术和以百万计的动物和成千上万的儿童已经诞生，从冻存胚胎。然而，与冷冻保存的重要缺陷，仍有待克服，由于冰晶体的形成，解决影响和渗透压冲击似乎导致解冻后的卵母细胞和胚胎中的几个cryoinjuries。慢速冷冻可编程冰柜使用低浓度的冷冻保护剂，通常与化学毒性和渗透压冲击相关的优势，但他们的能力是有限的，以避免在低浓度的冰晶体的形成。慢速冷冻也引起过冷的影响，必须避免使用手动或自动播种<sup&gt; 4</sup&gt;。在玻璃化过程中，高浓度的冷冻保护剂抑制冰晶的形成，并导致形成一个玻璃状的玻璃化冷冻水是凝固的状态，其中，但没有扩大。然而，由于在使用浓度cyroprotectants毒性，卵母细胞/胚胎只能暴露于冷冻保护剂的解决方案为一个很短的时间内，在一个体积最小解决方案，直接浸在液态氮的样品前<sup&gt; 5</sup&gt;。在过去十年中，玻璃化已成为更受欢迎，因为它是一个非常快的方法，它不需要昂贵的设备（可编程冰柜）。但是，慢速冷冻卵母细胞/胚胎冷冻保存最常用的方法。我们在这个视频文章显示，一步一步，如何收集，慢慢地冻结与解冻后的存活率仓鼠卵母细胞。同样的程序也可以应用，成功地冻结和解冻在不同阶段的小鼠胚胎着床前的发展。

Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

在这个视频文章中，我们提出了一个一步一步示范如何收集和仓鼠卵母细胞冻存与解冻后的存活率。同样的程序也可以应用，成功地冻结和解冻在不同阶段的小鼠胚胎着床前的发展。

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 ...

collection-and-cryopreservation-of-hamster-oocytes-and-mouse-embryos

Research

JoVE Journal

Biology

16.9K 次观看.  Universitat Autonoma de Barcelona. 在这个视频文章中，我们提出了一个一步一步示范如何收集和仓鼠卵母细胞冻存与解冻后的存活率。同样的程序也可以应用，成功地冻结和解冻在不同阶段的小鼠胚胎着床前的发展。

视频: Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

科研

生物学

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

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

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

从小鼠胚胎植入前胚胎干细胞系的推导

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

发育生物学

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

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

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

使用冷冻胚胎进行转基因小鼠高效生产

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

遗传学

Minimum Volume Vitrification of Immature Feline Oocytes

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

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

未成熟费林卵母细胞的最小体积玻璃化

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

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

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

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

小鼠卵母细胞显微注射，成熟和倍性评估

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

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

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 ...

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 ...

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.

鼠胚体外发育无血清全胚胎培养系统

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

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...