Name: collection and cryopreservation of hamster oocytes and mouse embryos
Uploaded: 2009-03-27T00:00:00
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。 </l...

<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?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French type mini-straw 0.25 ml</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>13407/0010</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Farma-Lepori, Spain</td>
<td>749036.4</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-3884</td>
<td>&nbsp;</td>
<tr>
<td>KSOM-H medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref. [11]</td>
</tr>
<tr>
<td>Liquid Nitrogen </td>
<td>&nbsp;</td>
<td>Air Liquide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic plugs</td>
<td>&nbsp;</td>
<td>Mini-tub</td>
<td>19046</td>
<td>&nbsp;</td>
<tr>
<td>PMSG</td>
<td>&nbsp;</td>
<td>Intervet, Spain</td>
<td>1.614/8447</td>
<td>&nbsp;</td>
<tr>
<td>Propilenglycol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>82280</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>107687</td>
<td>&nbsp;</td>
<tr>
<td>Surgical Scissors</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BC-060R; BG-061R</td>
<td>&nbsp;</td>
<tr>
<td>Thermo-sealer</td>
<td>&nbsp;</td>
<td>SIZ</td>
<td>220</td>
<td>&nbsp;</td>
<tr>
<td>35 and 90 mm culture dishes</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>153066; 150350</td>
<td>&nbsp;</td>
<tr>
<td>10 ml tubes</td>
<td>&nbsp;</td>
<td>Greiner Bio-one</td>
<td>163160</td>
<td>&nbsp;</td>
<tr>
<td>Nitrogen tank</td>
<td>&nbsp;</td>
<td>MVE, Cryogenics</td>
<td>XC 43/28</td>
<td>&nbsp;</td>
<tr>
<td>Programmable Freezer</td>
<td>&nbsp;</td>
<td>Planner Products Ltd.</td>
<td>Kryo-10 Series III</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Aesculap</td>
<td>BD-331R; BD-053R; OC-020R</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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 ...

Research

JoVE Journal

Biology

Nuclear Transfer into Mouse Oocytes

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

Dissection of 6.5 dpc Mouse Embryos

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 ...

Retrieval of Mouse Oocytes

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 ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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 ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Microinjection of Xenopus Laevis Oocytes

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Seawater Sampling and Collection

海水采样和收集

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

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 ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

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 ...