这里介绍的研究得到了查尔斯河。作者是基因工程模型和服务组，动物保健和激素注射胚胎的工作人员非常感谢。

1。小鼠精子冷冻保存<ol><li>对于每个冻存线，准备开始之前以下。 <ol><li> 1以及一个240升的试剂盒注册会计师4以及菜</li><li> 15精子冷冻保存如下吸管<ol><li>将0.25毫升吸管1毫升注射器，用注射器最接近的棉塞。在每个稻草，装入8厘米的试管培养基（约160μL），那么1厘米的空气。标签与行的名称被保留的救命稻草。 </li></ol></li></ol></li><li> 2号法律公告应该是15厘米深的泡沫箱。关闭盖子。 </li><li>安乐死从被冷冻保存的每行2个雄性小鼠。小鼠应在10和60周龄之间。取出两个尾鳍从每个鼠标输精管附睾。 </li><li>放入先前准备好的含注册会计师以及4盘（步骤1.1）的附睾。 </li><li>请在每个附睾5-7纵向切割，轻轻地挤压每个输精管推精子。 </li><li>动摇手工注册会计师和附睾的混合物在室温下轻轻地为3-5分钟。这将使精子游出。 </li><li>使用先前准备好的稻草，吸精子悬液5毫米（约10μL），然后1厘米的空气。 </li><li>重复这个过程中，共15个秸秆。装入密封最好的精子存活10分钟内的秸秆。 </li><li>热封的秸秆双方认真。第一次与精子列放置在冷冻罐的吸管。 </li><li>将1毫升吸管罐来延长处理套件的一部分提供。在泡沫箱，通过盖子上的孔放置在液氮罐。允许罐浮在LN 2，垂直为10分钟。 </li><li>沉浸在液氮罐。精子冷冻保存，可以转移到长期的液氮存储。 </li><li>不应该被用于罐下一行，直到它被加热到室温。 </li></ol> 2。小鼠超数排卵<ol><li>一般来说，年轻的雌性小鼠的精子捐献者使用相同的背景。 </li><li>小鼠应〜12克（年龄3-4周）。 </li><li>执行与腹腔注射孕马血清促性腺激素（PMSG）5-10 IU。 </li><li>等待46-48小时。 </li><li>执行与腹腔注射人绒毛膜促性腺激素（hCG）5-10 IU。 </li><li>收获的卵母细胞14-16 h后注射HCG如下所述。 </li></ol> 3。小鼠精子复苏和试管婴儿<ol><li>试管婴儿之前，为每个要解冻的秸秆，准备以下至少一小时在37℃CO 2培养箱平衡。 <ol><li>精子孵化盘：35毫米培养皿中，用90μL的精子处理液油覆盖（STM）的下降。 </li><li>切割菜：对于卵母细胞的收集最多10秒uperovulated女性，一个35毫米的Petri碟3毫升试管培养基。 </li><li>试管婴儿：对于每个组超排5位女性的试管婴儿，试管培养基500μL（如果您使用的是10超排的女性，例如，你将需要2井。）4以及菜</li><li>洗衣机洗涤每孔4以及用于体外受精菜盘：3毫升的钾单纯优化培养基（KSOM）一个35毫米的培养皿</li><li>培养皿：一个35毫米的Petri涵盖了从每个试管婴儿菜以及胚胎培养油的KSOM培养基50μL滴的菜。 </li></ol></li><li>从2号法律公告中取出的秸秆和地方在37℃水浴2-3分钟。 </li><li>从水浴中取出的秸秆和擦拭以除去多余的水分。请首次下调接近最近的棉塞的第一个空气柱。削减在试管培养基稻草，仍然会出现精子列前可见一个空气柱。 </li><li>然后切的秸秆年底在Second空气柱，小心，以避免削减精子列。在一个45度角的切割 - 这将使它更容易吸出精子。 </li><li>从末端的秸秆用200μL移液器吸精子悬液。 </li><li>广场中心的STM下降精子悬液，在37℃，5％的CO 2培养箱中孵育45-60分钟的菜。 </li><li>在精子孵育，解剖切割菜的超排女性输卵管。使用30克针或镊子撕每个输卵管的卵丘卵母细胞复合体（卵母细胞）释放。避免脏媒体额外的洗涤步骤，使得在体外受精中的脂肪和血液。 </li><li>一旦所有的输卵管已被撕裂，转让的卵母细胞，女5例，每个试管婴儿井。通过超数排卵产生的卵母细胞的数量是非常应变依赖。 </li><li>收集从周边的STM下降10-15μL精子和受精之一试管婴儿约14-16小时后绒毛膜促性腺激素注射ection。每口井使用重复施肥。最后精子浓度将约1万个/毫升。 </li><li>共培养精子和卵母细胞在37℃5％ 二氧化碳培养箱中培养4-6小时。 </li><li>洗净的胚胎，KSOM一个最低的2倍和文化发展的KSOM滴在37℃5％二氧化碳培养箱。 </li><li>两种细胞阶段的胚胎，培养过夜后使用总卵母细胞体外受精率计算。 </li></ol> 4。代表性的成果从5查尔斯河小鼠近交系的精子被冻结。保护比率的计算方法，新鲜精子评估值除以冷冻精子的评估值。精子活力的保护率分别为60.2％，81.3％，78.6％，66.5％，61.0％，129S2/SvPasCrl，FVB / NCrl，DBA/2NCrl和BALB / cAnNCrl C57BL/6NCrl，分别。快速进展蠕动的保护率分别为47.3％，69.4％，57.0％，52.8％，54.1％，129S2/SvPas C57BL/6NCrl<html> CRL，FVB / NCrl，DBA/2NCrl，和BALB / cAnNCrl，分别（图1）。 冻融精子体外受精率分别为55.7％，26.4％，87.3％，87.8％和26.2％C57BL/6NCrl，129S2/SvPasCrl，FVB / NCrl DBA/2NCrl，和BALB / cAnNCrl，分别为（图2） 。 超过1.5年期间，49通用鼠标线是由精子冷冻保存归档。这些生产线后来被成功回收（定义活幼仔出生）试管婴儿在女4株（C57BL / 6，BALB / C中，DBA / 1和129Sv）（图3）。 <img alt="图1。" src="/files/ftp_upload/3713/3713fig1.jpg" /> 图1的小鼠的精子活力和快速运动力的保护率。 <img alt="图2。" src="/files/ftp_upload/3713/3713fig2.jpg" /> 图2。冻融小鼠精子体外受精3713fig3.jpg“/&gt; 图3。冻融在转基因小鼠的精子在体外授精

<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>

作者查尔斯河，该试剂盒的制造商的员工。

自1990年以来，基本精子冷冻协议，使用18％的棉子糖和3％脱脂牛奶已被证明是可靠的小鼠多株和大量的鼠标线已冻存1,2,3,4,5,6,7。已与冰的形成8,9和活性氧10的损害精子细胞在冻融过程中的主要成因。补充自由基清除剂，如氨基酸，还原剂，如monothioglycerol在冻结介质，进行了调查，证明大幅提高冻融精子包括在近交系C57BL / 6 11,6的体外受精率。 在本议定书中，我们开发了一个新的I•冷冻试剂盒小鼠精子冷冻保存，通过优化与市售的抗氧化剂和冰阻滞剂的基本注册会计师和修改精子冷冻和体外受精过程。精子活力的保护率（&gt; 60％）和快速进步的活力（&gt; 45％），分别从5个近交系小鼠株，与我•低温套件冷冻精子。的2 -细胞期胚胎解冻精子在5小鼠近交系的发展速度明显提高相比，与注册会计师的基本 11,6 。 用户在使用试剂盒，由冷冻精子的每一行从2男性集中的精子悬液。只冻结15吸管的过程是快速，简便，占地少短期或长期存储的存储空间。大多数时候，鼠标线可以恢复解冻只有1或2个秸秆。

<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

转基因和基因敲除技术问世以来，已经创建了新的转基因（GM）株小鼠的数千。许多这些珍贵的动物只存在于活的动物，没有在紧急情况下的备份计划。胚胎冷冻保存，可以提供这样的备份，但费用昂贵，可以是一个漫长的过程，一般需要大量的动物成功。由于小鼠的精子，可以成功地与基本cryoprotective代理（CPA）的18％的棉子糖和3％脱脂牛奶组成冻存发现，精子冷冻保存已经成为一个可以接受的和具有成本效益的过程，归档，分发和回收这些珍贵的菌株。 在这里，我们展示了新开发的我•小鼠精子冷冻保存冷冻套件。从五个常用株近交系小鼠的精子被冻结使用这种试剂盒，然后恢复。精子活力更高的保护率（&gt;看到这种试剂盒在5近交系小鼠品系冷冻精子60％）和快速进步的活力（&gt; 45％）相比，控制（基本注册会计师）。一直在研究所有5个鼠标株两细胞阶段胚胎试管婴儿后恢复精子的发展进行了改进。超过1.5年期，49通用鼠标线精子冷冻保存归档与我•低温套件，后来通过体外受精收回。

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

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

在这里，我们展示了新开发的我•小鼠精子冷冻保存冷冻套件。使用本试剂盒在5个小鼠品系，两个解冻的精子细胞阶段胚胎发育的持续改善。超过1.5年期间，49转基因小鼠精子冷冻保存归档与我•低温套件，后来通过试管婴儿成功收回。

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

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Biology

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

39.4K 次观看.  Charles River. 在这里，我们展示了新开发的我•小鼠精子冷冻保存冷冻套件。使用本试剂盒在5个小鼠品系，两个解冻的精子细胞阶段胚胎发育的持续改善。超过1.5年期间，49转基因小鼠精子冷冻保存归档与我•低温套件，后来通过试管婴儿成功收回。

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

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.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

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

科研

生物学

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.

免疫组化：石蜡切片从Vector实验室使用Vectastain ABC试剂盒

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

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

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.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

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.

使用Sigma的提取物N -安培组织PCR试剂盒小鼠组织的快速基因分型

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.

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 ...

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.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

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.

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 ...

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.

使用软件和冷却装置进行鱼类精子评估

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

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

密度梯度离心法测定微分品质的精子

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...