Name: using mouse oocytes to assess human gene function during meiosis i
Uploaded: 2018-04-10T15:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Using Mouse Oocytes to Assess Human Gene Function During Meiosis I at JoVE.com

这项工作得到了来自美国生殖医学协会的研究补助金, 以及来自罗格斯大学的查尔斯和乔安娜. 布希纪念基金的支持, 新泽西州的 k.s.a. L.N. 得到了美国 (F31 HD0989597) 的资助。

1. 分子克隆
<ol><li>获得感兴趣基因的全长编码序列和质粒体外转录 (pIVT) 向量11。 注: 全长 cDNA 克隆可从各种供应商处获得, 也可通过反向转录聚合酶链反应 (RT PCR) 产生。国家生物技术信息中心 (NCBI) 在线资源提供核苷酸和表达序列标记 (EST) 数据库的基因转录序列。为了便于蛋白质可视化和表达水平分析, 克隆策略中可能需要融合到绿色荧光蛋白 (Gfp) 或其他合适的荧光标记。</li>
	<li>使用验证的克隆策略, 将感兴趣基因的编码序列插入 pIVT 的多个克隆区域。对分子克隆和所需步骤的详细描述以前已被描述为12。 注: 如果要生成基因融合产品, 请确保克隆在正确的阅读框架中。</li>
	<li>使用球蛋白引物对构造进行序列, 以确保正确的基因插入, 适当的帧内融合, 并没有产生聚合酶诱发的点突变。 注: 如果不提供桑格 dna 测序设备, 许多公司提供低成本的 dna 测序选项。</li>
	<li>Mutagenize DNA, 如果生成单核苷酸多态性 (SNPs) 或插入/删除 (INDELS)。DNA 突变在下面的步骤 1.5-1.7 中描述。对于野类型构造, 请继续线性化步骤1.7。</li>
	<li>设计诱变引物, 完成诱变 PCR 反应, 生成每个制造商协议的突变构造。PCR 介导的站点定向诱变以前曾被描述为13。此外, 许多公司提供网站定向的诱变套件, 其中包括协议。</li>
	<li>将 mutagenized DNA 转化为细菌宿主。分离纯化质粒。 注: 许多公司制作的套件, 可以用来方便地分离和纯化质粒 DNA。相应地遵循制造商协议。如果使用革兰阴性细菌菌株, 如大肠杆菌, 建议使用去除内毒素的试剂盒。</li>
	<li>使用标准的转化 dna 序列来确定分离的 dna 来自细菌的基因组, 以确认所需的 SNP 或 INDEL 的引入。</li>
	<li>进行线性化最终产品采用单限制酶消化纯化的 DNA。 注: pIVT 向量包含多个单酶切割点, 在 Addgene 14的质粒图中列出。</li>
	<li>确保产品通过琼脂糖凝胶电泳进行线性化。琼脂糖凝胶电泳方法以前已被定义为14。 注: 对于所有剩余的步骤, 使用屏障 (过滤器) 吸管提示和无 RNase 材料, 以确保生产稳定, 高质量的 RNA。</li>
	<li>使用经过验证的 DNA 清除协议或试剂盒纯化所消化的产品, 并在30µL RNase/DNase 水中洗脱。 注: 推荐采用硅基自旋柱基套件, 用于高质量、纯化的 DNA。相应地遵循制造商协议。</li>
	<li>
体外根据制造商的协议, 使用 T7 聚合酶转录 DNA。</li>
	<li>用硅基自旋柱基核酸纯化试剂盒在制造商的协议之后, 纯化和洗脱 RNA 在30µL 的 RNase/DNase 游离水中。</li>
	<li>用甲醛进行变性琼脂糖凝胶电泳, 确定最终的 RNA 产品尺寸。请参见14下的步骤 1.13-1. 22。(图 1)。</li>
	<li>在72毫升水中加热1克琼脂糖, 直到溶解, 然后冷却到60摄氏度。</li>
	<li>通过添加0.4 米拖把 (pH 7.0), 0.1 米醋酸钠和0.01 米 EDTA, 准备10X 拖把运行缓冲器。</li>
	<li>加入10毫升的10X 拖把运行缓冲器, 18 毫升37% 甲醛 (12.3 米) 的冷却琼脂糖溶液。</li>
	<li>将琼脂糖溶液倒入凝胶容器中, 使凝胶形成种姓。用梳子大到足以容纳10µL。凝胶设置后, 手感牢固, 轻轻取下梳子。</li>
	<li>将凝胶在电泳槽中组装, 加入足够的1X 拖把运行缓冲器, 确保凝胶完全覆盖。</li>
	<li>将1µg rna 添加到0.5X 容量的含甲醛负载染料中, 制备 rna 样品。</li>
	<li>在10µg/毫升浓度的 RNA 溶液中加入溴溴。</li>
	<li>变性 RNA 样品溶液在70°c 5 分钟。</li>
	<li>使用无菌, 10 µL 过滤器小贴士, 5 µL 分子量标记和5µL 的 RNA 样品 (s) 成单独的水井的凝胶和 electrophorese 在 5 V/厘米, 直到染料迁移至少2/3 的凝胶长度。</li>
	<li>在 UV 反式照明灯上可视化凝胶中的 RNA。通过比较分子量标记, 确保 RNA 的大小是正确的。</li>
	<li>为了测量 RNA 的浓度和纯度, 在紫外分光光度计上使用无菌10µL 过滤器将1.2 µL 纯化 rna 转移。 请注意, 高质量的 RNA 应该分别有 A260/280 (1.8-2.2) 和 260/230 (> 1.7) 的吸光度率。</li>
	<li>使用10µL 过滤器提示, 整除剩余的纯化 RNA 成无菌0.5 µL 离心管 (3-5 µL/管)。贮存管在-80 摄氏度, 直到准备注射。 注: 最终注射浓度为500µL 是最佳的起始点。建议在显微注射前稀释 1:2 RNase 游离 H20 中的 rna, 以减少微量注射吸管中 rna 的粘性。</li>
</ol>2. 动粒微管附着物测定
<ol><li>将卵母细胞分成相等的组进行显微注射。 注: 卵母细胞的收集、转移、显微注射和减数分裂成熟已在朱庇特以前的15中详细描述。</li>
	<li>Microinject 一组与实验性 mRNA 和其他组 (s) 与重量控制和 PBS 或Gfp mRNA。 注: 500 ng/µL 是建议的注射浓度从10开始。picoinjector 可用于控制注入量。建议使用 5-10 pL 的注入量15。</li>
	<li>使用手或嘴操作的吹打器具, 玻璃吸管的开口稍大于卵母细胞 (100-150 µm) 的直径, 将卵母细胞转移到胚胎质量矿物油覆盖的培养皿中的米力农培养基中的100µL 下降在 5% C02中孵化7小时37摄氏度的卵母细胞, 以使减数分裂的中期有足够的成熟 i (见 i) 15。</li>
	<li>孵化后, 使用与上述相同的吸管装置, 将卵母细胞转移到中心井器官培养皿中, 在中心井和500µL H20 在µL 中含有700的预冷最低基本介质 (内存) 收集介质, 并把盘子放在冰上7分钟。 注: 建议在治疗卵母细胞之前, 先将记忆介质和中心好的器官培养皿放在-20 摄氏度, 至少20分钟。</li>
	<li>用吸管装置将卵母细胞转移到400µL 2% 多聚甲醛的透明玻璃斑块的井中, 室温下进行20分钟固定。</li>
	<li>使用相同的吸管装置, 将卵母细胞转移到另一井中, 在含有400µL 阻塞溶液 (PBS + 0.3% BSA + 0.01% Tween-20 + 0.02% 南3) 的现货板上, 并存储在4°c, 直到准备好处理染色。 注: 用于将卵母细胞贮存在4摄氏度, 用 parafilm 盖上玻璃斑片以防止蒸发。</li>
	<li>使用相同的吸管装置, 在含有400µL 通透溶液 (PBS + 0.3% BSA + 0.1% TritonX-100 + 0.02% 南3) 的现货板上将卵母细胞转移到新井中, 并在室温下孵化20分钟. 迅速通过三转移卵母细胞滴400µL 堵塞溶液去除任何残留洗涤剂。 注: 一个9清楚的玻璃斑板工作良好的移动组通过多种处理, 在一个单一的菜 (步骤 2.4-2.5)。</li>
	<li>在湿室中执行剩余的免疫细胞化学程序, 在室温保护下不受光照。使用一个中型塑料容器衬里与潮湿的纸巾和覆盖铝箔。</li>
	<li>使用吸管装置, 将卵母细胞转移到96井板盖上的30µL 堵塞液, 在加湿室内放置10分钟。 注: 水滴放在盘子上的圆形磨损中。</li>
	<li>使用吸管装置, 将卵母细胞转移到含有抗 centromeric 抗原 (ACA、波峰) (一点半) 和抗乙酰蛋白 (1:1000) 的30µL 阻塞溶液中, 并至少1小时进行孵化, 以标记着丝粒和主轴。</li>
	<li>将卵母细胞通过三30µL 滴溶液转移至10分钟, 以冲洗卵子。</li>
	<li>使用吸管装置, 将卵母细胞转移到30µL 的阻断溶液中, 其中含有继以上抗体 (抗人 igg 1:200、抗鼠 igg 1:200) 和孵育1小时的免疫球蛋白。</li>
	<li>按照步骤2.11 中的说明, 重复30µL 阻塞解决方案中的3、10分钟漂洗步骤。</li>
	<li>使用吸管装置将卵母细胞转移到3-5 µL 安装介质中, 其中含有 DAPI (5.9 µg/µL) 在玻璃显微镜上滑动。</li>
	<li>放置4小滴 (针头大小) 的石油果冻在角落的盖子滑动, 以防止粉碎的卵母细胞。</li>
	<li>轻轻地把盖子滑到油果冻边放到滑梯上。</li>
	<li>用透明指甲油密封盖玻片边缘, 并允许干燥5分钟。</li>
	<li>存储幻灯片在4°c, 保护不受光, 直到处理通过共焦显微镜。 注: 确保安装介质的折射率和盖玻片, 符合所使用的显微镜目标。推荐的安装材料以前已被描述为15。</li>
	<li>图像卵母细胞在共焦显微镜上使用40-63x 目标, 在 z 平面捕获小步骤 (≤1µm), 为工作目标进行优化, 并对中期主轴的整个区域进行图像处理。 注: 确保根据步骤2.11 中使用的特定辅助抗体对所有3通道进行图像处理。图像平均≥4和变焦3是理想的足够的分辨率。1µm 步长提供了小鼠卵母细胞微管和动粒的清晰可视化。图像捕获的过程从显微镜到显微镜都不同。有关如何配置显微镜设置的具体步骤, 请参阅制造商共聚焦用户指南。</li>
	<li>在下面的步骤 2.21-2. 23 中, 使用成像软件识别动粒微管附件状态。 注: 以下步骤使用图像 J (NIH) 免费下载软件描述此过程, 但是可以使用备用成像软件。附件状态类型以前已定义为16 。</li>
	<li>通过将文件拖到图像 J 工具栏中打开图像。</li>
	<li>在打开的 z 系列中, 通过单击 "颜色" 子选项卡下的 "图像" 下拉菜单中的 "合并通道", 使所有通道可见 (DNA、动粒和主轴)。</li>
	<li>使用图像底部的切换, 移动每个 z 切片并确定动粒微管附件。详细的附件说明以前已被描述为16
</li>
</ol>3. 染色体对准挑战测定
<ol><li>根据步骤2.3 中所述的吸管装置, 将卵母细胞转移到100µL 的米力农培养基中, 并孵化卵母细胞7小时, 以使减数分裂的中期得以充分成熟 (见 i), 如上文步骤 2.3 17 中所述. 注: 卵母细胞的收集、显微注射和成熟过程概述了以前的15。有关控制和 RNA 显微注射浓度的步骤 2.1-2.2, 请参阅附件检测协议。</li>
	<li>使用相同的吸管装置, 将卵母细胞转移到一个中心井器官培养皿中, 其中含有700µL 培养基, 含有 monastrol [100 µM] 在中心井和400µL H20 在周围环和孵化在37°c 为 2 H。</li>
	<li>通过转移卵母细胞, 用上述的吸管装置冲洗出 monastrol, 通过三滴100µL CZB 培养基, 然后将卵母细胞转移到含有700µL 培养基 + MG132 [5 µM] 的中心-井器官培养皿中, 3 h。环。将400µL H20 添加到外圈。</li>
	<li>用吸管装置将卵母细胞转移到400µL 2% 多聚甲醛的透明玻璃斑块中, 室温下进行20分钟修复。</li>
	<li>固定后, 使用吸管装置将卵母细胞转移到一个含有400µL 阻塞溶液的透明玻璃斑块的新井中, 贮存在4摄氏度, 直到染色处理。 注: 用于将卵母细胞贮存在4摄氏度, 用 parafilm 盖上玻璃斑片以防止蒸发。</li>
	<li>Permeabilize 和标签卵母细胞通过免疫化学, 如步骤 2.6-2.16 所述。</li>
	<li>图像卵母细胞在共焦显微镜上使用40-63x 目标, 捕捉 < 1 µm 步骤在 z 平面, 确保图像的整个区域的中期主轴。</li>
	<li>用成像软件识别染色体对准状态打开图像文件。 注: 以下步骤使用图像 J (NIH) 免费下载软件描述此过程, 但是可以使用备用成像软件。</li>
	<li>将图像拖动到图像 J 控制面板中。</li>
	<li>在打开的 z 系列中, 使用 "点" 工具 (可通过单击图像 J 控制栏上的点工具图标), 通过将工具放置在图像中的主轴杆上并单击图像, 在各自的 z 切片上标记每个主轴柱尾的点。通过按键盘上的命令 + t 将这些点添加到 "感兴趣区域" (ROI) 管理器中。</li>
	<li>通过突出显示 ROI 管理器中的特定点并单击 "度量值" 按钮, 确定步骤3.10 中设置的每个位置的特定坐标。这将为每个点提供一个包含 x 和 y 坐标的结果表。这些将分别是 X1、Y1 和 X1、Y2 分。</li>
	<li>接下来, 确定真正的 Z 坐标。这是通过将特定 z 切片的切片数乘以在其中识别主轴极点 (即切片 #2 6), 每个单独点都由堆栈粗细 (即1 µm) 来实现 (例如: 切片 2 x 1 µm = 2)。这些将分别是 Z1 和 Z2 点。</li>
	<li>使用毕达哥拉斯定理方程 (2 + b2 = c2) 用以前定义的 x、y 和 z 坐标从步骤 3.11-3. 13 确定主轴长度。对于每个主轴, 最终长度等于: √ (X1-X2)2+ (Y1 Y2)2+ (Z1 Z2)2)</li>
	<li>确定主轴 midzone。计算为主轴长度的一半。使用图像 j 中的线条工具, 通过单击图像 j 工具栏中的线条图标, 绘制从一个主轴杆到主轴 midzone 的直线。长度将显示在图像 J 工具栏上。</li>
	<li>通过使用线测量工具, 通过评估主轴 midzone 的染色体距离来确定染色体对准状态。通过单击图像 J 工具栏中的线条图标, 可以使用直线工具, 从主轴 midzone 到染色体绘制一条直线。长度将显示在图像 J 工具栏上。从主轴 midzone 中超过4µm 的染色体被视为齐18。</li>
</ol>

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocyst+biopsy+with+comprehensive+chromosome+screening+and+fresh+embryo+transfer+significantly+increases+in+vitro+fertilization+implantation+and+delivery+rates:+a+randomized+controlled+trial.">Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.</a> Fertil Steril. 100 (3), 697-703 (2013).</li><li>Scott, R. T., Upham, K. M., Forman, E. J., Zhao, T., Treff, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage-stage+biopsy+significantly+impairs+human+embryonic+implantation+potential+while+blastocyst+biopsy+does+not:+a+randomized+and+paired+clinical+trial.">Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial.</a> Fertil Steril. 100 (3), 624-630 (2013).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+single+blastocysts+for+fresh+transfer+via+standard+morphology+assessment+alone+and+with+array+CGH+for+good+prognosis+IVF+patients:+results+from+a+randomized+pilot+study.">Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.</a> Mol Cytogenet. 5 (1), 24 (2012).</li><li>Rubio, C., et al. <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+preimplantation+genetic+diagnosis+for+aneuploidies+in+advanced+maternal+age:+a+randomized,+controlled+study.">In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.</a> Fertil Steril. 107 (5), 1122-1129 (2017).</li><li>Nguyen, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+Aurora+Kinase+B+and+C+variants+associated+with+maternal+aneuploidy.">Identification and characterization of Aurora Kinase B and C variants associated with maternal aneuploidy.</a> Mol Hum Reprod. , (2017).</li><li>Igarashi, H., Knott, J. G., Schultz, R. M., Williams, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+PLCbeta1+in+mouse+eggs+change+calcium+oscillatory+behavior+following+fertilization.">Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization.</a> Dev Biol. 312 (1), 321-330 (2007).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE. , (2017).</li><li>Carey, M. F., Peterson, C. L., Smale, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-mediated+site-directed+mutagenesis.">PCR-mediated site-directed mutagenesis.</a> Cold Spring Harb Protoc. 2013 (8), 738-742 (2013).</li><li>Armstrong, J. A., Schulz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols Essential Laboratory Techniques. , (2008).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+oocyte+microinjection,+maturation+and+ploidy+assessment.">Mouse oocyte microinjection, maturation and ploidy assessment.</a> J Vis Exp. (53), (2011).</li><li>Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+and+force+behind+kinetochore+orientation:+lessons+from+meiosis.">Geometry and force behind kinetochore orientation: lessons from meiosis.</a> Nat Rev Mol Cell Biol. 13 (6), 370-382 (2012).</li><li>Shuda, K., Schindler, K., Ma, J., Schultz, R. M., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+kinase+B+modulates+chromosome+alignment+in+mouse+oocytes.">Aurora kinase B modulates chromosome alignment in mouse oocytes.</a> Mol Reprod Dev. 76 (11), 1094-1105 (2009).</li><li>Lane, S. I., Yun, Y., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+anaphase-promoting+complex+activation+in+mouse+oocytes+is+predicted+by+microtubule-kinetochore+attachment+but+not+by+bivalent+alignment+or+tension.">Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension.</a> Development. 139 (11), 1947-1955 (2012).</li><li>Nguyen, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+threonine+3+on+histone+H3+by+haspin+kinase+is+required+for+meiosis+I+in+mouse+oocytes.">Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes.</a> J Cell Sci. 127 (Pt 23), 5066-5078 (2014).</li><li>Tsafriri, A., Chun, S. Y., Zhang, R., Hsueh, A. J., Conti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+maturation+involves+compartmentalization+and+opposing+changes+of+cAMP+levels+in+follicular+somatic+and+germ+cells:+studies+using+selective+phosphodiesterase+inhibitors.">Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors.</a> Dev Biol. 178 (2), 393-402 (1996).</li><li>Kapoor, T. M., Mayer, T. U., Coughlin, M. L., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+spindle+assembly+mechanisms+with+monastrol,+a+small+molecule+inhibitor+of+the+mitotic+kinesin,+Eg5.">Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.</a> Eg5. J Cell Biol. 150 (5), 975-988 (2000).</li><li>Jones, K. T., Lane, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+causes+of+aneuploidy+in+mammalian+eggs.">Molecular causes of aneuploidy in mammalian eggs.</a> Development. 140 (18), 3719-3730 (2013).</li><li>Fellmeth, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+three+Aurora+kinase+C+splice+variants+found+in+human+oocytes.">Expression and characterization of three Aurora kinase C splice variants found in human oocytes.</a> Mol Hum Reprod. 21 (8), 633-644 (2015).</li><li>Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+cold-stable+kinetochore+fiber+in+metaphase+PtK1+cells.">The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells.</a> Chromosoma. 84 (1), 145-158 (1981).</li><li>Brunet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+fibers+are+not+involved+in+the+formation+of+the+first+meiotic+spindle+in+mouse+oocytes,+but+control+the+exit+from+the+first+meiotic+M+phase.">Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase.</a> J Cell Biol. 146 (1), 1-12 (1999).</li><li>Joung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-Cas9+knockout+and+transcriptional+activation+screening.">Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.</a> Nat Protoc. 12 (4), 828-863 (2017).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li></ol>

作者没有什么可透露的。

由于与疾病有关的人类遗传变异的快速和不断增加的识别, 必须建立系统来评估其生物学意义。了解人类减数分裂中的蛋白质功能会带来特殊的挑战, 因为人类卵母细胞是珍贵的, 稀有的, 人类的精子不能服从基因的操纵。小鼠卵母细胞是哺乳动物模型系统, 对评估人类减数分裂基因功能有价值10,23。这个模型绕过了使用人类卵子的不切实际之处, 同时提供...

不孕是影响10-15% 人口的生育年龄1的一个条件, 近半数的人寻求医疗治疗2。虽然不孕的病因是多种多样的, 在许多情况下多因素, 最常见的遗传异常的人是胚胎体检3。体检被定义为细胞中正确数目的染色体的偏差 (增益或损耗)。体检在人类胚胎中的现象是常见的, 并随着高级孕产妇年龄的增长而增加4,5。四项随机对照试验突出了仅选择染色体正常 (euploid) 胚胎进行子宫移植的好处, 因为这一策略导致植入率增加, 流产率降低, 并且缩短了实现怀孕6,7,8,9。因此, 了解人类体检的病因学对辅助生殖有重要意义。
虽然植入前的 aneuploidies 基因检测对不孕症治疗是有益的, 但对 aneuploidies 起源的深入了解仍然缺乏。人们普遍认为, 减数分裂 aneuploidies (起源于配子生产期间) 与孕产妇年龄呈正相关, 然而, 有些妇女呈现出与其特定年龄的平均率相差4的胚胎体检率。这些病例表明, 单独年龄并不总是预示着产生异倍体胚胎的风险。其他因素可能会在增加胚胎体检的风险方面发挥作用, 如基因变异。
在卵母细胞减数分裂过程中, 研究基因变异对体检的潜在贡献的一个关键方面是设计一个快速评价减数分裂基因功能的系统。由于道德约束和有限的访问, 使用人类卵子进行这些实验是不切实际的。这些问题可以通过使用小鼠卵母细胞来规避, 这里描述了在减数分裂过程中评估人类基因功能的一系列检测方法。通过 microinjecting 的信使 RNA (mRNA) 编码的基因变异, 在小鼠卵中的人蛋白的定位可以可视化, 并用于确定野生型和变异人蛋白的异位表达是否导致任何可能导致体检的表型改变。这些表型包括增加微管, 附加到不适当的修女动粒和无法支持染色体排列在中期的减数分裂 i. 重要的是, 这个协议可以用来调查的增益和损失的功能遗传变异通过建立特定的实验条件来挑战卵母细胞减数分裂的关键事件, 如纺锤体构建和染色体对齐10。

<table><tbody><tr><td>0.2 mL Seal-Rite PCR tube</td><td>USA Scientific</td><td>1602-4300</td><td></td></tr><tr><td>1 kb DNA Ladder</td><td>Thermo Scientific</td><td>SM0313</td><td></td></tr><tr><td>100 bp DNA Ladder</td><td>Thermo Scientific</td><td>SM0243</td><td></td></tr><tr><td>6X DNA loading Dye</td><td>Thermo Scientific</td><td>R0611</td><td></td></tr><tr><td>9-well glass spot plate</td><td>Thomas Scientific</td><td>7812G17</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9539</td><td></td></tr><tr><td>Albumin from bovine serum&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3294&amp;nbsp;</td><td></td></tr><tr><td>Alexa-fluor-568 conjugated anti-mouse IgG</td><td>Thermo Scientific</td><td>A21050</td><td>1:200 dilution</td></tr><tr><td>Alexa-fluor-633 conjugated anti-human IgG</td><td>Thermo Scientific</td><td>A21091</td><td>1:200 dilution</td></tr><tr><td>Ampicillin</td><td>VWR</td><td>AA0356</td><td></td></tr><tr><td>Anti-vibration table</td><td>Technical Manufacturing Corp</td><td></td><td>any standard model</td></tr><tr><td>Anti-Acetylated Tubulin antibody</td><td>Sigma Aldrich</td><td>T7451</td><td>1:100 diution</td></tr><tr><td>Anti-centromeric (CREST) antibody</td><td>Antibodies Incorportated</td><td>15-234</td><td>1:30 dilution&amp;nbsp;</td></tr><tr><td>Barrier (Filter) Pipette tips</td><td>Thermo Scientific</td><td>AM12635</td><td>Make sure compatable with your brand of pipettors. These are compatible with Eppendorf brand pipettors.&amp;nbsp;</td></tr><tr><td>BD Difco Dehydrated Culture Media: LB Agar, Miller (Luria Bertani)</td><td>Fisher Scientific</td><td>DF0445-07-6</td><td></td></tr><tr><td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria Bertani)</td><td>Fisher Scientific</td><td>DF0446-07-5</td><td></td></tr><tr><td>Capillary tubing</td><td>Sutter</td><td>B100-75-10</td><td></td></tr><tr><td>Center Well organ culture dish</td><td>VWR</td><td>25381-141</td><td></td></tr><tr><td>CO2 tank</td><td></td><td></td><td>For incubator</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>any standard model</td></tr><tr><td>Centrifuge (With cooling ability)</td><td>Thomas Scientific</td><td></td><td>any standard model&amp;nbsp;</td></tr><tr><td>Cover Glass 11 x 22 mm</td><td>Thomas Scientific</td><td>6663F10</td><td></td></tr><tr><td>Coverslips</td><td>Thomas Scientific</td><td>6663-F10</td><td>thickness will vary for particular microscopes</td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>DEPC H20</td><td>Life Technologies</td><td>AM9922</td><td></td></tr><tr><td>Digital Dry Bath</td><td>Thermo Scientific</td><td>888700001</td><td></td></tr><tr><td>Easy A high fidelity cloning enzyme</td><td>Agilent</td><td>600400</td><td>For DNA cloning&amp;nbsp;</td></tr><tr><td>Enzymes for linearizing pIVT</td><td>New England Biolabs</td><td></td><td>NdeI or KasI can be used</td></tr><tr><td>Ethidium Bromide</td><td>Thermo Scientific</td><td>155585011</td><td></td></tr><tr><td>Fluorescent Microscope&amp;nbsp;</td><td></td><td></td><td>Any Fluorescent microscope may be used</td></tr><tr><td>Formaldehyde (37%)</td><td>Thermo Fisher Scientifc</td><td>9311</td><td></td></tr><tr><td>Formaldehyde RNA loading dye</td><td>Ambion</td><td>8552</td><td></td></tr><tr><td>Frosted Microscope Slides (Uncharged) 25X75 mm</td><td>Fisher Scientific</td><td>12-544-3</td><td></td></tr><tr><td>Full Length cDNA Clones</td><td></td><td></td><td>Can be obtained from any vendor that supplies open reading frame clones</td></tr><tr><td>Gel electrophoresis apparatus</td><td>Bio-Rad</td><td></td><td>any standard model</td></tr><tr><td>Glass Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-200</td><td></td></tr><tr><td>Globin Forward Primer for pIVT Construct</td><td></td><td></td><td>5&#039;- GAA GCT CAG AAT AAA CGC -3&#039;.&amp;nbsp; Can be purchased from any company that generates custom oligonucelotides</td></tr><tr><td>Globin Reverse Primer for pIVT Construct</td><td></td><td></td><td>5&#039;- ATT CGG GTG TTC TTG AGG CTG G -3&#039; Can be purchased from any company that generates custom oligonucelotides</td></tr><tr><td>Holding pipettes</td><td>Eppendorf</td><td>930001015</td><td>Vacutip</td></tr><tr><td>Humidified Chamber</td><td></td><td></td><td>Tupperware can be used</td></tr><tr><td>Illustra Ready-To-Go RT-PCR beads</td><td>GE Life Sciences</td><td>27925901</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td>any standard model with CO2 and water jacketed technology</td></tr><tr><td>Inverted Microscope</td><td>Nikon instruments</td><td></td><td>Any Standard model</td></tr><tr><td>Image J (NIH) Software</td><td>NIH</td><td></td><td>Image Analysis software</td></tr><tr><td>Lid of 96 well plate</td><td>Nalgene Nunc International</td><td>263339</td><td></td></tr><tr><td>Low Adhesion 0.5 mL microcentrifgue tube</td><td>USA Scientific</td><td>1405-2600</td><td></td></tr><tr><td>MacVector&amp;nbsp;</td><td>MacVector</td><td></td><td>Sequence analysis software</td></tr><tr><td>MG132</td><td>Selleckchem</td><td>S2619</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-544-3&amp;nbsp;</td><td></td></tr><tr><td>Millenium RNA Markers-Formaldehyde&amp;nbsp;</td><td>Ambion</td><td>AM7151</td><td></td></tr><tr><td>Milrinone</td><td>Sigma-Aldrich</td><td>M4659</td><td>Resuspend in DMSO at 2.5mM</td></tr><tr><td>Mineral Oil</td><td>Sigma-Aldrich</td><td>M5310</td><td>Only used embryo-tested, sterile-filtered</td></tr><tr><td>Monastrol</td><td>Sigma-Aldrich</td><td>M8515</td><td>Resuspend in DMSO at 100 mM</td></tr><tr><td>Mouthpiece</td><td>Biodiseno</td><td>MP-001-Y</td><td></td></tr><tr><td>N2 tank</td><td></td><td></td><td>for antivibration table</td></tr><tr><td>Nail Polish; Clear</td><td></td><td></td><td>Any clear nailpolish can be used</td></tr><tr><td>NanoDrop Microvolume UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>any standard model</td></tr><tr><td>NorthernMax 10X Denaturing Gel Buffer</td><td>Life Technologies</td><td>AM8676</td><td></td></tr><tr><td>NorthernMax 10X Running buffer</td><td>Life Technologies</td><td>AM8671</td><td></td></tr><tr><td>NuPAGE MOPS SDS Running buffer</td><td>Thermo Scnentific</td><td>NP0001</td><td></td></tr><tr><td>Organ Culture Dish 60x15mm</td><td>Life Technologies</td><td>08-772-12</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Polysciences, Inc.&amp;nbsp;</td><td>577773</td><td></td></tr><tr><td>PCR Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>4484075</td><td></td></tr><tr><td>Petri Dish 139 mm</td><td>Thermo Fisher Scientifc</td><td>501V</td><td></td></tr><tr><td>Petri dish 35 mm</td><td>Thermo Fisher Scientifc</td><td>121V</td><td></td></tr><tr><td>Petri Dish 60 mm</td><td>Falcon BD</td><td>351007</td><td></td></tr><tr><td>Picoinjector</td><td>XenoWorks Digital Microinjector</td><td></td><td>any standard model</td></tr><tr><td>Pipette puller</td><td>Flaming-Brown Micropipette puller</td><td>Model P-1000</td><td></td></tr><tr><td>pIVT plasmid</td><td>AddGene</td><td>32374</td><td>Empty vector suitable for oocyte expression.</td></tr><tr><td>Pregnant Mare Serum Gonadotropin</td><td>Lee BioSolutions</td><td>493-10</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td>purification of up to 20 uL of plasmid DNA</td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Quikchange II site directed mutagenesis kit</td><td>Agilent&amp;nbsp;</td><td>200523</td><td>mutagenesis kit for insertions and deletions</td></tr><tr><td>Quikchange lightning multi-site directed mutagenesis kit</td><td>Agilent&amp;nbsp;</td><td>210512</td><td>mutagenesis kit for single site changes</td></tr><tr><td>Scissors (Fine point)</td><td>Fine science tools</td><td>14393</td><td></td></tr><tr><td>Scissors (Medium point)</td><td>Fine science tools</td><td>WP114225</td><td></td></tr><tr><td>Seal-Rite 1.5 mL microcentrifuge tube</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>Slide Warmer</td><td></td><td></td><td>any standard model</td></tr><tr><td>Spectrophotometer (Nanodrop)</td><td>Thermo Fisher Scientific</td><td>ND-ONE-W</td><td></td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td>any standard model</td></tr><tr><td>Subcloning Efficiency DH5a Competent Cells</td><td>Thermo Fisher Scientifc</td><td>18265017</td><td></td></tr><tr><td>Syringe</td><td>BD Bioscienes</td><td>309623</td><td>1 ml, 27G(1/2)</td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>T7 mMessage Machine high-yield capped RNA transcription kit</td><td>Life Technologies</td><td>AM1340</td><td></td></tr><tr><td>TritonX-100</td><td>Sigma-Aldrich</td><td>x-100</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>274348</td><td></td></tr><tr><td>Tweezer (Fine point- Size 5)</td><td>Fine science tools</td><td>SN.743.12.1</td><td></td></tr><tr><td>UltraPure Dnase/Rnase-Free Distilled Water</td><td>Thermo Fisher Scientifc</td><td>10977015</td><td></td></tr><tr><td>UltraPure Ethidium Bromide 10mg/mL</td><td>Thermo Fisher Scientifc</td><td>15585011</td><td></td></tr><tr><td>UVP UV/White lite transilluminator</td><td>Fisher Scientific</td><td>UV95041501</td><td></td></tr><tr><td>Vectashield Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr></tbody></table>

using mouse oocytes to assess human gene function during meiosis i

胚胎体检是人类不孕症的主要遗传原因。这些事件大多来源于女性减数分裂, 尽管与孕产妇年龄呈正相关, 但年龄并不总是预示着产生异倍体胚胎的风险。因此, 基因变体可能会解释在卵子发生过程期间染色体分离的错误。鉴于对人类卵母细胞的访问有限的研究目的, 通过一系列的分析, 以研究人类基因功能在减数分裂 I 使用小鼠卵母细胞。首先, 基因的信使 RNA (mRNA) 和感兴趣的基因变异被杏仁入前期 I 被拘捕的老鼠卵母细胞。在允许表达时间后, 卵母细胞被同步释放到减数分裂成熟完成减数分裂 i。通过用荧光记者的序列标记 mRNA, 如绿色荧光蛋白 (Gfp), 除了表型改变外, 还可以评估人体蛋白的定位。例如, 可以通过建立挑战基因产物来修复减数分裂错误的实验条件来调查功能的增益或丧失。虽然这一系统有利于在卵子发生过程期间对人体蛋白质功能进行调查, 但由于蛋白质表达不在内源性水平, 而且除非受控(即敲), 小鼠同系物也存在于系统中。

在体外转录高品质的 RNA 将有一个 A260/A280 比 (1.8-2.2) 和 A260/A230 比≥1.7 时使用分光光度计。图 1中的图片显示了在电泳后变性琼脂糖凝胶上体外产生的 RNA 的迁移。在图案中涂抹的带或具有多个尺寸波段的样本可以指示样品的污染或降解。另外, 多波段可能表明 mRNA 不是完全变性, 或起始质粒没有完全线性化。mrna 的运行比预测的要大, 因?...

Watch this Scientific Journal Video about Using Mouse Oocytes to Assess Human Gene Function During Meiosis I at JoVE.com

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

随着与人类疾病相关的遗传变异开始显露出来, 开发系统以迅速评估那些被确定的变种的生物学意义变得越来越重要。本协议描述了在女性减数分裂过程中使用小鼠卵母细胞评估人类基因功能的方法。

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

using-mouse-oocytes-to-assess-human-gene-function-during-meiosis-i

Universidad de Cartagena UDEC

Education

Research

JoVE Core

JoVE Journal

Cell Biology

Molecular Biology

Genetics

Unit 2

Unit 1

Meiosis

SCIENTISTS IN ACTION

8.8K 次观看.  IVI-RMA New Jersey. 随着与人类疾病相关的遗传变异开始显露出来, 开发系统以迅速评估那些被确定的变种的生物学意义变得越来越重要。本协议描述了在女性减数分裂过程中使用小鼠卵母细胞评估人类基因功能的方法。

视频: Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

科研

遗传学

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

发育生物学

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

评估小鼠卵母细胞中纺锤体组件检查点完整性

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

鉴定小鼠前期停滞卵母细胞中的 DNA 损伤

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

作者聚焦：了解哺乳动物卵母细胞和植入前胚胎的 DNA 损伤反应 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

检测小鼠卵母细胞中的DNA双链断裂

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

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 ...

生物学

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 ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

成像前中期和减数分裂中期的我固定技术<em&gt;果蝇</em&gt;卵母细胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

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此视频中的章节

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

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