这项工作得到了研究院 (R01GM11755) 到 p.w. J 以及由国家癌症研究所 (CA009110) 向伦理和兰伯特提供的培训补助金奖学金的支持。

这里描述的所有方法都已得到约翰霍普金斯大学机构动物护理和使用委员会 (IACUC) 的批准。对野生型 C57BL/6J 小鼠进行了实验研究。
1. 采集胎儿或新生儿卵巢并制备前期染色质传播
<ol><li>提取胚胎在14.5–19.5 天后 coitum (dpc) 牺牲孕妇通过颈椎脱位或 CO2窒息根据 IACUC 的指导方针。 注: 产后1–5卵巢跳至步骤1.3。图 1总结了在不同胚胎和产后时代丰富的减数分裂前期阶段。</li>
	<li>用无菌剪刀打开盆腹腔结核腔, 形成 V 形开口。解剖出母体子宫角, 将胚胎与胎盘分离, 并将胚胎移植到35毫米培养皿中, 含3毫升预热的1x 磷酸盐缓冲盐水 (PBS), 37 摄氏度。 注: 一个 fliptop 孵化器设置在37°c 可以用来保持温度。此外, 温度调节玻璃的阶段也可以用来维持温度在卵巢操作。</li>
	<li>用3.5 英寸的手术剪刀, 根据 IACUC 的指导原则, 通过斩首牺牲胚胎或幼崽。在进一步解剖前, 将被斩首的胚胎或幼崽放置在预热的 PBS 中。</li>
	<li>一次解剖一个胚胎或小狗在一个单独的35毫米培养皿中含有3毫升的预热 PBS 在37摄氏度。按图 2A-B中概述的, 沿胚胎后半部的腹中线进行切割, 沿前肢的前半部分, 并直接位于后肢和尾部的上方。</li>
	<li>用3.5 英寸的手术剪刀打开腹部。使用细尖钳移位或移除肝脏和肠循环, 暴露卵巢在一个新的35毫米培养皿中含有3毫升的预热 PBS 在37摄氏度 (见指南在图 2B)。卵巢位于肾脏的下方和后面, 朝向腹膜腔的后壁 (图 2B-C)。在图 2D中提供了区分男性和女性性腺的指南。 注: 在最佳条件下, 在孕妇被牺牲后迅速解剖卵巢。</li>
	<li>从每个母胎中取出两个卵巢, 使用一对细尖钳在解剖范围内, 并放置在手表眼镜或一个多井板, 其中包含0.5 至1.0 毫升预热 PBS 和维持在37摄氏度。</li>
	<li>将每对卵巢放置在0.5 毫升的低萃取缓冲液中 (17 毫米柠檬酸三钠, 50 毫米蔗糖, 5 毫米 EDTA, 0.5 毫米, 30 毫米三盐酸, 蛋白酶抑制剂, pH 8.2) 在一个干净的手表玻璃或一个小井的9井板, 确保浸泡卵巢复合letely。孵育至少15分钟, 但不超过30分钟。 注: 在2小时内, 使低萃取缓冲器新鲜, 并使用。</li>
	<li>在孵化期间, 使用疏水性屏障 PAP 笔, 在干净的玻璃 25 x 75 毫米2, 1 毫米厚的显微镜幻灯片上绘制两个 22 x 22 毫米2方块, 如图 3A所示。 注意: 幻灯片可以提前准备好。</li>
	<li>吸管45–50µL 100 毫米蔗糖到干净的幻灯片和转移一对卵巢到下落。</li>
	<li>使用两个27克针的尖端, 逗卵巢分开, 释放细胞进入蔗糖溶液。用镊子, 取出大块的卵巢, 并小心地将蔗糖溶液移出分散的细胞。</li>
	<li>将40µL 固定液 (1% 多聚甲醛 (粉煤灰), 0.2% 洗涤剂 (参见材料表), pH 9.2) 放入准备好的幻灯片的每个正方形。用200µl 吸管尖端, 将固定液均匀地分布在滑动面上。</li>
	<li>吸管20µL 的蔗糖混合到每个正方形的幻灯片包含固定。 注意: 这等同于每张幻灯片使用一对卵巢。</li>
	<li>在室温下过夜, 在密闭潮湿的房间里孵化出滑梯。 注意: 夜间孵化可以在室温下 (12-15 小时) 左右。</li>
	<li>打开房间盖, 让幻灯片完全风干。</li>
	<li>在含有50毫升0.4% 润湿剂-PBS 解决方案 (参见材料表) 的 Coplin 罐子中, 清洗幻灯片2分钟, 风干, 然后进行 immunolabelling 协议 (步骤 6)。 注意: 可以将幻灯片存储在-80 摄氏度, 以便以后使用, 但这取决于感兴趣的蛋白质。为获得最佳结果, 请立即执行 immunolabelling 协议 (步骤 6)。</li>
</ol>2. 中期 I 卵母细胞收集
<ol><li>为了最大限度地增加从每只老鼠分离出的窦卵泡的数量, 腹腔注射性成熟的处女雌性小鼠与5内孕母马的血清促性腺激素 (PMSG, 也称为马绒毛膜促性腺激素 (eCG))。 注: 为了获得最佳卵母细胞产量, 老鼠的年龄应该是1到3个月, 因为卵母细胞的数量随着年龄的增加而减少。要准备 PMSG, 溶于100毫升的无菌 PBS (5 U/0.1 毫升) 存储在600摄氏度的单使用整除数-20 µL 瓶5000。</li>
	<li>44–48 h 后, 制备收集培养基, 最小必需培养基α (MEMα) 培养基, 辅以5% 胎牛血清 (血清) 和3毫克/毫升牛血清白蛋白 (BSA;MEMα/BSA/血清)。通过0.2 µm 孔过滤器消毒介质。醒酒在 5% CO2孵化器中, 将2.5 毫升的 MEMα/BSA/血清介质放入每只鼠标的一个玻璃盘中, 加热到37摄氏度。 注意: 其他商业上可用的收集和文化媒体 (M2 和 M16) 也经常使用。 注意: 我们建议将 5% CO2孵化器中的养殖菜肴一次性移除, 时间有限, 以尽量减少操作步骤中的环境空气暴露。</li>
	<li>根据 IACUC 指导原则, 通过宫颈脱位或 CO2进行窒息, 牺牲小鼠。</li>
	<li>用无菌剪刀打开盆腹腔结核腔, 形成大 V 形开口。用镊子, 将肠道移向老鼠头。定位每个子宫角;卵巢在肋骨笼的近端存在。使用解剖剪刀 (图 4), 用细钳握住输卵管, 并将脂肪切成卵巢。继续持有输卵管, 并与另一套罚款钳释放卵巢从囊和地方成收集皿含有 MEMα/BSA/血清。 注意: 在步骤1中, 必须将卵巢保持在37摄氏度, 并维持在 5% CO2。一个 fliptop 孵化器, 连接多达 5% CO2混合可用于允许最佳的维护温度和 CO2级别。另外, 在卵母细胞操作过程中, 还应使用温度调节玻璃舞台来保持温度。</li>
	<li>使用1毫升注射器与27口径针 (或类似的大小), 释放卵卵母细胞复合物手动穿刺大窦卵泡。</li>
	<li>在通过解剖显微镜观察时, 用嘴操作的玻璃吸管或毛细管, 或手操作的千分尺 (图 5) 来收集有时间的卵母细胞。只收集从窦卵泡释放的卵母细胞。µm 卵母细胞的直径约为 90. 可能会被2-3 层颗粒细胞包围, 总直径约为200µm。</li>
	<li>在一个干净的手表玻璃或9井板中培养卵母细胞, 其中含有 MEMα/BSA/血清中的6小时, 在 5% CO2孵化器中达到中期 I 37 摄氏度。</li>
	<li>继续执行步骤4。</li>
</ol>3. 中期二 (产业部) 卵母细胞收集
<ol><li>为了最大限度地提高与每只老鼠分离的卵母细胞, 腹腔注射 5 PMSG 的性成熟的处女雌性小鼠。44–48 h 后, 腹腔注射5人绒毛膜促性腺激素 (hCG)。 注: 为了获得最佳卵母细胞产量, 老鼠的年龄应该是1到3个月, 因为卵母细胞的数量随着年龄的增加而减少。要准备 hCG, 溶解一瓶 1万 u 在200毫升 PBS (5 U/0.1 毫升)。存储在单使用整除数600µL 在-20 摄氏度。</li>
	<li>12-14 小时后, 如上文步骤2.2 所述, 准备 MEMα/BSA/血清收集介质。</li>
	<li>根据 IACUC 指导原则, 通过宫颈脱位或 CO2进行窒息, 牺牲小鼠。</li>
	<li>用无菌剪刀打开盆腹腔结核腔, 形成大 V 形开口。用镊子, 将肠道移向老鼠头。定位每一个子宫角, 卵巢是存在近到肋骨笼。使用解剖剪刀 (图 4), 用细钳握住输卵管, 并将脂肪切成卵巢。然后将卵巢和输卵管移除, 放入含有 MEMα/BSA/血清培养基的收集皿中。 注意: 在步骤1和2中, 必须将卵巢保持在37摄氏度, 并维持在 5% CO2。一个 fliptop 孵化器, 连接多达 5% CO2混合可用于允许最佳的维护温度和 CO2级别。另外, 在卵母细胞操作过程中, 还应使用温度调节玻璃舞台来保持温度。</li>
	<li>使用1毫升注射器与27克 (或相似的大小) 或锋利的钳, 撕毁一个洞在腹部的输卵管释放的产业部卵母细胞。 注意: 小心不要破坏壶腹中的卵母细胞。壶腹将出现肿胀和半透明, 使卵母细胞可见 (图 4)。</li>
	<li>利用口腔操作的玻璃吸管或毛细管, 或手操作的千分尺 (图 5), 将输卵管的壶腹中的卵母细胞收获到一个新的盘子中。</li>
	<li>继续执行步骤4。</li>
</ol>4. 卵母细胞剥蚀和透明透明清除
<ol><li>在 MEMα培养基中制备300毫升的透明质酸酶, 辅以3毫克/毫升 BSA, 以观察玻璃中的掀卵母细胞 (2.5 毫升/小鼠), 并保持在37°c, 5% CO2。 注: 在1小时的准备后, 透明质酸酶的功效急剧下降。对于产业部卵母细胞, 不需要透明质酸酶治疗。</li>
	<li>将卵母细胞卵丘细胞复合物暴露在 MEMα/BSA 中的透明质酸酶的300毫升/mL 中, 掀周围卵丘细胞的卵母细胞的3分钟。 注意: 不要超过3分钟接触透明质酸酶, 因为它会损害卵母细胞。</li>
	<li>要清洗卵母细胞, 将卵母细胞转移到新鲜温暖的 MEMα/BSA 中菜。使用小口操作的玻璃吸管或毛细管 (略大于卵母细胞的直径, 大约90µm), 吸管的配合物上下完全分离卵丘细胞。在准备下一步时, 允许卵母细胞在孵化器中恢复。</li>
	<li>温酸性 Tyrode 的溶液, MEMα/BSA, Waymouth 的介质到37°c (500 µL/小鼠)。放置300µL 在每个井小9井玻璃板材。</li>
	<li>将5–10卵母细胞的透明透明 (ZP) 转移到 Tyrode 的溶液中。将卵母细胞暴露在溶液中只有30-45 秒。 注意: 太少暴露在溶液中不会完全去除 ZP, 这将防止卵母细胞爆裂和染色体传播。太多的暴露会伤害和杀死卵母细胞。观察 ZP 溶解在解剖显微镜下可以帮助优化暴露时间。 注意: 使用新鲜 Tyrode 的解决方案是至关重要的, 因为它的功能随着年龄的推移而下降。对每组卵母细胞使用 Tyrode 的新鲜井, 以确保 ZP 的均匀消化。</li>
	<li>ZP 清除后, 立即将卵母细胞转移到温暖的 MEMα/BSA/血清中。重复这个洗涤步骤。 注意: 转移时要小心, 因为卵母细胞会很容易地粘在一起, 然后移除 ZP 后的玻璃吸管或毛细管。预润湿的玻璃吸管或毛细管在 Waymouth 的培养基将减少卵母细胞粘在一起。</li>
	<li>在 5% CO2孵化器中, 转移并允许卵母细胞在 Waymouth 的培养基中以37摄氏度恢复30分钟。</li>
	<li>继续执行步骤5。</li>
</ol>5. MI 和产业部卵母细胞染色质传播
<ol><li>使用 PAP 笔, 在玻璃幻灯片上绘制一个 11 x 44 mm2矩形, 如图 6A所示。</li>
	<li>将滑块的薄层固定 (1% 多聚甲醛, 0.2% 海卫-X 100 在H2 O, pH 9.2) 通过吹打100µL 固定在幻灯片上, 来回摇晃滑动。点击幻灯片以摆脱多余的固定器。</li>
	<li>在5-10 卵母细胞之间拿起一个小口吸管针与尽可能小的媒体, 并拖动针在一条线横跨固定涂层的幻灯片, 同时把卵母细胞从1厘米以上的幻灯片到固定解决方案。使用解剖显微镜执行此步骤, 以确保卵母细胞已沉积, 并已爆裂。 注意: 卵母细胞应立即爆裂在幻灯片上。卵母细胞沉积的高度影响染色质的传播程度。有关详细信息, 请参见图 6中的代表结果。</li>
	<li>夜间在密闭的湿润室中孵化出室温, 允许染色质粘附。</li>
	<li>允许幻灯片完全风干, 并在含有50毫升0.4% 润湿剂-H2O、pH 8.0 的 Coplin 罐子中冲洗幻灯片。</li>
	<li>继续执行步骤6。</li>
</ol>6. Immunolabelling
<ol><li>准备表 1中描述的抗体稀释缓冲 (ADB)。</li>
	<li>准备两个 Coplin 罐, 其中包含50毫升的洗涤缓冲液 (10% 亚行稀释在 PBS), 一个含有50毫升和0.05% 洗涤剂溶液的 Coplin 罐子 (例如, 添加250µL 的10% 洗涤剂到50毫升)。</li>
	<li>将本协议1和5节中准备的幻灯片洗净10分钟, 其中一 Coplin 罐子中含有50毫升的世界银行。 注意: 不要让幻灯片在 immunolabelling 期间的任何时刻干燥。</li>
	<li>在含有50毫升和0.05% 洗涤剂溶液的 Coplin 罐中, 清洗幻灯片10分钟。然后, 在剩余的 Coplin 罐子中洗掉包含50毫升的10分钟的工务解决方案的幻灯片。</li>
	<li>用100µL 在亚行稀释的选择的原发抗体, 轻拍多余的液体和覆盖滑动。在封闭潮湿的室内一夜之间孵化4摄氏度。 注: 孵化可以缩短到2到3小时, 在37摄氏度。用盖玻片或 parafilm 覆盖幻灯片, 使用较小数量的抗体(例如、50µL)。</li>
	<li>在含有50毫升0.2% 润湿剂-PBS 溶液, pH 8.0 的 Coplin 罐子中冲洗幻灯片。</li>
	<li>重复步骤6.2 到6.5。</li>
	<li>用100µL 稀释在亚行中的二次抗体, 利用多余的液体和覆盖滑动。孵化1到2小时的幻灯片在37°c 在一个封闭的潮湿的房间。</li>
	<li>在含有50毫升0.2% 润湿剂-PBS 溶液, pH 值8.0 的 Coplin 罐子中, 2 次清洗幻灯片10分钟。</li>
	<li>在含有50毫升0.2% 润湿剂-H2O 溶液, pH 8.0 的 Coplin 罐子中, 2 次清洗幻灯片5分钟。</li>
</ol>7. 安装滑轨
<ol><li>在步骤2中制备的前期染色质扩散, 添加100µL 的安装培养基, 含 4 ', 6-diamidino-2-苯基吲哚 (DAPI, 1.5µg/毫升)。对于在步骤5中制备的 MI 和产业部染色质扩散, 添加50µL 包含 DAPI (1.5µg/毫升) 的安装介质。轻轻地涂抹过量的液体。</li>
	<li>将22毫米 x 60 毫米盖玻片放在顶部, 用透明指甲油封住。在4°c 或-20 °c 的幻灯片框中存储, 直到通过荧光显微镜进行评估。 注: 轻敲盖板滑动, 以消除多余的安装介质, 然后用指甲油密封。</li>
</ol>

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K., 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=Localization+of+MMR+proteins+on+meiotic+chromosomes+in+mice+indicates+distinct+functions+during+prophase+I.">Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.</a> J Cell Biol. 171 (3), 447-458 (2005).</li><li>Rankin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+elaboration:+making+sense+of+meiotic+cohesin+dynamics.">Complex elaboration: making sense of meiotic cohesin dynamics.</a> FEBS Journal. 282 (13), 2426-2443 (2015).</li><li>Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Cohesin+and+Condensin+in+Chromosome+Dynamics+During+Mammalian+Meiosis.">Roles of Cohesin and Condensin in Chromosome Dynamics During Mammalian Meiosis.</a> J. Reprod Dev. 59 (5), 431-436 (2013).</li><li>Verver, D. E., Hwang, G. H., Jordan, P. W., Hamer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+complex+chromosome+structures+during+meiosis:+versatile+deployment+of+Smc5/6.">Resolving complex chromosome structures during meiosis: versatile deployment of Smc5/6.</a> Chromosoma. 125 (1), 15-27 (2016).</li><li>Hopkins, 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=Meiosis-specific+cohesin+component,+Stag3+is+essential+for+maintaining+centromere+chromatid+cohesion,+and+required+for+DNA+repair+and+synapsis+between+homologous+chromosomes.">Meiosis-specific cohesin component, Stag3 is essential for maintaining centromere chromatid cohesion, and required for DNA repair and synapsis between homologous chromosomes.</a> PLoS Genet. 10 (7), e1004413 (2014).</li><li>Hwang, G., 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=SMC5/6+is+required+for+the+formation+of+segregation-competent+bivalent+chromosomes+during+meiosis+I+in+mouse+oocytes.">SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.</a> Development. 144 (9), 1648-1660 (2017).</li><li>Kota, S. K., Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+transitions+in+germ+cell+development+and+meiosis.">Epigenetic transitions in germ cell development and meiosis.</a> Dev Cell. 19 (5), 675-686 (2010).</li><li>Taketo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspread+ovarian+cell+preparations+for+the+analysis+of+meiotic+prophase+progression+in+oocytes+with+improved+recovery+by+cytospin+centrifugation.">Microspread ovarian cell preparations for the analysis of meiotic prophase progression in oocytes with improved recovery by cytospin centrifugation.</a> Methods Mol Biol. 825 (1), 173-181 (2012).</li><li>Sun, X., Cohen, P. 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作者没有什么可透露的。

染色质传播制剂允许研究人员按时间顺序研究雌性哺乳动物的减数分裂和相关蛋白质的动态定位。胚胎和新生儿染色质的传播允许密切分析整个减数分裂前期的事件。中期 I 和中期 II. 染色质传播可用于区分单亲姐妹条染色单体和配对同源染色体, 以及评估倍性。相比之下, 这里描述的协议可以比整个卵母细胞 immunolabelling 更有利, 它经常产生高水平的背景信号, 从而降低了染色质绑定蛋白的分辨率?...

在精子发生过程中, 在青春期开始和整个成年期的1中, 减数分裂生殖细胞的大的半同步波在睾丸中迅速和持续补充。与男性不同的是, 雌性的减数分裂只在胎儿发育期间开始。出生后, 卵母细胞在前期 I 期的长期 dictyate 阶段仍被逮捕, 并有完整的生发泡 (如: 核信封) 直到青春期。在青春期开始时, 卵母细胞的一个子集被周期性地选择来经历生长和成熟, 标志着减数分裂恢复的开始。完全生长的卵母细胞减数分裂的恢复表现为在一个称为生发泡破裂 (GVBD) 的过程中, 该方法的消失。卵母细胞随后进行染色体凝结和分离, 其次是极性体挤压。卵母细胞在进化到产业部后被逮捕, 并被刺激以完成受精后的第二和最后减数分裂。
女性生育力是高度依赖于减数分裂前期 I. 进展的成功。关键是形成一个物理联系之间的同源染色体称为交叉, 这是调解通过修复诱导 DNA 双链断裂 (DSBs) 通过交叉重组2。这个过程发生在一个动态富含蛋白质的脚手架的背景下, 称为联会复合体 (SC), 在同源染色体之间形成, 以促进它们的联会3。SC 是一种拉链状的三方结构, 由由中央区域蛋白质连接的两个平行的侧向元素组成, 在整个 DNA 修复过程中同系物在一起。在联会之前, 侧向元素的前体称为轴向元素, 在姐妹条染色单体之间形成。联会复杂的蛋白质, 如 SYCP2 和 SYCP3 形成轴向元素, colocalize 的姐妹-染色单体凝聚轴在早期前期。这些以后担当捆绑的站点为横向长丝蛋白质 SYCP1, 促进中央元素汇编和联会在被排列的同系物4之间。在小鼠卵母细胞中, 完整的联会是由20完全重叠的 SYCP3 和 SYCP1 伸展的存在所表明的, 它可以用染色质传播制剂进行可视化。联会是在进入粗线期 substage 后完成的, 由此, 注定要在同系物之间形成交叉的成熟分频者用 mutL 同源 (MLH1/3) 脂肪酸进行装饰, 以促进其精确处理5, 6, 7. 染色体 (SMC) 复合物的结构维护, 包括内聚力联合体、condensin 和 SMC5/6 复合体, 对于整个减数分裂过程中染色体动力学和结构的调控非常重要 8, 9, 10,11,12。总的来说, 这些事件确保了同源染色体在 SC 拆卸后与主轴两极的正确双向定位。
减数分裂细胞周期是一个强大的模型, 以检查不同的蛋白质在基因组维护的作用, 由于程序性诱导和后续修复 DNA DSBs。此外, 哺乳动物减数分裂也是研究表观遗传修饰和印迹13的相关模型。然而, 在技术上很难评估这些事件在女性减数分裂期间发生在胎儿和新生儿卵巢在哺乳动物 (图 1)。前期我可以分为 5 substages: leptonema, zygonema, pachynema, diplonema 和 dictyate。在这里, 我们描述如何隔离和区分胎儿和新生儿的卵巢和睾丸 (图 2)。根据以前描述的方法, 本手稿还概述了一个协议 (步骤 1) 与视频演示准备女性减数分裂前期 I 染色质传播14,15,16,17.当与 immunolabelling 结合, 如步骤6-7 所述, 本协议对卵母细胞前期 I 事件进行详细的显微分析。
卵子发生过程是易出错的, 染色体 missegregation 事件在第一次减数分裂分裂期间代表最常见的来源的遗传疾病的后裔18, 19.在这篇手稿 (协议步骤 2) 中, 我们描述了一个协议, 其中从成年雌性小鼠的引物卵巢提取成熟的自已的牙分期卵母细胞。在支持条件下, 完全生长的卵母细胞在分离和培养后接受促黄体激素-独立恢复减数分裂20。在减数分裂恢复后, 卵母细胞在第二中期被拘捕。卵母细胞在中期 II 中仍被逮捕, 除非受精的.在步骤2–5中, 我们将以前报告的协议与视频演示相适应, 以描述如何收集、培养和准备用于染色质传播准备的 MI 和产业部卵母细胞21。这种染色质传播技术允许明确 immunolabelling 与染色体相关的蛋白质。此外, 该协议还可以用来区分二价和单叶染色体, 并可以进一步解决单亲姐妹条染色单体, 以促进评估卵母细胞的倍性。因此, 除了揭示减数分裂蛋白的定位模式外, 该协议还可以作为一个宝贵的工具, 以阐明在 MI 和产业部的染色体 missegregation 的潜在原因。

<table>
	<tbody>
		<tr>
			<td>10X Phosphate buffered saline (PBS) pH 7.4</td>
			<td>Quality Biological</td>
			<td>119-069-161</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm x 15 mm petri dishes</td>
			<td>Denville</td>
			<td>T1106</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm x 12mm petri dishes</td>
			<td>Denville</td>
			<td>T1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>VWR</td>
			<td>300-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissection scissors</td>
			<td>Ted Pella</td>
			<td>1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160;scissors, sharp tip, 41/2&#34;</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass square 1 5/8</td>
			<td>Carolina Biological Supply Company</td>
			<td>742300</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium Citrate Dihydrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>646563</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>50x Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td>
			<td>Becton, Dickinson (BD)</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td>
			<td>Thermo</td>
			<td>3063-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP pen, large (liquid blocker)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>71310</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde aqueous</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Detergent in manuscript</td>
		</tr>
		<tr>
			<td>Immuno stain moisture chamber</td>
			<td>Evergreen</td>
			<td>240-9020-Z10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jars</td>
			<td>Fisher</td>
			<td>08-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-flo 200</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>74257</td>
			<td>Wetting agent in manuscript</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>Sigma</td>
			<td>G4877</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (HCG)</td>
			<td>Sigma</td>
			<td>C0434</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>PYREX Spot Plates with nine concave cavities</td>
			<td>Fisher</td>
			<td>13-748B</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Fisher</td>
			<td>22260943</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Brand Parafilm M</td>
			<td>Sigma</td>
			<td>BR701501</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5B</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5D</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Mitutoyo micrometer head</td>
			<td>Mituoyo</td>
			<td>150-208</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe filter 0.45 &#956;m filter</td>
			<td>Corning</td>
			<td>431220</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipette 5 3/4&#34;</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>83 x 0.5 mm pipette tip</td>
			<td>Denville</td>
			<td>P3080</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>11415049</td>
			<td></td>
		</tr>
		<tr>
			<td>Waymouth&#39;s media</td>
			<td>Life Technologies</td>
			<td>11220035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher</td>
			<td>A3160602</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td>For oocyte culture media</td>
		</tr>
		<tr>
			<td>500ml filter units 0.22um</td>
			<td>Denville</td>
			<td>F5227</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s solution</td>
			<td>Sigma</td>
			<td>T1788</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H-1270</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides (22mmx60mm)</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SteREO Discovery.V8</td>
			<td>Zeiss</td>
			<td>495015-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Observer Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-MLH1</td>
			<td>Life Technologies</td>
			<td>MA5-15431</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-SYCP1</td>
			<td>Life Technologies</td>
			<td>PA1-16763</td>
			<td>Dilution factor- 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-SCP3</td>
			<td>Santa Cruz</td>
			<td>sc-20845</td>
			<td>Dilution factor- 1:50</td>
		</tr>
		<tr>
			<td>Human anti-centromere protein</td>
			<td>Antibodies Incorporated</td>
			<td>15-235</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti- TOPOII</td>
			<td>Abcam</td>
			<td>ab109524</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td>
			<td>Abcam</td>
			<td>ab78517</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Rec8</td>
			<td>Courtesy of Dr. Karen Schindler</td>
			<td>N/A</td>
			<td>Dilution factor- 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11057</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-21202</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-31573</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11004</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11013</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11011</td>
			<td>Dilution factor- 1:500</td>
		</tr>
	</tbody>
</table>

chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii

染色质传播技术被广泛用于评估配子期间各种蛋白质的动态定位, 尤其是精子发生。这些技术允许在减数分裂事件中, 如同源染色体配对, 联会和 dna 修复的蛋白质和 dna 定位模式的可视化。虽然在文献中已经描述了一些协议, 但由于胚胎卵巢中减数分裂的时机成熟, 使用哺乳动物前期卵母细胞的一般染色质传播技术是有限的和困难的。比较而言, 前期精母细胞可以从具有较高产量的幼雄性小鼠身上收集, 而不需要进行显微解剖。然而, 由于幼睾丸减数分裂和减数分裂后生殖细胞的异质性, 在特定阶段很难获得纯同步的细胞群。在减数分裂的后期阶段, 对正在进行减数分裂 I (MI) 或减数分裂 II (产业部) 的卵母细胞进行评估是有利的, 因为成熟的卵母细胞可以从成年雌性小鼠身上收集, 并刺激恢复培养的减数分裂。在这里, 用伴随的视频演示, 描述了用胎儿、新生儿和成人卵巢解剖的卵母细胞进行减数分裂的染色质扩张制剂的方法。哺乳动物卵母细胞的染色体 missegregation 事件是频繁的, 特别是在 MI 期间。这些技术可以用来评估和描述不同的突变或环境暴露在卵子发生过程的不同阶段的影响。由于卵子发生过程和精子发生之间存在着明显的差异, 其中所描述的技术对于提高我们对哺乳动物卵子发生过程的认识和减数分裂过程中染色体和蛋白质动态的性二形特征是非常宝贵的。.

我们描述了两种方法来可视化和评估卵母细胞减数分裂染色体。第一种技术是评估胚胎和新生儿卵巢的前期进展。前期染色质传播制剂对于在减数分裂过程中可视化许多动态过程非常有价值, 包括染色体配对、联会和 desynapsis、同源重组和表观遗传染色体重塑。在这里, 我们已经证明了这种方法的效用, 以稳健的可视化和定量分析的交叉形成卵母细胞从 C57BL/6J 胚胎 (<strong cla...

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

用于分析小鼠卵母细胞从前期到中期 II 进展的染色质扩散制剂遗传学 |视频

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

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

哺乳动物中的卵子发生过程已知易出错, 特别是由于染色体 missegregation。本手稿描述了小鼠前期、中期 I 和 II. 期卵母细胞的染色质传播制备方法。这些基本技术允许研究整个哺乳动物卵子发生过程的染色质束缚蛋白和染色体形态学。

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

chromatin-spread-preparations-for-analysis-mouse-oocyte-progression

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15.2K 次观看.  Johns Hopkins University Bloomberg School of Public Health. 哺乳动物中的卵子发生过程已知易出错, 特别是由于染色体 missegregation。本手稿描述了小鼠前期、中期 I 和 II. 期卵母细胞的染色质传播制备方法。这些基本技术允许研究整个哺乳动物卵子发生过程的染色质束缚蛋白和染色体形态学。

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

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

科研

遗传学

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...