我们感谢木聪岸上、沙耶香和歌山、博明 Nagatomo、木聪 Kamimura 和假名田文雄, 提供了重要的评论和技术支持。这项工作由教育、文化、体育、科学和技术部部分资助, 以促进国家大学对作案手法的改革;日本促进科学学会 (16H02593)、田真央科学基金会和武田科学基金会 T.W。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。

这项研究是严格按照《山梨大学实验室动物护理和使用指南》的建议进行的。该议定书获得了山梨大学动物实验伦理学委员会 (许可证号: A24-50) 的批准。所有的手术都是在 tribromoethanol 麻醉下进行的, 并尽一切努力尽量减少痛苦。图 1和表 1分别显示了过程和时间表的插图概述。
1. 信使 RNA 的制备 (eGFP-H2B mRNA 的体外转录)
<ol><li>进行线性化5µg 模板脱氧核糖核酸 pTOPO-eGFP-H2B3,7与 30 U不I 在50µL 在37°c 隔夜。</li>
	<li>收集1.5 µL 的消化 DNA, 以确认是否经电泳完全消化。<ol>
<li>将1.5 µL 和 3-5 µL 的未消化 DNA 与载染料分别应用于2% 琼脂糖凝胶井中, 并经电泳处理。 注: 如果完全消化, 则观察单个波段 (约 5 kb)。未消化的 DNA 被用作控制, 出现在不同的位置。</li>
	</ol>
</li>
	<li>添加151.5 µL ddH2O 和200µL 苯酚/氯仿/异戊醇 (25:24:1) 的剩余的消化 DNA。<ol>
<li>由涡旋强烈混合。</li>
		<li>离心机以最大速度为15分钟。</li>
	</ol>
</li>
	<li>恢复上清, 然后添加200µL 氯仿。<ol>
<li>由涡旋强烈混合。</li>
		<li>离心机以最大速度15分钟。</li>
	</ol>
</li>
	<li>恢复上清, 然后添加20µL 3M 醋酸钠和1.5 µL 的 ethachinmate。<ol>
<li>由涡旋强烈混合。</li>
		<li>加入550µL 的100% 乙醇, 然后用漩涡大力搅拌。</li>
		<li>离心机以最大速度15分钟。</li>
		<li>去掉上清, 然后用70% 乙醇清洗颗粒。</li>
		<li>蒸发 5-10 分钟, 使颗粒干燥。</li>
	</ol>
</li>
	<li>将沉淀质粒 DNA 溶于8µL 的核酸酶水中。</li>
	<li>按照制造商的指示, 评估质粒浓度 (预期浓度约为 300-500 µL) 与 nanodrop。</li>
	<li>按照制造商的指示, 在总反应量的20µL 中, 用1µg 纯化 DNA 作为模板, 对 mRNA 编码 eGFP-H2B 进行体外转录。<ol>
<li>在体外转录后, 添加 36 ul 的水, 然后从56µL 的合成 mRNA 中恢复1.5 µL, 进行电泳分析 (如无聚 A)。</li>
	</ol>
</li>
	<li>按照制造商的指示, 在100µL 反应量中对合成 mRNA 进行聚一尾矿。</li>
	<li>将60µL 的氯化锂沉淀液添加到 eGFP-H2B 的多尾 mRNA 中, 然后进行强力搅拌。<ol>
<li>-30 摄氏度冷却30分钟。</li>
		<li>离心机以最大速度15分钟。</li>
		<li>去掉上清, 然后用70% 乙醇清洗颗粒。</li>
		<li>蒸发 5-10 分钟, 使颗粒干燥。</li>
	</ol>
</li>
	<li>用20µL 核酸酶的水, 用55摄氏度的热量将颗粒溶解30分钟。</li>
	<li>用 nanodrop 评价沉淀 mRNA 的浓度。稀释至500µL 与核酸酶的水和存储在-80 °c, 直到使用。</li>
	<li>确认首选 mRNA 生产;主题1µL 的 mRNA 与/没有 (获得1.8.1 和1.12 步骤, 分别) 聚一尾混合1.5 µL 0.1 毫克/毫升溴溴和3µL 样品加载染料的电泳分析。应用体积为5.5 µL。 注: 如果聚 a 尾矿成功, 则应观察转移带与没有聚尾矿的 mRNA 相比较 (图 2A)。</li>
</ol>2. 输精管切除术雄性小鼠的制备
<ol><li>用体重秤在 8-12 月的时间内称其为雄性小鼠。</li>
	<li>腹腔注射 0.7-0.9 毫升 1.2% tribromoethanol 麻醉雄性小鼠。 注: 麻醉量取决于鼠标大小。例如, 一只重量为40克的老鼠注射了0.8 毫升的 tribromoethanol 麻醉。</li>
	<li>用70% 乙醇将浆果淋湿。</li>
	<li>在浆果上做一个小切口 (3-5 毫米), 然后在剪刀的帮助下在肌肉壁上做一个切口。 注: 在开始手术前, 应用70% 乙醇清洗剪刀和镊子。</li>
	<li>用镊子夹住脂肪垫, 然后轻轻地拔出。然后, 睾丸和输精管出现。 注: 输精管为 U 形管, 有红色血管。</li>
	<li>用手术缝线将输精管栓在两个点上, 然后在栓点之间切开中间部分。</li>
	<li>轻轻地把脂肪垫和睾丸放回浆果里。<ol>
<li>用剩下的睾丸重复手术。</li>
	</ol>
</li>
	<li>用两针缝合肌肉壁缝合手术。</li>
</ol>3. IVF
<ol><li>注射 8-10 周的老女性 B6D2F1 (BDF1) 小鼠腹腔7.5 的马绒毛膜促性腺激素 (eCG) 和人绒毛膜促性腺激素 (hCG) 在 46-50 h 间隔。</li>
	<li>准备300µL 人输卵管液 (HTF)8种培养基进行精子和人工授精1天前, 在30毫米的盘子中受精, 用矿物油在实验室的长凳上盖上这些水滴, 然后在一夜之间在孵化箱中 preincubate。</li>
	<li>通过颈椎脱位牺牲成熟的雄性小鼠。用27克针解剖马尾附睾, 收集精子。培养他们为精子在300µL HTF 媒介为 1-2 h 在38°c。</li>
	<li>16小时后 hCG 注射, 牺牲超级排卵雌性小鼠颈椎脱位。将输卵管壶腹部转移到 HTF 培养基旁的矿物油中, 然后用30克针将卵丘细胞和卵母细胞从这个输卵管壶腹中抽出到 preincubated 受精-HTF 培养基中。 注:一些雌性小鼠不经常显示排卵, 尽管超级排卵治疗。扩大输卵管壶腹是排卵的标志。</li>
	<li>精子后, 将约束精子的 2-3 µL 转移到受精-HTF 培养基中, 排卵卵母细胞被收集。</li>
	<li>1 h 受精后, 收集受精的受精卵, 并在100µg/毫升的透明质酸酶治疗, CZB9培养基10分钟。 注:当受精完成后, 1 小时的受精后, 受精的受精卵与2和极地的身体被观察到。如果使用较少或低质量的精子, 只观察与2和极性身体的小受精卵。此外, 如果许多精子用于人工授精, 多精受精发生。适当的人工授精导致受精卵与男性和女性 pronuclei。通过使用这些标准, 有可能发现人工授精是否做得很好。<ol>
<li>在 CZB 介质中洗涤后, 受精卵与第二极性体进行显微注射 eGFP-H2B mRNA。</li>
	</ol>
</li>
</ol>4. 室和微注射吸管的制备
<ol><li>稀释 RNA 编码 eGFP-H2B 使用无核酸酶水获得浓度为 250 ng/µL。</li>
	<li>准备 2-3 滴 PVP HTF 和 eGFP-H2B 滴 mRNA, 5-6 滴 Hepes 缓冲 CZB (H CZB) 滴在60毫米的盘子上。用矿物油盖住这些水滴。 注: 这些准备工作可以在实验室的长凳上进行。不需要使用层流气流柜。</li>
	<li>拉硼玻璃与微拉出 (例如, P = 500, 热 = 825, 拉 = 30, 威 = 120, 时间 = 200)</li>
	<li>在打破了吸管的尖端, 弯曲的拉玻璃微注射吸管接近尖端 (−300µm 背部) 在周围的30°使用 microforge。</li>
</ol>5. 显微注射
<ol><li>关闭机器人台上的板式加热器, 以防止在 mRNA 注射过程中用压电法杀死受精卵。</li>
	<li>在含有 10% pvp (pvp-HTF) 的 HEPES 缓冲 HTF 中, 清洗并涂上注射针的内侧。</li>
	<li>收集受精的受精卵与2和极性身体 (图 2B) 和转移 20-25 受精卵到显微镜阶段的室附有机器人。</li>
	<li>将稀释后的 eGFP-H2B mRNA 从小贴士中填充到硼硅酸盐玻璃毛细血管中。</li>
	<li>将受精卵与持有的吸管保持在一起, 然后用压电驱动来打破透明的透明和胞浆膜。</li>
	<li>将10的 mRNA 注入受精卵的细胞质中。在10分钟内完成 mRNA 注射, 以防止在孵化器外培养受精卵所造成的损害。 注:注入量应与2和极性体一样大 。如果注射的 mRNA 量太大, 就有可能导致游离 eGFP-H2B 分数, 这可能会影响移动分数。</li>
	<li>注射10分钟后, 至少3滴洗涤, 然后培养注射受精卵在 CZB 38 摄氏度, 直到 zFRAP 分析。</li>
</ol>6. zFRAP 分析
<ol><li>1 h 在 zFRAP 分析之前, 打开激光和热板连接到共焦显微镜的舞台上。将温度设置为摄氏38摄氏度。</li>
	<li>将 6-10 µL 的 HEPES 缓冲的 CZB 介质覆盖在60毫米的玻璃底盘上, 加热加热器直到合子收集。</li>
	<li>受精后 8-12 小时, 收集 5-10 eGFP-H2B-expressing 受精卵并转入 6-10 µL 预热 HEPES 缓冲 CZB 中滴。 注意: 为避免在孵化器外培养较长时间, 每项分析使用 5-10 eGFP-H2B-expressing 受精卵。5到10受精卵可以在1小时内进行分析。</li>
	<li>根据制造商的说明设置漂白和成像条件。本案为共焦激光扫描显微镜 (材料表) 如下所示:<ol>
<li>使用60X 油透镜 (60X 60 x/1.35 油)。 注: 放大倍数 (高数值孔径) 的透镜显示较高的灵敏度。</li>
		<li>设置扫描速度为4.0 µs/像素和大小为 "512" 上的 "采集设置" (补充图 1)。</li>
		<li>增加数字变焦到 "5" (补充图 1)。 注意: 需要更高的缩放来识别焦点漂移是否发生。</li>
		<li>打开染料列表 (补充图 1), 然后选择 "EGFP"。将观察时间设置为1.6 秒 (间隔设置为 "自由运行" 设置) (补充图 1) 注意: 更多的观察点可能会导致更详细的数据, 但也可能导致毒性。在 zFRAP 分析中, 1.6 的观察时间似乎足以检测出染色质松动的父母不对称性。</li>
		<li>将图片总数设置为 12 (补充图 1)。</li>
		<li>启动 "刺激设置" 面板, 然后单击 UseScanner 上的 "Main"。将扫描速度设置为10.0 µs/像素。单击 "串联激活", 然后将体细胞设置为 "3 帧", 激活时间为 "5 秒" (补充图 1)。</li>
		<li>单击 "焦点 x 2" 并设置焦点, 然后 "停止"。</li>
		<li>通过调整 "激光测量功率" (补充图 1), 分别将漂白和成像激光功率 (477 nm) 设为110和15µW。 注: 非常强的漂白可能会造成较大的漂白面积, 这会给定量分析带来困难。对于激光功率的测量, 请使用功率计。确定激光功率以避免激光功率与时间相关的劣化效果是很重要的。</li>
		<li>在 "刺激设置" 面板中单击 "剪辑矩形" (补充图 1)。设置感兴趣的区域 (ROI; 红色;ROI #1B) 为 40 x 40 像素 (7.6 µm2)。准备参考区域 (REF; 绿色;ROI #2) 和背景 (BG; 蓝色;roi #3) 使用 "复制 roi" 和 "粘贴 roi" (鼠标右键单击按钮)。 注意: 像素大小可以通过 "roi 管理器" 设置, 当光标位于 roi 上时, 单击鼠标的右键即可调用它。更大的 ROIs 被认为是更好的, 因为他们可以标准化的分散 zFRAP 评分。然而, 太大的 ROI 不能用于这种分析, 因为它使得很难避免核仁前体 (NPB)。eGFP-H2B 在这个车厢被认为是无染色质和显示显着更高的机动性3。ROI 和 REF 区域应该尽可能地分开。如果这两个区域接近, 漂白也可能影响到 REF 区。</li>
		<li>单击工具栏上的 "实时", 然后开始 "实时绘图" (补充图 1)。 注: 现场图表示每个 ROI 内的荧光信号强度, 用于用高压调整激光功率。请注意, 如果未选择 ROI, 则在 "实时绘图" 面板上不会检测到强度。</li>
		<li>确定 ROI 位置 注意: 可以通过拖动到 pronuclei 中的所需位置来移动 ROI。(1) 一定要避免核仁, (2) 适合整个 ROI 的 nucleoplasm。不包含区域外的核膜 (细胞质) 在 ROI。eGFP-H2B 的定位高度限制在 nucleoplasm 中, 所以很容易发现 ROI 是否含有细胞质, 或者不是由 eGFP-H2B 的荧光。雄性 pronuclei 往往比雌性的大, 通常在2和极体附近进行定位.在使用这些标准时, 要判断男性或女性的 pronuclei。</li>
		<li>单击 "焦点 x 2" (补充图 1), 然后将通道的高压功率计调整为 EGFP 到大约 2000 (荧光强度可在 "活图" 窗口中看到)。</li>
	</ol>
</li>
	<li>点击 "时间", 然后 "XY" (补充图 1) 开始 zFRAP 分析。 注: 如果 "时间" 按钮被适当选中, "t" 将出现在 "XY" 按钮上。<ol>
<li>点击 "完成" (补充图 1), 它出现在酶成像附近的 "XY 按钮", 然后获得酶分析的数据。</li>
		<li>通过 "另存为" (可以使用 ctrl+ Shift + S), 将文件 "2D 视图-图像的 oib" 保存为首选的命名文件 (文件格式)。</li>
		<li>选择 ROI、REF 和 BG, 然后在 "2D 视图图像" 窗口中单击 (可同时使用 Ctrl + A) "系列分析"。</li>
		<li>获取行酶数据, 然后单击 "实时绘图" 窗口中的 "保存" 按钮。 注意: 行酶量化数据保存为 csv 文件。</li>
	</ol>
</li>
	<li>对于没有 zFRAP 的控制, 把一些受精卵注入 mRNA 的下降, 这是接近边缘的玻璃底部菜肴, 以避免从荧光观察的效果。</li>
</ol>7. 数据计算
<ol><li>在使用获得的量化数据时, Excel 将 "恢复曲线" 和 "移动分数" 的图表显示在参考3、7和一些修改 (见下文和补充 Excel 文件) 中。</li>
	<li>计算相对强度, 方法是将 BG 的值从 ROI 和 REF 中减去每个时间点的12张图片。</li>
	<li>将 ROI 的价值除以 REF 的值。因此, 可以获得12标准的相对强度分数在每个时间点。</li>
	<li>计算在照片漂白前3张图像中 ROI 的相对分数的平均值。</li>
	<li>计算回收率。在每一个时间点 (7.1.2 获得的) 图像中, 除以相对分数的平均值 (在7.1.3 获得), 将12获得的 ROI 的相对分数分到。使用这些分数绘制恢复曲线 (图 2E)。</li>
	<li>计算漂白率。 注: 漂白率 (%) = 100-恢复率为 4次图像。</li>
	<li>使用公式计算移动分数 (MF), 如下所示10、11、12 MF (%) = 12回收率 (在终点)-4% 回收率/漂白率 x 100。</li>
</ol>8. 胚胎移植
<ol><li>交配成熟的女性在 8-12 月与输精管切除术雄性小鼠在胚胎移植前的前一天晚上让他们在同一个笼子过夜。</li>
	<li>收集 zFRAP 分析的胚胎, 并确定其发育为两细胞阶段。</li>
	<li>腹腔注射雌性小鼠 0.7-0.9 毫升 1.2% tribromoethanol 麻醉或 0.35-0.45 毫升 0.75/4/5 毫克/千克 medetomidine/咪唑仑/布托啡诺混合剂在胚胎移植之前。 注: 麻醉量由小鼠的体重决定。Tribromoethanol: 体重 0.2 mL/10 克;medetomidine/咪达唑仑/布托啡诺混合剂: 0.1 毫升/10 克的体重。<ol>
<li>在 coitum 后0.5 天, 将这两个细胞阶段的胚胎转移到 pseudopregnant 雌性小鼠的输卵管中, 并使用阴道插头。</li>
		<li>胚胎移植后, 将雌性小鼠放在加热器上设置为37摄氏度, 直到它们醒来。</li>
	</ol>
</li>
	<li>在 18.5 dpc, 用宫颈脱位牺牲代孕母, 从 zFRAP 分析受精卵中提取的幼崽进行剖宫产。一些被恢复的幼崽被送到寄养母亲身上, 他们的体重每周都被评估。</li>
</ol>

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作者没有什么可透露的。

如本研究所揭示的, zFRAP 分析不会对足月发展造成严重损害, 这表明该方法是揭示分子事件与胚胎发育潜能之间关联的非常有用的工具。在重新编程过程中, 进入 ES 细胞的双细胞状状态, 这些细胞的差异电位与两细胞阶段胚胎的高度一样高, 染色质松动改变为与两细胞级胚胎5的可比性。因此, 有可能胚染色质松动对胚胎发育潜力很重要。体细胞细胞核转移到卵母细胞的细胞质显示, ...

受精后, 染色质结构动态改变, 胚染色质结构最终建立1,2。在此期间, 在父系 pronuclei, 显性染色质蛋白从精蛋白转化为组蛋白。由此产生的染色质与精子和雌性卵母细胞在几个点上的差别非常大 (例如, 组蛋白变异组成, 组蛋白修饰)。因此, 形成的胚染色质被认为是重要的后续胚胎发育。然而, 尽管努力揭示胚染色质结构的细节, 在长时间内, 评价受精卵质量的方法或预测其在单细胞阶段的全面发展, 通过分析其染色质结构从来没有建立。
在先前的研究中, 发现受精卵有一个非常松散的染色质结构3。目前, 染色质松动或开放性被认为是一个重要因素的细胞分化潜能的胚胎干细胞 (ES) 细胞4。ES 细胞在自然界中并不表现出同质性, 而是相当异构;在 ES 细胞菌落中, 一些瞬时获得的分化电位较高, 可与卵裂球的两细胞阶段胚胎相媲美。在这种向双细胞状状态的过渡过程中, ES 细胞的染色质松动改变为与两细胞阶段胚胎5相媲美的东西。因此, 染色质松动似乎是重要的细胞分化潜力, 这是可能的广泛开放染色质在受精卵是有益的评价胚发展潜力。
实时成像是一个强大的工具, 允许分析在个体发育期间的分子事件, 因为这种方法允许随后的发展, 甚至是6的足月发展。作为一种实时成像方法, 酶分析已用于检查前体细胞和 ES 细胞3,4,5的染色质松动。如果通过酶分析对胚染色质松动进行分析, 对足月发育无不良影响, 可能是评价单细胞期胚胎质量的重要工具。然而, 这一实验方法对足月发展的影响还未被研究。最近, 建立了一个利用酶评价胚染色质松动的实验系统。因为这是一个新的观察系统的受精卵, 它被称为胚酶 (zFRAP)。zFRAP 没有严重影响全面发展, 并已在别处报告7。在本报告中, 介绍了该实验方法的协议。

<table><tbody><tr><td>Confocal laser scaning microscope</td><td>Olympus</td><td>FV1200</td><td>FV1000 can be also used for FRAP analysis (reference #7)</td></tr><tr><td>Thermo plate</td><td>Tokai Hit</td><td>TP-110RH26</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Micro manupilator</td><td>Narishige</td><td>MMO-202ND</td><td></td></tr><tr><td>Pieze Micro Micromanipulator</td><td>Prime tech</td><td>PMAS-CT150</td><td></td></tr><tr><td>35 mm culture dish</td><td>Falcom</td><td>351008</td><td>35 x 100 mm style; for IVF</td></tr><tr><td>60 mm culture dish</td><td>Falcom</td><td>351007</td><td>60 x 15 mm style; for embryo culture</td></tr><tr><td>50 mm culture dish</td><td>Falcom</td><td>351006</td><td>50 x 9 mm style; for manipulation on the stage</td></tr><tr><td>50 mm glass bottom dish</td><td>Matsunami</td><td>D910400</td><td>50 mm dish, 27 mm &amp;phi; hole size; for FRAP analysis</td></tr><tr><td>Mineral oil</td><td>Organic spceiality chemicals</td><td>625071</td><td>For FRAP analysis on the glass bottom dishes</td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M8410-1L</td><td>For IVF and embryo culture</td></tr><tr><td>glass capillary</td><td>Drummond</td><td>1-000-1000</td><td>For handling mouse zygotes</td></tr><tr><td>Borosilicate glass</td><td>Prime tech</td><td>B100-75-10-PT</td><td>For microinjection of mRNA</td></tr><tr><td>Micropipett puller</td><td>Sutter instrument</td><td>P-97/IVF</td><td>For preparation of the injection or holding neadle (out side diameter: 80 &amp;micro;m, inner diameter: 10 &amp;micro;m)&amp;nbsp;</td></tr><tr><td>Microforge</td><td>&amp;nbsp;Narishige</td><td>MF-900</td><td></td></tr><tr><td>mMESSAGE MACHINE SP6 Transcription kit</td><td>Thermo&lt;br/&gt; Fisher scientific</td><td>AM1340</td><td></td></tr><tr><td>Poly (A) tailing kit</td><td>Thermo&lt;br/&gt; Fisher scientific</td><td>AM1350</td><td></td></tr><tr><td>PVP solution 10% (PVP-HTF)</td><td>IrvineSceientific</td><td>99311</td><td>HEPES buffered HTF containing 10% PVP</td></tr><tr><td>Sodium HEPES</td><td>Sigma</td><td>H3784</td><td></td></tr><tr><td>pTOPO eGFP-H2B</td><td></td><td></td><td>Template plasmid for eGFP-H2B mRNA (reference #6)</td></tr><tr><td>NorthernMax Gly Sample loading Dye&amp;nbsp;</td><td>Thermo&lt;br/&gt; Fisher scientific</td><td>AM8551</td><td>For electrophoresis of in vitro transcribed mRNA</td></tr><tr><td>Phenol chloroform isamyl alcohol</td><td>nacalai tesque</td><td>25970-14</td><td></td></tr><tr><td>Chloroform</td><td>nacalai tesque</td><td>&amp;nbsp;08401-65</td><td></td></tr><tr><td>Not I</td><td>TOYOBO</td><td>NOT-111X</td><td></td></tr><tr><td>Ethachinmate</td><td>WAKO</td><td>318-01793</td><td></td></tr><tr><td>3664 Otical power meter</td><td>Hioki</td><td>3664</td><td>&amp;nbsp;Power meter for laser power</td></tr><tr><td>Stereomicmicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr></tbody></table>

zygotic fluorescence recovery after photo-bleaching analysis for chromatin looseness that allows full-term development

实时成像是一个强大的工具, 允许分析分子事件在个体发育期间。最近, 染色质松动或开放性已被证明参与了细胞分化潜能的多潜能胚胎干细胞。此前据报道, 与胚胎干细胞相比, 受精卵港的染色质结构非常松动, 暗示其与全能的关系。然而, 直到目前为止, 还没有讨论这一极其松动/开放的染色质结构对胚胎发育潜力是否重要。在本研究中, 为了检验这一假说, 本文研制了一种实验系统, 在受精卵的光漂白后, 通过荧光恢复分析, 得到了不明显的损伤。重要的是, 除了共焦激光扫描显微镜, 这个实验系统还需要一个热水瓶加热器。这项研究的结果表明, 光漂白分析 (酶) 分析后的荧光恢复可以用来调查胚染色质中的分子事件是否对足月的发展是重要的。

zFRAP 分析与 eGFP-H2B
适当产生的 mRNA 编码 eGFP-H2B, 这是被视为一个转移带引起的聚一条尾矿 (图 2A), 被注入到受精卵胞浆与第二极性身体在 1-3 小时后受精 (图 2B)。八至12小时人工授精后, 受精卵与 2 pronuclei 显示 eGFP-H2B 的表达, 并受到 zFRAP 分析 (图 2C)。酶分析包括?...

Watch this Scientific Journal Video about Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development at JoVE.com

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

染色质松动似乎参与了卵裂球的发展潜力。然而, 目前尚不清楚染色质松动是否可以作为胚胎发育潜能的可靠指标。在这里, 我们描述了一个实验系统, 其中染色质松动评价受精卵可以发展到足月。

胚荧光恢复后的染色质松动的光漂分析, 允许足月发展

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been ...

zygotic-fluorescence-recovery-after-photo-bleaching-analysis-for

Research

JoVE Journal

Developmental Biology

7.9K 次观看.  University of Yamanashi. 染色质松动似乎参与了卵裂球的发展潜力。然而, 目前尚不清楚染色质松动是否可以作为胚胎发育潜能的可靠指标。在这里, 我们描述了一个实验系统, 其中染色质松动评价受精卵可以发展到足月。

视频: Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

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 ...

遗传学

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

色质域新形成研究方法

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

生物学

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.