Name: chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii
Uploaded: 2018-02-26T13:00:00
Description: Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

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?...

<ol>
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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

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 ...

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SCIENTISTS IN ACTION

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

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

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

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

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

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

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

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

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

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

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

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

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

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 ...