Name: preparation of meiotic chromosome spreads from mouse spermatocytes
Uploaded: 2017-11-22T12:30:00
Description: Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes at JoVE.com

作者承认阿比盖尔. 哈里斯的技术援助。他们还感谢保拉. 科恩和金-哈威, 帮助优化了从老鼠精中制备表面扩散核的协议。作者也欣赏凯伦. 辛德勒对手稿的批判性阅读。
这项工作得到了 NIH R01 GM106262 对 K.M.B。

所有涉及小鼠的方法都使用了高道德和福利标准, 并得到了尔大学医学院的动物保育和使用委员会的批准。
1. 拟订所需的解决办法和文书
<ol><li>在50毫升的超纯实验室级水中制备低渗萃取缓冲液, pH 值 8.2-8.4 (表 1)。每一次使溶液新鲜, 保持在冰上, 并在2小时内使用准备利差;这是必要的, 以优化减少和蛋白酶抑制活动的 PMSF, 分别。每对老鼠睾丸使用10毫升的希伯来人。</li>
	<li>?...

<ol>
	<li>Handel, M. A., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mammalian+meiosis:+regulation,+dynamics+and+impact+on+fertility.">Genetics of mammalian meiosis: regulation, dynamics and impact on fertility.</a> Nat Rev Genet. 11, 124-136 (2010).</li><li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Counce, S. J., Meyer, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+synaptonemal+complex+and+the+kinetochore+in+Locusta+spermatocytes+studied+by+whole+mount+electron+microscopy.">Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy.</a> Chromosoma. 44, 231-253 (1973).</li><li>Speed, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis+in+the+foetal+mouse+ovary.+I.+An+analysis+at+the+light+microscope+level+using+surface-spreading.">Meiosis in the foetal mouse ovary. I. An analysis at the light microscope level using surface-spreading.</a> Chromosoma. 85, 427-437 (1982).</li><li>Ashley, T., 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=Dynamic+changes+in+Rad51+distribution+on+chromatin+during+meiosis+in+male+and+female+vertebrates.">Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.</a> Chromosoma. 104, 19-28 (1995).</li><li>Baker, S. M., 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=Involvement+of+mouse+mLh1+in+DNA+mismatch+repair+and+meiotic+crossing+over.">Involvement of mouse mLh1 in DNA mismatch repair and meiotic crossing over.</a> Nat Genet. 13, 336-342 (1996).</li><li>Plug, A. W., Xu, J., Reddy, G., Golub, E. I., Ashley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+association+of+Rad51+protein+with+selected+sites+in+meiotic+chromatin.">Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.</a> Proc Natl Acad Sci U S A. 93, 5920-5924 (1996).</li><li>Berkowitz, K. M., 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=Disruption+of+CHTF18+causes+defective+meiotic+recombination+in+male+mice.">Disruption of CHTF18 causes defective meiotic recombination in male mice.</a> PLoS Genet. 8, e1002996 (2012).</li><li>Gomez, R., 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=Sororin+loads+to+the+synaptonemal+complex+central+region+independently+of+meiotic+cohesin+complexes.">Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes.</a> EMBO Rep. 17, 695-707 (2016).</li><li>Brooker, A. S., Berkowitz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+cohesins+in+mitosis,+meiosis,+and+human+health+and+disease.">The roles of cohesins in mitosis, meiosis, and human health and disease.</a> Methods Mol Biol. 1170, 229-266 (2014).</li><li>McNicoll, F., Stevense, M., Jessberger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cohesin+in+gametogenesis.">Cohesin in gametogenesis.</a> Curr Top Dev Biol. 102, 1-34 (2013).</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 J. 282, 2426-2443 (2015).</li><li>Holloway, J. K., Sun, X., Yokoo, R., Villeneuve, A. M., Cohen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+CNTD1+is+critical+for+meiotic+crossover+maturation+and+deselection+of+excess+precrossover+sites.">Mammalian CNTD1 is critical for meiotic crossover maturation and deselection of excess precrossover sites.</a> J Cell Biol. 205, 633-641 (2014).</li><li>La Salle, S., Sun, F., Handel, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+short-term+culture+of+mouse+spermatocytes+for+analysis+of+meiosis.">Isolation and short-term culture of mouse spermatocytes for analysis of meiosis.</a> Methods Mol Biol. 558, 279-297 (2009).</li><li>Wiltshire, T., Park, C., Caldwell, K. A., Handel, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+premature+G2/M-phase+transition+in+pachytene+spermatocytes+includes+events+unique+to+meiosis.">Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis.</a> Dev Biol. 169, 557-567 (1995).</li></ol>

为了获得大量的良好传播, 优良的质量精核, 该议定书的最关键步骤包括适当的孵化时间的精细管在希伯来, 适当的切细精小管, 和技术使用将蔗糖细胞悬浮在定影膜上。我们从从出生日15到成年的野生型雄性的睾丸中孵化出的生精小管45分钟, 而从可比较年龄的Chtf18--空老鼠只孵育30分钟, 因为睾丸是一半大小, 包含明显较少的生殖细胞8。在我们的经验中, Chtf18的孵育--?...

小鼠被广泛用作动物的模型有机体来研究哺乳动物的减数分裂。正常的发育过程和在减数分裂过程中出现的缺陷都可以评估。在前期 I 和阶段中发生的显著特征的时间轴和进展, 以及在调控减数分裂过程中所涉及的许多特定的蛋白质, 都可以在野生类型和突变小鼠身上进行表征。在减数分裂期间发生的几个特殊的过程, 我可以详细研究 (在亨德尔和 Schimenti1中进行了回顾)。这些包括 DNA 双断裂 (争端) 的形成和修复, 重组, 联会复杂的形成, 和染色体分离。
研究减数分裂过程的一个重要方面是能够检查含有同源染色体的细胞核, 它们的视觉和光学分辨率能更好地区分和识别阶段前期 i. 前期 i. 5 阶段具有特殊特征, 包括联会复合体 (SC) 的形成。SC 是一个三方蛋白支架, 使配对和 DNA 双断裂修复的同系物。在 leptonema 期间, 同系物对齐作为 SC 的轴向元素被放下。然后在 zygonema 期间, 将中心元素附着在联会复合体上, 有助于配对和物理连接 (联会) 对同系物。在 pachynema, 联会同系物变得完整, dna 分频是由一个选择的种群 dna 双断裂通过同源重组修复。SC 分解在 diplonema, 允许同系物 desynapse, 但仍然附着在他们的丝和在 DNA 分频的地点。最后在变期间, 同系物 recondense 和过渡到中期发生了1。
从历史上看, 减数分裂染色质的表面扩散产生了少量的细胞核, 尤其是早期 I 期2。因此, 这些方法进行了改进, 以改善细胞从组织 (即睾丸或卵巢) 分离, 传播减数分裂染色质的技术, 和高品质的减数分裂细胞核的产量评价2,3,4. 此外, 这一方法证明是有用的保存核和染色质结合蛋白在减数分裂核如显示的 immunolocalization 技术5,6,7。因此, 最初由彼得斯2描述的方法, 并在这里演示了许多单独的突发精核, 其中包含同系物经历期、偶、线、双和变阶段的前期 i。
制备表面扩散核 (也称为染色质传播分析) 技术被广泛应用于生殖和细胞生物学领域的减数分裂研究。含有表面扩散细胞核的幻灯片可以随后 immunostained 与感兴趣的蛋白质抗体或受银染色, 然后用显微镜分析。该方法对雄性和雌性小鼠都是相似的, 虽然对雌性小鼠的修饰已经被描述为2。不 overmince 生精小管是至关重要的。细胞核也必须很好地传播, 以便以下免疫同系物和相关的蛋白质在显微镜下清楚地看到。表面扩散核可以在短短的几个小时内, 或 immunostained 立即或储存在-80 ° c, 最多3周解冻和免疫。然而, 如果免疫立即或在准备表面扩散核的2周内进行, 最好得到一些蛋白质的最佳结果。幻灯片可以 immunostained 与一个标准协议使用抗体的蛋白质的兴趣, 其次是孵化与第二抗体共轭荧光蛋白或染料, 然后成像荧光显微镜8。在产后一天 15-21 有足够的组织每对野生类型的睾丸产生 6-10 幻灯片, 和年长的老鼠 (包括成年人) 将产生更多的幻灯片。然而, 野生型小鼠6周龄和年龄较大的人也会产生更多的 post-meiotic 细胞 (即精子) 的幻灯片后, 免疫。此外, 所有阶段的前期 I 可以观察到的幼鼠和成年老鼠。雄性在产后10到15的睾丸会产生更多的期和偶细胞。早于产后14至15的小鼠会产生更多的线和双核。
在这里, 我们描述和演示了一个广泛使用的方法制备表面扩散核从小鼠精。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Photo-Flo 200</td>
			<td>Kodak</td>
			<td>1464510</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (formaldehyde aqueous solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
			<td>Dilute 16% solution into 4% working stocks with 1X PBS and freeze aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Teflon printed 3 ring slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>63418-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium uncharged frosted end glass slides</td>
			<td>Several commercial brands</td>
			<td></td>
			<td>Clean slides in ethyl or isopropyl alcohol and allow to drip dry prior to use; label slides on frosted end with a permanent marker or pencil depending on subsequent use of slides</td>
		</tr>
		<tr>
			<td>Surgical scissors (sharp and blunt tips)</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Fine tip surgical scissors (sharp tips)</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Straight fine tip forceps</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Curved medium tip forceps</td>
			<td>Fisher</td>
			<td>16100110</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Mouse anti-SYCP3</td>
			<td>Abcam</td>
			<td>ab97672</td>
			<td>Use at 1:300</td>
		</tr>
		<tr>
			<td>Anti-centromere protein (derived from human CREST patient serum)</td>
			<td>Antibodies Incorporated</td>
			<td>15-234-0001</td>
			<td>Use at 1:50</td>
		</tr>
		<tr>
			<td>Humidified chamber</td>
			<td></td>
			<td></td>
			<td>We use Nunc square culture dishes 500 cm2/well with moistened paper towels</td>
		</tr>
		<tr>
			<td>Widefield microscope with epifluorescence</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>Any standard model</td>
		</tr>
		<tr>
			<td>Coplin jars</td>
			<td>Several commercial brands</td>
			<td></td>
			<td>50 ml capacity; 40 mm (1.6 in.) diameter, holds 10 slides (back to back)</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Several commercial brands</td>
			<td></td>
			<td>100 x 15 mm diameter, polystyrene sterile untreated</td>
		</tr>
	</tbody>
</table>

preparation of meiotic chromosome spreads from mouse spermatocytes

哺乳动物减数分裂是一种动态发展过程, 发生在生殖细胞, 可以研究和表征。我们使用一种方法在幻灯片表面上传播细胞核 (而不是从高处掉落), 我们展示了一项在1997年首次描述的小鼠精的优化技术。该方法在实验室中被广泛应用于研究哺乳动物减数分裂, 因为它产生了大量的高品质核, 并在前期阶段 i. 生精小管首先放在低渗溶液中以膨胀精。然后, 精被释放到蔗糖溶液中, 形成细胞悬浮, 细胞核被涂在固定的玻璃片上。在免疫之后, 可以检查与减数分裂过程密切相关的蛋白质多样性。例如, 联会复合体的蛋白质, 一个三方结构连接的染色体轴/核心同系物一起可以很容易地可视化。减数分裂重组蛋白通过同源重组参与 DNA 双的修复, 也可 immunostained 评价前期进展。在这里, 我们详细描述和演示了一种技术广泛用于研究哺乳动物减数分裂的精从少年或成年雄性小鼠。

这种方法提供大量的表面扩散核包含完整的同系物的能力取决于三主要因素: 1) 细精小管在低渗缓冲液中的适当孵育时间 (即,) 获得适当膨胀的精核爆裂, 但不解体从长期孵化在 2) micropipetting 的蔗糖细胞, 以获得一个分离的悬浮细胞核, 不成群在一起, 和 3) 倾斜每一个方向的固定涂层的幻灯片在一个时间而不是来回。一旦准备好, 表面扩散核可以 immunostained 与荧光标记抗体的...

Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes at JoVE.com

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

减数分裂是通过单轮 DNA 复制和连续两轮染色体分离形成配子的发展过程。哺乳动物减数分裂可以通过利用一种技术来进行减数分裂染色体的研究。在此, 我们展示了一种从小鼠精中制备表面扩散核的方法。

小鼠精减数分裂染色体的制备

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

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游离的小鼠皮层神经元文化的制备

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Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

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Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

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In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Flow Cytometry Purification of Mouse Meiotic Cells

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Chromosome Preparation From Cultured Cells

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

快速风干滴染色体制备方法适用于FISH在植物

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

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 ...