这项工作得到了 NIH 授予 GM59354 颁发给沙龙 e. Bickel 的支持。我们感谢 Huy, 阮协助开发的协议, 产生荧光手臂探头, Ann Lavanway 的帮助与共焦显微镜, j. 因苏阿芦苇技术援助。我们还感谢果蝇社区的许多同事提供有益的讨论和建议。

<ol>
	<li>Peters, J. M., Nishiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sister+chromatid+cohesion.">Sister chromatid cohesion.</a> CSH Perspect Biol. 4 (11), (2012).</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>Buonomo, S. B., 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=Disjunction+of+homologous+chromosomes+in+meiosis+I+depends+on+proteolytic+cleavage+of+the+meiotic+cohesin+Rec8+by+separin.">Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.</a> Cell. 103 (3), 387-398 (2000).</li><li>Bickel, S. E., Orr-Weaver, T., Balicky, E. 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+sister-chromatid+cohesion+protein+ORD+is+required+for+chiasma+maintenance+in+Drosophila+oocytes.">The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes.</a> Curr. Biol. 12 (11), 925-929 (2002).</li><li>Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMC1beta-deficient+female+mice+provide+evidence+that+cohesins+are+a+missing+link+in+age-related+nondisjunction.">SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction.</a> Nat Genet. 37 (12), 1351-1355 (2005).</li><li>Freeman, S. B., 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=The+National+Down+Syndrome+Project:+design+and+implementation.">The National Down Syndrome Project: design and implementation.</a> Public Health Rep. 122 (1), 62-72 (2007).</li><li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nat Rev Genet. 13 (7), 493-504 (2012).</li><li>Angell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-meiotic-division+nondisjunction+in+human+oocytes.">First-meiotic-division nondisjunction in human oocytes.</a> Am. J. Hum. Genet. 61, 23-32 (1997).</li><li>Tsutsumi, 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=Age-related+decrease+of+meiotic+cohesins+in+human+oocytes.">Age-related decrease of meiotic cohesins in human oocytes.</a> PLoS One. 9 (5), e96710 (2014).</li><li>Liu, L., Keefe, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+cohesin+is+associated+with+age-dependent+misaligned+chromosomes+in+oocytes.">Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes.</a> Reprod Biomed Online. 16 (1), 103-112 (2008).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+predisposes+oocytes+to+meiotic+nondisjunction+when+the+cohesin+subunit+SMC1+is+reduced.">Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced.</a> PLoS Genet. 4 (11), e1000263 (2008).</li><li>Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, 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=Evidence+that+weakened+centromere+cohesion+is+a+leading+cause+of+age-related+aneuploidy+in+oocytes.">Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes.</a> Curr. Biol. 20 (17), 1522-1528 (2010).</li><li>Lister, L. 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=Age-related+meiotic+segregation+errors+in+mammalian+oocytes+are+preceded+by+depletion+of+cohesin+and+Sgo2.">Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2.</a> Curr. Biol. 20 (17), 1511-1521 (2010).</li><li>Duncan, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+cohesion+decreases+in+human+eggs+with+advanced+maternal+age.">Chromosome cohesion decreases in human eggs with advanced maternal age.</a> Aging Cell. 11 (6), 1121-1124 (2012).</li><li>Yun, Y., Lane, S. I., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+dyad+separation+in+meiosis+II+is+the+major+segregation+error+with+maternal+age+in+mouse+oocytes.">Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes.</a> Development. 141 (1), 199-208 (2014).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-mediated+association+of+achiasmate+homologues+declines+with+age+when+cohesion+is+compromised.">Heterochromatin-mediated association of achiasmate homologues declines with age when cohesion is compromised.</a> Genetics. 181, 1207-1218 (2009).</li><li>Jeffreys, C. A., Burrage, P. S., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+system+for+increased+meiotic+nondisjunction+in+older+oocytes.">A model system for increased meiotic nondisjunction in older oocytes.</a> Curr. Biol. 13 (6), 498-503 (2003).</li><li>Weng, K. A., Jeffreys, C. A., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rejuvenation+of+Meiotic+Cohesion+in+Oocytes+during+Prophase+I+Is+Required+for+Chiasma+Maintenance+and+Accurate+Chromosome+Segregation.">Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation.</a> PLoS Genetics. 10 (9), e1004607 (2014).</li><li>Perkins, A. T., Das, T. M., Panzera, L. C., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+in+oocytes+during+midprophase+induces+premature+loss+of+cohesion+and+chromosome+segregation+errors.">Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors.</a> Proc Natl Acad Sci U S A. 113 (44), E6823-E6830 (2016).</li><li>Dernburg, A. F., Sedat, J. W., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+a+role+for+heterochromatin+in+meiotic+chromosome+segregation.">Direct evidence of a role for heterochromatin in meiotic chromosome segregation.</a> Cell. 86, 135-146 (1996).</li><li>Dernburg, A. F., Sedat, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Method Cell Biol. 53, 187-233 (1998).</li><li>Guo, Z., Batiha, O., Bourouh, M., Fifield, E., Swan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Securin,+Separase+and+Cohesins+in+female+meiosis+and+polar+body+formation+in+Drosophila.">Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.</a> J Cell Sci. 129 (3), 531-542 (2016).</li><li>Giauque, C. C., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-Associated+Proteins+HP1a+and+Piwi+Collaborate+to+Maintain+the+Association+of+Achiasmate+Homologs+in+Drosophila+Oocytes.">Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.</a> Genetics. 203 (1), 173-189 (2016).</li><li>Joyce, E. F., Apostolopoulos, N., Beliveau, B. J., Wu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+progenitors+escape+the+widespread+phenomenon+of+homolog+pairing+during+Drosophila+development.">Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development.</a> PLoS Genet. 9 (12), e1004013 (2013).</li><li>Gilliland, W. D., Hughes, S. F., Vietti, D. R., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congression+of+achiasmate+chromosomes+to+the+metaphase+plate+in+Drosophila+melanogaster+oocytes.">Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes.</a> Dev Biol. 325 (1), 122-128 (2009).</li><li>Gyuricza, M. 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=Dynamic+and+Stable+Cohesins+Regulate+Synaptonemal+Complex+Assembly+and+Chromosome+Segregation.">Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation.</a> Curr Biol. 26 (13), 1688-1698 (2016).</li><li>Radford, S. J., McKim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+Imaging+Prometaphase+and+Metaphase+of+Meiosis+I+in+Fixed+Drosophila+Oocytes.">Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes.</a> J Vis Exp. (116), (2016).</li></ol>

作者声明没有竞争的金融利益。

使用鱼探针评估中期 i 和中期 i果蝇卵母细胞的臂部凝聚状态是在果蝇减数分裂领域的一个重要进展。从历史上看,果蝇研究人员已经被限制在成熟的卵母细胞中推断出过早丧失手臂的凝聚力11,18,19。现在, 用这里所提出的方法, 可以直接使用鱼来测定手臂的内聚力状态。获得身体证据的能力过早失去了手臂的凝聚力, 大大扩大了方法, 以研究的机制, 导致过早失去手臂的凝聚力和染色体 missegregation 在果蝇卵母细胞。
关键步骤 虽然我们没有对在成熟的果蝇卵母细胞中沿染色体臂 hybridizes 单拷贝序列的荧光探针的成功可视化所需的关键参数进行系统分析, 但我们提供了以下列出了可能有助于这项技术成功的因素。为了生成 arm 探针, 我们使用了六重叠的 BAC 探针的组合, 它在X染色体臂上覆盖了大约 0.8 Mb 的间隔。我们最初尝试使用更少的巴克斯来跨越较小的区域是不成功的。此外, 我们还使用了一种方法来分割和放大的 BAC DNA 前端标记与 Alexa-647。被标记的多数限制片段是 100-200 bp 长 (图 2), 与150核苷酸的目标大小一致, 这是有效地通过厚组织扩散所必需的,21。保持大的果蝇卵母细胞形态的固定条件, 但也允许探针穿透是必不可少的。我们修改了以前使用的固定方法23 , 将室温庚烷添加到预热到37° c 的固定解决方案中。我们认为有可能增加的庚烷通过卵黄膜的渗透, 以及降低固定的温度, 有助于成功的固定条件的可视化手臂的凝聚力。
当分析过早失去凝聚力时, 你需要一个样本大小, 允许定量的缺陷, 目前在低到中等水平。为了获得大量成熟的卵母细胞, 其他人用搅拌器扰乱成年雌性与筛分26,27。在这里, 我们描述一种方法, 手解剖卵巢从 20-25 女性, 与人工分离晚期卵母细胞的移。虽然搅拌机/筛分方法也应与该协议, 手解剖的优势之一是, 没有筛选步骤, 卵母细胞在固定前的缓冲时间较少, 这减少了后期 I 发病的机会, 由于人工卵激活.此外, 这种方法需要较少的女性作为起始材料, 使用更小的试剂量, 并有一个更简单的工作流程, 所有这些都有利于处理多个基因型。
手解剖和人工分离成熟卵母细胞提供了足够数量的卵母细胞 "滚" 的绒毛膜和卵黄膜去除, 结果约 75-150 卵母细胞在年底的杂交洗。一个诀窍, 以最大限度地提高产量中期我逮捕了卵母细胞是在解剖25之前, 在没有雄性的情况下, 在 yeasted 瓶中举行处女雌性。卵母细胞滚动, 以消除 chorions 和卵黄膜必须进行温和的触摸, 以避免破坏卵母细胞。然而, 从100% 的卵母细胞中去除 chorions 和卵黄膜并不是一个现实的目标, 过度滚动只会导致卵母细胞的丧失。因此, 当大约 75-85% 的卵母细胞缺乏 chorions 和卵黄膜时, 每个滚动周期都应该停止。
限制 尽管我们在生成和使用 arm 探针来监测成熟的果蝇卵母细胞中的内聚力状态方面取得了成功, 但该过程仍然受到限制。虽然我们很少检测不到信号的 pericentromeric 探针, 手臂探头信号微弱或缺席约 15-20% 的卵母细胞, 我们成像。一个促成因素可能是第9寡核苷酸探针 (22-28 核苷酸) 与较大的臂探头 (100-200 核苷酸) 的差分穿透。此外, 第9探针所识别的大重复序列会产生比 arm 探头更亮的信号。卵母细胞在卵母细胞中的定位和减数分裂染色体的放置也给成像带来了挑战。虽然减数分裂染色体与细胞皮质相对较近, 但在片上安装时, 无法控制卵细胞的方向。因此, 在卵母细胞的某些部分, 减数分裂的染色体可能是 100-200 µm 从片, 信号强度和质量可能会受到负面影响, 当试图通过大量的卵母细胞的图像。这些限制意味着在最后的定量分析中包括的卵母细胞数量大大低于该协议中处理的卵母细胞数量。确定100卵母细胞的手臂内聚力状态很可能需要两个独立的准备。用激光扫描共聚焦显微镜来量化内聚力缺陷的另一个限制因素是捕获一个典型的 Z 系列 (大约5分钟) 所需的时间, 即使捕获的区域限于 1024 x 512 像素。这意味着, 对照组和实验性基因型 (100 个卵母细胞) 的内聚力将需要大约16小时的图像采集时间。
修改和故障排除 我们已经能够监测的手臂的凝聚力的状态使用一个探测器, 承认X染色体在细胞学位置 6 e-7 b19。hybridizes 到X染色体上较远端位置的探针可能更适合于检测交叉维护的故障。另外, 其他研究人员可能希望分析其他染色体上的手臂内聚力。虽然我们没有试图探测染色体, 初步数据表明, 一个探测器, 承认 2 3 c 区的X染色体也会导致一个可检测的信号。然而, 根据有限的初步数据, 2 f-3 c 探针的信号可能比 6 e 7 b 探针的 "紧凑" 更少。虽然鱼的信号是紧密和脆的一些卵母细胞染色体, 杂交信号的染色体上的其他卵母细胞是相当多的扩散。这些信号的差异是否反映了两个细胞学位置之间染色体形态的真正差异, 两个探针杂交效率的变异性, 或者只是不同卵母细胞之间的随机变异需要更透彻的分析。
重要的是, 即使对于 6 e 7 b 探针, 我们观察到了一些卵母细胞中的弥漫性染色体信号, 这常常需要他们排除在分析之外。此外, 我们注意到信号质量和亮度在 6 e 7 b 探头不同的准备之间的变化, 这就要求对成像参数进行相应的调整。将杂交温度提高到42° c 并不能减少具有漫反射信号的卵母细胞数量, 但对第9寡核苷酸探针的信号有严重影响。为了更好地保存染色体形态学24, 我们还尝试了较长的变性步骤在较低的温度 (80 ° c), 但发现这种治疗既没有增加信号强度, 也没有减少的卵母细胞的数量扩散染色体鱼信号。我们还试图获得一个更明亮的手臂信号, 使用12重叠巴克斯对应的约 1.5 Mb 间隔, 跨区的细胞学区域 5 e-7 b。当使用 12 BAC 探针时, 信号强度的变化程度以及卵母细胞缺乏信号的百分比与六巴克斯用于做手臂探针的情况没有什么不同。在使用 12 BAC 探针时, 由于鱼的信号更大, 所以对粘着性缺陷的评分也更具挑战性, 这使得解决与单个染色相对应的斑点更加困难。在没有用新准备的探针获得信号的情况下, 研究人员应该检查放大和限制消化的 BAC DNA 的大小, 以确认用于结束标记的片段主要在 100-200 bp 范围内。这可能是最关键的因素之一, 成功的探针准备。
未来方向 未来的工作, 以加强图像获取, 以及适应协议, 包括免疫将是主要的改进, 将有利于其他研究人员。在图像采集过程中, 在轴向维数中采集更多的图像, 以及更快的图像采集将有利于量化凝聚缺陷。在优化激光扫描共焦的成像参数时, 我们发现0.25 µm 是最小的步长, 可用于 Z 系列, 以避免鱼信号的可观的漂白。但是, 对于在 Z 堆栈中被小于0.25 µm 的鱼信号, 此步长可能会导致无法捕获焦点之间的 "空间", 因此可能会低估聚合缺陷的数量。旋转圆盘共焦的图像采集速度越快, 就可以使用较小的步长, 而无需进行信号漂白。这将提供更准确的图像重建的轴向尺寸, 以及允许更多的卵母细胞的凝聚力缺陷分析。此外, 结合免疫与鱼类可视化的手臂凝聚力状态的能力将大大增强该协议。对于一些准备工作, 我们试图执行的纺锤免疫后, 杂交过程。然而, 强健的纺锤染色在仅一小部分卵母细胞是可看见的。此外, 由于不清楚的原因, 当鱼类和免疫结合时, 更高水平的杂散鱼信号包围了减数分裂染色体。进一步的工作, 以优化协议, 使免疫无论是在鱼的过程之前或之后, 将大大扩大该技术的能力。

在有丝分裂和减数分裂过程中适当的染色体分离需要建立、维护和以协调的方式释放姊妹染色单体的内聚力1,2。衔接是在 S 阶段建立的, 由内聚力复合体介导, 形成物理联系, 把姐妹染色在一起。在减数分裂, 衔接远端的交叉也功能举行重组同系物一起, 这个物理协会有助于确保正确定位的二价在中期 I 轴 (图 1)3,4, 5。在后期 I 释放手臂的内聚力允许同系物分离到相反的主轴杆。然而, 如果手臂的凝聚力过早丢失, 重组同系物将失去他们的物理连接和随机隔离, 这可能导致体配子 (图 1)。
在人类卵母细胞中, 染色体分离的错误是导致流产和先天缺陷的主要原因, 如唐氏综合症6, 其发病率随着产妇年龄的增加呈指数级的7。胎卵母细胞中建立了姊妹染色单体的内聚力, 在出生前完成减数分裂重组。卵母细胞, 然后在中期前逮捕我, 直到排卵和在此逮捕期间, 重组同系物的持续物理关联依赖于姐妹染色单体的内聚力。因此, 在减数分裂和正常妊娠结局中准确的分离需要五年的内聚力保持完好。
在人类卵母细胞长期减数分裂的过程中, 过早失去凝聚力已被建议为产妇年龄效应和多行证据支持这一假设8,9。然而, 考虑到研究人类卵母细胞减数分裂凝聚的挑战, 我们对这种现象的理解大多依赖于模型生物体的使用5,10,11,12, 13,14,15。
果蝇卵母细胞为研究减数分裂凝聚和染色体分离提供了许多优势。一个简单的基因分析允许一个人从体配子中恢复后代, 并在大规模的11、16、17中测量X-染色体隔离的保真度。此外, 你也可以确定是否出现染色体偏析错误, 因为重组同系物 missegregate 在减数分裂 I, 一个表型, 是一致的过早失去手臂的凝聚力11,18, 19. 在果蝇卵母细胞中的减数分裂状态的直接观察也可以使用荧光原位杂交 (fish)。虽然荧光寡核苷酸, 杂交重复的卫星序列已经使用了十几年来监测 pericentromeric 凝聚在成熟的果蝇卵母细胞4,20, 分析手臂凝聚力更具挑战性。手臂内聚力状态的可视化需要一个探针, 它横跨一个大区域的单拷贝序列, 并且足够明亮, 可以在缺乏手臂内聚力的情况下为单个的姐妹染色产生可见的信号。此外, 卵母细胞的固定条件和大小的标记 dna 必须促进渗透21到大成熟的果蝇卵母细胞 (200 µm 宽500µm 长)。最近, 一个手臂探针成功地用于可视化果蝇卵母细胞染色在后期 i, 但作者说, 他们无法检测信号在中期或中期我逮捕了卵母细胞22。在这里, 我们提供了一个详细的协议, 以生成X-染色体臂鱼探针和卵母细胞的制备条件, 使我们能够检测早期丢失的姊妹染色单体的内聚力在中期 i 和中期 i 卵母细胞。这些技术, 使我们能够识别基因产品, 这是需要维护的减数分裂的凝聚力, 将允许其他人的姐妹染色单体内聚力缺陷的成熟的果蝇卵母细胞不同基因型。

<table>
	<tbody>
		<tr>
			<td>Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midi Prep kit</td>
			<td>Qiagen</td>
			<td>12143</td>
			<td>Prep BAC clone DNA</td>
		</tr>
		<tr>
			<td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td>
			<td>Sigma</td>
			<td>WGA2</td>
			<td>Amplify BAC clone DNA</td>
		</tr>
		<tr>
			<td>ARES Alexa Fluor 647 DNA labeling kit</td>
			<td>Invitrogen</td>
			<td>A21676</td>
			<td>Label BAC clone DNA</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Remove non-conjugated dye following labeling of BAC clone DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals &#38; Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td>Prepare 10% stock 
			Freeze aliquots</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C75-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Toxic: wear appropriate protection. Prepare 100&#181;g/ml stock in 100% ethanol, store in aliquots at -20 &#176;C. Prepare 1 &#181;g/ml solution in 2X SSCT before use.</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>Sigma</td>
			<td>D-8906</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite</td>
			<td>Drierite Company</td>
			<td>23001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Invitrogen</td>
			<td>15508-013</td>
			<td>Prepare 10 mM stock</td>
		</tr>
		<tr>
			<td>dTTP (10 &#181;mol, 100 &#181;l)</td>
			<td>Boehringer Mannheim</td>
			<td>1277049</td>
			<td>Prepare 1 mM stock</td>
		</tr>
		<tr>
			<td>EDTA (Disodium ethylenediamine tetraacetic acid)</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>Prepare 250 mM stock</td>
		</tr>
		<tr>
			<td>100% ethanol (molecular grade, 200 proof)</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Ultra Pure)</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)</td>
			<td>Sigma</td>
			<td>E-3889</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Ted Pella, Inc.</td>
			<td>18505</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>AM9342</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>901393</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Boehringer Mannheim</td>
			<td>737-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Fisher Scientific</td>
			<td>H350-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore</td>
			<td>HX0603-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydroxylamine</td>
			<td>Sigma</td>
			<td>438227</td>
			<td>Prepare 3 M stock</td>
		</tr>
		<tr>
			<td>4.9 M Magnesium chloride</td>
			<td>Sigma</td>
			<td>104-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 &#376; 7H2O</td>
			<td>Fisher Scientific</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 &#376; 2H2O</td>
			<td>Fisher Scientific</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (0.1mg/ml)</td>
			<td>Sigma</td>
			<td>P8920-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher Scientific</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S209-500</td>
			<td>Prepare 3M stock</td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Polysciences, Inc.</td>
			<td>1131</td>
			<td>Toxic: wear appropriate protection. Prepare 400mM stock</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td>BP327-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>Thermo Scientific</td>
			<td>28320</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>10% Triton X-100</td>
			<td>Thermo Scientific</td>
			<td>28314</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 1 mM EDTA, pH = 8.0</td>
		</tr>
		<tr>
			<td>20X SSC (Saline Sodium Citrate)</td>
			<td></td>
			<td></td>
			<td>3 M NaCl, 300 mM sodium citrate</td>
		</tr>
		<tr>
			<td>2X cacodylate fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA</td>
		</tr>
		<tr>
			<td>1.1X Hybridization buffer</td>
			<td></td>
			<td></td>
			<td>3.3X SSC, 55% formamide, 11% dextran sulfate</td>
		</tr>
		<tr>
			<td>Fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution</td>
		</tr>
		<tr>
			<td>PBSBTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.5% BSA, 0.1% Trition X-100</td>
		</tr>
		<tr>
			<td>PBSTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100</td>
		</tr>
		<tr>
			<td>Extraction buffer (PBSTx + Rnase)</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100, 100 &#181;g/mL RNase</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>2X SSCT + 20% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 40% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 50% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AluI</td>
			<td>New England Biolabs</td>
			<td>R0137S</td>
			<td></td>
		</tr>
		<tr>
			<td>HaeIII</td>
			<td>New England Biolabs</td>
			<td>R0108S</td>
			<td></td>
		</tr>
		<tr>
			<td>MseI</td>
			<td>New England Biolabs</td>
			<td>R0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>MspI</td>
			<td>New England Biolabs</td>
			<td>R0106S</td>
			<td></td>
		</tr>
		<tr>
			<td>RsaI</td>
			<td>New England Biolabs</td>
			<td>R0167S</td>
			<td></td>
		</tr>
		<tr>
			<td>BfuCI</td>
			<td>New England Biolabs</td>
			<td>R0636S</td>
			<td></td>
		</tr>
		<tr>
			<td>100X BSA</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>10X NEB buffer #2</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Restriction enzyme digestion buffer. Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase (TdT) 400 U/&#181;l</td>
			<td>Roche/Sigma</td>
			<td>3333566001</td>
			<td></td>
		</tr>
		<tr>
			<td>TdT buffer</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>Cobalt chloride</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Toxic: wear appropriate protection. Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>RNase A (10 mg/mL)</td>
			<td>Thermo-Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cytology Tools etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>#5 INOX, Biologie</td>
		</tr>
		<tr>
			<td>9&#8221; Disposable glass Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td>Autoclave to sterilize</td>
		</tr>
		<tr>
			<td>Shallow glass dissecting dish</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Deep well dish (3 wells)</td>
			<td>Pyrex</td>
			<td>7223-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium microscope slides</td>
			<td>Fisher Scientific</td>
			<td>22-038-104</td>
			<td>Used to cover deep well dishes</td>
		</tr>
		<tr>
			<td>Frosted glass slides, 25 x 75 mm</td>
			<td>VWR Scientific</td>
			<td>48312-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, 3 x 1 in, 1 mm thick</td>
			<td>Thermo-Scientific</td>
			<td>3051</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 10 x 10 mm, No. 1.5</td>
			<td>Thermo-Scientific</td>
			<td>3405</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needle</td>
			<td></td>
			<td></td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Prolong GOLD mounting media</td>
			<td>Molecular Probes</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed-air in can</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000, spectrophotometer</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at room temperature</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at 4 &#176;C</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven or incubator with rotator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A1RSi laser scanning confoal</td>
			<td>Nikon</td>
			<td></td>
			<td>40X oil Plan Fluor DIC (NA 1.3)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;l microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l PCR tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic container with tight fitting lid</td>
			<td>Various</td>
			<td></td>
			<td>To hold Drierite</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Various</td>
			<td></td>
			<td>disposable wipes</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Various</td>
			<td></td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC purified 5&#39;-labeled oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Cy3-labeled probes that recognize the 359 bp satellite repeat of the X chromosome</td>
		</tr>
		<tr>
			<td>Volocity 3D Image Analysis Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Version 6.3</td>
		</tr>
	</tbody>
</table>

using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes

在人类中, 卵母细胞的染色体分离错误是导致大多数流产和先天缺陷的原因。此外, 随着妇女年龄的增长, 她们受孕体胎儿的风险急剧增加, 这种现象被称为孕产妇年龄效应。在减数分裂的过程中, 准确的染色体分离的一个要求是在卵母细胞经历的延长的前期阶段保持姐妹染色单体的内聚力。人类和模型生物体的细胞学证据表明, 在衰老过程中, 减数分裂的凝聚力会恶化。此外, 人类卵母细胞中的偏聚错误在减数分裂 I 中最为普遍, 与过早失去手臂的内聚力一致。模型生物体的使用对于解开依赖于年龄的丧失凝聚力的机制至关重要。果蝇为研究卵母细胞减数分裂凝聚的调节提供了一些优势。然而, 直到最近, 在不同基因型的卵母细胞或不同的实验条件下, 只有遗传试验才能检测到手臂的内聚力丧失。在这里, 提供了一个详细的协议, 使用荧光原位杂交 (fish) 直接可视化缺陷的手臂凝聚力在中期 i 和中期我逮捕了果蝇卵母细胞。通过生成一个鱼探针, hybridizes 到X染色体的远端臂并收集共焦 Z 栈, 研究人员可以在三维度中可视化单个鱼信号的数量, 并确定姊妹染色单体的手臂是否分离.这一过程使人们有可能在数以百计的果蝇卵母细胞中量化手臂的内聚力缺陷。因此, 这种方法提供了一个重要的工具, 以调查的机制, 促进凝聚力的维护, 以及导致其消亡的因素在老化过程中。

图 5显示了在X染色体上 hybridizes 到细胞学区域 6 e-7 b 的臂探针中获得的图像。该探针的结果是与 DAPI 的本地化的信号, 很容易区分与背景, 并已成功地用于量化的手臂凝聚力缺陷的不同基因型的19。粘聚缺陷的量化仅限于中期 i. 期和中期 i 阶段;核包膜破裂之前的卵母细胞被排除在分析19之外。一个完整的核信封是明显的, 当扫描在 DAPI 通道, 因为卵母细胞染色体将周围的一个离散的圆形区域, 比周围的细胞质更暗。
图 5B显示了反褶积和对比增强后单个 Z 系列的最大强度投影。聚合缺陷的量化要求每个探针确定与 DAPI 信号重叠的鱼信号的数量。对于这样的分析, 在三维的合并图像的可视化是当务之急。在分析中, 只有与所有三维度中的 DAPI 信号明显相关的鱼点才应包括在内。
完整的姐妹染色单体的内聚力证明为两个鱼点的手臂和第9探针 (图 5B, 左)。含有三或四分离鱼斑的卵母细胞被认为具有内聚力缺陷 (图 5B, 右)。将两个鱼的信号分类为单独的斑点, 在所有三维度中, 必须用与最小光斑直径对应的距离最小分隔。连接两个鱼点的微弱细线并不少见。这种信号不被认为是完全分离的, 因此不算为衔接缺陷的实例。
与 6 e 7 b 臂杂交探针, 大约 15-20% 的卵母细胞成像完全缺乏信号或信号太弱, 以自信的评分。此外, 大约5% 的卵母细胞成像显示了一个大面积的弥漫性染色体信号, 这些被丢弃的分析。相比之下, 很少遇到卵母细胞, 第9杂交信号缺失或过于弥漫而无法得分。
6 e-7 b 臂探头已被广泛应用于量化中期和中期 I 逮捕了不同基因型的果蝇卵母细胞的手臂内聚力缺陷19。当内聚力亚单位 SMC3 在中前期被撞倒时, 16.3% 的成熟卵母细胞分析了超过两个手臂的斑点, 表明过早失去手臂的内聚力。相比之下, 我们观察到只有5.1% 的卵母细胞分离的手臂斑点, 其中包含正常水平的 SMC319。有趣的是, 虽然在多重击倒基因型中, 手臂内聚力缺陷的频率提高了, 但姐妹染色在他们的第9区域19中很少分离。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56802/56802fig1.jpg"> 图 1: 在延长的前期 I 期内, 由于女性减数分裂导致的内聚力丧失, 造成染色体分离和倍的错误.凝聚力由黑酒吧代表在姐妹染色之间。臂凝聚功能, 以保持重组同系物在一起, 并要求在后期精确离析如果手臂的内聚力过早丢失, 染色体 missegregation 会发生, 导致体卵。此图是从引用19改编的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56802/56802fig2.jpg"> 图 2: DNA 分子在 BAC 碎裂和扩增后 (左) 和限制性酶消化后 (右) 三不同巴克斯的大小.400 ng 的放大 DNA 产品显示在前三车道。最后三条小巷在隔夜制约摘要以后显示脱氧核糖核酸片段。扩增的 DNA 产品范围从 200 bp 到 3 kb 不等。在限制文摘之后, 多数片段从 100-200 bp, 但更大的片段 (至多 500 bp) 是可看见的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56802/56802fig3.jpg"> 图 3: 细胞学工具协议中使用的工具: (1) 对镊子 (#5);(2) 钨针;(3) 带盖的深井盘;(4) 浅玻璃解剖盘;(5) 拔出巴斯德吸管。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56802/56802fig4.jpg"> 图4。滚动卵母细胞的排列和运动.(A)图, 用于在协议中描述的卵母细胞滚动的幻灯片。较暗的区域表示幻灯片的磨砂玻璃部分。(B)在卵母细胞滚动过程中滑动 #2 的运动矢量方向。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56802/56802fig5.jpg"> 图5。鱼化验结果。(A)示意图描述了果蝇 X染色体和两个臂探针 (6 巴克斯) 和在协议杂交中描述的22寡核苷酸 pericentromeric 探针。为简单起见, 6 e 7 b 探头被标记为 7B, 2 f-3 c 探头标记为3C。(B)鱼的代表性图像 (6 e-7 B 探针), 用于完整的臂部内聚力 (两个臂探头点, 一个同源), 以及中断的手臂内聚力 (三到四臂探头点)。脱氧核糖核酸是蓝色的, 胳膊探针信号是黄色的, 并且第9探针信号是红色的。在反褶积和对比增强后, 图像对应于 Z 系列的最大强度投影。缩放条 = 2 µm。此图是从引用19改编的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

使用荧光原位杂交 FISH 监测前中期和中期 I 果蝇卵母细胞臂内聚状态发展|生物学 |视频

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

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

本手稿提出了一个详细的方法生成X-染色体臂探针和执行荧光原位杂交 (鱼), 以检查中期和中期我被捕的姐妹染色单体的内聚力状态果蝇卵母细胞。本协议适用于确定不同基因型的减数分裂手臂的内聚力是否完好或中断。

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

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

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11.3K 次观看.  Dartmouth College. 本手稿提出了一个详细的方法生成X-染色体臂探针和执行荧光原位杂交 (鱼), 以检查中期和中期我被捕的姐妹染色单体的内聚力状态果蝇卵母细胞。本协议适用于确定不同基因型的减数分裂手臂的内聚力是否完好或中断。

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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

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发育生物学

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

斑马鱼原位杂交染色的基因分型与定量

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...