Name: live cell imaging of chromosome segregation during mitosis
Uploaded: 2018-03-14T15:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

我们要感谢郭 Chiuan 和 Bharatkumar 博士的宝贵意见, 改进了手稿。

在这些研究中进行的所有实验都是按照《食品和药物管理局 (FDA) 研究机构机构动物保育和使用委员会批准的议定书》进行的。
1. 小鼠胚胎成纤维细胞的分离培养 (MEFs)
<ol><li>通过标准协议7,8,9, 从 PP2A-B56γ鼠株和野生型窝中分离出小鼠胚成纤维细胞 (MEFs)。</li>
	<li>ExpandMEFs 3 通道, 冻结和存储, 直到实验需要<...

<ol>
	<li>Funk, L. C., Zasadil, L. M., Weaver, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+CIN:+Mitotic+Infidelity+and+Its+Consequences+for+Tumor+Promotion+and+Suppression.">Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression.</a> Dev Cell. 39, 638-652 (2016).</li><li>Etemad, B., Kops, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+issues:+kinetochore+transformations+and+spindle+checkpoint+silencing.">Attachment issues: kinetochore transformations and spindle checkpoint silencing.</a> Curr Opin Cell Biol. 39, 101-108 (2016).</li><li>Lara-Gonzalez, P., Westhorpe, F. G., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle+assembly+checkpoint.">The spindle assembly checkpoint.</a> Curr Biol. 22, 966-980 (2012).</li><li>Jallepalli, P. V., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+segregation+and+cancer:+cutting+through+the+mystery.">Chromosome segregation and cancer: cutting through the mystery.</a> Nat Rev Cancer. (2), 109-117 (2001).</li><li>Zhu, L., 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=Mitotic+protein+CSPP1+interacts+with+CENP-H+protein+to+coordinate+accurate+chromosome+oscillation+in+mitosis.">Mitotic protein CSPP1 interacts with CENP-H protein to coordinate accurate chromosome oscillation in mitosis.</a> J Biol Chem. 290 (45), 27053-27066 (2015).</li><li>Pikor, L., Thu, K., Vucic, E., Lam, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+detection+and+implication+of+genome+instability+in+cancer.">The detection and implication of genome instability in cancer.</a> Cancer Metastasis Rev. 32 (3-4), 341-352 (2013).</li><li>Varadkar, P., Despres, D., Kraman, M., Lozier, J., Phadke, A., Nagaraju, K., McCright, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+phosphatase+2A+B56&#947;+regulatory+subunit+is+required+for+heart+development.">The protein phosphatase 2A B56&#947; regulatory subunit is required for heart development.</a> Dev Dyn. 243 (6), 778-790 (2014).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Mouse+Embryonic+Fibroblast+Cells+Suitable+for+Culturing+Human+Embryonic+and+Induced+Pluripotent+Stem+Cells.">Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells.</a> J. Vis. Exp. (64), e3854 (2012).</li><li>Varadkar, P., Abbasi, F., Takeda, K., Dyson, J. J., McCright, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PP2A-B56&#947;+is+required+for+an+efficient+spindle+assembly+checkpoint.">PP2A-B56&#947; is required for an efficient spindle assembly checkpoint.</a> Cell Cycle. 18 (12), 1210-1219 (2017).</li><li>Rosner, M., Schipany, K., Hengstschl&#228;ger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+high-quality+biochemical+fractionation+with+a+refined+flow+cytometry+approach+to+monitor+nucleocytoplasmic+protein+expression+throughout+the+unperturbed+mammalian+cell+cycle.">Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.</a> Nat Protoc. 8 (3), 602-626 (2013).</li><li>Jonkman, J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+Way+You+Slice+It-A+Comparison+of+Confocal+Microscopy+Techniques.">Any Way You Slice It-A Comparison of Confocal Microscopy Techniques.</a> J Biomol Tech. 26 (2), 54-65 (2015).</li><li>Versaevel, M., Braquenier, J. B., Riaz, M., Grevesse, T., Lantoine, J., Gabriele, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+reveals+LINC+complex+recruitment+at+nuclear+indentation+sites.">Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites.</a> Sci Rep. 8 (4), 7362 (2014).</li><li>Wild, T., Larsen, M. S., Narita, T., Schou, J., Nilsson, J., Choudhary, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Spindle+Assembly+Checkpoint+Is+Not+Essential+for+Viability+of+Human+Cells+with+Genetically+Lowered+APC/C+Activity.">The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.</a> Cell Rep. 1 (8), 1829-1840 (2016).</li><li>Iuso, D., 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=Exogenous+Expression+of+Human+Protamine+1+(hPrm1)+Remodels+Fibroblast+Nuclei+into+Spermatid-like+Structures.">Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures.</a> Cell Rep. 1 (9), 1765-1771 (2015).</li><li>Ratcliffe, E., Glen, K. E., Naing, M. W., Williams, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+perspectives+on+stem+cell-based+therapies+undergoing+clinical+trials+for+regenerative+medicine:+case+studies.">Current status and perspectives on stem cell-based therapies undergoing clinical trials for regenerative medicine: case studies.</a> Br Med Bull. 108, 73-94 (2013).</li></ol>

细胞周期控制检查点, 确保准确的染色体隔离防止体检和细胞转换1,2,3。在本研究中, 我们发现 PP2A-B56γ的失活导致了一个薄弱的主轴装配检查点。活体细胞成像允许我们观察染色体错误隔离在有丝分裂期间 PP2A-B56γ MEFs 治疗 nocodazole 9。
该协议利用 BacMam 2.0 技术生成的 Histone2B-GFP 标签来标?...

在下面的协议中, 我们描述了一个方便的方法来可视化的染色体分离和有丝分裂进展在小鼠胚胎成纤维细胞使用组蛋白 2 b-GFP, BacMam 2.0 标记和活细胞成像。
细胞周期控制检查点监视染色体隔离, 并在维护细胞的基因完整性方面发挥重要作用1,2,3。分离的染色体的积累可能导致体检, 这是大多数实体肿瘤的标志4。因此, 对滞后染色体的检测可以作为一种研究染色体不稳定性的方法。
荧光标记的蛋白质可用于可视化活染色体分离和染色体滞后, 但 mCherry 标记或 H2B-GFP 标记蛋白的产生需要大量的基因传递和分子生物学知识5。在这里, 我们描述使用 CellLight Histone2B-GFP BacMam 2.0 试剂, 此后称为 CL-HB 摄政, 为了简单。这种试剂可以立即使用, 从而消除了对矢量质量和完整性的关注。此外, 该试剂不需要使用潜在的有害治疗或脂质和染料加载化学品。不同于传统的荧光标签, CL-HB 摄政污点独立功能 (i. e, 膜电位)。CL-HB 摄政可以简单地添加到细胞和孵化过夜的蛋白质表达。CL-HB 不复制哺乳动物细胞, 可用于生物安全水平 (BSL) 1 实验室设置。此外, 这种瞬变转染可以检测后, 夜间孵化长达5天, 足够的时间进行最动态的细胞分析。
另外, 可以通过流式细胞术、免疫组化或荧光原位杂交 (鱼) 6等多种技术研究染色体异常。流式细胞术可用于研究体检, 可根据细胞周期中 DNA 含量和细胞相的相位进行测量。虽然流式细胞术可以用来测量体检, 但它并没有提供染色体错误隔离的信息。鱼类和免疫组化技术使用荧光探针绑定到 DNA 或染色体。虽然这些技术提供了一个细胞种群状态的快照, 但它们不允许活细胞成像, 从而错过了任何信息, 通过时空可视化胞质分裂在特定的细胞后一段时间。
本协议用于研究 nocodazole 处理的小鼠胚胎成纤维细胞 (MEFs) 离体 PP2A-B56γ的滞后染色体或染色体错隔离。除上述应用外, 本协议还提供了一种简单的工具, 用于标记和可视化各种细胞类型的染色体分离, 可用于研究肿瘤细胞的细胞周期调节或染色体不稳定性。此外, 它还可用于研究各种药物治疗引起的染色体不稳定性, 或研究基因敲出导致染色体错误隔离的效果。

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			<td height="21" style="height:21px;width:191px;">DMEM/F12</td>
			<td style="width:135px;">Gibco</td>
			<td align="right" style="width:128px;">11320082</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Gibco</td>
			<td align="right">25030081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">MEM Non-Essential amino acids solution (100X)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">11140050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Dulbecco&#8217;s Phosphate Buffered saline</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Trypsin-EDTA</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Fetal bovine serum</td>
			<td style="width:135px;">Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:135px;">EMD Millipore</td>
			<td>MX 1458-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryogenic vial storage boxes</td>
			<td style="width:135px;">Fisherbrand</td>
			<td style="width:128px;">10-500-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Cryogenic vials</td>
			<td style="width:135px;">Corning/costar</td>
			<td align="right">431416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">T75 flasks</td>
			<td style="width:135px;">Cellstar</td>
			<td align="right" style="width:128px;">658175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">2 well chambered cover glass</td>
			<td style="width:135px;">Nunc</td>
			<td style="width:128px;">155380PK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Cellometer Vision Cell Profiler</td>
			<td style="width:135px;">Nexcelom Bioscience LLC</td>
			<td style="width:128px;">Cellometer Vision Trio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Nocodazole</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:128px;">M1404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">CellLight Histone 2B-GFP, BacMam 2.0</td>
			<td style="width:135px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:128px;">C10594</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Zeiss Cell Observer Spinning Disk Confocal Microscope system</td>
			<td style="width:135px;">Carl Zeiss Microscopy</td>
			<td style="width:128px;">Zeiss Cell Observer SD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Water Bath</td>
			<td style="width:135px;">Thermoscientific</td>
			<td style="width:128px;">280 series</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Incubator</td>
			<td style="width:135px;">Sanyo commercial solutions</td>
			<td style="width:128px;">MCO-18AIC (UV)</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Class II Biological safety cabinet</td>
			<td style="width:135px;">The Baker Company</td>
			<td style="width:128px;">SterilGard</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Axiovision software</td>
			<td style="width:135px;">Zeiss</td>
			<td style="width:128px;">Ver.4.8.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">ImageJ software</td>
			<td style="width:135px;">National Institute of Health</td>
			<td style="width:128px;">Ver. 1.51r</td>
			<td></td>
		</tr>
	</tbody>
</table>

live cell imaging of chromosome segregation during mitosis

染色体在有丝分裂细胞分裂过程中必须可靠地和均匀地隔离成子细胞。染色体隔离的保真度由包括主轴装配检查点 (SAC) 在内的多个机制控制。该囊是一个复杂的反馈系统的一部分, 负责防止细胞的进展, 通过有丝分裂, 除非所有染色体动粒连接到纺锤管。染色体滞后和异常染色体分离是功能障碍细胞周期控制检查点的指示器, 可用于测量细胞的基因组稳定性。解除对囊的放松会导致正常细胞通过染色体分离过程中的错误积累而转化为恶性细胞。该囊的实施和动粒复合体的形成由激酶和磷酸酶 (如蛋白磷酸酶 2A (PP2A)) 之间的相互作用紧密调节。该协议描述了小鼠胚胎成纤维细胞中的滞后染色体的活细胞成像, 这些老鼠的 PP2A-B56γ调节亚基被挖空。这种方法克服了其他细胞周期控制成像技术的缺点, 如流式细胞术或免疫细胞化学, 只提供一个胞质分裂的状态的快照, 而不是一个动态的时空可视化的染色体有丝分裂期间。

MEFs 从野生型和 PP2A-B56γ小鼠种子在2井室盖玻璃, 并允许附加。在2天, MEFs 的同步使用0.1% 的血清24小时。在3天, MEFs 在 200 ng/毫升 nocodazole 和 30 PPC CL regentwere 的介质中, 在 37°c 和 5% CO2上孵化18小时。在4天, 这些细胞是使用旋转圆盘共聚焦显微镜系统 (图 1) 进行成像的。活细胞成像被用来可视化的命运, 个别细胞, 因为他们从 NEBD 的进展, ...

Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

Live Cell Imaging of Chromosome Segregation During Mitosis

该协议描述了一种简便、方便的方法, 用 Histone2B-GFP BacMam 2.0 标签和旋转椎间盘共焦显微系统对有丝分裂细胞中的活染色体进行标记和可视化。

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

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