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 ...

The overall goal of this method is to label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system. This method can be used to visualize mitotic chromosomes in cultured cells to study the effects of genetic modifications or drug treatments on chromosome segregation. The main advantage of this technique is that it provides an easy and convenient method to label and visualize live chromosomes in mitotic cells.Demonstrating the live imaging procedure will be Doctor Kazuyo Takeda who maintains the Microscopy and Imaging Core Facility in the Division of Viral Products at the FDA Center for Biologics Evaluation and Research. The mouse embryonic fibroblasts, or MEFs, used in this experiment are grown in a two-well chambered cover glass at 37 degrees Celsius and 5%CO2 overnight, as described in the text protocol. On the following day, synchronize the MEFs in G0/G1 phase by incubating in DMEM/F12 growth medium containing 0.1%FBS.On the day of labeling, prepare a 1.5 milligram per milliliter stock solution of nocodazole in DMSO. A critical step is to calculate the right amount of CL-HB particles per cells to achieve enough labeling for chromosome visualization without toxicity to cells. We have previously determined, by titration experiments, that 30 PPC works best.Add to the MEFs, 200 microliters of growth medium with 10%FCS, 200 nanograms per milliliter of nocodazole, and CL-HB reagent at 30 particles per cells. Incubate the MEFs at 37 degrees Celsius and 5%CO2 for 18 hours. Visualization of the MEFs is accomplished using a spinning disk confocal microscope system equipped with an environmental chamber and an oil immersion, 63X objective lens.One day before imaging, turn the environmental chamber power on and warm up the entire chamber at 37 degrees Celsius overnight. On the following day, turn the power on for the microscope stand, camera, spinning disk unit, illuminator, argon laser, computer, and motorized stage. After letting the system warm up for three minutes, start the argon laser by turning on the ignition key.Switch the toggle switch for the argon laser from standby to laser run. Launch the data acquisition and processing software. Initiate the CO2 controller for the stage top incubator, and set the concentration of CO2 at 5%Remove the chambered cover glass from the incubator, place it on the microscope stage, and select the oil immersion 63X objective lens.View through the ocular lenses, focus the image, and identify a cell in the stage of nuclear envelope breakdown, or NEBD. Initiate the 488-nanometer argon laser to visualize Histone 2B-GFP. Open the acquisition control window, and set exposure time for the GFP channel.Identify a cell that is in NEBD. Then, manually determine the cell's top and bottom focal plane, and enter the XYZ optical sectioning settings. Observe the cell for 20 minutes.It is important to be aware that since not all cells will go through mitosis, mitotic progress must be actively monitored. If the cell does not proceed through cell division after 20 minutes, stop image acquisition and move on to the next cell that is in NEBD. To obtain data for a movie, take pictures every three minutes.Approximately one hour per cell is needed to image lagging chromosomes during mitosis in PP2A-B56-gamma cells that escaped from the spindle assembly checkpoint. Save images in ZVI file format for further analysis. Live cell imaging was used to visualize the fate of individual cells as they progressed from NEBD through mitosis.Post-nocodazole treatment, all 21 wild-type cells observed were arrested at metaphase. In contrast, from the 21 cells lacking PP2A-B56-gamma observed, 16 were able to go through mitosis after nocodazole treatment. In addition, abnormal chromosomal segregation was detected in 13 of those MEFs.An example of a PP2A-B56-gamma cell undergoing chromosomal mis-segregation is shown in this video clip. Once mastered, this technique will take three to four days depending on the cell treatment prior to the labeling protocol. It is important to remember that this is a transient transfection and signals can be detected only up to five days.This protocol can be used to answer additional questions related to the cell cycle regulation, genomic instability, and the quality of the cell therapy products. After its development, this technique could be used to study live imaging of chromosomes in a wide variety of cell lines, such as primary cell lines, stem cells, neurons, and immortalized T-cells. After watching this video, you should have a good understanding of how to easily label and visualize live chromosomes in mitotic cells using Histone 2B-GFP BacMam 2 label and a spinning disc confocal microscopy system.

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Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

成像核内作用棒在活热应力 果蝇 胚胎

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...