Name: micromanipulation of chromosomes in insect spermatocytes
Uploaded: 2018-10-22T12:30:00
Description: Watch this Scientific Journal Video about Micromanipulation of Chromosomes in Insect Spermatocytes at JoVE.com

我们感谢杰西卡霍尔的宝贵讨论。

1. 初级昆虫精母细胞细胞培养的显微操作制备
<ol><li>滑动准备<ol>
<li>获得一个75毫米 x 25 毫米的玻璃滑块, 在幻灯片的中心切出20毫米直径的圆孔。 注意: 这些都是从一张窗玻璃切割成一个玻璃滑块的大小在中心的一个洞。</li>
		<li>运行一个25毫米 x 25 毫米 #1 5 盖玻片通过本生燃烧火焰为 2 s。</li>
		<li>在玻璃滑块的孔边缘周围涂抹真空润滑脂</li>
		<li>将盖玻片放在孔上, 如图 1</st...

<ol>
	<li>Chambers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+studies+II.+The+cell+aster:+a+reversible+gelation+phenomenon.">Microdissection studies II. The cell aster: a reversible gelation phenomenon.</a> Journal of Experimental Zoology. 23 (3), 483-505 (1917).</li><li>Carlson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+studies+of+the+dividing+neuroblast+of+the+grasshopper,+Chortophaga+viridifasciata.">Microdissection studies of the dividing neuroblast of the grasshopper, Chortophaga viridifasciata.</a> Chromosoma. 5 (3), 199-220 (1952).</li><li>Nicklas, R. B., Staehly, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+I.+The+mechanics+of+chromosome+attachment+to+the+spindle.">Chromosome micromanipulation. I. The mechanics of chromosome attachment to the spindle.</a> Chromosoma. 21 (1), 1-16 (1967).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+II.+Induced+reorientation+and+the+experimental+control+of+segregation+in+meiosis.">Chromosome micromanipulation. II. Induced reorientation and the experimental control of segregation in meiosis.</a> Chromosoma. 21 (1), 17-50 (1967).</li><li>Nicklas, R. B., Koch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+3.+Spindle+fiber+tension+and+the+reorientation+of+mal-oriented+chromosomes.">Chromosome micromanipulation. 3. Spindle fiber tension and the reorientation of mal-oriented chromosomes.</a> Journal of Cell Biology. 43 (1), 40-50 (1969).</li><li>Nicklas, R. B., Ward, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+error+correction+in+mitosis:+microtubule+capture,+release,+and+tension.">Elements of error correction in mitosis: microtubule capture, release, and tension.</a> Journal of Cell Biology. 126 (5), 1241-1253 (1994).</li><li>Li, X., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+forces+control+a+cell-cycle+checkpoint.">Mitotic forces control a cell-cycle checkpoint.</a> Nature. 373 (6515), 630-632 (1995).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+the+force+produced+by+the+mitotic+spindle+in+anaphase.">Measurements of the force produced by the mitotic spindle in anaphase.</a> Journal of Cell Biology. 97 (2), 542-548 (1983).</li><li>Nicklas, R. 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+forces+that+move+chromosomes+in+mitosis.">The forces that move chromosomes in mitosis.</a> Annual Review of Biophysics and Biophysical Chemistry. 17, 431-449 (1988).</li><li>Nicklas, R. B., Koch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation.+IV.+Polarized+motions+within+the+spindle+and+models+for+mitosis.">Chromosome micromanipulation. IV. Polarized motions within the spindle and models for mitosis.</a> Chromosoma. 39 (1), 1026 (1972).</li><li>Zhang, D., Nicklas, R. 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+impact+of+chromosomes+and+centrosomes+on+spindle+assembly+as+observed+in+living+cells.">The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.</a> Journal of Cell Biology. 129 (5), 1287-1300 (1995).</li><li>Nicklas, R. B., Lee, G. M., Rieder, C. L., Rupp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+cut+mitotic+spindles:+clean+cuts+and+stable+microtubules.">Mechanically cut mitotic spindles: clean cuts and stable microtubules.</a> Journal of Cell Science. 94 (Pt 3), 415-423 (1989).</li><li>Zhang, D., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomes+initiate+spindle+assembly+upon+experimental+dissolution+of+the+nuclear+envelope+in+grasshopper+spermatocytes.">Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes.</a> Journal of Cell Biology. 131 (5), 1125-1131 (1995).</li><li>Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+distribution:+experiments+on+cell+hybrids+and+in+vitro.">Chromosome distribution: experiments on cell hybrids and in vitro.</a> Philosophical Transactions of the Royal Society of London B. 227 (955), 267-276 (1977).</li><li>Paliulis, L. V., Nicklas, R. 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+reduction+of+chromosome+number+in+meiosis+is+determined+by+properties+built+into+the+chromosomes.">The reduction of chromosome number in meiosis is determined by properties built into the chromosomes.</a> Journal of Cell Biology. 150 (6), 1223-1232 (2000).</li><li>Church, K., Nicklas, R. B., Lin, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulated+bivalents+can+trigger+mini-spindle+formation+in+Drosophilamelanogaster+spermatocyte+cytoplasm.">Micromanipulated bivalents can trigger mini-spindle formation in Drosophilamelanogaster spermatocyte cytoplasm.</a> Journal of Cell Biology. 103 (6), 2765-2773 (1986).</li><li>Forer, A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+autosome+movements+and+of+sex-chromosome+movements+on+sex-chromosome+segregation+in+crane+fly+spermatocytes.">Influence of autosome movements and of sex-chromosome movements on sex-chromosome segregation in crane fly spermatocytes.</a> Chromosoma. 40 (4), 417-442 (1973).</li><li>Camenzind, R., Nicklas, R. 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+non-random+chromosome+segregation+in+spermatocytes+of+Gryllotalpa+hexadactyla.+A+micromanipulation+analysis.">The non-random chromosome segregation in spermatocytes of Gryllotalpa hexadactyla. A micromanipulation analysis.</a> Chromosoma. 24 (3), 324-335 (1968).</li><li>Ault, J. G., Felt, K. D., Doan, R. N., Nedo, A. O., Ellison, C. A., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-segregation+of+sex+chromosomes+in+the+male+black+widow+spider+Latrodectus+mactans+(Araneae,+Theridiidae).">Co-segregation of sex chromosomes in the male black widow spider Latrodectus mactans (Araneae, Theridiidae).</a> Chromosoma. 126 (5), 645-654 (2017).</li><li>Felt, K. D., Lagerman, M. B., Ravida, N. A., Qian, L., Powers, S. R., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+the+amphitelically+attached+univalent+X+chromosome+in+the+spittlebug+Philaenus+spumarius.">Segregation of the amphitelically attached univalent X chromosome in the spittlebug Philaenus spumarius.</a> Protoplasma. 254 (6), 2263-2271 (2017).</li><li>Golding, A. E., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype,+sex+determination,+and+meiotic+chromosome+behavior+in+two+pholcid+(Araneomorphae,+Pholcidae)+spiders:+implications+for+karyotype+evolution.">Karyotype, sex determination, and meiotic chromosome behavior in two pholcid (Araneomorphae, Pholcidae) spiders: implications for karyotype evolution.</a> PLoS One. 6, e24748 (2011).</li><li>Doan, R. N., Paliulis, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+reveals+an+XO-XX+sex+determining+system+in+the+orb-weaving+spider+Neoscona+arabesca+(Walckenaer).">Micromanipulation reveals an XO-XX sex determining system in the orb-weaving spider Neoscona arabesca (Walckenaer).</a> Hereditas. 146 (4), 180-182 (2009).</li><li>Paliulis, L. V., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+chromosomes+reveals+that+cohesion+release+during+cell+division+is+gradual+and+does+not+require+tension.">Micromanipulation of chromosomes reveals that cohesion release during cell division is gradual and does not require tension.</a> Current Biology. 14 (23), 2124-2129 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology Micromanipulator. DIY High Precision Micromanipulator. , (2018).</li><li>Ellis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezoelectric+micromanipulators.">Piezoelectric micromanipulators.</a> Science. 138 (3537), 84-91 (1962).</li><li>Ellis, G. W., Begg, D. A., Zimmerman , . A. M., Forer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+micromanipulation+studies.">Chromosome micromanipulation studies.</a> Mitosis/Cytokinesis. , 155-179 (1981).</li><li>Powell, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microforge+attachment+for+the+biological+microscope.">A microforge attachment for the biological microscope.</a> Journal. Royal Microscopical Society. 72 (4), 214-217 (1953).</li><li>Alsop, G. B., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+continuously+dictate+distribution+of+actin+filaments+and+positioning+of+cell+cleavage+in+grasshopper+spermatocytes.">Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes.</a> Journal of Cell Science. 117 (Pt 8), 1591-1602 (2004).</li><li>Zhang, D., Nicklas, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphase'+and+cytokinesis+in+the+absence+of+chromosomes.">Anaphase' and cytokinesis in the absence of chromosomes.</a> Nature. 382, 466-468 (1996).</li></ol>

通过练习, 在细胞周围移动染色体可以成为第二天性。针是足够僵硬和足够薄的尖端是很难 "得到的诀窍" 捏造, 但这种能力也附带实践。针是如此之好, 它们在卤烃油中移动时会变形, 对推动细胞中的染色体并不有用。针是如此钝, 他们的提示是可见的, 大到1/3 的宽度的染色体 (或更大) 很可能杀死细胞。这项协议的关键步骤是创建一个足够精细的针来推动染色体, 但不像杀死细胞那样钝。
<p class=...

显微操作揭示了染色体 congression、主轴检查点和后期染色体运动的部分机制。描述显微操作实验结果的最早出版物是罗伯特. 钱伯斯1。腔室使用带有连接玻璃针的机械微操作机器人来探测多种不同细胞类型的细胞质。不幸的是, 允许在活细胞中的染色体和许多其他细胞成分的可视化的对比方法当时是不可用的, 因此钱伯斯的实验无法显示重新定位这些蜂窝组件的效果。早期 micromanipulations 改变染色体位置使用腔室装置扫描后期细胞中的主轴安备, 表明这种操作可以改变染色体臂在后期蝗虫果蝇2的位置.本特纳和他的合作者是第一个执行精细 micromanipulations 染色体, 伸展染色体3, 分离他们从主轴和诱导方向3,4, 稳定一个malorientation 通过对5、6、7的染色体施加张力, 并测量主轴在后期8、9中产生的力。本特纳实验室的其他工作表明 , 细胞质颗粒也可以纵10 , 中心体可以通过显微操作11重新定位。显微操作不仅适用于移动染色体和其他蜂窝组件。显微操作针可以通过 demembranated 细胞中的有丝分裂主轴清晰地切割12或可用于溶解核包络13。此外, 相邻的细胞可以通过显微操作14,15进行融合。
使用微操作可以完成如此广泛的有趣实验, 乍一看, 很少有染色体生物学家做过显微操作实验。这一缺陷的一个原因是, 从脊椎动物组织派生的有丝分裂培养细胞和通常用于研究染色体运动是极其困难的 micromanipulate。这些组织培养细胞通常有皮质细胞骨架, "得到在方式" 的显微操作针, 和染色体要么无法通过针或针通过细胞研磨, 导致细胞破裂和死亡。我们, 以及其他使用显微操作的实验者, 发现了节肢动物细胞能够顺应显微操作。节肢动物精母细胞很容易在一层卤烃油中传播, 在细胞分裂过程中, 细胞膜上的皮质细胞骨架也很不稳定。因此, 节肢动物睾丸提供了一个良好的来源 meiotically 分裂细胞 (精母细胞) 和有丝分裂分裂细胞 (精原细胞) 与易于访问的染色体的显微操作。一个在操作过程中固定的蚱蜢精母细胞的序列切片表明, 针头从未穿透细胞膜;细胞膜在针周围变形 (本特纳右岸导流洞, 个人通信)。精母细胞从一些昆虫和蜘蛛分类 micromanipulated 成功, 包括蚱蜢, 祈祷螳螂, 果蝇, 鹤蝇, 蟋蟀, spittlebugs, 蛾, 黑寡妇蜘蛛, 地窖蜘蛛, 和 orb 编织蜘蛛3、7、17、18、19、20、21、22。从昆虫中培养、有丝分裂细胞可以 micromanipulated。例如, 蝗虫果蝇中的染色体在初级文化中有可以很容易地 micromanipulated2,23的染色体。我们怀疑, 来自果蝇和其他昆虫的可用培养线也将 micromanipulatable, 虽然我们没有测试这些细胞的技术。我们将展示如何为显微操作准备分裂的蝗虫和蟋蟀细胞。在一年中的任何时候, 蟋蟀都很容易从大多数宠物商店获得。蚱蜢只有在夏天才容易获得, 除非研究员可以进入实验室菌落, 但使用的物种 (Melanoplus sanguinipes) 有很容易扁平的细胞和长, 易于操作的染色体。
另一个原因, 微操作实验是由少数生物学家做的, 是并联, 移动染色体很好, 很少在市场上可用。我们发现, 操纵杆控制的压电微操作机器人控制在操纵杆运动和针头运动之间没有振动、漂移或滞后的针运动, 但其他类型的机械手也可以成功地推动染色体在牢房里由埃利斯和 Begg25,26并联设计的是染色体显微操作的理想选择, 尽管它们使用较旧的技术。压电并联目前可用, 并常用于生理学;但是, 这些并联通常不受操纵杆控制。操纵杆控制是成功的显微操作所需的平滑运动的关键, 因此, 应构建自定义操纵杆, 使当前可用的压电并联工作于染色体显微操作。操纵杆控制的压电并联的工作最好有直接位置控制, 在其中的操纵杆的移动直接转换为针运动。
新设计的压电微操作机器人可从可轻松更换的商用部件和一些小的3维印刷元件中构造, 适用于染色体显微操作24。微操作机器人具有可调节的灵敏度, 手动粗定位, 无振动, 漂移, 或滞后的针运动, 和直接位置控制的针。科学家可以使用在线24上提供的指令构造微操作机器人。以下是制备主要精母细胞细胞培养的方法和微操作细胞内的染色体。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VWR micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366 249</td>
			<td style="width:167px;">25x25 mm, no 1.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dow Corning High Vacuum Grease</td>
			<td style="width:71px;">VWR</td>
			<td>AA44224-KT</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">KEL-F Oil #10</td>
			<td style="width:71px;">Ohio Valley Specialty Chemical</td>
			<td align="right" style="width:119px;">10189</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microdissecting Scissors, Stainless Steel</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>S3271-1EA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dumont #5 fine foreceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">0.85 mm outer diameter, 0.65 mm inner diameter Pyrex glass tube&#160;</td>
			<td style="width:71px;">Drummond Scientific</td>
			<td style="width:119px;">Custom order--call to request</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Inverted, Phase contrast microscope with 10X or 16X low magnification objective and 60X or 100X high magnification objective</td>
			<td style="width:71px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">microforge</td>
			<td style="width:71px;">either custom built or Narashige</td>
			<td style="width:119px;">MF-900</td>
			<td></td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">micromanipulator</td>
			<td style="width:71px;">either custom built or Burleigh PCS-6000 with custom piezo-controlling joystick</td>
			<td style="width:119px;">PCS-6300</td>
			<td></td>
		</tr>
	</tbody>
</table>

micromanipulation of chromosomes in insect spermatocytes

染色体显微操作是照亮染色体 congression、主轴检查点和后期染色体运动机制的重要方法, 是了解控制染色体运动的关键。在细胞分裂期间。一个熟练的生物学家可以使用微操作机器人从主轴分离染色体, 重新定位细胞内的染色体, 并使用一个小的玻璃针和一个非常细的尖端对染色体施加作用力。虽然可以使用其他方法 (如光学陷印和激光的其他用途) 对染色体进行摄动, 但迄今为止, 没有任何其他方法允许将蜂窝组件重新定位在十到成百上千微米的尺度上, 对细胞几乎没有损伤。.
为染色体显微操作选择和准备合适的细胞, 特别是描述蚱蜢和板球精母细胞主要文化的制备, 用于活体细胞成像和显微操作, 是这里描述。此外, 我们还展示了一种用于在细胞内移动染色体的针的构造, 以及使用带有玻璃针的操纵杆控制压电微操作机器人在分裂细胞内重新定位染色体。样本结果显示使用微操作机器人分离染色体从主轴在主精母细胞和重新定位该染色体在细胞内。

图 6显示了2个相邻蚱蜢主精母细胞的显微操作, 其中几个例子说明了显微操作的可能用法。这个实验是使用倒置的相位对比显微镜完成的。0:00 (显示的时间在 min: s) 图像显示在操作之前的两个单元格。底部细胞中的一条染色体显示在由显微操作针 (0:05; 黑色箭头) 所施加的张力下, 然后完全脱离主轴 (0:10; 黑色箭头)。底部细胞中的一条染色体 (6:25; 黄色...

Watch this Scientific Journal Video about Micromanipulation of Chromosomes in Insect Spermatocytes at JoVE.com

Micromanipulation of Chromosomes in Insect Spermatocytes

在本协议中, 我们描述了用于显微操作的合适细胞的选择和制备, 以及使用压电微操作机器人在这些细胞中重新定位染色体。

昆虫精母细胞中染色体的显微操作

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and ...

这种方法可以帮助回答细胞生物学中的关键问题，例如，张力在满足主轴检查点方面有多重要，在miosis的染色体行为中，主轴附件的作用是什么？这种技术的主要优点是，它是一种非致命性技术，允许实验者对活细胞中的染色体施加张力或重新定位染色体。首先，准备一个75由25毫米的幻灯片与一个直径20毫米的圆形孔切出中心。然后，运行一个25由25毫米数1.5盖通过一个本森燃烧器火焰两秒钟。在玻璃滑梯的孔边缘周围涂抹真空润滑脂。接下来，将盖滑过孔并按下，形成紧密的密封。翻转滑梯，用卤化油填充新创建的解剖井。获得一些成年雄性板球后，使用解剖剪刀切割平行于腹部长轴的后侧表面，直接在翼芽后面。然后，轻轻挤压腹部，将睾丸推入外骨骼的切口。使用钳子，将隔离的睾丸放入解剖井中。在解剖显微镜下，使用细指钳将睾丸分成更小的碎片，并去除任何脂肪。然后，将睾丸的内装物铺在盖缝表面的油下。继续传播睾丸的内容，直到肉眼几乎看不到扩散部分。如有必要，可以使用额外的装油解剖井。首先，将玻璃管的末端放在本森燃烧器的火焰中。握住管子的端，形成 150 度角和窄玻璃管的扩展区域。打破薄区域中的油管，使从以前创建的角度延伸的薄区域大约 10 毫米长。使用微福，熔化玻璃针的尖端，在针和微叉的铂丝之间形成45度角。然后，将玻璃从电线上拉出，同时关闭热量。将先前准备的幻灯片放在倒置相对比度显微镜的舞台上。然后，找到分割的单元格，并使用尽可能低的放大倍率在视场中居中。将微针放入微操纵器的针架中。然后，手动将微对象置于显微镜的光路径中。聚焦显微镜，使包含细胞的平面上方的几个焦点平面。接下来，使用操纵手柄控制器多次重新定位微针，以在 x 轴和 y 轴中查找针的阴影。重新调整针的位置，直到尖端的位置可见，然后，沿 z 轴调整针的位置，直到尖端对焦。接下来，重新聚焦在细胞上，将显微镜聚焦在细胞平面的上方。然后，重新调整针头的位置，使尖端在此焦点平面中处于焦点位置。使用较高的放大率目标和更高的灵敏度设置重复此过程。重新聚焦细胞，并调整它们的位置，使它们保持在视野的中心。使用操纵杆控制微需要，因为它推动细胞内的染色体。确保针尖保留在细胞上方的平面中。要移动染色体，请聚焦在细胞顶部附近的染色体上。然后，调整操纵手柄上的 z 轴，使针尖对焦，并将指针与操纵手柄一起移动 x 和 y。将针头的尖端直接放在感兴趣的染色体上，然后按所需方向推动。施加足够的张力将染色体与主轴分离。最后，将染色体放在细胞内的任何地方。使用此协议，可以重新定位、应用张力，并使用微操纵将染色体从主轴上完全分离。在此示例中，两个纵的染色体被不断用微操纵针轻推，使两个纵的染色体无法重新连接到主轴上。一旦掌握了样品，就可以准备操作，微型操纵器可以在15到20分钟内定位。微管理实验可能需要几秒钟到几个小时。在尝试此过程时，重要的是要记住在离盖滑最远的细胞一侧操作染色体。该技术开发后，为细胞生物学领域的研究人员探索蝗虫和祈祷蟑螂的主轴检查点铺平了道路。

Research

JoVE Journal

Developmental Biology

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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

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

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 ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

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

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

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 ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...