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

所有涉及小鼠的方法都使用了高道德和福利标准, 并得到了尔大学医学院的动物保育和使用委员会的批准。
1. 拟订所需的解决办法和文书
<ol><li>在50毫升的超纯实验室级水中制备低渗萃取缓冲液, pH 值 8.2-8.4 (表 1)。每一次使溶液新鲜, 保持在冰上, 并在2小时内使用准备利差;这是必要的, 以优化减少和蛋白酶抑制活动的 PMSF, 分别。每对老鼠睾丸使用10毫升的希伯来人。</li>
	<li>将0.342 克蔗糖添加到10毫升的超纯实验室级水中, 准备一个 100 mM 的蔗糖溶液 (每次新鲜)。调整 pH 值为8。2</li>
	<li>准备定影液 (1% 甲醛 (粉煤灰), 0.15% 海卫 X-100, pH 值调整到 9.2, 1 n HCl 或 1 n 氢氧化钠) 新鲜每月和存储在室温下保护免受光。稀释16% 的商业可用的粉煤灰起始库存到4% 在 1X PBS 用作工作库存, 然后存储4% 等分在-20 ° c。<ol>
<li>要准备固定液, 添加10毫升4% 的煤灰溶液和600µL 10% 海卫 X-100 到30毫升的 1X PBS, 混合好, 并调整 pH 值 9.2, 1 n HCl 或 1 n 氢氧化钠。储存在室温下用铝箔包裹的50毫升锥形管中的定影液 (不超过4周)。</li>
	</ol>
</li>
	<li>准备洗涤解决方案 (当幻灯片烘干最后30分钟) 与相片弗洛 200: 0.4% 相片弗洛 200/pbs (320 µL 的照片弗洛200在80毫升 1X pbs) 和0.4% 相片弗洛 200/dH2O (160 µL 的照片弗洛200在40毫升的超纯水实验室级水)。使用一个50毫升的 Coplin 罐充满40毫升的解决方案, 以洗涤10或更少的幻灯片一次。</li>
	<li>准备下列仪器: 1 对手术剪刀 (切皮), 1 对精尖手术剪刀 (切开腹膜), 2 对直细尖钳 (解剖每个睾丸), 1 直刃刀片 (切割每个睾丸),2对弯曲尖端钳 (固定每个睾丸和挤出精细管), 50 毫升 Coplin 罐子, 聚四氟乙烯印刷3环幻灯片, 1 手术刀与大小11刀片, 湿化室, 100 x 15 mm 培养皿, 罚款提示永久标记或铅笔, 和平台旋转振动筛。<ol>
<li>地方溢价不 precleaned 磨砂结束玻璃幻灯片直立在一个50毫升 Coplin 罐充满固定解决方案, 直到使用。 注: 如果随后在需要乙醇的过程中使用幻灯片, 例如荧光原位杂交, 则首选铅笔。</li>
	</ol>
</li>
</ol>2. 精核扩散
<ol><li>根据机构指导方针 (如颈椎脱位或无 CO2窒息) 安乐雄性小鼠。</li>
	<li>使用 P10 P20 雄性在第一波精子发生中评价减数分裂细胞;我们经常使用 P15 P17 雄性从前期的所有阶段中获取细胞. 成人睾丸也将提供前期 i 细胞, 但他们也会产生更多的 postmeiotic 细胞, 如精子。</li>
	<li>收获和称量睾丸 (记录配对的重量);然后把它们放进一个培养皿里, 里面有几滴冷的, 让它们保持湿润。</li>
	<li>用弯曲的尖端钳把睾丸靠在盘子的底部, 用一条直刃刀片在睾丸的蒴果上做一个小的垂直中线切口。虽然仍然保持睾丸对底部的盘子与一个镊子, 使用第二套弯曲尖端钳或直刃刀片, 轻轻地挤压从睾丸的精曲小管。<ol>
<li>将小管放入15毫升的锥形管内, 内含10毫升冷的, 注意避免将胶囊放入试管。重复这些步骤与第二个睾丸。</li>
	</ol>
</li>
	<li>在冰上的锥形管内孵育 decapsulated 精小管 30-60 分钟, 视睾丸的大小而定。从一对30毫克或少于30分钟的睾丸中孵育生精小管, 从一对睾丸重31到50毫克, 30-45 分钟, 和更大的睾丸60分钟。不要改变一个单一的睾丸的时间。 注意: 根据实验者的经验、所使用的试剂和正在分析的小鼠, 最佳潜伏期可能会有所不同。</li>
	<li>当生精小管正在孵化的时候, 用记号笔或铅笔用鼠标编号、滑动号和日期标注 precleaned 滑块的结霜端。然后将 pre-cleaned 的幻灯片放在含有定影液的 Coplin 罐中。</li>
	<li>在孵化后, 将小管和, 倒入一个干净的培养皿中, 并将细管分成大约3毫米直径的块状。每个丛产生2张幻灯片, 所产生的幻灯片数量取决于睾丸的大小。 注意: 我们从野生类型和6张幻灯片获得8张幻灯片从 subfertile Chtf18-空 P16 睾丸 (这是较小的由于减数分裂和有丝分裂生殖细胞数量减少8)。一个成人睾丸重达100毫克或以上, 预计将产生16幻灯片。</li>
	<li>将23µL 100 mM 的蔗糖溶液放入聚四氟乙烯印刷的一环中, 3 环滑动, 并在环中加入一丛精曲小管。 然后用弯曲的尖端钳夹住的丛, 轻轻地将小管剁碎的大小为11刀片, 直到溶液变得浑浊。 注意: 不要 overmince, 因为这会导致染色体分裂。请注意, 碎片染色体也可能是突变型的指示。</li>
	<li>去除碎片, 再添加23µL 的蔗糖溶液, 并使用微轻轻地将混合物向上和向下移几次, 以帮助分离悬浮液中的细胞。</li>
	<li>在包含固定液的 Coplin 罐中取下一张幻灯片, 并将滑倒在罐子上, 这样就能在幻灯片的下角收集到一个慷慨的水滴。<ol>
<li>吸管20µL 的蔗糖细胞悬浮从聚四氟乙烯的环上印制3环滑入固定液滴在一个溢价磨砂玻璃幻灯片, 和吸管的悬浮上和下 2-3 倍。 注: 固定液滴应足够大, 以增加20μ l 的蔗糖细胞悬浮到它允许覆盖整个幻灯片时, 混合物是传播 (见下文 2.11)。</li>
	</ol>
</li>
	<li>缓慢地将滑块倾斜到液滴的一侧, 沿幻灯片的长度将单元格悬架展开。然后慢慢地倾斜幻灯片, 沿幻灯片的宽度将单元格悬挂完全覆盖。避免在幻灯片的同一区域中多次传播悬浮, 否则单元格将相互重叠。</li>
	<li>立即将滑动平放在湿化室中, 重复步骤2.8 至 2.11, 直到所有的细精小管都被用来制作幻灯片。重要的是不要在干燥过程中移动湿化室, 以避免撕裂和重叠的细胞。</li>
	<li>在室温下, 允许幻灯片在一个覆盖的湿润的室内孵育2.5 小时, 使其干燥缓慢而不完全。高湿度和慢干在这一步是重要的, 以确保足够的传播染色质。然后去除腔室的盖子, 并允许幻灯片完全风干额外的30分钟。</li>
	<li>将幻灯片 (回退或以锯齿模式) 放在包含0.4% 张照片 200/1 x PBS 解决方案的 Coplin 罐子中, 并将两次幻灯片分别洗净5分钟, 通过在平台旋转振动筛上轻轻地搅动 Coplin 罐。使用新鲜的解决方案为每个洗涤和保持幻灯片从不同的小鼠在分开的罐子为每个洗涤。</li>
	<li>在搅拌时, 将0.4% 张 200/dH2O 溶液的 Coplin 罐中的玻片冲洗一遍。</li>
	<li>从 Coplin 罐中取出幻灯片 (最后一次洗涤), 仔细擦拭滑块的边缘和底部以去除多余的液体, 并将幻灯片置于倾斜的、几乎直立的位置以晾干。精通这种技术的实验者可以在一天内从4或5只老鼠中处理和制备表面扩散核。</li>
	<li>一旦干燥 (15-20 分钟), 立即处理幻灯片的免疫荧光染色或存储在-80 ° c 在一个紧密封闭的幻灯片框保护, 最多3周 (取决于所评估的目标蛋白; 可以获得最佳结果, 如果在同一天或2周内染色)。<ol>
<li>如果储存在-80 ° c, 然后允许幻灯片到室温和洗涤在一个 Coplin 罐子包含 1X PBS 或块在染色之前5分钟。 注意: 许多不同的免疫荧光染色协议将提供良好的结果;有关免疫协议8, 请参阅贝尔科维奇 et al.。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

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

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

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

preparation of meiotic chromosome spreads from mouse spermatocytes

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

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

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

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

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

小鼠精减数分裂染色体的制备

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

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11.7K 次观看.  Drexel University College of Medicine. 减数分裂是通过单轮 DNA 复制和连续两轮染色体分离形成配子的发展过程。哺乳动物减数分裂可以通过利用一种技术来进行减数分裂染色体的研究。在此, 我们展示了一种从小鼠精中制备表面扩散核的方法。

视频: Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

科研

生物学

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

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

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

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

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

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

果蝇聚乙烯南瓜抗体标记染色体的制备

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

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

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

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

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

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

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

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

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

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

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

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

快速风干滴染色体制备方法适用于FISH在植物

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

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.