Name: polysome profiling in leishmania, human cells and mouse testis
Uploaded: 2018-04-08T15:00:00.000+00:00
Duration: 14 min 32 s
Description: Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

作者感谢清李的录音帮助。这项研究得到了来自德州理工大学健康科学中心的启动基金的支持, 以及由翻译神经科学和治疗中心 (CTNT) 授予 PN-CTNT 2017-05 AKHRJDHW A.L.K.;部分由 NIH 授予 R01AI099380 对 K.Z. 詹姆斯 c. 霍夫曼和克里斯汀 r 巴卡是喜赛 (干细胞教育 & 研究的整合中心) 学者并且由节目支持。

所有动物的治疗和组织的处理获得的研究是根据协议的机构动物护理和使用委员会在德州理工大学健康科学中心按照国家研究所健康动物福利准则, 96005 号议定书。请牺牲脊椎动物, 根据动物保育和使用委员会的指导方针准备组织。如果缺乏这样一个委员会, 请参考国家卫生研究院动物福利指南。成人 (> 60 天) C57BL/6 小鼠使用。所有动物和组织都是根据美国德州理工大学卫生科学中心的机构动物护理和使用委员会批准的协议获得的, 根据国家卫生动物福利研究所的准则。对于安乐死, 一只老鼠被放在一个小房间里, 空气逐渐偏移, 大约有30% 二氧化碳麻醉, 并将动物的痛苦降到最低。在呼吸停止后, 我们用颈椎脱位来确认动物在收割组织前的死亡。
注意: 所有与活的利什曼原虫和培养的人类细胞的工作都是在 BSL-2 认证实验室的生物安全柜中完成的。
1. 从利什曼原虫少校、培养的人体细胞和小鼠组织中制备细胞质裂解物
注: 不同来源材料的裂解制剂有几个不同之处。其他步骤, 包括蔗糖梯度准备和 polysomal 分馏是相同的, 不依赖于样品来源。
<ol><li>
利什曼原虫主要细胞质裂解制剂
	<ol>
<li>接种利什曼原虫少校(FV1 应变) 细胞在30毫升 1x M199 中29含有10% 胎牛血清 (血清) 和青霉素/链霉素混合物 (100 单位和100微克/毫升相应) 在密度 1x105细胞/毫升. 注意: 涉及利什曼原虫主要单元格的所有步骤都必须在生物安全柜中进行。</li>
		<li>将细胞放置在孵化器中, 并将其生长在27摄氏度, 直到对数相 (中间的日志对应于 5x106细胞/mL)。它通常需要两天的时间来成长。</li>
		<li>将放线菌酮添加到利什曼原虫主要区域性, 最终集中100微克/毫升以在翻译的基因上逮捕核糖体。将细胞放回孵化器中10分钟, 在27摄氏度。</li>
		<li>放线菌酮治疗完成后, 将细胞转移到50毫升圆锥管, 并将其旋转 1800 x g 和4°c, 以8分钟为单位丢弃上清。</li>
		<li>用30毫升 Dulbecco 磷酸盐缓冲盐水 (DPBS) 冲洗细胞。离心机在 1800 x g 和4°c 为8分钟。</li>
		<li>放弃上清。并用重悬细胞在1毫升的 DPBS。</li>
		<li>整除细胞, 并将其与3.5% 甲醛溶液混合。</li>
		<li>通过 hemocytometer 计数细胞并确定它们的浓度。将所需的细胞数转移到离心管中。从 0.5x108-2x108细胞/mL 中制备的裂解液足够用于一个蔗糖梯度加载。</li>
		<li>旋转细胞在 1800 x g 和4°c 为8分钟. 丢弃上清。</li>
		<li>并用重悬在1毫升含有蛋白酶抑制剂和 RNase 抑制剂 (20 毫米 HEPES) 的裂解缓冲液中, 将细胞颗粒放在冰上, pH 值 7.4, 100 毫米氯化钾, 10 毫米氯化镁2, 2 毫米, 1% NP-40, 1x 蛋白酶抑制剂鸡尾酒 (无 EDTA), 200 单位/毫升 RNase 抑制剂)。</li>
		<li>通过23口径针三倍的裂解液。在通过针后, 裂解液应变得透明。</li>
		<li>离心机在 11200 x g 和4°c 为10分钟澄清裂解。将澄清的裂解液转移到新鲜的管子上, 并将其保存在冰上直到蔗糖梯度离心。</li>
		<li>收集 400-500 ul 的裂解剂作为输入 (稍后分析), 冻结它在液氮中的未来蛋白质分析或添加 rna 纯化试剂冷冻前的 rna 分析。</li>
	</ol>
</li>
	<li>
人 HeLa 细胞培养的细胞质裂解制剂
	<ol>
<li>将 HeLa 细胞分裂成20毫升的 DMEM 培养基, 其中含有10% 的血清和青霉素/链霉素混合物 (100 个单位和100微克/毫升相应), 细胞计数 2x105细胞/毫升在15厘米板中。</li>
		<li>生长 HeLa 细胞在37°c, 5% CO2为 20-24 h. 根据制造商的协议进行质粒 DNA 转染。</li>
		<li>在37°c、5% CO2转染后, 将细胞传播24小时。</li>
		<li>添加放线菌酮到生长的 HeLa 细胞到最后浓度的100微克/毫升, 以逮捕核糖体在翻译基因和孵化细胞为10分钟37摄氏度, 5% CO 2.吸入培养基。用冷 DPBS 在冰上洗两次细胞。</li>
		<li>添加 500 ul 的裂解缓冲 (20 毫米 HEPES-KOH pH 7.4, 100 毫米氯化钾, 5 毫米氯化镁2, 1 毫米, 0.5% NP-40, 1x 蛋白酶抑制剂鸡尾酒 (无 EDTA), 200 单位/毫升 RNase 抑制剂或1毫克/毫升肝素) 到板上, 并刮在冰上的细胞。</li>
		<li>将裂解细胞转移到离心管。根据样品体积的增加, 将 NP-40 的浓度调整为0.5% 和氯化镁2到5毫米。</li>
		<li>通过23口径针3-6 倍的裂解液。</li>
		<li>旋转 11200 x g 和4°c 8 分钟, 以澄清裂解。离心后, 将上清液转移到新管上。使用分光光度计评估细胞裂解效率, 并确定在梯度上加载的样本量。添加 10 ul 样品到0.5 毫升的0.1% 月桂酸钠 (SDS)。空白对 0.1% SDS。测量吸光度在 260 nm。预期吸光度值约为15-20 单位/毫升。</li>
		<li>在蔗糖梯度离心前稀释所有与裂解缓冲剂相同的吸光度值。将样品保存在冰上直到蔗糖梯度离心。</li>
	</ol>
</li>
	<li>
小鼠睾丸胞浆裂解制剂的制备
	<ol>
<li>解剖小鼠睾丸。在膜中做一个小切口, 收集睾丸的精管, 并将其转移到含有5毫升 DPBS 的15毫升圆锥管内, 辅以0.1 毫米 phenylmethylsulfonyl 氟化物 (PMSF)。</li>
		<li>多次反转, 使组织剧烈混合。允许组织在冰上以单位重力沉降5分钟。</li>
		<li>除去和丢弃含有结缔组织和组织片段的多云缓冲区。重复这个过程2-3 次。其余的白色颗粒被丰富的精矿小管和生殖细胞。</li>
		<li>将精管小球转移到2毫升离心管内, 在 500 x g 处旋转1分钟。</li>
		<li>添加500µL 裂解缓冲液 (20 毫米三盐酸, pH 值 7.4, 100 毫米氯化钾, 5 毫米氯化镁2, 1 毫米, 0.5% NP-40, 1x 蛋白酶抑制剂鸡尾酒 (无 EDTA), 1 毫克/毫升肝素或200单位/毫升 RNase 抑制剂) 到小管。用吸管 triturate 组织。</li>
		<li>将悬浮液转移至小 (0.5-1.0 毫升) Dounce 均质机。用玻璃杵的七到八冲程扰乱组织。</li>
		<li>将裂解液转移到1.5 毫升离心管。</li>
		<li>离心样品在 1.2万 x g 和4°c 8 分钟, 以清除裂解。将上清液转移到新管上, 储存在冰上, 直到在蔗糖梯度上加载。</li>
		<li>收集 50 ul 的裂解液作为输入样本, 冻结它马上在-80 °c 为未来的蛋白质分析;或者在冷冻前加入 rna 纯化试剂进行 rna 分析。</li>
	</ol>
</li>
</ol>2. 蔗糖梯度的制备和离心
<ol><li>准备两个蔗糖梯度溶液 (20 毫米 HEPES, pH 值 7.4, 100 毫米氯化钾, 10 毫米氯化镁2, 1 毫米, 1x 蛋白酶抑制剂鸡尾酒), 含有10% 蔗糖或50% 蔗糖。(三盐酸, pH 值 7.4, 可以使用而不是 HEPES)。根据实验设计, 添加200个单位/毫升 RNase 抑制剂或1毫克/毫升肝素。将 ultracentrifuge 管用于 SW 41 转子进入标记块并沿该块的上层绘制线。把管子转到一个稳定的架子上。</li>
	<li>采取10毫升注射器与分层设备连接, 并填补注射器与10% 蔗糖溶液 (准备如上)。轻轻松开它在 ultracentrifuge 管的底部, 直到它到达管上的标记。</li>
	<li>用50% 蔗糖溶液填充另一注射器, 并小心地将其分层装置通过10% 蔗糖层插入管底部。从底部开始轻轻地释放蔗糖溶液, 直到它到达管子上的标记。用所提供的瓶盖封住管子。</li>
	<li>要准备蔗糖渐变, 请打开 "渐变制造器" 设备ON。使用向上或下拉按钮对板进行水平设置, 然后按完成。调平对渐变的线性度很重要。</li>
	<li>在对板进行平整后, 按研究生打开渐变菜单。转到 "渐变" 菜单上的列表, 然后选择 SW 41 Ti 转子。然后使用向上和下拉按钮从菜单列表中选择所需的蔗糖渐变。按使用。</li>
	<li>将渐变管架放在渐变制造板上。把管子转到支架上。可同时准备多达6个渐变。按运行。梯度发生器按编程速度和角度旋转管子, 形成线性梯度。准备渐变要花几分钟时间。</li>
	<li>完成过程后, 将管子放在机架中。把帽子摘下来。从 ultracentrifuge 管顶部取出与试样体积相同的体积。</li>
	<li>仔细加载 400-500 ul 的裂解液, 其中包含15-20 个在顶部的 polysomes 的260单位。把管子放在转子桶里, 平衡它们。</li>
	<li>离心机在 26万 x g 和4°c 2 小时使用 SW 41 转子。</li>
</ol>3. Polysome 分馏和样品收集
注: 虽然裂解制剂有一定的差异, 根据来源, 梯度准备和 polysome 分馏协议是相同的所有类型的裂解物。
<ol><li>离心完成后, 将转子桶与管子放在冰上。打开分数收集器和渐变分馏塔ON。单击 "分馏塔" 菜单上的扫描。把24收集管的架子放进分数收集器。</li>
	<li>用去离子水填满分馏塔一侧的冲洗池。按下十年代的冲洗键以在分馏塔上冲洗泵。将冲洗适配器与装满水的注射器连接在活塞上进行校准。</li>
	<li>打开计算机上的分馏塔软件。按校准。使用默认设置并按确定。准备从注射器中注入水。</li>
	<li>按确定进行校准。立即开始为下5秒注入水。在这段时间内, 水会流经紫外线探测器的流动细胞, 仪器将被校准。将出现符号零校准完成。该仪器已准备好进行分馏。</li>
	<li>用注射器取出冲洗适配器。将尖端连接到分馏塔的活塞上。</li>
	<li>打开黄铜空气阀, 并按空气键十年代, 以干油管和流动电池。关闭气阀。</li>
	<li>轻轻地从转子桶中取出渐变管并将其放在机架中。将管座帽涂在管子的顶部, 并小心地将管子移入管架内, 并将其锁定到位。</li>
	<li>将持有人置于分馏塔的活塞下。通常, polysomal 带可以通过眼睛看到。介绍所需的分数和体积的设置 (24 分数在 500 ul/分数通常是足够的)。适当地命名该文件。按确定, 然后转到图形按钮。在下一个窗口中, 按开始扫描。设置将显示, 请按确定。收集器将从排水沟移动到第一个分数, 活塞将进入管。当活塞到达梯度的顶端, 它会减慢到选定的速度和分数将收集。完成后, 活塞从渐变管中移出。</li>
	<li>打开黄铜气阀并按下分馏塔上的air键以检索最后一小部分。</li>
	<li>把管子从架子上移到冰上。</li>
	<li>将2卷 rna 纯化试剂添加到每一个分数, 并在液氮中快速冷冻直至 RNA 纯化。或者, 如果需要分析蛋白质, 添加乙酸酸到最终浓度为 10%, 将其浓缩为西方印迹 (见8节)。</li>
</ol>4. qPCR 数据分析中基因水平规范化的合成 RNA体外制备
注意: 本协议中使用了大肠杆菌欧帕 mRNA 进行规范化。任何其他 RNA 没有广泛的身份与研究的有机体的基因 (哺乳动物或利什曼原虫) 可以使用。
<ol><li>用含有欧帕基因30的质粒的标准 PCR 反应制备含有 SP6 启动子序列的欧帕 DNA 片段。</li>
	<li>准备100µL 的混合物:80 毫米 HEPES, pH 值 7.5, 16 毫米氯化镁2, 2 毫米胺, 10 毫米多线, 3 毫米 ATP, 3 毫米 CTP, 3 毫米 UTP, 3 毫米 GTP, 0.5 U/ul RNase 抑制剂, 1 微克欧帕 PCR DNA, 3 ul SP6 RNA 聚合酶, 0.005 U/ul 焦磷酸酶。</li>
	<li>孵育40°c 为2小时。</li>
	<li>rna 纯化试剂盒纯化 rna。</li>
	<li>用分光光度法测定浓度, 用琼脂糖凝胶电泳检查。</li>
</ol>5. RNA 分离从梯度组分和 cDNA 准备
注: 如果使用 RNase 抑制剂作为核糖核酸抑制剂, 则直接使用此协议进行 RNA 纯化。然而, 当用作核糖核酸抑制剂时, 肝素会抑制逆转录酶在 cDNA 制备中的应用。因此, 如果用肝素在溶解缓冲和梯度中使用, 则需要额外的 RNA 纯化。请参阅6节, 以制备 RNA 为 cDNA 合成如果使用肝素。
<ol><li>将含有 rna 纯化试剂的样品解冻, 加入20的合成 RNA 作为 qPCR 结果规范化的内部控制。根据制造商的协议进行 RNA 的准备, 除了一个修改。添加1µL 的 RNA 级糖原 (20 µg) 前异丙醇沉淀。将 RNA 颗粒溶于20-25 µL 的 RNase 水中。 注意: 糖原作为载体, 有助于避免损失和可视化 RNA 颗粒在纯化过程中。欧帕 mRNA 用于 qPCR 反应的进一步规范化。</li>
	<li>用分光光度法测定 RNA 浓度, 确保产量充足。将含有40S、60S 和 monosomes 的 RNA 分数的相等体积组合为 prepolysomes。含有2-4 核糖体的分数作为轻 polysomes 和5-8 核糖体的分数组合为重 polysomes。</li>
	<li>使用5-10 µL 的 RNA 从组合的分数, 以准备基因使用套件和以下制造商的建议。</li>
	<li>添加80µL 核酸酶游离水到20µL 的 cDNA。将 cDNA 样本冷冻-20 摄氏度。</li>
</ol>6. 从肝素污染中提取 RNA
注: 肝素抑制核酸处理酶 (如逆转录)。因此, 当肝素用于溶解缓冲和/或梯度时, 使用这个附加的纯化协议。
<ol><li>添加 LiCl 到1米最终浓度的纯化 RNA 样品。</li>
	<li>将样品混合在冰上孵育1小时。</li>
	<li>旋转样品在 1.6万 x g 和4°c 为15分钟。</li>
	<li>用吸管将上清液取出即可完成。</li>
	<li>空气干燥的颗粒约5分钟。</li>
	<li>在 RNase 水的初始体积内重新悬浮颗粒。</li>
	<li>执行分光光度法测量 260 nm, 以确定 RNA 的浓度。通常, 样品的损失是极小的。</li>
</ol>7. qPCR 和基因分布的数据分析
<ol><li>结合 10.2 ul 的水, 20 ul 的 SYBR 绿色, 4.8 ul 的基因特异性引物 (2.5 微米每套), 5 ul 的 cDNA, 混合良好和负载 10 ul 每井在 triplicates 成384井板块。</li>
	<li>盖板与胶膜紧密和离心板在 1800 x g 5 分钟。</li>
	<li>使用实时 PCR 仪在表 1中显示的条件下设置 qPCR 反应。</li>
	<li>使用循环阈值 (CT) 和比较 Ct (ΔΔCT) 方法31计算 prepolysomes、轻型和重 polysomes 中 mRNA 分布的百分比 (%), 如所述32与一个修改。在 qPCR 数据分析中使用合成 RNA (欧帕) 进行数据规范化。合成 RNA 提供了一个规范化的控制, 允许计算相对基因水平, 并比较在不同的梯度分数。</li>
</ol>8. 西方印迹法分析 Polysomal 馏分中的蛋白质
<ol><li>从 100% (瓦特/v) 股票, 添加乙酸酸 (TCA) 到选定的分数 (500 ul) 到最后浓度为 10%, 保持在冰上至少15分钟, 离心机在一个离心为5分钟, 放弃上清, 洗两次与冰冷的丙酮和溶解在 25 ul S用于电泳的 DS 页样本加载缓冲区。</li>
	<li>负载在 SDS 页和进行标准电泳与以下转移到 PVDF 膜。继续进行西方印迹33。</li>
</ol>

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作者没有什么可透露的。

蔗糖梯度结合 RNA 和蛋白质分析 Polysome 分馏是分析个体基因或整个 translatome 的平移状态以及调节平移的蛋白质因子作用的有力方法。机械在正常的生理或疾病状态。Polysomal 分析是一种特别适合于研究生物体中的平移调节的技术, 如 trypanosomatids 包括利什曼原虫, 其中转录控制很大程度上缺乏, 基因表达调控主要发生在翻译过程中。
在这里, 我们描述了一个 polysome 分馏协?...

细胞基因表达调控由转录、转录后水平和翻译后机制控制。在深 RNA 测序方面的进展允许在一个空前的水平上研究基因组范围内的稳态 mRNA 水平。然而, 最近的研究发现, 稳态 mRNA 水平并不总是与蛋白质生产相关1,2。一个单独的成绩单的命运是非常复杂的, 取决于许多因素, 如内部/外部刺激, 压力,等。在蛋白质合成过程中, 基因表达调控为在变化条件下快速反应提供了另一层表达控制。Polysome (或 "polyribosome") 剖面分析, 积极翻译核糖体的分离和可视化, 是研究蛋白质合成调控的有力方法。虽然, 它的第一个实验应用出现在二十世纪六十年代3中, polysome 分析目前是蛋白质翻译研究4中最重要的技术之一。单基因可由多个核糖体转化导致形成 polysome。转录可以停滞在核糖体与放线菌酮5和基因包含不同数量的 polysomes 可以在 polysome 分馏过程中分离的蔗糖梯度离心 6, 7 ,8,9. polysomal 分数的 RNA 分析然后允许测量基因组范围内单个基因的平移状态的变化, 在不同的生理条件下4,7,10. 该方法还用于揭示 5 ' UTR 和 3 ' UTR 序列在 mRNA 可译性的控制中的作用11, 检查 miRNAs 在平移抑制中的角色12, 揭开核糖体合成13 的缺陷, 并了解核糖体相关蛋白与人类疾病的作用14,15。在过去的十年中, 在翻译过程中对基因表达调控的作用越来越大, 这说明了它在人类疾病中的重要性。在癌症、代谢和神经退行性疾病中进行平移控制的证据非常惊人15,16,17,18。例如, eIF4E-dependent 平移控制的失调有助于自闭症相关的赤字15和 FMRP 参与停滞的核糖体基因链接到自闭症14。因此, polysomal 分析是研究多种人类疾病的翻译调节缺陷的重要工具。
不同生理条件下 polysomal 分数的蛋白质分析, 对翻译过程中核糖体相关因素的作用进行了剖析。polysome 分析技术已用于许多物种, 包括酵母, 哺乳动物细胞, 植物和原生动物10,19,20,21。像锥和利什曼原虫这样的原生动物寄生虫表现出有限的基因表达转录控制。他们的基因组被组织成 polycistronic 基因簇, 缺乏启动子调控的转录22。相反, 发展基因表达主要控制在蛋白质的翻译水平和 mRNA 稳定性的 trypanosomatid 物种23,24。因此, 在缺乏转录调控的情况下理解平移控制对这些生物体尤为重要。Polysomal 分析是研究利什曼原虫25、26、27、28中基因表达转录后水平调控的有力工具。
通过实时定量 PCR (RT-qPCR) 和转录的下一代测序以及蛋白质组学技术检测个体基因水平的最新进展, 将 polysomal 分析的解决方案和优势带到了一个新的层次。通过对单个 polysomal 分数的分析, 结合蛋白质组学分析的方法, 可以进一步扩大对各细胞的使用, 以监测整个基因型范围内体细胞的平移状态。这使得在不同的生理和病理条件下, 识别新的分子玩家调节翻译。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型: 寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。本文就不同生物体的细胞裂解物的制备、梯度条件的优化、RNase 抑制剂的选择、qPCR、印迹和 RNA 电泳等方面的应用提出建议, 以分析 polysome 分数。

<table><tbody><tr><td>&lt;strong&gt;Instruments:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gradient master</td><td>Biocomp Instruments Inc.</td><td>108</td><td></td></tr><tr><td>Piston Gradient Fractionator</td><td>Biocomp Instruments Inc.</td><td>152</td><td></td></tr><tr><td>Fraction collector</td><td>Gilson, Inc.</td><td>FC203B</td><td></td></tr><tr><td>NanoDrop One</td><td>Thermo Scientific</td><td>NanoDrop One</td><td></td></tr><tr><td>Nikon inverted microscope</td><td>Nikon</td><td>ECLIPSE Ts2-FL/Ts2</td><td></td></tr><tr><td>2720 Thermal Cycler</td><td>Applied Biosystems by Life Technologies</td><td>4359659</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Panasonic Healthcare Co.</td><td>MCO-170A1CUV</td><td></td></tr><tr><td>HERATHERM incubator</td><td>Thermo Scientific</td><td>51028063</td><td></td></tr><tr><td>Biological Safety Cabinet, class II, type A2</td><td>NuAire Inc.</td><td>NU-543-400</td><td></td></tr><tr><td>Revco freezer</td><td>Revco Technologies</td><td>ULT1386-5-D35</td><td></td></tr><tr><td>Beckman L8-M Ultracentifuge</td><td>Beckman Coulter</td><td>L8M-70</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Ultracentrifuge Rotor SW41</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Swing-bucket rotor</td><td>Eppendorf</td><td>A-4-62</td><td></td></tr><tr><td>Fixed angle rotor</td><td>Eppendorf</td><td>F-45-30-11</td><td></td></tr><tr><td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td><td>Applied Biosystems by life technologies</td><td>4470661</td><td></td></tr><tr><td>TC20 Automated cell counter</td><td>Bio-Rad</td><td>145-0102</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>02-671-51B</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Triax software&amp;nbsp;</td><td>Biocomp Instruments Inc.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Counting slides, dual chamber for cell counter</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td><td>Seton Scientific</td><td>7030</td><td></td></tr><tr><td>Syringe, 5 mL</td><td>BD</td><td>309646</td><td></td></tr><tr><td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td><td>BD</td><td>10020439</td><td></td></tr><tr><td>Nunclon Delta Surface plate, 14 cm</td><td>Thermo Scientific</td><td>168381</td><td></td></tr><tr><td>Nunclon Delta Surface plate, 9 cm</td><td>Thermo Scientific</td><td>172931</td><td></td></tr><tr><td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td><td>Thermo Scientific</td><td>569-0020</td><td></td></tr><tr><td>BioLite 75 cm&lt;sup&gt;3&lt;/sup&gt; flasks</td><td>Thermo Scientific</td><td>130193</td><td></td></tr><tr><td>Nunc 50 mL conical centrifuge tubes</td><td>Thermo Scientific</td><td>339653</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trizol LS</td><td>Ambion by Life Technologies</td><td>10296028</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1378-5KG</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium-high glucose (DMEM)</td><td>Sigma</td><td>D6429-500ML</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F0926-50ML</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S)</td><td>Sigma</td><td>P0781-100ML</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Sigma</td><td>D8537-500ML</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td><td>Acros Organics</td><td>AC413415000</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma</td><td>P9541-500G</td><td></td></tr><tr><td>Nonidet P 40 (NP-40)</td><td>Fluka (Sigma-Aldrich)</td><td>74385</td><td></td></tr><tr><td>Recombinant Rnasin Ribonuclease Inhibitor</td><td>Promega</td><td>N2511</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3993-1MU</td><td></td></tr><tr><td>cOmplete Mini EDTA-free protease inhibitors</td><td>Roche Diagnostics</td><td>11836170001</td><td></td></tr><tr><td>Glycogen</td><td>Thermo Scientific</td><td>R0551</td><td></td></tr><tr><td>Water</td><td>Sigma</td><td>W4502-1L</td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C7698-1G</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>194002</td><td></td></tr><tr><td>Dithiotreitol (DTT)</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Fisher Scientific</td><td>BP-1302-10</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td><td>Fisher Scientific</td><td>S316-212</td><td></td></tr><tr><td>Optimem</td><td>Life Technologies</td><td>22600050</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma</td><td>P8833-100MG</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3KG</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T4049-100ML</td><td></td></tr><tr><td>Hgh Capacity cDNA Reverse Transcriptase Kit</td><td>Applied Biosystems by life technologies</td><td>4368814</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>Applied Biosystems by life technologies</td><td>4367659</td><td></td></tr><tr><td>HCl</td><td>Fisher Scientific</td><td>A144SI-212</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP26324</td><td></td></tr><tr><td>Potassium Hydroxide (KOH)</td><td>Sigma</td><td>221473-500G</td><td></td></tr><tr><td>Anti-RPL11 antibody</td><td>Abcam</td><td>ab79352</td><td></td></tr><tr><td>Ribosomal protein S6 (C-8) antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-74459</td><td></td></tr><tr><td>1xM199</td><td>Sigma</td><td>M0393-10X1L</td><td></td></tr><tr><td>Lithium cloride</td><td>Sigma</td><td>L-9650</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-500</td><td></td></tr><tr><td>Gel Loading Buffer II</td><td>Thermo Scientific</td><td>AM8546G</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Thermo Scientific</td><td>16500-100</td><td></td></tr><tr><td>Trichloracetic acid (TCA)</td><td>Fisher Scientific</td><td>A322-100</td><td></td></tr><tr><td>SuperSignal West Pico PLUS chemiluminescent substrate</td><td>Thermo Scientific</td><td>34580</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher Scientific</td><td>BP531-500</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma</td><td>L5750-1KG</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma</td><td>P7626-5G</td><td></td></tr><tr><td>RNeasy Mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate (ATP)</td><td>Sigma</td><td>A1852-1VL</td><td></td></tr><tr><td>Cytosine 5&#039;-triphosphate disodium salt hydrate (CTP)</td><td>Sigma</td><td>C1506-250MG</td><td></td></tr><tr><td>Uridine 5&#039;-triphosphate trisodium salt hydrate (UTP)</td><td>Sigma</td><td>U6625-100MG</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate (GTP)</td><td>Sigma</td><td>G8877-250MG</td><td></td></tr><tr><td>SP6 RNA Polymerase</td><td>NEB</td><td>M0207S</td><td></td></tr><tr><td>Pyrophoshatase</td><td>Sigma</td><td>I1643-500UN</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>S0266-1G</td><td></td></tr></tbody></table>

polysome profiling in leishmania, human cells and mouse testis

正确的蛋白质表达在适当的时间和正确的数量是正常细胞功能和生存在一个快速变化的环境的基础。长期以来, 基因表达研究以转录水平的研究为主。然而, 基因的稳态水平与蛋白质的生产没有很好的关联, 基因的可译性因条件而变化很大。在某些有机体, 象寄生虫利什曼原虫, 蛋白质表达在翻译水平上主要被调控。最近的研究表明, 蛋白质翻译失调与癌症、新陈代谢、神经变性和其他人类疾病有关。Polysome 分析是研究蛋白质翻译规律的有力方法。它允许测量个体基因的平移状态或在全基因组范围内检查翻译。这种技术的基础是分离 polysomes, 核糖体, 他们的亚基和自由基因在离心的细胞质裂解过程中通过蔗糖梯度。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型-寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。利什曼原虫细胞在悬浮体中自由生长, 培养的人细胞生长在粘附单层, 而小鼠睾丸代表动物组织样本。因此, 该技术适用于所有这些来源。polysomal 分数分析的协议包括通过 rt-pcr 检测个体 mRNA 水平、qPCR 蛋白和用电泳分析核糖体 rna。通过对基因与核糖体在转录水平上的研究, 通过对核糖体相关蛋白的分型质谱分析, 可以进一步扩展该方法。该方法可以很容易地调整到其他生物模型。

在本研究中, 我们描述了 polysomal 分析技术在三种不同来源的应用: 寄生性的利什曼原虫少校、培养的人体细胞和小鼠睾丸。利什曼原虫细胞在悬浮液培养基中自由生长, 培养的人细胞生长在板上的贴膜上, 小鼠睾丸代表组织样本。该方法可以很容易地调整到其他类型的自由生长细胞在悬浮, 不同类型的组织, 或从另一个有机体, 和不同类型的培养细胞。该方法由...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

polysome 分析技术的总体目标是分析个体基因或转录基因在蛋白质合成过程中的平移活动。该方法对健康和多人疾病的蛋白质合成调控、翻译活化和抑制的研究具有重要意义。

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

polysome-profiling-in-leishmania-human-cells-and-mouse-testis

Research

JoVE Journal

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

17.9K 次观看.  Texas Tech University Health Sciences Center. polysome 分析技术的总体目标是分析个体基因或转录基因在蛋白质合成过程中的平移活动。该方法对健康和多人疾病的蛋白质合成调控、翻译活化和抑制的研究具有重要意义。

视频: Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

科研

生物化学

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

免疫与感染

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

具有eGFP组成型表达的 利什曼原虫 菌株的发展

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

遗传学

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

发育生物学

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

生物学

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

翻译起始的分析在应力条件由多聚核糖体剖析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

使用多体分析在发育中的鼠标大脑中进行翻译分析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

神经科学

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

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真核Polyribosome剖面分析

翻译起始的分析在应力条件由多聚核糖体剖析

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

使用多体分析在发育中的鼠标大脑中进行翻译分析

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

摘要

探索更多视频

此视频中的章节

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Antileishmanial筛选对细胞内的寄生虫救援和转化实验

具有eGFP组成型表达的 利什曼原虫 菌株的发展

无需梯度测定器或分馏系统的多聚体分析

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

真核Polyribosome剖面分析

翻译起始的分析在应力条件由多聚核糖体剖析

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

使用多体分析在发育中的鼠标大脑中进行翻译分析

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

具有eGFP组成型表达的利什曼原虫菌株的发展