这项工作得到了2012毒理学学会的支持--高露洁榄替代研究基金 (m) 和大韩民国动植物检疫机构国际合作研究基金会 (m. 方) 和 NIEHS 赠款P30ES005022 (h. Zarbl)。我们要感谢 Dr. (在休斯敦德克萨斯健康科学中心的麦戈文医学院) 为他的有益的讨论, Mr. 绍-一个胡安为他的实验协助, 和 Ms. 基米中田为她的校对阅读。

1. 构建 PER2 启动器驱动的不稳定荧光报告矢量 <ol> <li> 购买自定义的 pLS [ hPER2P / rLuc /普洛] 向量, 其中含有 cDNA 编码 rLuc 和人类 PER2 启动子片段 (hPER2P, 941 bp) 在多个克隆区域 17 中的 Sac I 和 后 III 之间的站点上. </li>
	<li> 将人类的 PER2 启动子片段 (hPER2P, 941 bp) 从向量中剪切出来. 
	<ol> <li> 添加2.5 和 #181; 限制酶缓冲, 10 和 #181; l (180 ng) 请 [ hPER2P / rLuc /普洛] 向量, 1 和 #181; l 每一个限制酶, Sac I (10 单位/和 #181; l) 和后 III (10 单位/和 #181; l), 成 DNA/RNA/无核苷酸离心管。添加超纯水 (10.5 和 #181; l) 最多25和 #181; 我轻轻地混合移. </li>
		<li> 在加热块中孵育37和 #176; C 为90分钟. </li>
		<li> 运行整卷 (25 #181; L) 的限制酶反应对0.7% 琼脂糖凝胶含有0.0001% 溴溴 (EtBr) 分离 hPER2P 从矢量. 
		注: EtBr, 38-氨基-1-乙基 6-phenylphenantridinium 溴化铵 (CAS 注册号: 1239-45-8), 是一种无嗅的红色液体。它是一种插剂, 通常用作琼脂糖凝胶电泳中的荧光标记 (核酸染色), 在紫外光下可见为桔色。虽然没有任何管理机构将其归类为致癌物质, 但在实验动物中发生了不可逆转的诱变效应的可能风险 (18 。因此, 我们收集了含有 EtBr 的琼脂糖凝胶和电泳缓冲剂, 作为化学危险废物, 并要求罗格斯大学环境健康和 #38; 安全 (REHS), 以便能够安全地拾取和处置. </li>
		<li> 在 941 bp 切割一条含有 EtBr 荧光带的琼脂糖凝胶, 并用 DNA 凝胶萃取试剂盒从凝胶中提纯 hPER2P 片段, 然后通过测量 260 nm 波长的吸收密度 (OD) 对分光光度计进行量化. </li>
	</ol> </li> <li> 线性1.0 和 #181; 不稳定的萤火虫荧光表达载体的 L (1 和 #181; g), pGL [ Luc2P /Neo] 使用与步骤 1.2. 1-1. 2.2 中所述相同的方法。提取的载体与 DNA 清除试剂盒后, 量化如上所述使用分光光度计. </li>
	<li> 结扎 hPER2P 到线性化向量 pGL [ Luc2P /Neo]. 
	注: 根据制造商和 #39 的指示, 理想的摩尔比的插入和矢量是2:1。对于 50 ng 向量, 理想的插入量是计算为 23.6 ng 与公式, [(50 ng 向量 x 1.0 kb 插入)/4.242 kb 向量] x (2/1) = 23.6 ng 插入]。根据 hPER2P 的浓度 (7.26 ng/和 #181; l) 和 pGL [ Luc2P /新] (50 ng/和 #181; l), 50 ng pGL [ Luc2P /新] 和 23.6 ng hPER2P 的体积计算为1和 #181; l 和3.26 和 #181; 分别。
	<ol> <li> 添加2和 #181; L T4 DNA 连接反应缓冲器 (10X) (50 毫米三-HCl, 10 mm 氯化镁 2 , 1 毫米 ATP, 和10毫米的德勤), 3.26 和 #181; l (23.6 ng) hPER2P, 1 和 #181; l (50 ng) pGL [ Luc2P /新尼], 13.74 和 #181; l 水高达20和 #181;, 轻轻地混合. </li>
		<li> 添加1和 #181; T4 DNA 连接 (400 单位/和 #181; l), 轻轻地混合, 孵育在16和 #176; C 隔夜在 thermocycler, 然后冷却的结扎向量冰每制造商和 #39 的指示. </li>
	</ol> </li> <li> 将已结扎的矢量 pGL [ hPer2P / Luc2P /Neo] 转换为具有化学能力的 大肠杆菌 19 . 
	<ol> <li> 设置42和 #176 的水浴; C、加热超级优选汤与物镇压 (soc) 中等 (2% 胰, 0.5% 酵母提取物, 10 毫米氯化钠, 2.5 毫米氯化钾, 10 毫米氯化镁 2 , 10 毫米 MgSO 4 和20毫米葡萄糖) 在室温, 预热 Luria-Bertani (LB) 琼脂板 (含100和 #181; 氨苄西林, 80 和 #181; g/毫升 X gal, 0.5 和 #181; M IPTG) 在37和 #176; C 孵化器, 解冻在冰上每一个转换的一个合格的 大肠杆菌 细胞. </li>
		<li> 添加6和 #181; (21 ng) 结扎向量或1和 #181; l (30 ng) 空向量 (负控制) 到一管化学合格的大肠杆菌 (50 和 #181; l) 然后轻轻地翻转管混合。在冰上孵育30分钟. </li>
		<li> 42 #176 热冲击; C 水浴三十年代不摇晃, 然后返回冰. </li>
	</ol> </li> <li> 选择一个 大肠杆菌 菌落, 使用蓝/白筛选器将其与结扎的向量进行转换. 
	<ol> <li> 添加250和 #181; 在转换后的 大肠杆菌 管中 soc 培养基, 在37和 #176 中孵育1小时; C 以 200 rpm 晃动. </li>
		<li> 传播10-50 和 #181; 转换 大肠杆菌 均匀地在 LB 琼脂板的表面上。把盘子倒过来放在37和 #176; C 恒温箱过夜. 
		注意: 有效的克隆反应应该产生几百个菌落。电镀两个不同的卷建议, 以确保至少有一个板块将有大, 清晰的白色或蓝色的颜色, 和匀整的殖民地。当殖民地太小, 颜色是不可区分的, 使用显微镜 (10X 或 50X) 是有帮助的. </li>
		<li> 从板上取3-6 白色的菌落, 用无菌的牙棒, 并将每个菌落释放到一个含有4毫升培养基的培养基中, 50 和 #181; g/毫升氨苄西林. </li>
		<li> 在37和 #176 上孵育管, 在夜间以 200 rpm 晃动。用2毫升的4毫升培养的 大肠杆菌 提取的 dna 质粒 dna 提取迷你准备试剂盒, 每个制造商和 #39 的指令. </li>
	</ol> </li> <li> 验证并分析插入 (hPER2P, 941 bp) <ol> <li> 以限制性酶消化质粒 DNA, 并按照步骤1.2 中的描述运行电泳. 1-1. 2.3 确认是否有插入. 
		注: 阳性对照 (hPER2P 纯化的步骤 1.2.4) 和负控制 ( 大肠杆菌 转化为空向量) 包括在内. </li>
		<li> 使用 M13 前向和 M13 反转引物序列选定的质粒 DNA, 以确认在质粒中插入的方向和顺序 19 . 
		注: 在测序结果中, 只有一个具有正确尺寸的 hPER2P 在电泳和正确的方向和顺序的一个菌落被选中. </li>
		<li> 分析人类 PER2 启动子片段 (hPER2P, 941 bp) 序列的昼夜调节元素, 包括电子盒主题 (Bmal1 绑定网站, 猫/CGTG), CCAATC, GC 框, 转录开始网站 (CAGCGG) 20 。
	</ol> </li> <li> 通过将剩余的 2 ml 培养的 大肠杆菌 添加到包含50和 #181 的200毫升培养基中, 从而放大所选的 大肠杆菌 。
	<li> 提取 DNA 与无内毒素质粒最大准备试剂盒每制造商和 #39 的指令, 然后用分光光度计测量 260 nm 波长的外径来量化。分和存储质粒 DNA, pGL [hPer2P/Luc2P/Neo], 在-80 和 #176; C 冷冻以备将来使用. </li>
</ol> 2。瞬态转染 <ol> <li> 购买和区域性 MCF10A 单元格 <ol> <li> 从商业细胞库中购买一个永生的、non-transformed 正常的人类乳腺上皮细胞系 (MCF10A), 其中细胞 cytogenetically在冻结前对短串联重复分析进行测试和验证。解冻和保持每瓶冷冻细胞在文化中最多8周. 
		注意: 与其他细胞不同, 这些细胞一旦到达 ~ 70% 汇合点, 就会有接触抑制。它要求亚文化在 ~ 70% 进行. </li>
		<li> 培养细胞与乳腺上皮细胞生长培养基 (MEGM), 含有乳腺上皮基底培养基 (MEBM), 生长补充剂和霍乱毒素在37和 #176; C, 95% 湿度, 5% CO 2 . 
		注: 购买 SingleQuot (平方米) 提供的即食生长因子, 并分别获得霍乱毒素。最后的生长补充剂是0.4% 牛垂体提取物, 0.1% 胰岛素, 0.1% 可的松, 0.1% 人表皮生长因子, 100 ng/毫升霍乱毒素, 0.05% 硫酸庆大霉素和0.05% 两性霉素 B. </li>
		<li> 将 10 cm 培养皿中的细胞与10毫升胰蛋白酶/EDTA 分离为20分钟, 然后通过加入10毫升胰蛋白酶中和溶液来中和胰蛋白酶。收集所有细胞与 HEPES 缓冲盐水溶液 (HBSS). </li>
		<li> 在倒置显微镜下将例计数, 计算单个细胞悬浮液的浓度, 然后根据需要对细胞进行一定数量的种子。将板材彻底旋转, 以获得均匀分布在每个板块中的细胞。孵育细胞培养池在37和 #176; C, 95% 湿度, 5% CO 2 . 
		注: 购买含有胰蛋白酶/EDTA 的亚文化试剂包, 胰蛋白酶中和溶液, 并 HBSS 使用. </li>
	</ol> </li> <li> 瞬态转染细胞与构建的昼夜矢量。
	<ol> <li> 种子 2 x 10 5 MCF10A 细胞均匀地在35毫米培养皿与 MEGM 并且增长到20-30% 汇合 (次日). </li>
		<li> 预热降低的血清介质 ( 例如 , 优化) 在37和 #176; C 水浴和转染试剂在室温下30分钟稀释的质粒 DNA (pGL [ hPer2P / Luc2P /Neo]), 以 30 ng/和 #181; 我与内毒素免费三 EDTA (TE) 缓冲并保持室温. </li>
		<li> 在1.5 毫升离心管中制备质粒转染混合物, 加入63.6 和 #181; 先加热还原血清培养基, 然后加入33.4 和 #181; l (0.1 和 #181; g) 质粒 DNA, 然后轻轻移, 最后加入3和 #181; 转染试剂直接进入管中心, 不接触墙壁。用移轻轻搅拌后, 在室温下保持30分钟. </li>
		<li> 将整个质粒混合物 (100 和 #181; L) 直接放到盘中介质的中心表面, 然后通过摇动盘子轻轻地混合。将 CO 2 孵化器中的单元格培养为额外的 48-72 h. 
		注: 据预测, 由于其在 ~ 70% 的接触抑制, 其转染效率最高为40-70%。因此, 转染工作最好从〜20% 开始, 并停止在〜70% 汇合. </li>
	</ol> </li> </ol> 3。瞬时转染 MCF10A 细胞中的 体外生物荧光检测法的建立 <ol> <li> 在 MEBM 70% 汇合处 (在 48-72 h) 中, 在没有生长因子的情况下, 将培养的细胞饿死在 24 h. </li>
	<li> 在 CO 2 孵化器中, 使用同步代理 ( (如 , 50% 马血清 (HS)) 在 MEBM 中处理 1.5 h 的单元格. 
	注: 对于理想的同步剂的选择, 10 和 #181; m 佛司可林, 1 nM 褪黑素, 0.1 和 #181; m 地塞米松, 50% HS, 100% 平方在昼夜节律向量转染细胞中的昼夜振幅和周期进行比较。空矢量转染细胞处理100% 平方为负控制. </li>
	<li> 预准备20毫升记录介质 (10 的 30 mm 培养皿), 加入5毫升的 MEGM, 15 毫升的 MEBM, 150 和 #181; 碳酸氢钠, 200 和 #181; l HEPES, 40 和 #181; l 素库存解决方案 (50 毫米) 在50毫升灭菌离心管. 
	注: 每个成分的最终浓度: 20% 平方和 20 ng/毫升霍乱毒素, 0.06% 碳酸氢钠, 1% 青霉素/链霉素, 和10毫米 HEPES。预热准备记录介质和灭菌的 1x PBS 30 分钟, 在37和 #176; C 水浴。在使用前立即添加100和 #181; M 素. </li>
	<li> 在与同步代理进行孵育后, 用温热的 1x Dulbecco #39 的磷酸缓冲盐水 (D-PBS) 清洗电池3次, 然后添加2毫升记录介质. </li>
	<li> 用硅脂来密封这道灭菌的盖子, 防止蒸发, 然后将其贴在侧面. </li>
	<li> 将密封盘放在计内的座位上, 并打开保存文件夹中文件位置的选项 (有关详细信息, 请参阅6节). </li>
</ol> 4。稳定转染细胞的选择 <ol> <li> 根据制造商和 #39 对稳定转染细胞的选择, 优化理想的 G418 浓度 (800 和 #181; g/毫升), 并采用 kill 曲线法. </li>
	<li> 在瞬时转染48小时或72小时后, 亚文化和种子细胞与 MEGM 在更大的盘子在5000细胞或盘 (100 毫米)。24 h 培养在细胞培养孵化器 (37 和 #176; C, 95% 湿度, 5% CO 2 ), 用800和 #181 对单元格进行处理; 用含有800和 #181 的新介质替换旧介质, G418; 克/毫升 G418 每3-4 天2周. </li>
	<li> 亚文化生存稳定转染的殖民地1-3 通道与 MEGM 包含500和 #181; g/毫升 G418 保持 hPer2P/Luc2P 的稳定表达, 并在液氮中冷冻以供将来使用. </li>
	<li> 治疗500和 #181; 在一般亚文化 G418 后, 重新细胞的稳定转染量, 如果在体外的结果中的生物发光强度在许多段落使用后, 生物荧光检测逐渐降低. </li>
</ol> 5。使用化学药剂处理 <ol> <li> 在48小时或72小时 post-transfection, 将 MEBM 中的细胞从生长因子中饿死24小时. </li>
	<li> 同步后与 50% HS 2 小时, 治疗0.25 毫米或0.5 毫米 nitrosomethylurea (NMU), 20 nM EX527, 或1和 #181; M cambinol 为 1 h, 在 MEBM 含有20% 平方米的细胞. </li>
	<li> 使用 1x D PBS 冲洗后, 添加包含12.5 和 #181 的记录介质; 单独或与 20 nM EX527 或 1 #181; m cambinol, 对细胞。用计监测生物发光. </li>
	<li> 将稳定转染的 MCF10A/ PER2 - dLuc 单元格处理为 0.5, NMU 1, 或2毫米, EX527 在 40 nM, 或 Cambinol 在2.0 和 #181; m, 在与 1 HS 同步后 50% h, 然后在含有12.5 和 #181 的记录介质中孵育细胞; MSCn-乙酰半胱氨酸 (NAC) 的不同剂量 (5, 10, 或20和 #181; M)。将印版放在计中, 记录并保存计4-8 天的生物发光, 如3.6 节所述. 
	注: NMU 是一种甲基化-n-化合物, 具有致突变、致癌和致畸性质。NMU 是一种直接作用的烷剂, 半衰期很短 (T 1/2 = ~ 30 min)。在酸性条件下 (ph 值为 4-5), 但在碱性条件下 (ph 9-10) 不稳定, 温度在20和 #176 ° C 以上;因此, 10% 的漂白剂可以作为一种有效的停用剂, 用于清洁可能被 NMU 方案污染的工作台面和实验室用具。上.剩余的库存解决方案是提取和安全处置的 REHS。根据化学药剂的作用方式, 在记录培养基培养前, 同步细胞可以进行较短时间的处理。细胞也可以在记录介质中处理较长时间 (几天). </li>
</ol> 6。数据收集、分析和演示文稿 注意: 需要使用标准方法 (如 "MTT 法") 在相同浓度的相同时间内对细胞进行治疗后确定细胞活力。在 体外 生物荧光检测中使用的所有治疗浓度均低于30% 的致死浓度 (LC30)。所有实验均进行了三份, 并提出了有代表性的结果. 
<ol> <li> 在密封盘 (步骤 3.5) 后, 将盘子装在计的座椅上, 并将其放在37和 #176 的恒温箱内; C 没有 H 2 O 和 CO 2 , 并连接到计算机. </li>
	<li> 单击和 #34; 保存和 #34; 并输入文件夹和文件的名称, 从相应的盘中记录的发光信号将被保存. 
	注意: 系统检测 real-time 中的发光信号来自于单个位置的盘中的细胞。信号通过4光子计数光电倍增管传输到计算机, 并通过数据采集软件保存并显示在计算机中. </li>
	<li> 在录制4-7 天后对信号进行分析, 如果需要, 可以在第二周进行中等变化和连续录制. 
	注意: 为了获得昼夜节律参数, 包括相位、周期长度、节律振幅和阻尼率, 我们利用计分析软件对生物发光数据进行了分析. </li>
	<li> Detrend 原始数据的运行平均值, 并分析 best-fits 到正弦波以获得周期、相位、振幅和阻尼率 21 、 22 . </li>
	<li> 由于处理和介质变化时的高瞬态生物发光, 排除了分析数据的第一个循环. </li>
	<li> 用于数据演示, 根据时间 (h 或天) 绘制原始数据 (生物发光、计数/秒)。必要时, 可以绘制基线减去数据来比较振幅和相位. </li>
</ol>

<ol>
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作者没有什么可透露的。

在哺乳动物细胞中, 昼夜节律时钟的周期性由相互关联的转录/平移反馈回路调节。Heterodimers 的 Bmal1 和时钟或 Npas2 调节昼夜转录通过绑定到电子盒元素的推动者的核心厘米, 包括Per2和哭, 和许多 CCGs4。当它们在细胞中堆积时, heterodimers 的每一个: 哭 post-translationally 被修饰并传送到细胞核以抑制 Clock:Bmal1 的转录活性。这个负反馈回路允许核心厘米调节自己的转录, 并?...

生物钟昼夜节律调节着一系列的生物学过程, 从昼夜的基因表达到睡眠行为, 周期约24小时. 流行病学研究强烈表明慢性扰乱昼夜节律节奏增加了轮班工作人员的乳腺癌和前列腺癌的风险, 包括护士和空勤人员1,2,3。这些发现得到了啮齿动物研究的证实, 证明暴露在恒定的光照、light-at 夜间或模拟时差的光周期会增加肿瘤发病率并加速肿瘤的生长4,5。根据人类和啮齿类研究的数据, 国际癌症研究机构在 2010年6将轮班工作归类为可能的人类致癌物 (类型 2A)。
此前, 我们证明, 一个单一的致癌剂量的乳腺肿瘤特异性致癌物, n-硝基-n-脲 (NMU), 扰乱了主要昼夜节律基因 (厘米) 的昼夜表达 (如,期间2, Per2) 和几个昼夜节律控制的基因 (CCGs), 包括靶乳腺 (但不是肝脏) 中的关键 DNA 损伤应答和修复 (复员) 基因。此外, 通过化学饮食的膳食L-甲基胱氨酸 (MSC), 将Per2和复员基因的昼夜节律表达复位为正常, 将肿瘤发病率降低63%。这些发现首次显示了昼夜节律, 化学致癌和化学7,8之间的机械联系。暴露于其他环境毒物显示扰乱昼夜基因表达在体内也与增加环境疾病的风险相关9,10。了解环境毒物和致病机制将昼夜节律紊乱联系起来的机理, 可能导致 mechanistically-based 的疾病预防方法。然而, 旨在确定暴露和昼夜节律之间相互作用的研究通常在体内进行.一个典型的体内实验研究对昼夜节律的影响需要大量的动物, 因为至少有三控制的组织和三暴露的动物必须在24或48小时内每3-4 小时收集一次。开发一个验证的体外系统, 重述在体内的观察和机制, 因此不仅减少了所需的动物数量, 而且大大降低了实验成本, 并要求研究人员在24-48 小时内连续工作。此外, 经验证的体外系统可用于高吞吐量的化合物筛选和/或影响昼夜节律的基因改变, 或对环境压力源或毒物的反应。因此, 需要对体外和体内模型和实验的策略性结合, 以获得不同焦点的不同见解。
在哺乳动物中, 昼夜节律振荡器不仅存在于 SCN 的专门神经元中, 而且还出现在大多数外围细胞类型中。这些分子钟类似于已建立成纤维细胞系和从胚胎或成年动物的原代成细胞;但是, 需要组织类型特定的蜂窝模型11。因此, 传统的运动活动的研究在体内, SCN外植体的ex 体内, 和细胞的在永生化成纤维细胞中的检测被广泛应用于研究自主性的昼夜生理缺陷。然而, 没有证据表明, 一个体外成纤维细胞的检测可以重述其他周边器官的细胞中的昼夜节律机制和反应,体内。不同的细胞类型可以有不同的基因表达模式, 异代谢, 和复员, 以及毒性和昼夜基因表达之间的联系可能是细胞类型的具体和/或不同的生理参数调制。此外, fibroblast-based 系统中的昼夜振荡器尚未得到充分评估, 以应对环境毒物、压力源和预防性药物, 它们将暴露与疾病发展和预防机制联系起来。因此, 需要简便、有效的细胞类型特异性、体外生物荧光检测来研究器官特定的环境昼夜干扰物。虽然各种细胞时钟模型 (例如, 在肝脏, 角质形成细胞, 和脂肪细胞, 以及骨肉瘤干细胞线) 已发展在最近几年12,13,14,15, 这里所描述的是乳腺上皮细胞中的第一个细胞时钟模型, 也是第一次在体内对环境压力源、毒物、药物和化学剂的反应进行复述的示范。
Renilla 荧光 (rLuc) 和萤火虫荧光是 30-61 kDa 单体蛋白, 不需要后处理酶活性, 并可以作为一个遗传学记者后立即翻译。一旦基体与荧光酶相关联, 生物化学反应就会产生闪光。因此, 荧光结构被广泛应用于基因表达报告系统体外和体内。然而, 在昼夜节律研究中, 荧光记者的效用受荧光蛋白 (T1/2 = 3.68 h) 相对较长的半衰期的限制, 因为它相对于昼夜节律基因的变化 (特别是短周期)。表达;然而, 多年来许多研究成功地使用了荧光基因在 pGL3 传染媒介, 表明快速地可降解的荧光可能不是必要的为报告昼夜节律, 特别是为节奏以较长的期间, 例如24小时因此, 一个记者质粒使用不稳定的荧光载体, pGL [Luc2P/Neo], 其中包含 hPEST (蛋白质失稳序列) 已经开发, 使我们可以使用它作为一个昼夜报告矢量我们目前的体外生物荧光测定法。由Luc2P编码的蛋白质具有更短的半衰期 (T1/2 = 0.84 h), 因此, 对转录活性的变化比对野生类型的反应更快和更大的程度, 我ndicating 可用于监视PER2启动器在 real-time16中精确调节的荧光的节律表达式。

<table>
	<tbody>
		<tr>
			<td>pLS[hPER2P/rLuc/Puro] vector</td>
			<td>SwitchGear Genomics</td>
			<td>S700000</td>
			<td>customized vector</td>
		</tr>
		<tr>
			<td>pGL4.18[Luc2P/Neo] vector</td>
			<td>Promega</td>
			<td>9PIE673</td>
			<td>destabilized luciferase expression vector</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen</td>
			<td>15224041</td>
			<td>For subcloning</td>
		</tr>
		<tr>
			<td>TOPO TA cloning kit</td>
			<td>Invitrogen</td>
			<td>K4500-02</td>
			<td>with One Shot TOP10 Chemically Competent E. coli</td>
		</tr>
		<tr>
			<td>Sac I</td>
			<td>New England BioLabs</td>
			<td>R0156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Hind III-HF</td>
			<td>New England BioLabs</td>
			<td>R3104S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>CutSmart buffer</td>
			<td>New England BioLabs</td>
			<td>B7204S</td>
			<td>Restriction enzyme buffer</td>
		</tr>
		<tr>
			<td>DNA gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Purify DNA fragments from agarose gel</td>
		</tr>
		<tr>
			<td>PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>DNA clean up</td>
		</tr>
		<tr>
			<td>LB Miller&#39;s modification</td>
			<td>TEKNOVA</td>
			<td>L8600</td>
			<td>For transformed E. Coli culture</td>
		</tr>
		<tr>
			<td>LB Agar Plate</td>
			<td>TEKNOVA</td>
			<td>L1902</td>
			<td>For white/blue selection</td>
		</tr>
		<tr>
			<td>QIAprep spin miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>For extraction of plasmide DNA</td>
		</tr>
		<tr>
			<td>EndoFree plasmid maxi prep Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>For extraction of plasmide DNA</td>
		</tr>
		<tr>
			<td>FuGene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium</td>
			<td>Invitrogen</td>
			<td>31985-062</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>MCF10A cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRL-10317</td>
			<td>Mammary Epithelial Cells</td>
		</tr>
		<tr>
			<td>MEGM BulletKit</td>
			<td>Lonza</td>
			<td>CC-3150</td>
			<td>Mammary Epithelial Growth Medium</td>
		</tr>
		<tr>
			<td>MEBM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
			<td>Mammary Epithelial Basal Medium</td>
		</tr>
		<tr>
			<td>MEGM SingleQuot Kit</td>
			<td>Lonza</td>
			<td>CC-4136</td>
			<td>Suppliments &#38; Growth Factors</td>
		</tr>
		<tr>
			<td>ReagentPack</td>
			<td>Lonza</td>
			<td>CC-5034</td>
			<td>Reagent for subculture</td>
		</tr>
		<tr>
			<td>D-PBS (10X)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>Wash cells in culture dishes</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977</td>
			<td>Dilute 10X D-PBS</td>
		</tr>
		<tr>
			<td>Tissue culture dish (35 mm)</td>
			<td>BD/Falcon</td>
			<td>353001</td>
			<td>cell culture dish suitable to LumiCycle</td>
		</tr>
		<tr>
			<td>Silicon grease</td>
			<td>Fisher</td>
			<td>NC9044707</td>
			<td>For sealing dish with recording medium</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>Harvard Bioscience</td>
			<td>64-1500 (CS-40R)</td>
			<td>For sealing dish with recording medium</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>Supplement for growth medium</td>
		</tr>
		<tr>
			<td>G418 sulfate (Geniticin)</td>
			<td>Invitrogen</td>
			<td>10131035</td>
			<td>Antibiotic for selection of stabliy transfected cells</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A9393</td>
			<td>For colony selection</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Melatonin</td>
			<td>Sigma</td>
			<td>M5250</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>d-Luciferin, sodium salt</td>
			<td>Invitrogen</td>
			<td>L2912</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>IC261</td>
			<td>Sigma</td>
			<td>10658</td>
			<td>Positive control for circadian disruptor</td>
		</tr>
		<tr>
			<td>Methylnitrosourea (NMU)</td>
			<td>Sigma</td>
			<td>N1517</td>
			<td>Mammary specific carcinogen</td>
		</tr>
		<tr>
			<td>Methylselenocysteine (MSC)</td>
			<td>Sigma</td>
			<td>M6680</td>
			<td>Organic selenium (chemopreventive agent)</td>
		</tr>
		<tr>
			<td>EX527</td>
			<td>Sigma</td>
			<td>E7034</td>
			<td>SIRT1 specific inhibitor</td>
		</tr>
		<tr>
			<td>Cambinol</td>
			<td>Sigma</td>
			<td>C0494</td>
			<td>SIRT1 &#38; SIRT2 inhibitor</td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 8000</td>
			<td>Quantify nucleotide</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 9700</td>
			<td>Applied Biosystems</td>
			<td>N805-0200</td>
			<td>For molecular biology experiment</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NAPCO</td>
			<td>Series 8000 DH</td>
			<td>For cell culture at 5% CO2 at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Desktop centrifuge with refrezerator</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td>For molecular biology experiment</td>
		</tr>
		<tr>
			<td>Centrifuge with swing bucket</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>80124</td>
			<td>Phase contrast optional</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>Labconco</td>
			<td>Class II A2</td>
			<td>BSL-2 certified</td>
		</tr>
		<tr>
			<td>LumiCycle 32</td>
			<td>Actimetrics</td>
			<td>Not Available</td>
			<td>Luminoscence detector</td>
		</tr>
	</tbody>
</table>

in vitro bioluminescence assay to characterize circadian rhythm in mammary epithelial cells

昼夜节律是所有生物体中的基本生理过程, 它们调节着从基因表达到睡眠行为等生物过程。在脊椎动物中, 昼夜节律由一个分子振荡器控制, 它在交叉核 (SCN; 中央起搏器) 和包括大多数外围组织的单个细胞中起作用。更重要的是, 暴露于 light-at 夜间、环境压力源和/或毒物的昼夜节律的破坏与慢性疾病和衰老的风险增加有关。能够识别能扰乱中枢和/或周边生物生物钟的药物, 以及能预防或减轻昼夜节律紊乱影响的药物, 对预防慢性病具有重要意义。虽然啮齿类动物模型可以用来识别诱发或预防/减轻昼夜节律紊乱的暴露和药物, 但这些实验需要大量的生物。在体内研究还需要大量的资源和基础设施, 并要求研究人员整夜工作。因此, 迫切需要一个细胞类型的适当的体外系统, 以筛选环境昼夜干扰和细胞类型的促进剂从不同的器官和疾病状态。我们构建了一个向量, 驱动转录的不稳定的荧光在真核细胞的控制下的人的周期 2基因启动子。这种昼夜节律的报告结构被稳定地转染为人乳腺上皮细胞, 并选择了昼夜节律性的记者细胞来发展体外生物荧光检测。在这里, 我们提出了一个详细的协议, 建立和验证的化验。我们进一步提供详细的概念实验证明的能力, 我们的体外化验, 以重述的体内的影响, 各种化学品的细胞生物钟。结果表明, 该方法可以适应各种细胞类型, 以筛选的环境干扰和化学促进剂的昼夜节律时钟。

昼夜生物发光报告载体: 人的PER2不稳定荧光变体的启动子驱动表达式
DNA 序列包括一个 941 bp 片段从人类的PER2启动器, 用于构建的昼夜报告矢量, pGL [hPer2P/Luc2P/Neo, 首先分析了存在的监管元素已知的调节昼夜基因表达。生物信息学分析表明, 在这个启动子片段, 有三不同的 Bmal1 结合位点 (电子盒) (猫/CGTG), ?...

Watch this Scientific Journal Video about In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells at JoVE.com

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

一个体外生物荧光法测定乳腺上皮细胞的细胞昼夜节律。该方法利用哺乳动物细胞报告质粒表达不稳定的荧光在周期 2基因启动子的控制下。它可以适应其他细胞类型, 以评估器官特定的影响昼夜节律。

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

in-vitro-bioluminescence-assay-to-characterize-circadian-rhythm

Research

JoVE Journal

Genetics

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

9.7K 次观看.  Rutgers University. 一个体外生物荧光法测定乳腺上皮细胞的细胞昼夜节律。该方法利用哺乳动物细胞报告质粒表达不稳定的荧光在周期 2基因启动子的控制下。它可以适应其他细胞类型, 以评估器官特定的影响昼夜节律。

视频: In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

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植物病原体的遗传操作