Name: in vitro bioluminescence assay to characterize circadian rhythm in mammary epithelial cells
Uploaded: 2017-09-28T12:30:00.000+00:00
Duration: 11 min 56 s
Description: Watch this Scientific Journal Video about In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells at JoVE.com

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>

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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&#039;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 &amp;amp; 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 &amp;amp; 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 &amp;deg;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 ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

生物学

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

癌症研究

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

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

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

毛因神经发生期间诺奇信号动力学的实时生物发光成像

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

发育生物学

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

神经科学

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

优化人肠道类肠生物用于昼夜节律研究

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

作者聚焦：多细胞系统中昼夜节律的体外研究

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

使用类器官中的生物发光报告基因监测昼夜节律振荡

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...