| Generation of Luciferase-expressing Tumor Cell Lines
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Author:
Date:
2018-04-20
[Abstract] Murine tumor models have been critical to advances in our knowledge of tumor physiology and for the development of effective tumor therapies. Essential to these studies is the ability to both track tumor development and quantify tumor burden in vivo. For this purpose, the introduction of genes that confer tumors with bioluminescent properties has been a critical advance for oncologic studies in rodents. Methods of introducing bioluminescent genes, such as firefly luciferase, by viral transduction has allowed for the production of tumor cell lines that can be followed in vivo longitudinally over long periods of time. Here we describe methods for the production of stable luciferase expressing tumor cell lines by lentiviral transduction.
[摘要] 鼠肿瘤模型对于我们对肿瘤生理学知识和有效肿瘤治疗方法发展的进展至关重要。 这些研究的关键是能够跟踪肿瘤发展并量化体内肿瘤负荷。 为此，引入赋予肿瘤生物发光特性的基因已经成为啮齿动物肿瘤研究的重要进展。 通过病毒转导引入生物发光基因（例如萤火虫萤光素酶）的方法已经允许产生可以在体内纵向长时间地进行的肿瘤细胞系。 在这里我们描述了通过慢病毒转导产生稳定表达荧光素酶的肿瘤细胞系的方法。
【背景】体内跟踪细胞最重要的是能够通过微创方法从外部检测它们。使用来自萤火虫的荧光素酶（Photinus pyralis ）的酶促生物发光是用于体内基于图像的细胞追踪的广泛使用的方法。生物发光已被用于各种体内应用，包括报告基因表达的无创成像（Herschman，2004），研究昼夜节律（Southern and Millar，2005），成像脑卒中（Vandeputte
萤火虫荧光素酶氧化物萤光素在分子氧，镁和三磷酸腺苷存在下在560nm产生黄绿色光（Wilson和Hastings，1998; ...
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| A Chemical Genetic Screening Procedure for Arabidopsis thaliana Seedlings
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Author:
Date:
2015-07-05
[Abstract] Unbiased screening approaches are powerful tools enabling identification of novel players in biological processes. Chemical genetic screening refers to the technique of using a reporter response, such as expression of luciferase driven by a promoter of interest, to discover small molecules that affect a given process when applied to plants. These chemicals then act as tools for identification of regulatory components that could not otherwise be detected by forward genetic screens due to gene family redundancy or mutant lethality.
This protocol describes a chemical genetic screen using Arabidopsis thaliana seedlings, which has led to recognition of novel players in the plant general stress response.
[摘要] 无偏的筛选方法是能够识别生物过程中的新玩家的有力工具。 化学遗传筛选是指使用报告物应答（例如由感兴趣的启动子驱动的荧光素酶的表达）来发现当应用于植物时影响给定过程的小分子的技术。 这些化学物质因此作为用于鉴定由于基因家族冗余或突变致死性而不能通过正向遗传筛选检测的调节组分的工具。
此协议描述使用拟南芥幼苗的化学遗传屏幕，这导致在植物一般胁迫反应中的新玩家的识别。
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| Longitudinal Bioluminescent Quantification of Three Dimensional Cell Growth
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Author:
Date:
2013-12-05
[Abstract] The use of three-dimensional (3D) cell culture systems is widely accepted as representing a more physiologically relevant means to propagate mammary epithelial and breast cancer cells. However, 3D cultures systems are plagued by several experimental and technical limitations as compared to their traditional 2D counterparts. For instance, quantifying the growth of mammary epithelial or breast cancer organoids longitudinally is particularly troublesome using standard [3H]thymidine or MTT assay systems, or using computer-assisted area calculations. Likewise, the nature of the multicellular aggregates and organoids formed by breast cancer cells under 3D conditions precludes efficient recovery of the cells from 3D matrices, an event that is time consuming and leads to spurious ...
[摘要] 使用三维（3D）细胞培养系统被广泛接受为代表更多生理相关手段以增殖乳腺上皮和乳腺癌细胞。然而，与其传统的2D对应物相比，3D培养系统受到若干实验和技术限制的困扰。例如，使用标准的[3 H]胸苷或MTT测定系统或使用计算机辅助面积计算，纵向量化乳腺上皮或乳腺癌组织的生长是特别麻烦的。同样，在3D条件下由乳腺癌细胞形成的多细胞聚集体和类器体的性质阻止了细胞从3D基质的有效回收，这是耗时的并且导致假的结果的事件。这里描述的测定利用萤火虫荧光素酶的稳定表达作为定量在3D矩阵内传播的细胞的纵向生长的手段。该技术的主要优点包括其高通量性质和在规定的时间段内纵向跟踪单个孔的能力，从而降低与测定性能相关的成本。最后，该技术可以容易地与药物治疗和/或遗传操作组合以测定它们对3D类器官生长的影响。
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