An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines.
An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines.
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DOI：
10.1039/c4mb00155a
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发表时间：
2014-10
影响因子：
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通讯作者：
Zartman J
中科院分区：
文献类型：
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作者：
Burnette M;Brito-Robinson T;Li J;Zartman J
果蝇细胞培养物被用作具有多种应用的模型系统，包括在筛选中鉴定新的治疗靶点、保守信号转导途径机制的研究以及作为重组蛋白的表达系统。然而，果蝇细胞和器官培养的体外方法相对不发达。为了表征果蝇细胞系长期维持的最低要求，我们开发了一种反向筛选策略来识别小分子和在化学成分确定的培养基中刺激增殖的协同作用。在这种化学遗传学方法中，化合物-蛋白质相互作用数据库用于在屏幕范围内系统地对遗传目标进行评分，以提取有关细胞生长的更多信息。在试点筛选中，我们重点关注两种特征明确的细胞系：Clone 8 (Cl.8) 和 Schneider 2 (S2)。研究了经过验证的因子在化学成分确定的培养基 (CDM) 中维持细胞多次传代生长的能力。多胺亚精胺被证明是 CDM 能够支持 Cl.8 细胞长期维持的关键成分。补充亚精胺可上调 Cl.8 和 S2 细胞的 DNA 合成，并增加 Cl.8 细胞的 MAPK 信号传导。 CDM 还支持 Kc167 细胞的长期生长。我们的目标评分方法验证了多胺的重要性，并富集了两种细胞系中发现的多种多胺本体。屏幕的未来迭代将能够识别针对特定应用（新细胞系的维护和生成或重组蛋白的生产和纯化）优化的化合物组合，从而增加果蝇细胞培养作为遗传和生化模型系统的多功能性。我们的累积目标评分方法改进了传统的化学遗传学方法，并且可扩展到其他物种的生物过程。
Drosophila cell culture is used as a model system with multiple applications including the identification of new therapeutic targets in screens, the study of conserved signal transduction pathway mechanisms, and as an expression system for recombinant proteins. However, in vitro methods for Drosophila cell and organ culture are relatively undeveloped. To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. In this chemical-genetics approach, a compound-protein interaction database is used to systematically score genetic targets on a screen-wide scale to extract further information about cell growth. In the pilot screen, we focused on two well-characterized cell lines, Clone 8 (Cl.8) and Schneider 2 (S2). Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support the long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines, with enrichment for multiple polyamine ontologies found for both cell lines. Future iterations of the screen will enable the identification of compound combinations optimized for specific applications—maintenance and generation of new cell lines or the production and purification of recombinant proteins— thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. Our cumulative target scoring approach improves on traditional chemical-genetics methods and is extensible to biological processes in other species.