Enhancement of Vivid-based photo-activatable Gal4 transcription factor in mammalian cells.
Enhancement of Vivid-based photo-activatable Gal4 transcription factor in mammalian cells.
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DOI：
10.1247/csf.22074
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发表时间：
2023-02-08
影响因子：
1.5
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中科院分区：
文献类型：
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在许多模式生物中，Gal4/UAS系统是操纵细胞空间和时间外源基因表达的多功能工具。随着光激活（PA）分子开关的发展和这些工具的集成，许多光可控Gal4/UAS系统现在都是可用的。然而，即使在黑暗条件下，许多PA-Gal4转录因子也具有不期望的背景转录活性，这严重削弱了可靠的光控基因表达。因此，开发可靠的PA-Gal4转录因子具有强大的光诱导基因表达和有限的背景活性是很重要的。通过对合成的PA-Gal4转录因子进行优化，验证了Gal4 DNA结合域、转录激活域和蓝光依赖性二聚体形成分子Vivid （VVD）的构型，并应用转录激活域类型开发了新的PA-Gal4转录因子eGAV （enhanced Gal4-VVD转录因子）。eGAV在黑暗条件下的背景活性明显低于常用的PA-Gal4转录因子hGAVPO，并且最大光诱导基因表达水平也有所提高。在质粒瞬时转染培养的HEK293T细胞和慢病毒载体介导的小鼠EpH4细胞中证实了光控基因的表达。此外，光控eGAV介导的转录在发育和成年小鼠脑和鸡脊髓的神经干细胞和祖细胞以及成年小鼠肝细胞中得到证实，这表明eGAV可以应用于广泛的实验系统和模式生物。
The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.
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影响因子：
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