Modelling how genes act in both space and time is critical to understand animal development, which can potentially drive intervention in gene expression. Gene regulation is examined using many techniques; however, challenges such as cell delivery, invasiveness, toxicity, and efficacy limit our ability to fully probe gene networks. Recent advances have led to the development of tunable, titratable, and reversible tools that can be genetically-encoded into animal model systems to modulate genes with temporal and spatial control. This study compares such tools, testing several aptazyme-based switches that can be expressed inside cells and controlled through the addition of non-toxic small molecules. Three switches responsive to different small molecules were compared for switching activity in mammalian cells. The most efficient switches in terms of activity gauged by their modulation of gene expression were then further assayed. Finally, the specificity of the hypoxanthine switch was tested based on chemical structure and classification. The comparisons revealed the importance of both timing and small molecule concentrations on switch activity, while the specificity testing demonstrated switch activity inside the cell correlated to the aptamer binding properties that were measured biochemically. This work demonstrates the suitability of aptazyme-based switches for application in diverse genetic environments, and in controlling and studying gene networks in animals.
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Copyright (c) 2023 Janeva Shahi, Maureen McKeague