Abstract
Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R).
Highlights
Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings
To take advantage of the large array of methods to deliver Cre and the diverse model systems that rely on Cre-driven cancer initiation, we developed a ‘double-floxed’ gene trap that can be stably inverted by Cre, to conditionally inactivate virtually any gene of interest
The widespread practice of restoring gene function in established tumours within their natural setting has been greatly limited by previous approaches that are incompatible with specific genes of interest or by strategies that require multiple technically challenging steps to implementation[1,2,3,4,5,31]
Summary
Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R). Restoration of tumour suppressor gene function has the potential to identify relevant programmes of tumour suppression in physiologically diverse settings. To drive reproducible and stable DNA inversions, mutant loxP and FRT sites have been used to facilitate the permanent inversion of DNA sequences containing gene traps, to create conditional alleles in mouse embryonic stem (ES) cells and adult mice[7,8,9,10]. Designed for broad use and compatibility with established recombinase-based tools, XTR alleles provide a powerful method to establish causal relationships between genes and the greater physiological programmes they regulate in specific contexts
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