Abstract
BackgroundThe tetracycline-controlled transactivator system is a powerful tool to control gene expression in vitro and to generate consistent and conditional transgenic in vivo model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator.FindingsIn this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA) in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results.ConclusionsThe purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time.
Highlights
The tetracycline-inducible system is a binary transgenic system that enables spatial and temporal regulation of gene expression
The purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time
It consists of an inducible transcriptional activator and a tetracycline-responsive promoter element (TRE element, tetop) that controls the transcription of a target gene sequence [1,2]
Summary
The tetracycline-inducible system (tet-ON/OFF) is a binary transgenic system that enables spatial and temporal regulation of gene expression It consists of an inducible transcriptional activator (tTA or rtTA) and a tetracycline-responsive promoter element (TRE element, tetop) that controls the transcription of a target gene sequence [1,2]. Our laboratory has generated transgenic mice with different minigenes of wildtype and mutant human lamin A, under the control of the TRE element (tetop), to study the molecular mechanisms underlying Hutchinson-Gilford progeria syndrome (HGPS) [9,10,11]. Targeted expression of the lamin A minigene carrying the HGPS mutation (tetop-LAG608G) to the skin in mice, using the keratin 5 transactivator (K5tTA) [14], leads to epidermal disease with similar clinical features as have been reported in HGPS patients [10]. It offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator
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