Priyadharsini Nagarajan1, Satrajit Sinha. 1. Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, NY 14214, USA. pn7@buffalo.edu
Abstract
BACKGROUND: The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and/or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. OBJECTIVE: To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. METHODS: We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. RESULTS: Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2alpha in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet-inducibility upon continuous culturing. CONCLUSION: We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies.
BACKGROUND: The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and/or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. OBJECTIVE: To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. METHODS: We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. RESULTS: Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2alpha in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet-inducibility upon continuous culturing. CONCLUSION: We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies.