(466a) Design and Characterization of a Salicylic Acid-Inducible Gene Expression System for Mammalian Cells

Engineering mammalian cells requires reliable and predictable control over gene expression. Ligand-inducible bacterial repressor proteins are commonly used to regulate genes in mammalian hosts. These repressors can tune transcription through direct interaction with operator sequences placed within or around promoter elements. Proper functioning of bacterial repressors in mammalian systems often requires several modifications to the regulatory proteins, in addition to incorporation of hybrid promoters in the mammalian host. Further, the small-molecule inducer should be well tolerated by the host, and the repressor/inducer pair should show minimal cross-talk with other transcriptional machinery in the host. While a variety of repressor systems have been adapted to work in various mammalian cell lines, these often show high leakiness or inducer mediated toxicity in the host cell lines, thus hampering applications. Here we describe the design and characterization of a salicylic acid (SA)-inducible gene expression system in human Jurkat cells, using the ~17kDa, E. coli multiple antibiotic resistance repressor protein, MarR. These efforts included testing the effects of MarR protein modifications like codon optimization, fusion to nuclear localization sequence (NLS) and fusion to GFP, in E. coli and mammalian cell lines. Changes in MarR mediated gene regulation, using different operator sequence combinations in native/chimeric promoters and using mono-cistronic /bi-cistronic transcripts, were also tested. Further steps to improve the dynamic range of the regulation system will also be described.