Impact of Fluorophore and Epitope Position on Destabilized Reporter Performance in C. elegans

ABSTRACT Determining spatial and temporal gene expression is crucial for understanding animal development and physiology. Promoter reporters are powerful tools used to dissect how cis-regulatory elements and trans-acting factors control gene expression. Many fluorescent proteins used in promoter reporters, however, have long half-lives (>24 hr) which limit the study of dynamic expression. Destabilizing sequences like PEST reduce the half-life of reporter proteins to provide a more representative readout of gene expression. mlt-11 is a putative protease inhibitor known to oscillate in expression at the mRNA and protein level, yet a mlt-11p::mNeonGreen::3xFLAG::PEST::tbb-2 3’UTR promoter reporter did not detectably oscillate. We systematically dissected this transgene, finding that placement of a 3xFLAG tag adjacent to a PEST sequence severely hampered oscillatory expression of a mNeonGreen promoter reporter. Surprisingly, reporter designs that effectively oscillate with a GFP or mNeonGreen fluorophore fail to oscillate when mStayGold is used, with these reporters remaining detectable over 24 hours following promoter inactivation. In addition, other tested epitopes (Myc, ALFA, OLLAS) did not hamper PEST-dependent destabilization but led to varying levels of reporter expression. This study details key considerations for designing destabilized fluorescent promoter reporters.