Tumour Necrosis Factor (TNF) is a pro-inflammatory cytokine with a diverse range of biological functions, and dysregulation of TNF expression is associated with various autoimmune and inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. TNF expression is dependent upon both transcriptional and post-transcriptional mechanisms, but a reliable reporter of TNF expression in vivo is still lacking. For the purpose of generating novel transgenic TNF translational reporter mouse strains, we have developed and characterised two fluorescent TNF reporter constructs for validation in vitro. We have successfully expressed a tdTomato-TNF fusion protein in cell lines, and have demonstrated membrane localisation and TACE-dependent cleavage of the fusion protein to produce soluble TNF. Our data support physiological behaviour of the tdTomato-TNF fusion protein, indicating this fusion strategy represents a viable approach for a knock-in fluorescent TNF reporter mouse strain where functional TNF protein is preserved.
Cellular accumulation of stable fluorescent protein prevents real-time observation of target gene expression and is a limitation in many fluorescent reporter models. To address this limitation, we have developed a second TNF reporter system utilising Halo-PROTAC technology which enables targeted degradation of a HaloTag-tdTomato reporter protein. Our results demonstrate dose- and time-dependent degradation of the fluorescent HaloTagged reporter protein in vitro following inducible Halo-tdTomato expression and administration of a VHL-recruiting HaloPROTAC compound. Collectively, our study has identified two systems that we may utilise for the generation of TNF translational reporter mouse strains. These TNF reporter strains will permit the visualisation and observation of TNF-producing cells by a variety of approaches including intravital and live imaging, constituting novel models for in vivo characterisation of TNF expression in physiological settings as well as in disease states.