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. We therefore sought to develop a fluorescent TNF translational reporter mouse where functional TNF protein is preserved, permitting accurate visualisation and observation of TNF-producing cells by a variety of approaches including intravital and live imaging.
We have generated two knock-in fluorescent TNF reporter mouse strains utilising tdTomato-TNF fusion protein expression constructs we have previously characterised and validated in vitro. In cell lines, the positioning of tdTomato within the TNF open reading frame significantly altered TNF expression between our two reporter constructs, however, similar patterns of TNF expression were observed in vivo between our two reporter mouse strains. Our TNF reporter animals, while expressing robust fluorescence, exhibit reduced TNF protein expression in a gene dose-dependent manner where both heterozygosity and homozygosity for the reporter allele lead to clear reductions in the amount of TNF protein produced. Despite successful expression in vitro, preliminary results also suggest homozygosity for the reporter allele yields near-complete loss of TNF expression, however, characterisation of homozygote reporter animals is ongoing. Collectively, our results highlight the complexity of developing reporter models for the study of TNF in vivo, and we are now investigating alternatives to the TNF fusion protein approach to develop our translational TNF reporter.