Abstract
Tumour necrosis factor (TNF) is a key cytokine during inflammatory responses and its dysregulation is detrimental in many inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. Here, we used a bacterial artificial chromosome (BAC) construct that expresses luciferase under the control of the human TNF locus to generate a novel transgenic mouse, the hTNF.LucBAC strain. In vitro stimulation of hTNF.LucBAC cells of different origin revealed a cell specific response to stimuli demonstrating the integrated construct’s ability as a proxy for inflammatory gene response. Lipopolysaccharide was the most potent luciferase inducer in macrophages, while TNF was a strong activator in intestinal organoids. Lipopolysaccharide-induced luciferase activity in macrophages was downregulated by inhibitors of NF-κB pathway, as well as by Interleukin-10, a known anti-inflammatory cytokine. Moreover, the transgene-dependent luciferase activity showed a positive correlation to the endogenous murine soluble TNF secreted to the culture medium. In conclusion, the hTNF.LucBAC strain is a valuable tool for studying and screening molecules that target TNF synthesis and will allow further functional studies of the regulatory elements of the TNF locus.
Highlights
Tumour necrosis factor (TNF) is an important pro-inflammatory cytokine produced by the majority of the cells of the immune system
The reporter activity was initially tested on cell-lines by transiently transfecting the hTNF.LucBAC into human neuroblastoma cell-line SK-N-AS, and human colon carcinoma cell-line HCT116
Whilst SK-N-AS cells demonstrated luciferase activation in the presence of PAM3CSK4 treatment (p < 0.001), HCT116 cells did not. These data indicate that the hTNF.LucBAC is able to report on TNF gene activation in vitro
Summary
Tumour necrosis factor (TNF) is an important pro-inflammatory cytokine produced by the majority of the cells of the immune system. The use of a human BAC on a mouse background allows for the study of human gene activation in a surrogate physiological system Encoded reporters, such as luciferases and fluorescent proteins, are powerful tools to detect gene expression, providing both accurate and quantifiable dynamic measurement of activity in gene expression. We have generated a reporter mouse by utilising a BAC that carries the firefly luciferase coding sequence under the control of the human TNF promoter. This system allowed us to directly measure human TNF promoter activity in ex-vivo and in vitro approaches
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