Biochimica et biophysica acta. Molecular cell research · 2024
A fluorescent protein C-terminal fusion knock-in is functional with TRPA1 but not TRPC5.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Tragl Aaron, Ptakova Alexandra, Sinica Viktor, Meerupally Rathej, König Christine, Roza Carolina, Barvík Ivan, Vlachova Viktorie, Zimmermann Katharina
Аннотация
OBJECTIVE: Transgenic mice with fluorescent protein (FP) reporters take full advantage of new in vivo imaging technologies. Therefore, we generated a TRPC5- and a TRPA1-reporter mouse based on FP C-terminal fusion, providing us with better alternatives for studying the physiology, interaction and coeffectors of these two TRP channels at the cellular and tissue level.
METHODS: We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging.
RESULTS: Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function.
CONCLUSIONS: Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons.
METHODS: We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging.
RESULTS: Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function.
CONCLUSIONS: Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons.