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A fluorescent protein C-terminal fusion knock-in is functional with TRPA1 but not TRPC5
A. Tragl, A. Ptakova, V. Sinica, R. Meerupally, C. König, C. Roza, I. Barvík, V. Vlachova, K. Zimmermann
Jazyk angličtina Země Nizozemsko
Typ dokumentu časopisecké články
- MeSH
- červený fluorescenční protein MeSH
- ganglion trigeminale metabolismus MeSH
- genový knockin * MeSH
- kationtové kanály TRPC * genetika metabolismus MeSH
- kationtový kanál TRPA1 * genetika metabolismus MeSH
- luminescentní proteiny * genetika metabolismus MeSH
- myši transgenní * MeSH
- myši MeSH
- rekombinantní fúzní proteiny metabolismus genetika MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
Citace poskytuje Crossref.org
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- $a Tragl, Aaron $u Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Anesthesiology, Krankenhausstraße 12, 91054 Erlangen, Germany
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- $a 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.
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- $a Ptakova, Alexandra $u Department of Cellular Neurophysiology, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic
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- $a Sinica, Viktor $u Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Anesthesiology, Krankenhausstraße 12, 91054 Erlangen, Germany; Department of Cellular Neurophysiology, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic
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- $a Roza, Carolina $u Departamento de Biología de Sistemas, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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- $a Barvík, Ivan $u Department of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic
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- $a Vlachova, Viktorie $u Department of Cellular Neurophysiology, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic. Electronic address: viktorie.vlachova@fgu.cas.cz
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- $a Zimmermann, Katharina $u Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Anesthesiology, Krankenhausstraße 12, 91054 Erlangen, Germany. Electronic address: katharina.zimmermann@fau.de
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