We fused rat TRPV1 (NM_031982), containing the E600K mutation that increases sensitivity to Csn by over 10–fold 21 (link), rat TRPM8 (NM_134371), and Crotalus atrox (rattlesnake) TRPA1 (GU562967), to TagRFPT 22 (link) at their C–termini. For expression in trigeminal and Rohon–Beard sensory neurons, we cloned the 4 kb islet1 sensory neuron specific enhancer 23 (link) upstream of the GAL4VP16 transcriptional activator 24 (link), followed by 4xUAS:E1b minimal promoter–TRP channel–RFPT. We used the 1 kb zebrafish hcrt promoter 25 (link) to express TRPV1–RFPT in Hcrt neurons. Each open reading frame was followed by an SV40 polyA sequence and each cassette was flanked by ISce1 meganuclease sites and Tol2 transposase arms. We generated the Tg(hcrt:TRPV1–RFPT)ct824 transgenic line using the Tol2 transposase method 26 (link). We generated the Tg(islet1:GAL4VP16,4xUAS:TRPV1–RFPT)ct825 and Tg(islet1:GAL4VP16,4xUAS:TRPA1–RFPT)ct826 transgenic lines using the ISce1 method 27 (link). TRPM8 experiments used transient injection of an islet1:GAL4VP16,4xUAS:TRPM8–RFPT transgene. The Tg(elavl3:GCaMP5G)a4598 28 (link), Et(e1b:GAL4VP16)s1102t 29 (link), Tg(14xUAS:EGFP–Aequorin)a127 30 (link), Tg(–2.0Tru.Hcrt:EGFP)zf11Tg 16 (link) and Tg(qrfp:EGFP)ct820 15 (link) transgenic lines have been described.
Protocol detail
Genetically Engineered Zebrafish Sensory Neurons
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Aequorin
Arms
Hcrt
Kb 23
Mutation
Neurons
Polya
Rattlesnake
Sensitivity
Sensory neuron
Sv40
Transcriptional
Transgene
Transgenic
Transient
Transposase
Zebrafish
Corresponding Organization :
Other organizations : California Institute of Technology, Cornell University
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Variable analysis
independent variables
- Rat TRPV1 (NM_031982) containing the E600K mutation
- Rat TRPM8 (NM_134371)
- Crotalus atrox (rattlesnake) TRPA1 (GU562967)
- Sensitivity to Csn (increased over 10-fold for TRPV1 E600K)
- Sensory neuron expression using the 4 kb islet1 sensory neuron specific enhancer
- Hcrt neuron expression using the 1 kb zebrafish hcrt promoter
- TagRFPT fluorescent protein fused to the C-termini of the TRP channels
- SV40 polyA sequence following each open reading frame
- ISce1 meganuclease sites and Tol2 transposase arms flanking each cassette
- Tg(elavl3:GCaMP5G)a4598
- Et(e1b:GAL4VP16)s1102t
- Tg(14xUAS:EGFP–Aequorin)a127
- Tg(–2.0Tru.Hcrt:EGFP)zf11Tg
- Tg(qrfp:EGFP)ct820
- Not explicitly mentioned
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