We have uploaded the verified vector information for the published Drosophila KCR constructs to Zenodo. Files can be assessed under: https://zenodo.org/records/15074206
The file contains vector maps for the below constructs:
pJFRC7_20xUAS_HcKCR1_AAA_YFP
pJFRC7_20xUAS_HcKCR1_C29D_YFP
pJFRC7_20xUAS_HcKCR1_ET_YFP
pJFRC7_20xUAS_HcKCR1_GS_YFP
pJFRC7_20xUAS_HcKCR2_AAA_YFP
pJFRC7_20xUAS_HcKCR2_ET_YFP
pJFRC7_20xUAS_HcKCR2_GS_YFP
pJFRC7_20xUAS_WiChR_ET_YFP
These were reported in:
Ott, S., Xu, S., Lee, N. et al. Kalium channelrhodopsins effectively inhibit neurons. Nat Commun 15, 3480 (2024). https://doi.org/10.1038/s41467-024-47203-w
Also:
Kalium channelrhodopsins effectively inhibit neurons in the small model animals
Stanislav Ott, Sangyu Xu, Nicole Lee, Ivan Hee Kean Hong, Jonathan Anns, Danesha Devini Suresh, Zhiyi Zhang, Xianyuan Zhang, Raihanah Harion, Weiying Ye, Vaishnavi Chandramouli, Suresh Jesuthasan, Yasunori Saheki, Adam Claridge-Chang
bioRxiv 2024.01.14.575538; doi: https://doi.org/10.1101/2024.01.14.575538
Fly constructs and genetics
UAS-KCR1-ET, UAS-KCR2-ET, UAS-KCR1-GS and UAS-WiChR transgenic lines were generated by de novo synthesis (Genscript) of Drosophila codon-optimized HcKCR insert sequences 43 (Genbank #MZ826861 and #MZ826862) or the WiChR sequence 45 (Genbank #OP710241) as eYFP fusions. After Sanger sequencing verification (Genscript), the fragments were cloned into an pJFRC7-20XUAS-IVS-mCD8::GFP vector (Addgene plasmid # 26220), replacing the mCD8::GFP insert via restriction enzyme digest (XhoI, Xba I). For UAS-KCR1-GS, a 3×GGGGS sequence was used to link the opsin with the fluorophore. For the KCR-ET and WiChR constructs, an AAA linker sequence was used as the starting point, to which two modifications were made: (1) an FCYENEV motif was added to the C terminus of eYFP to boost protein export from the endoplasmic reticulum and prevent potential aggregate formation 51; and (2) a KSRITSEGEYIPLDQIDINV trafficking signal from Kir 2.1 52 was added to the linker at C terminus of the opsin to boost protein expression 10. The KCR1-C29D variant 45 was obtained by site-directed mutagenesis of the KCR1-ET sequence, where the cysteine at position 29 was replaced by aspartic acid (Genscript). The synthesized constructs were injected into flies and targeted to attP1 or attP2 insertion sites on the second or third chromosomes respectively and the transgenic progeny were balanced either over CyO or TM6C (BestGene). Expression was verified by imaging of eYFP fluorescence with a Leica TCS SP8 STED confocal microscope. Opsin transgenic flies were crossed with relevant Gal4 driver lines to produce F1 offspring for use as test subjects. Driver Gal4 lines and UAS-opsin responder lines were each crossed with an otherwise wild-type w1118 line and the F1 progeny (e.g. UAS-KCR1-ET/+ or elav-Gal4/+) were used as control subjects.
The file contains vector maps for the below constructs:
pJFRC7_20xUAS_HcKCR1_AAA_YFP
pJFRC7_20xUAS_HcKCR1_C29D_YFP
pJFRC7_20xUAS_HcKCR1_ET_YFP
pJFRC7_20xUAS_HcKCR1_GS_YFP
pJFRC7_20xUAS_HcKCR2_AAA_YFP
pJFRC7_20xUAS_HcKCR2_ET_YFP
pJFRC7_20xUAS_HcKCR2_GS_YFP
pJFRC7_20xUAS_WiChR_ET_YFP
These were reported in:
Ott, S., Xu, S., Lee, N. et al. Kalium channelrhodopsins effectively inhibit neurons. Nat Commun 15, 3480 (2024). https://doi.org/10.1038/s41467-024-47203-w
Also:
Kalium channelrhodopsins effectively inhibit neurons in the small model animals
Stanislav Ott, Sangyu Xu, Nicole Lee, Ivan Hee Kean Hong, Jonathan Anns, Danesha Devini Suresh, Zhiyi Zhang, Xianyuan Zhang, Raihanah Harion, Weiying Ye, Vaishnavi Chandramouli, Suresh Jesuthasan, Yasunori Saheki, Adam Claridge-Chang
bioRxiv 2024.01.14.575538; doi: https://doi.org/10.1101/2024.01.14.575538
Fly constructs and genetics
UAS-KCR1-ET, UAS-KCR2-ET, UAS-KCR1-GS and UAS-WiChR transgenic lines were generated by de novo synthesis (Genscript) of Drosophila codon-optimized HcKCR insert sequences 43 (Genbank #MZ826861 and #MZ826862) or the WiChR sequence 45 (Genbank #OP710241) as eYFP fusions. After Sanger sequencing verification (Genscript), the fragments were cloned into an pJFRC7-20XUAS-IVS-mCD8::GFP vector (Addgene plasmid # 26220), replacing the mCD8::GFP insert via restriction enzyme digest (XhoI, Xba I). For UAS-KCR1-GS, a 3×GGGGS sequence was used to link the opsin with the fluorophore. For the KCR-ET and WiChR constructs, an AAA linker sequence was used as the starting point, to which two modifications were made: (1) an FCYENEV motif was added to the C terminus of eYFP to boost protein export from the endoplasmic reticulum and prevent potential aggregate formation 51; and (2) a KSRITSEGEYIPLDQIDINV trafficking signal from Kir 2.1 52 was added to the linker at C terminus of the opsin to boost protein expression 10. The KCR1-C29D variant 45 was obtained by site-directed mutagenesis of the KCR1-ET sequence, where the cysteine at position 29 was replaced by aspartic acid (Genscript). The synthesized constructs were injected into flies and targeted to attP1 or attP2 insertion sites on the second or third chromosomes respectively and the transgenic progeny were balanced either over CyO or TM6C (BestGene). Expression was verified by imaging of eYFP fluorescence with a Leica TCS SP8 STED confocal microscope. Opsin transgenic flies were crossed with relevant Gal4 driver lines to produce F1 offspring for use as test subjects. Driver Gal4 lines and UAS-opsin responder lines were each crossed with an otherwise wild-type w1118 line and the F1 progeny (e.g. UAS-KCR1-ET/+ or elav-Gal4/+) were used as control subjects.
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