JellyOp (obtained from Addgene) and human OPN1MW amplified from retinal cDNA was cloned in front of the IRES site of a pIRES2-TurboFP635 vector or in the position of the IRES site of a pIRES2-mKate vector to create fusion proteins22 (link). The JellyOp(K72T) mutation was introduced to the pIRES2-JellyOp-TurboFP635 vector using a Phusion mutagenesis kit (ThermoFisher).
For viral production, JellyOp was cloned into a pAAV-770En_454P(hGRM6)-JellyOp-IRES2-TurboFP635-WPRE-BGHpA plasmid20 (link). Viral vectors were produced in HEK293 cells by the triple plasmid co-transfection method using the pXX80 helper plasmid and the rep-cap plasmid encoding AAV(7m8)45 (link) as described in detail elsewhere46 (link). The viral titre was 4 × 1013 GC/ml. The virus was stored in aliquots at −80 °C until the day of use.
For viral production, JellyOp was cloned into a pAAV-770En_454P(hGRM6)-JellyOp-IRES2-TurboFP635-WPRE-BGHpA plasmid20 (link). Viral vectors were produced in HEK293 cells by the triple plasmid co-transfection method using the pXX80 helper plasmid and the rep-cap plasmid encoding AAV(7m8)45 (link) as described in detail elsewhere46 (link). The viral titre was 4 × 1013 GC/ml. The virus was stored in aliquots at −80 °C until the day of use.
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