Igs7^{tm230(tetO-XCaMPG,CAG-ChRmine*/oScarlet*,-tTA2)Tasic}

The targeting vector has a Tet response element (tetO), loxP-STOP-loxP cassette, XCaMP-G-WPRE, a CAG promoter, Lox2272-STOP-Lox2272 cassette, ChRmine*/oScarlet* sequence, and IRES-tTA2-WPRE sequence - all inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high cre and/or Tet inducibility). ChRmine is a pump-like cation-conducting channelrhodopsin, derived from Rhodomonas lens, that exhibits extremely high current and light sensitivity, as well as a markedly red-shifted spectrum. This is fused with a membrane trafficking signal (TS) and oScarlet fluorescent protein, which is based on the sequence of mScarlet with an additional E95D mutation to increase neuronal cell trafficking. XCaMP-G is an EGFP fluorescent calcium detector of neuronal activity with high signal-to-noise ratio, linear property, and high-frequency spike resolution suitable for in vivo interrogation of complex neural dynamics. XCaMP-G incorporates a second calmodulin-binding peptide as a "protection motif", which decreases calcium channel disruption and nuclear accumulation. Of note, the Ai229 allele was designed with an IRES upstream of the tTA2 sequence to reduce/eliminate leakiness in the absence of cre recombinase. (J:101977)