Ai218 mice harbor the TITL-Ace2N-4AA-mNeonGreen-TS-ER-WPRE-ICL-mRuby3-IRES2-tTA2-WPRE conditional allele, designed with a tetO promoter, loxP-STOP-loxP cassette and Ace2N-4AA-mNeonGreen-WPRE sequence, and then a CAG promoter, Lox2272-STOP-Lox2272 cassette, mRuby3, and IRES-tTA2-WPRE sequence. Ace2N-4AA-mNeonGreen is an indicator variant of Acetabularia acetabulum-derived rhodopsin (Ace) with an inactivated proton pump (Ace2N), with four extra amino acids in a linker domain that fuses to mNeonGreen fluorescent protein to enable voltage-sensitive fluorescence resonance energy transfer (FRET). These mice have blue-shifted absorption spectra compared with Mac and Arch, yielding superior FRET acceptors when paired with green or yellow emitters. Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (Ace2N-4AA-mNeonGreen, mRuby3, and tTA2). Ai218 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (January 2023), it has not been attempted to make this strain homozygous. When bred to mice expressing Cre recombinase, the offspring will have both STOP cassettes deleted in the cells where Cre is expressed, resulting in Ace2N-4AA-mNeonGreen, mRuby3, and tTA2 expression. Specifically, the donating investigator reports Ai218 exhibits no visible Ace2N-4AA-mNeonGreen or mRuby3 fluorescence in the absence of Cre. Of note, the Ai218 allele was designed with an IRES upstream of the tTA2 to reduce/eliminate leakiness in the absence of Cre recombinase (Graybuck et al. 2021 Neuron [<a href="https://pubmed.ncbi.nlm.nih.gov/33789083">PMID:33789083</a>]). The donating investigator reports when Ai218 mice are bred with a Cux2-CreERT2 driver line, robust native Ace2N-4AA-mNeonGreen and mRuby3 expression has been observed in fixed brain sections from double transgenic mice with tamoxifen treatment, with more widespread mRuby3 expression seen in the cortex. This may be due to selective silencing of the TREtight promoter relative to CAG which is more ubiquitous. In vivo voltage imaging has been performed and Ace2N-4AA-mNeonGreen appears functional in cortex.

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Ai218 (also called Ai218(TITL-Ace2N-4AA-mNeonGreen-TS-ER-WPRE-ICL-mRuby3-IRES2-tTA2-WPRE)) is a Cre recombinase-dependent, Tet-controllable, optogenetic and voltage-sensitive fluorescent tool allele created by targeted insertion at the Igs7 locus (TIGRE). Exposure to Cre recombinase results in expression of Ace2N-4AA-mNeonGreen, mRuby3, and tTA2 in those cells where Cre is expressed.

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