The Ai90(TITL-Chronos)-D allele (also called Ai90Δhygro or Ai90D) has a Tet response element, floxed-STOP cassette and the Chronos/EGFP fusion protein 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).
Specifically, the pTRE-LSL-Chronos/EGFP replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a modified Tet response element (TRE or tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the channelrhodopsin/EGFP fusion protein coding sequence (Chronos/EGFP; details below), a WPRE sequence (to enhance mRNA stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5’hygro cassette, an RNA splice donor and a FRT5 site.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3’hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-Chronos/EGFP replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME).
Chimeric mice were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743) to remove the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL).
The Ai90D allele is therefore TIGRE-frt3-Ins-TRE-LSL-Chronos/EGFP-WPRE-bGHpA-Ins-AttL.
The resulting Ai90D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed). In 2015, black males from generation N6F1 were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
The Chronos/EGFP fusion protein, designed by Dr. Ed Boyden (MIT), is composed of a mammalian codon-optimized Stigeoclonium helveticum-derived channelrhodopsin ShChR (Chronos) fused in-frame via a 18 nucleotide linker to the N-terminus of enhanced green fluorescent protein (EGFP). Chronos is a blue-to-green light-driven (~470-530 nm) cation channel that depolarizes the cell and causes action potentials. Chronos has high light sensitivity and faster kinetics than previous channelrhodopsins.
The TRE promoter used here is Tet-responsive Ptight; containing a modified Tet response element (TREmod) composed of seven direct 36 bp repeats (each containing the 19 bp tet operator sequence [tetO]). TREmod is just upstream of a minimal human cytomegalovirus promoter (PminCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, Ptight is silent in the absence of TetR or rTetR binding to tetO.
