These Ai62(TITL-tdT)-D allele (also called Ai62(TITL-tdT)Δhygro or Ai62D) has a Tet response element, floxed-STOP cassette and tdTomato fluorescent protein, 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).
Specifically, the pTRE-LSL-tdTomato replacement vector was designed with (from 5' to 3') an FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce expression of the reporter gene in the absence of transactivator protein), a modified Tet response element (TRE or (tetO); described in detail below), a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to a synthetic pA-hGHpA-PGKpA unit), a sequence encoding the tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the 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-tdTomato 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 Ai62D allele is therefore TIGRE-frt3-Ins-TRE-LSL-tdTomato-WPRE-bGHpA-Ins-AttL. The resulting Ai62D mice were bred with C57BL/6J wildtype mice for at least three additional generations prior to sending to The Jackson Laboratory Repository in 2013. Upon arrival, mice were bred to C57BL/6J inbred animals (Stock No. 000664) at least once to remove the PhiC31 gene. The Ai62D cryopreserved sperm is the equivalent of six generations backcrossed onto C57BL/6J [June 2014].
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.
