Igs7^{tm62.1(tetO-tdTomato)Hze}

The targeting constructed introduced by recombinase mediated cassette exchange at the existing site inserted anan 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. PhiC31-mediated recombination removed the hygromycin cassette and replaced it with the recombined AttB/AttP site (AttL). The Ai62D allele is therefore TIGRE-frt3-Ins-TRE-LSL-tdTomato-WPRE-bGHpA-Ins-AttL. The modified TRE promoter used in these transgenic mice is the Tet-responsive Ptight promoter. This Ptight promoter contains a modified Tet response element (TREmod), which consists of seven direct repeats of a 36-bp sequence each containing the 19-bp tet operator sequence (tetO). The TREmod is just upstream of a minimal human cytomegalovirus (CMV) promoter (PminCMVDelta), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P>tight< is silent in the absence of binding of TetR or rTetR to the tetO sequences. (J:101977, J:136987, J:219930)