| Primary Identifier | MGI:5558064 | Allele Type | Targeted |
| Attribute String | Conditional ready, Inducible, Reporter | Gene | Igs7 |
| Transmission | Germline | Strain of Origin | (129S6/SvEvTac x C57BL/6NCrl)F1 |
| Induced With | doxycycline/tetracycline | Is Recombinase | false |
| Is Wild Type | false |
| molecularNote | The targeting vector has a Tet response element, floxed-STOP cassette and improved Halorhodopsin/EGFP fusion protein (Halo57*K200R*W214F/EGFP or Jaws) 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). The replacement vector was designed with 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 Jaws coding sequence (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 spice donor and a FRT5 site. The hop/EGFP fusion protein (Halo57*K200R*W214F/EGFP)is composed of a modified Halobacterium halobium (strain shark)-derived halorhodopsin (Halo57) fused in-frame via a 15 nucleotide linker to the N-terminus of enhanced green fluorescent protein (EGFP). The Halo57 sequence has K200R and W214F mutations to substantially boost photocurrents. An endoplasmic reticulum exporting sequence (ER2) from the Kir2.1 inward rectifier potassium channel gene (amino acids FCYENEV) at the C-terminal of EGFP improves membrane trafficking/prevents aggregation. Embryonic stem cells, previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3' hygro cassette::SV40 polyA:AttPin the same locus, were re-transfected with the targeting vector and Flp recombinase vector. Mutant mice were bred with Gt(ROSA)26Sortm3(phiC31*)Sor mice to remove to remove the AttB/AttP-flanked PGK-hygro-SV40pA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai79D allele is therefore TIGRE-frt3-Ins-TRE-LSL-Jaws-WPRE-bGHpA-Ins-AttL. |
