Igs7^{tm79.1(tetO-hop/EGFP)Hze}

基础信息

表型特征

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这个靶向载体包含了Tet响应元件、floxed-STOP cassette以及改进的Halorhodopsin/EGFP融合蛋白（Halo57*K200R*W214F/EGFP，或称Jaws），插入到了9号染色体的T1 TIGRE位点（一个位于AB124611和Carm1之间的低基底转录活性的非基因间区，允许高Cre和/或Tet诱导）。载体设计从5'至3'包括FRT3序列、两个鸡β-珠蛋白HS4绝缘子元件（以减少在无转录激活剂时报告基因的表达）、修改的Tet响应元件（TRE或tetO）、floxed-STOP cassette（三个读框的终止子与合成的pA-hGHpA-PGKpA相连）、Jaws编码序列（详情如下）、WPRE序列（增强mRNA稳定性）、BGHpolyA、两个鸡β-珠蛋白HS4绝缘子元件、AttB位点、PGK 5'hygro cassette、RNA spice捐赠者以及FRT5位点。融合蛋白Halo57*K200R*W214F/EGFP由改造自Halobacterium halobium的Halorhodopsin（Halo57）通过15个核苷酸链接到增强绿色荧光蛋白（EGFP）的N端组成，K200R和W214F突变提高了光电流。载体末端包含内向整流钾通道Kir2.1基因的ER2序列，用于优化膜运输和防止聚集，位于EGFP的C端。先前在相同位点用FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3' hygro cassette::SV40 polyA:AttPin处理的胚胎干细胞，现在再次转染了靶向载体和Flp重组酶载体。通过与Gt(ROSA)26Sortm3(phiC31*)Sor小鼠杂交，去除了AttB/AttP介导的PGK-hygro-SV40pA片段，并用重组的AttB/AttP位点（AttL）替换，产生了Ai79D等位基因，其序列是TIGRE-frt3-Ins-TRE-LSL-Jaws-WPRE-bGHpA-Ins-AttL。（信息来源：J:101977, J:219930）

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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. (J:101977, J:219930)