Igs7^{tm210(tetO-GCaMP7f,CAG-tTA2)Tasic}

基础信息

表型特征

文献报道

这个靶向载体包含了从5'到3'的方向：Bxb1 attL 立即位点、一段非功能的500bp绿色荧光蛋白序列（用于增加Bxb1 attL 与绝缘子序列之间的空间，以提高载体的稳定性），两个鸡β-珠蛋白HS4绝缘子元件（在没有转录激活因子时减少报告基因的表达），tetO响应元件/启动子，一段由loxP标记的终止子（包含三个读框的终止密码子，连接到pA-hGHpA-PGKpA的合成序列），一个GCaMP7敏感快速变体钙指示器序列（jGCaMP7f，详情如下），后跟着6xHis标签、T7标签和XPress标签序列，木鼠肝病毒的后转录调控元件（WPRE，以增强mRNA的稳定性），一段BGHpolyA，两个鸡β-珠蛋白HS4绝缘子元件，CMV-IE增强子/鸡β-肌动蛋白/兔β-珠蛋白的混合启动子（CAG，来自pCAGGS），一段FRT标记的终止子（与pA-hGHpA-TKpA的合成序列相连的终止密码子），一个内部核糖体进入位点（IRES）序列，一个合成的修改型四环素调控转录激活器基因（tTA2(S)），再一个WPRE，一段BGHpolyA，一个ΦC31 attB位点，一段PGK-5'hygro插入片段，一个mRNA剪接供体，一个Bxb1 attR位点，SA，3'hygro，SV40pA，以及另一个ΦC31 attP。"Janelia GCaMP7"敏感快速变体钙指示器是一种对神经活动极为敏感的超快速检测器，具有更快的衰减和响应速度。这个等位基因携带有鸡平滑肌的M13肌动蛋白轻链激酶片段（也称为钙结合肽(CBP)），一个环转的绿色荧光蛋白（cpEGFP，149-238序列后跟1-144序列（通过变异来增加动态范围和基线荧光，以及提高敏感性和减缓反应速度）），以及一段鼠钙调素DNA片段（CaM，2-148序列，通过变异来增加小钙信号变化的荧光响应）。cpEGFP到CaM的链接处氨基酸替换有助于传感器功能的提升。此外，此等位基因还包含GFP的H78K突变和CaM的A317L、R381T、T383S和G392R突变（来源：J:101977）。

查看原文参与反馈

The targeting vector contains (from 5' to 3') a Bxb1 attL site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attL site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a Tet response element/promoter (tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a GCaMP7 sensitive and fast variant calcium indicator sequence (jGCaMP7f; details below) followed by a 6xHis tag, T7 tag and XPress tag sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a FRT-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), an internal ribosome entry site (IRES) sequence, a synthetic modified tetracycline-regulated transactivator gene (tTA2(S)), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an mRNA splice donor, a Bxb1 attR site, SA, 3'hygro, SV40pA, and PhiC31 attP. The "Janelia GCaMP7" sensitive and fast variant calcium indicator is an ultrasensitive detector of neuronal activity with faster decay and response kinetics. jThe allele carries a chicken smooth muscle M13 fragment of myosin light chain kinase (also called calmodulin-binding peptide [CBP]), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations to increase dynamic range/baseline fluorescence, as well as increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations to increase the fluorescence change for small calcium transients]). Amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function. In addition, this allele has the H78K mutation in GFP and the A317L, R381T, T383S, and G392R mutations in CaM. (J:101977)