Igs7^{tm170.1(tetO-GFP/TPTE2,CAG-tTA2)Hze}

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这个向量设计包含了从5'到3'的方向：一个FRT3位点，两个鸡β-珠蛋白HS4绝缘子元件（用于减少在没有转录激活因子时的报告基因表达），一个Tet响应元件/启动子（TRE2，详情如下），一个由loxP标记的终止子（包含所有三种阅读框的终止密码子，连接到合成的pA-hGHpA-PGKpA），一个ASAP2s绿色荧光电压指示蛋白的序列，带有 soma 和近端树突特异性导向序列（ASAP2s-Kv，详情如下），一个WPRE（增强mRNA转录稳定性），一个BGHpolyA，两个鸡β-珠蛋白HS4绝缘子元件，一个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），一个由lox2272标记的终止子（同样连接到合成的pA-hGHpA-TKpA），一个合成的修饰的四环素响应转录激活器基因（tTA2S），一个WPRE，一个BGHpolyA，一个AttB位点，一个PGK-5'hygro插入片段，一个RNA剪接供体以及一个FRT5位点。使用的TRE2启动子是Tet响应的P>hCMV*-1<，它包含7个拷贝的19bp tet操作子序列（tetO）上游，紧邻一个无增强子的CMV最小启动子（P>min CMV<）。因此，没有tTA或rtTA与tetO结合时，P>hCMV*-1<是沉默的。GFP*/TPTE2*包含了Gallus gallus电压敏感磷脂酶TPTE2的VSD（电压传感器域）序列，包括R154Q变异以实现所需的电压响应。在S3-S4之间插入了环化翻转的超级folder GFP 1-10 OPT变异体（cpsfGFP-OPT）。GgVSD包括R154Q变异，与原始描述中的R153Q等效。GFP 1-10 OPT通过折叠报告GFP的替换（F99S, M153T, V163A, F64L, S65T）和超级folder GFP的变异（S30R, Y145F, I171V, A206V）以及额外的7个替换（N39I, T105K, E111V, I128T, K166T, I167V, S205T）来优化亮度和动态范围，同时保持膜上的高效表达。此外，这个电压传感器还附带了一个65氨基酸的鼠Kv2.1细胞质段，以限制蛋白质定位到细胞体和近端树突。通过Phic31介导的重组，移除了AttB/AttP-开头序列，并替换为重组的AttB/AttP位点（AttL）。(来源：J:260362)

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The vector is 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 Tet response element/promoter (TRE2; details below), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a green fluorescent voltage indicator protein ASAP2s sequence with soma and proximal dendrite targeting sequences (ASAP2s-Kv; details below), a 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 lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a synthetic modified tetracycline-regulated transactivator gene (tTA2S), a WPRE, a BGH polyA, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. The TRE2 promoter used here is Tet-responsive P>hCMV*-1<; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P>min CMV<), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P>hCMV*-1< is silent in the absence of tTA or rtTA binding to tetO. GFP*/TPTE2* has VSD (voltage sensor domwin) sequences (GgVSD) from the Gallus gallus voltage-sensitive phosphatase TPTE2, including the R154Q mutation for desired voltage response. A circularly permuted superfolder GFP 1-10 OPT variant (cpsfGFP-OPT) is inserted into the GgVSD extracellular loop between the third and fourth transmembrane segments (S3-S4). The GgVSD includes the R154Q mutation for desired voltage response (equivalent to R153Q in original descriptions). The GFP 1-10 OPT includes the substitutions from folding reporter GFP (F99S, M153T, V163A, F64L, S65T) and superfolder GFP (S30R, Y145F, I171V, A206V), and also has seven additional substitutions (N39I, T105K, E111V, I128T, K166T, I167V, S205T). Together, these mutations result in improved brightness and dynamic range, while maintaining efficient expression at the membrane. In addition, this voltage sensor is tagged with a 65-amino-acid cytosolic segment of a rat Kv2.1 to restrict protein localization to the soma and proximal dendrites. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence and replaced it with the recombined AttB/AttP site (AttL). (J:260362)