A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse
A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse
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DOI：
10.1093/nar/29.7.e39
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发表时间：
2001-04-01
影响因子：
14.9
通讯作者：
Sprengel, R.
中科院分区：
文献类型：
--
作者：
Krestel, H. E.;Mayford, M. R.;Sprengel, R.
在小鼠中研究了两种转录偶联转基因的多西环素（Dox）敏感性共调节。为此，我们产生了四个独立的小鼠品系，其在双顺反子双向模块中携带绿色荧光蛋白（GFP）和β-半乳糖苷酶的编码区。在所有四个系中，表达模块是沉默的，但当转录因子tTA由α-CaMKII-tTA转基因提供时被激活。GFP荧光、β-半乳糖苷酶和免疫化学染色的体内分析揭示了不同品系之间GFP和β-半乳糖苷酶水平的差异，但表达模式相当。在嗅觉系统、新皮层、边缘叶和基底节结构的神经元中发现了强信号。较弱的表达仅限于丘脑、脑桥和髓质结构、脊髓、眼睛和小脑中的一些浦肯野细胞。强烈的GFP信号总是伴随着强烈的β-半乳糖苷酶活性，这两者都可以被Dox共调节。我们的结论是，tTA敏感的双向表达模块是非常适合表达的目的基因在一个受管制的方式和绿色荧光蛋白可用于跟踪在活小鼠的模块的转录活性。
Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and beta-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the alpha-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, beta-galactosidase and immunochemical stainings revealed differences in GFP and beta-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense beta-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.