Ectopic transgene expression in the retina of four transgenic mouse lines.
Ectopic transgene expression in the retina of four transgenic mouse lines.
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DOI：
10.1007/s00429-015-1128-2
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发表时间：
2016-09
影响因子：
3.1
通讯作者：
Wilhelm M
中科院分区：
文献类型：
--
作者：
Gábriel R;Erdélyi F;Szabó G;Lawrence JJ;Wilhelm M
在四个小鼠品系中检查转基因的视网膜表达。两种构建体由胆碱乙酰转移酶（ChAT）启动子驱动：与tau蛋白缀合的绿色荧光蛋白（tau-GFP）或通过CRE重组酶诱导的Rosa 26表达产生的胞质黄色荧光蛋白（YFP）（ChAT-CRE/Rosa 26 YFP）。另外两种构建体靶向抑制性中间神经元：通过谷氨酸脱羧酶（GAD 65-GFP）或小清蛋白（PV）细胞（PV-CRE/Rosa 26 YFP）鉴定的GABA能水平和无长突细胞。经心脏灌注动物并制备视网膜切片。使用抗PV、钙视网膜蛋白（CALR）、钙结合蛋白（CALB）和酪氨酸羟化酶（TH）的抗体对转基因表达细胞进行复染。在PVxRosa和ChAT-tauGFP构建体中，染色出现在类似于Müller细胞的垂直取向的突起行中。在ChATxRosa构建体中，标记神经节细胞层中的无长突细胞和神经元群体。一些视锥细胞也表现出GFP荧光。CALR、PV和TH在上述细胞中均未检出。偶尔，我们在神经节细胞层（GCL）中发现GFP/ CALR和GFP/PV双染色细胞。在GAD 65-GFP构建体中，除了光感受器之外，神经视网膜的所有层都被标记。并非所有水平细胞都表达GFP。我们没有发现GFP/TH双标记的细胞，GFP很少出现在CALR和CALB的细胞。许多PV阳性神经元也被标记为GFP，包括小直径的无长突。在GCL中，确定了GFP和PV的单标记，以及几个CALR/PV双染色的神经元。在GCL中，GFP/CALR/ CALB三重标记的细胞稀疏。总之，四个转基因构建体中只有一个表现出与内源性视网膜蛋白表达一致的表达模式，而其他的强烈建议异位基因表达。
Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/ Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/ CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR-and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/ CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression.
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作者：
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通讯作者：
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