Bai Qing, Garver Jessica A, Hukriede Neil A, Burton Edward A
Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Nucleic Acids Res. 2007;35(19):6501-16. doi: 10.1093/nar/gkm608. Epub 2007 Sep 25.
The aim of this study was to isolate cis-acting regulatory elements for the generation of transgenic zebrafish models of neurodegeneration. Zebrafish enolase-2 (eno2) showed neuronal expression increasing from 24 to 72 h post-fertilization (hpf) and persisting through adulthood. A 12 kb eno2 genomic fragment, extending from 8 kb upstream of exon 1 to exon 2, encompassing intron 1, was sufficient to drive neuronal reporter gene expression in vivo over a similar time course. Five independent lines of stable Tg(eno2 : GFP) zebrafish expressed GFP widely in neurons, including populations with relevance to neurodegeneration, such as cholinergic neurons, dopaminergic neurons and cerebellar Purkinje cells. We replaced the exon 2-GFP fusion gene with a cDNA encoding the 4-repeat isoform of the human microtubule-associated protein Tau. The first intron of eno2 was spliced with high fidelity and efficiency from the chimeric eno2-Tau transcript. Tau was expressed at approximately 8-fold higher levels in Tg(eno2 : Tau) zebrafish brain than normal human brain, and localized to axons, neuropil and ectopic neuronal somatic accumulations resembling neurofibrillary tangles. The 12 kb eno2 promoter drives high-level transgene expression in differentiated neurons throughout the CNS of stable transgenic zebrafish. This regulatory element will be useful for the construction of transgenic zebrafish models of neurodegeneration.
本研究的目的是分离顺式作用调控元件，以构建神经退行性疾病的转基因斑马鱼模型。斑马鱼烯醇化酶-2（eno2）在受精后24至72小时（hpf）神经元表达增加，并持续至成年期。一个12 kb的eno2基因组片段，从外显子1上游8 kb延伸至外显子2，包括内含子1，足以在相似的时间进程内驱动体内神经元报告基因的表达。五个独立的稳定Tg(eno2:GFP)斑马鱼品系在神经元中广泛表达GFP，包括与神经退行性疾病相关的神经元群体，如胆碱能神经元、多巴胺能神经元和小脑浦肯野细胞。我们用编码人类微管相关蛋白Tau的4重复异构体的cDNA替换了外显子2-GFP融合基因。eno2的第一个内含子从嵌合的eno2-Tau转录本中以高保真度和效率进行剪接。在Tg(eno2:Tau)斑马鱼脑中，Tau的表达水平比正常人脑高约8倍，并定位于轴突、神经纤维网和类似神经原纤维缠结的异位神经元体细胞聚集处。12 kb的eno2启动子在稳定转基因斑马鱼的整个中枢神经系统的分化神经元中驱动高水平的转基因表达。该调控元件将有助于构建神经退行性疾病的转基因斑马鱼模型。
