Analysis of the upstream regulatory elements of Nolz-1 gene and construction of the conditional gene targeting construct of Nolz-1 gene
| Autor: | Ting-Hao Huang, 黃鼎皓 |
|---|---|
| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 The striatum is the major recipient of inputs to the basal ganglia from the cerebral cortex. The striatum is known to be involved in processing multiple neurological functions, like the planning and modulation of movement, cognitive processes, and the reward learning. The malfunctions of striatum are involved in the pathogenesis of several neurological diseases, including Parkinson’s disease, Huntington’s disease, obsessive-compulsive disorder, and drugs addiction. In order to understand the development of such important structure, our laboratory has previously identified a novel gene, Nolz-1, that expressed in the striatal anlage, the lateral ganglionic eminence (LGE). In this study, I analyzed a series of luciferase reporter gene constructs containing different lengths of the 5’-flanking regions of Nolz-1 upstream of the translation initiation site. Reporter constructs were transiently transfected into Neuro2A and ST14A cells to test the cell type specificity of the promoter activity. Transient transfection of the series of reporter gene constructs showed that, in both Neuro2A and ST14A cells, the constructs containing -2.4 kb region displayed similar pattern of promoter activity. Transfection of the constructs containing 5’-flanking region between -0.2 kb and -0.8 kb showed gradually increased promoter activities. The two fragments from -0.8 kb to -1.1 kb and from -1.5 kb to -1.6 kb enhanced the promoter activity; however, the other two regions from -1.1 kb to -1.5 kb and -1.6 kb to -2.4 kb repressed the promoter activity. Moreover, there is a specificity of increased promoter activity in ST14A cells with the 5’-flanking region between -2.4 kb and -6.7 kb. I also tried to test which 5’-flanking region regulated the promoter activity that was involved in Nolz-1 expression during neuronal differentiation. I transfected a series of deletion 5’-flanking region constructs into P19 cells, and studied the changes of the promoter activity during neural induction by aggregation and retinoic acid treatment. I found that there is a significant enhancement of promoter activity after neural induction in the region between -0.2 kb and -0.45 kb. In order to identify evolutionally conserved DNA sequences that regulate Nolz-1, I compared the genomic sequences of the homologs of Nolz-1 among different species, and found that the 3’-UTR of Nolz-1 was highly conserved between vertebrate animals. To characterize the conserved 3’-UTR function, I inserted the conserved 3’-UTR into the 3’-UTR region of luciferase gene. Transfection this construct into Neuro2A cells showed that the conserved 3’-UTR reduced the expression of the reporter gene at both mRNA and protein levels. Taken together, my studies provide an initial characterization of the regulatory regions in 5’- and 3’-flanking regions of Nolz-1 gene. In order to further study the roles of Nolz-1 during striatal development, I planned to generate Nolz-1 conditional knockout mice. Therefore, I successfully used the recombineering-based method to construct the Nolz-1 conditional gene targeting construct in which the exon3 of Nolz-1 were flanked by loxP sites. In the future, when Nolz-1 condition knockout mice are crossed with Cre transgenic mice under control of different tissue-specific promoters, we can analyze neural development in the mutant mice to elucidate the biological function of Nolz-1 gene. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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