In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene

碩士 === 國立陽明大學 === 神經科學研究所 === 96 === SCN8A gene encodes voltage-gated sodium channel Nav1.6 �� subunit, which is broadly expressed throughout the central and peripheral nervous system (CNS and PNS). The absence or reduced SCN8A expression are associated with the cognitive and motor deficits in human...

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Main Authors: Sih-Huei Wu, 吳思慧
Other Authors: Huey-Jen Tsay
Format: Others
Language:en_US
Published: 2008
Online Access:http://ndltd.ncl.edu.tw/handle/24539769838022659568
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spelling ndltd-TW-096YM0052910062015-10-13T13:51:29Z http://ndltd.ncl.edu.tw/handle/24539769838022659568 In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene 以斑馬魚搜尋調控神經特異性鈉離子通道scn8aa基因之調控序列 Sih-Huei Wu 吳思慧 碩士 國立陽明大學 神經科學研究所 96 SCN8A gene encodes voltage-gated sodium channel Nav1.6 �� subunit, which is broadly expressed throughout the central and peripheral nervous system (CNS and PNS). The absence or reduced SCN8A expression are associated with the cognitive and motor deficits in humans, movement disorders in mice and locomotor movement impairments in zebrafish. The brain-specific regulatory mechanism of mouse SCN8A has been published. Zebrafish scn8aa and scn8ab are orthologous to mammalian SCN8A gene. Although scn8aa and scn8ab displayed overlapping expression patterns, scn8aa mRNA is more broad and abundant than scn8ab. Scn8ab present in a subset of cells that express scn8aa. The precise mechanism regulating the spatial-temporal expression of zebrafish scn8aa in the CNS and PNS has not been studied. 15-kb genomic fragment of scn8aa driven GFP reporter was characterized by the transient injection assay and transgenic stable line. Stable and transient transgenic expression of scn8aa:GFP showed that the 15-kb fragment contained cis-regulatory elements recapitulated the endogenous expression profile of scn8aa mRNA during the maturation of the nervous system, with the exception of the inner nuclear layer of retina. The limited GFP-positive cell numbers of Tg(scn8aa:GFP) line in comparing with widely scn8aa mRNA expression pattern in the inner nuclear layer was observed. It suggested that 15-kb regulatory fragment was not sufficient to drive the GFP expression in certain cell types of the inner nuclear layer. The deletion analysis of 15-kb fragment of zebrafish scn8aa revealed that the fragment +52/+525 containing the evolutionarily conserved element shared with mouse SCN8A, and three novelty identified fragments including +525/+851, +851/+1340 and +6373/+7631 were important for controlling the scn8aa neuronal expression levels. These regulatory elements located in exon 1 and intron 1 functioned cooperatively on enhancing the expression of GFP reporter in the nervous system. The deletion of each positive element reduced the level of neuronal-specific expression without inducing the ectopic expression of GFP in the non-neuronal cells. The regulatory elements of mouse SCN8A did not share with the identified regulatory elements of the other neuronal-specific genes of mice. Although the expression profiles of zebrafish neurogenin1 and islet1 resembled to scn8aa mRNA, the regulatory sequences of neurogenin1and islet1 was not found in 15-kb genomic sequence of scn8aa. It suggested distinct regulatory mechanisms responsible for different neuronal genes expression in one single neuron type. The search neuronal-specific regulatory mechanism is at the infant stage. The enhancing elements identified from those published literatures could modulate a group of neuronal genes as more neuronal-specific regulatory mechanism revealed. Huey-Jen Tsay 蔡惠珍 2008 學位論文 ; thesis 0 en_US
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language en_US
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description 碩士 === 國立陽明大學 === 神經科學研究所 === 96 === SCN8A gene encodes voltage-gated sodium channel Nav1.6 �� subunit, which is broadly expressed throughout the central and peripheral nervous system (CNS and PNS). The absence or reduced SCN8A expression are associated with the cognitive and motor deficits in humans, movement disorders in mice and locomotor movement impairments in zebrafish. The brain-specific regulatory mechanism of mouse SCN8A has been published. Zebrafish scn8aa and scn8ab are orthologous to mammalian SCN8A gene. Although scn8aa and scn8ab displayed overlapping expression patterns, scn8aa mRNA is more broad and abundant than scn8ab. Scn8ab present in a subset of cells that express scn8aa. The precise mechanism regulating the spatial-temporal expression of zebrafish scn8aa in the CNS and PNS has not been studied. 15-kb genomic fragment of scn8aa driven GFP reporter was characterized by the transient injection assay and transgenic stable line. Stable and transient transgenic expression of scn8aa:GFP showed that the 15-kb fragment contained cis-regulatory elements recapitulated the endogenous expression profile of scn8aa mRNA during the maturation of the nervous system, with the exception of the inner nuclear layer of retina. The limited GFP-positive cell numbers of Tg(scn8aa:GFP) line in comparing with widely scn8aa mRNA expression pattern in the inner nuclear layer was observed. It suggested that 15-kb regulatory fragment was not sufficient to drive the GFP expression in certain cell types of the inner nuclear layer. The deletion analysis of 15-kb fragment of zebrafish scn8aa revealed that the fragment +52/+525 containing the evolutionarily conserved element shared with mouse SCN8A, and three novelty identified fragments including +525/+851, +851/+1340 and +6373/+7631 were important for controlling the scn8aa neuronal expression levels. These regulatory elements located in exon 1 and intron 1 functioned cooperatively on enhancing the expression of GFP reporter in the nervous system. The deletion of each positive element reduced the level of neuronal-specific expression without inducing the ectopic expression of GFP in the non-neuronal cells. The regulatory elements of mouse SCN8A did not share with the identified regulatory elements of the other neuronal-specific genes of mice. Although the expression profiles of zebrafish neurogenin1 and islet1 resembled to scn8aa mRNA, the regulatory sequences of neurogenin1and islet1 was not found in 15-kb genomic sequence of scn8aa. It suggested distinct regulatory mechanisms responsible for different neuronal genes expression in one single neuron type. The search neuronal-specific regulatory mechanism is at the infant stage. The enhancing elements identified from those published literatures could modulate a group of neuronal genes as more neuronal-specific regulatory mechanism revealed.
author2 Huey-Jen Tsay
author_facet Huey-Jen Tsay
Sih-Huei Wu
吳思慧
author Sih-Huei Wu
吳思慧
spellingShingle Sih-Huei Wu
吳思慧
In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
author_sort Sih-Huei Wu
title In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
title_short In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
title_full In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
title_fullStr In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
title_full_unstemmed In vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
title_sort in vivo identification of regulatory elements mediating the neuronal-specific expression of zebrafish scn8aa sodium channel gene
publishDate 2008
url http://ndltd.ncl.edu.tw/handle/24539769838022659568
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