建構基因表達載體以觀察形成長期記憶神經元的基因調控

Developing promoter-driven constructs for visualizing neural activity

張瑞予(Jhang, Ruei Yu)

指導教授 ： 陳令儀

國立清華大學/生命科學院/分子醫學研究所/碩士(2015年)

摘要

有鑒於長期記憶的形成與短期記憶不同，需要新蛋白的合成參與，提供神經元調整或是強化突觸的能力，增加與其它神經元間的溝通。並且這樣的生理變化可能發生於一個相當小，如腦中的數顆神經元左右的範圍，我們需要更有效的工具，提供我們能在單顆神經元的層次上，觀察神經元中經刺激後特定與記憶形成相關的重要蛋白個別受調控的情形。於是我們致力在構築帶有各種重要記憶蛋白啟動子的果蠅表現載體，使用從果蠅中放大的各種記憶蛋白的候選啟動子片段，放入表現載體中，甚或加上可受光調控的變色螢光蛋白。利用觀察螢光的表達位置，還有進一步使用帶有啟動子載體去表達RNA干擾的果蠅株，對特定記憶蛋白的表現抑制，觀察果蠅的學習與記憶表現，來証實啟動子載體的正確性。一方面我們也針對完成的基因載體，製作多株或單株抗體來幫助辨認載體引導螢光表現位置的準確性，以及幫助接下來的相關研究。在目前已完成的基因之中，我們已觀察到血清素受器的表現會顯著影響少數次訓練後長期記憶的生成，為了更加研究其中的機制，我們將設計帶有對酸鹼敏感的螢光蛋白標記的血清素受器基因載體，幫助我們觀察受器藉由胞吞的回收與記憶生成的相關性。在已知許多蛋白對於長期記憶的形成有不可或缺的作用的基礎上，使用這樣的基因工具我們可以更進一步瞭解，在學習與記憶的過程中，神經元的刺激會造成這些記憶蛋白在表達的時間點以及位置上受到怎麼樣的調控，幫助解答記憶迴路在腦中的全貌。

關鍵字

長期記憶 ； 果蠅 ； 啟動子 ； 血清素受體

並列摘要

Formation of long term memory (LTM) formation requires delicate regulation of numerous molecules in order to change the plasticity of neurons. To elucidate the expression of memory genes and neural activity, we are dedicated to providing molecular tools for investigating the temporal and spatial expression of memory regulated genes during the formation of learning and memory at single cell resolution. We first generated a series of promoter driven lines for the purpose of reporting spatial expressions of specific memory genes in response to neuronal activity. After the expression pattern is confirmed either via in situ hybridization or antibody staining, Gal4 sequence will subsequently be replaced by photoconvertible fluorescent protein to visualize the transcriptional activity real time. Thus far, we have generated several constructs, including a 5.5 kb and a 3 kb regions of N-methyl-D-aspartate receptor 2 (NR2) promoter in random insertion vector, pPTGAL, and a modified version replacing GAL4 with fluorescent protein EOS. And constructs of NR1, NR2, cAMP response element-binding protein A (CREBA), cAMP response element-binding protein 2 (CREB2), dopamine receptor (DopR1), DopR2, 5-hydroxytryptamine receptor (5-HT1A), 5-HT7 serotonin transporter (SERT), dopa-decarboxylase (Ddc), slow border cells (Slbo), ephrin genes with different length of regulatory elements on specific insertion vector pBPGAL4 have also been generated and sequenced. These constructs were used to generate promoter driven fly lines. Thus far, we have obtained several promoter lines expressing pattern of GFP (ex. CREBA 8k, Slbo 5.4k) that needed further confirmation and one confirmed construct of 5.2 kb 5-HT1A on pBPGAL4 that expresses GFP fluorescence in  and  lobes of the mushroom body, AMMC and the optic lobe of fly brain. The expression pattern of 5-HT1A promoter line correlates well with published in situ hybridization result and its known anatomical function. To better monitor the activity of 5-HT1A in response to learning within short period of time, promoter constructs with photoconvertible fluorescent protein-PSmOrange and Kaede have also been generated. Two different pH-sensitive 5-HT1A constructs were made for evaluating the protein trafficking of 5-HT1A during memory formation. These two constructs have been made and are in process of generating transgenic fly lines.

並列關鍵字

Drosophila ； long term memory ； promoter ； CREB ； 5HT1A ； NMDAR

參考文獻

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