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

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研究生：	張瑞予 Jhang, Ruei Yu
論文名稱：	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
指導教授：	陳令儀 Chen, Linyi
口試委員:	江安世 Chiang, Ann Shyn 張壯榮 Chang, Chuang Rung
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	53
中文關鍵詞：	長期記憶、果蠅、啟動子、血清素受體
外文關鍵詞：	5HT1A, NMDAR
相關次數：	點閱：1 下載：0
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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。

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.

Index
Abstract    2
中文摘要    4
誌謝    5
Index    7
Introduction    9
Material and methods    11
Genomic DNA extraction    11
Generation of promoter lines    12
Generation of pH-sensitive 5-HT1A constructs    13
Results and Discussion    15
N-methyl-D-aspartate receptor (NMDA receptor)    15
cAMP response element-binding protein A (CREB)    16
Dopamine receptors and Dopa-decarboxylase proteins    17
Oo18 RNA binding protein (Orb)    18
Slow border cells (Slbo)    19
Ephrin    19
Serotonin receptor type 1A, 7 and serotonin transporter    20
Figures    23
Figure 1. Genomic structure, map and expression pattern of NMDAR2 constructs    24
Figure 2. Gel data of NR2 3, 5.5, 9k promoter construct    24
Figure 3. Genomic structure, map and gel data of NMDAR1 construct    25
Figure 4. Genomic structure, map, gel data and expression pattern of CREBA    27
Figure 5. Genomic structure, map and gel data of CREB2 constructs    29
Figure 6. Genomic structure, map and gel data of DopR1 constructs    31
Figure 7. Genomic structure, map and gel data of DopR2 constructs    32
Figure 8. Genomic structure, map, gel data and expression pattern of Ddc construct    33
Figure 9. Genomic structure, map and gel data of Orb construct    34
Figure 10. Genomic structure, map and gel data of Orb2 construct    35
Figure 11. Genomic structure, map, gel data and expression pattern of Slbo construct    36
Figure 12. Genomic structure, map and gel data of Ephrin construct    37
Figure 13. Genomic structure, map and gel data of SERT construct    38
Figure 14. Genomic structure, map, gel data and expression pattern of 5-HT7 constructs    39
Figure 15. Genomic structure, map, gel data and expression pattern of 5-HT1A constructs    41
Figure 16. Gel data of 5HT1A-Kaede and 5HT1A-PSmOrange construct    43
Figure 17. Maps and gel data of the UAS-pH-sensitive 5-HT1A constructs    44
Table 1.    45
References    50

                                

1. Quinn WG, Harris WA, & Benzer S (1974) Conditioned behavior in Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America 71(3):708-712.
2. Tully T & Quinn WG (1985) Classical conditioning and retention in normal and mutant Drosophila melanogaster. Journal of comparative physiology. A, Sensory, neural, and behavioral physiology 157(2):263-277.
3. Tempel BL, Bonini N, Dawson DR, & Quinn WG (1983) Reward learning in normal and mutant Drosophila. Proceedings of the National Academy of Sciences of the United States of America 80(5):1482-1486.
4. Quinn WG & Dudai Y (1976) Memory phases in Drosophila. Nature 262(5569):576-577.
5. Isabel G, Pascual A, & Preat T (2004) Exclusive consolidated memory phases in Drosophila. Science 304(5673):1024-1027.
6. Tully T, Preat T, Boynton SC, & Del Vecchio M (1994) Genetic dissection of consolidated memory in Drosophila. Cell 79(1):35-47.
7. Yu D, Akalal DB, & Davis RL (2006) Drosophila alpha/beta mushroom body neurons form a branch-specific, long-term cellular memory trace after spaced olfactory conditioning. Neuron 52(5):845-855.
8. Yin JC, et al. (1994) Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79(1):49-58.
9. Yin JC, Del Vecchio M, Zhou H, & Tully T (1995) CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81(1):107-115.
10. Perazzona B, Isabel G, Preat T, & Davis RL (2004) The role of cAMP response element-binding protein in Drosophila long-term memory. The Journal of neuroscience : the official journal of the Society for Neuroscience 24(40):8823-8828.
11. Pascual A & Preat T (2001) Localization of long-term memory within the Drosophila mushroom body. Science 294(5544):1115-1117.
12. Akalal DB, Yu D, & Davis RL (2010) A late-phase, long-term memory trace forms in the gamma neurons of Drosophila mushroom bodies after olfactory classical conditioning. The Journal of neuroscience : the official journal of the Society for Neuroscience 30(49):16699-16708.
13. Chen CC, et al. (2012) Visualizing long-term memory formation in two neurons of the Drosophila brain. Science 335(6069):678-685.
14. Wu CL, et al. (2007) Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body. Nature neuroscience 10(12):1578-1586.
15. Miesenbock G, De Angelis DA, & Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394(6689):192-195.
16. Sankaranarayanan S, De Angelis D, Rothman JE, & Ryan TA (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophysical journal 79(4):2199-2208.
17. Blackmore CG, et al. (2001) Measurement of secretory vesicle pH reveals intravesicular alkalinization by vesicular monoamine transporter type 2 resulting in inhibition of prohormone cleavage. The Journal of physiology 531(Pt 3):605-617.
18. Tantama M, Hung YP, & Yellen G (2011) Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor. Journal of the American Chemical Society 133(26):10034-10037.
19. Koivusalo M, et al. (2010) Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. The Journal of cell biology 188(4):547-563.
20. Nowak L, Bregestovski P, Ascher P, Herbet A, & Prochiantz A (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307(5950):462-465.
21. McBain CJ & Mayer ML (1994) N-methyl-D-aspartic acid receptor structure and function. Physiological reviews 74(3):723-760.
22. Xia S, et al. (2005) NMDA receptors mediate olfactory learning and memory in Drosophila. Current biology : CB 15(7):603-615.
23. Brindle PK & Montminy MR (1992) The CREB family of transcription activators. Current opinion in genetics & development 2(2):199-204.
24. Dash PK, Karl KA, Colicos MA, Prywes R, & Kandel ER (1991) cAMP response element-binding protein is activated by Ca2+/calmodulin- as well as cAMP-dependent protein kinase. Proceedings of the National Academy of Sciences of the United States of America 88(11):5061-5065.
25. Hardingham GE, Chawla S, Johnson CM, & Bading H (1997) Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature 385(6613):260-265.
26. Gonzalez GA & Montminy MR (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59(4):675-680.
27. Kim YC, Lee HG, & Han KA (2007) D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila. The Journal of neuroscience : the official journal of the Society for Neuroscience 27(29):7640-7647.
28. Tempel BL, Livingstone MS, & Quinn WG (1984) Mutations in the dopa decarboxylase gene affect learning in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 81(11):3577-3581.
29. Qin H, et al. (2012) Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila. Current biology : CB 22(7):608-614.
30. Xu S, Hafer N, Agunwamba B, & Schedl P (2012) The CPEB protein Orb2 has multiple functions during spermatogenesis in Drosophila melanogaster. PLoS genetics 8(11):e1003079.
31. Keleman K, Kruttner S, Alenius M, & Dickson BJ (2007) Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nature neuroscience 10(12):1587-1593.
32. Pai TP, et al. (2013) Drosophila ORB protein in two mushroom body output neurons is necessary for long-term memory formation. Proceedings of the National Academy of Sciences of the United States of America 110(19):7898-7903.
33. Kruttner S, et al. (2012) Drosophila CPEB Orb2A mediates memory independent of Its RNA-binding domain. Neuron 76(2):383-395.
34. Montell DJ, Rorth P, & Spradling AC (1992) slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP. Cell 71(1):51-62.
35. Starz-Gaiano M, Melani M, Wang X, Meinhardt H, & Montell DJ (2008) Feedback inhibition of Jak/STAT signaling by apontic is required to limit an invasive cell population. Developmental cell 14(5):726-738.
36. Murphy AM, Lee T, Andrews CM, Shilo BZ, & Montell DJ (1995) The breathless FGF receptor homolog, a downstream target of Drosophila C/EBP in the developmental control of cell migration. Development 121(8):2255-2263.
37. Dubnau J, et al. (2003) The staufen/pumilio pathway is involved in Drosophila long-term memory. Current biology : CB 13(4):286-296.
38. Rorth P, Szabo K, & Texido G (2000) The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation. Molecular cell 6(1):23-30.
39. Boyle M, Nighorn A, & Thomas JB (2006) Drosophila Eph receptor guides specific axon branches of mushroom body neurons. Development 133(9):1845-1854.
40. Singh AP, et al. (2013) Sensory neuron-derived eph regulates glomerular arbors and modulatory function of a central serotonergic neuron. PLoS genetics 9(4):e1003452.
41. Savelieva KV, et al. (2008) Learning and memory impairment in Eph receptor A6 knockout mice. Neuroscience letters 438(2):205-209.
42. Becnel J, Johnson O, Luo J, Nassel DR, & Nichols CD (2011) The serotonin 5-HT7Dro receptor is expressed in the brain of Drosophila, and is essential for normal courtship and mating. PloS one 6(6):e20800.
43. Johnson O, Becnel J, & Nichols CD (2011) Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila melanogaster. Neuroscience 192:372-381.
44. Lee PT, et al. (2011) Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 108(33):13794-13799.

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