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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 閔明源(Ming-Yuan Min) | |
dc.contributor.author | Ching-Tsuey Chen | en |
dc.contributor.author | 陳景萃 | zh_TW |
dc.date.accessioned | 2021-06-17T08:11:09Z | - |
dc.date.available | 2019-08-18 | |
dc.date.copyright | 2019-08-18 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-15 | |
dc.identifier.citation | Alfaro-Ruiz, R., Aguado, C., Martin-Belmonte, A., Moreno-Martinez, A. E., & Lujan, R. (2019). Expression, Cellular and Subcellular Localisation of Kv4.2 and Kv4.3 Channels in the Rodent Hippocampus. Int J Mol Sci, 20(2). doi:10.3390/ijms20020246
Amarillo, Y., De Santiago-Castillo, J. A., Dougherty, K., Maffie, J., Kwon, E., Covarrubias, M., & Rudy, B. (2008). Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons. J Physiol, 586(8), 2093-2106. doi:10.1113/jphysiol.2007.150540 Bae, S., Kweon, J., Kim, H. S., & Kim, J. S. (2014). Microhomology-based choice of Cas9 nuclease target sites. Nat Methods, 11(7), 705-706. doi:10.1038/nmeth.3015 Barrangou, R. (2015). The roles of CRISPR-Cas systems in adaptive immunity and beyond. Curr Opin Immunol, 32, 36-41. doi:10.1016/j.coi.2014.12.008 Bibikova, M., Golic, M., Golic, K. G., & Carroll, D. (2002). Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics, 161(3), 1169-1175. Chen, X., Yuan, L. L., Zhao, C., Birnbaum, S. G., Frick, A., Jung, W. E., . . . Johnston, D. (2006). Deletion of Kv4.2 gene eliminates dendritic A-type K+ current and enhances induction of long-term potentiation in hippocampal CA1 pyramidal neurons. J Neurosci, 26(47), 12143-12151. doi:10.1523/JNEUROSCI.2667-06.2006 Cho, S. W., Kim, S., Kim, J. M., & Kim, J. S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 31(3), 230-232. doi:10.1038/nbt.2507 Clark, B. D., Kwon, E., Maffie, J., Jeong, H. Y., Nadal, M., Strop, P., & Rudy, B. (2008). DPP6 Localization in Brain Supports Function as a Kv4 Channel Associated Protein. Front Mol Neurosci, 1, 8. doi:10.3389/neuro.02.008.2008 Coetzee, W. A., Amarillo, Y., Chiu, J., Chow, A., Lau, D., McCormack, T., . . . Rudy, B. (1999). Molecular diversity of K+ channels. Ann N Y Acad Sci, 868, 233-285. doi:10.1111/j.1749-6632.1999.tb11293.x Eric R. Kandel, J. H. S., Thomas M. Jessell, Steven A. Siegelbaum, A. J. Hudspeth, & Mack, S. (2012). Principles of Neural Science (5 ed.). Gonzalez, C., Baez-Nieto, D., Valencia, I., Oyarzun, I., Rojas, P., Naranjo, D., & Latorre, R. (2012). K(+) channels: function-structural overview. Compr Physiol, 2(3), 2087-2149. doi:10.1002/cphy.c110047 Hebb, D. O. (1949). The Organization of Behavior. Heidenreich, M., & Zhang, F. (2016). Applications of CRISPR-Cas systems in neuroscience. Nat Rev Neurosci, 17(1), 36-44. doi:10.1038/nrn.2015.2 Hoffman, D. A., Magee, J. C., Colbert, C. M., & Johnston, D. (1997). K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons. Nature, 387(6636), 869-875. doi:10.1038/43119 Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278. doi:10.1016/j.cell.2014.05.010 Hu, H. J., Alter, B. J., Carrasquillo, Y., Qiu, C. S., & Gereau, R. W. t. (2007). Metabotropic glutamate receptor 5 modulates nociceptive plasticity via extracellular signal-regulated kinase-Kv4.2 signaling in spinal cord dorsal horn neurons. J Neurosci, 27(48), 13181-13191. doi:10.1523/JNEUROSCI.0269-07.2007 Hu, H. J., Carrasquillo, Y., Karim, F., Jung, W. E., Nerbonne, J. M., Schwarz, T. L., & Gereau, R. W. t. (2006). The kv4.2 potassium channel subunit is required for pain plasticity. Neuron, 50(1), 89-100. doi:10.1016/j.neuron.2006.03.010 Huang, H.-Y., Cheng, J.-K., Shih, Y.-H., Chen, P.-H., Wang, C.-L., & Tsaur, M.-L. (2005). Expression of A-type K+ channel α subunits Kv4.2 and Kv4.3 in rat spinal lamina II excitatory interneurons and colocalization with pain-modulating molecules. European Journal of Neuroscience, 22(5), 1149-1157. doi:10.1111/j.1460-9568.2005.04283.x Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821. doi:10.1126/science.1225829 Kerti, K., Lorincz, A., & Nusser, Z. (2012). Unique somato-dendritic distribution pattern of Kv4.2 channels on hippocampal CA1 pyramidal cells. Eur J Neurosci, 35(1), 66-75. doi:10.1111/j.1460-9568.2011.07907.x Kiselycznyk, C., Hoffman, D. A., & Holmes, A. (2012). Effects of genetic deletion of the Kv4.2 voltage-gated potassium channel on murine anxiety-, fear- and stress-related behaviors. Biol Mood Anxiety Disord, 2, 5. doi:10.1186/2045-5380-2-5 Lockridge, A., Su, J., & Yuan, L. L. (2010). Abnormal 5-HT modulation of stress behaviors in the Kv4.2 knockout mouse. Neuroscience, 170(4), 1086-1097. doi:10.1016/j.neuroscience.2010.08.047 Lockridge, A., & Yuan, L. L. (2011). Spatial learning deficits in mice lacking A-type K(+) channel subunits. Hippocampus, 21(11), 1152-1156. doi:10.1002/hipo.20877 Lugo, J. N., Brewster, A. L., Spencer, C. M., & Anderson, A. E. (2012). Kv4.2 knockout mice have hippocampal-dependent learning and memory deficits. Learn Mem, 19(5), 182-189. doi:10.1101/lm.023614.111 Mark F. Bear, B. W. C., Michael A. Paradiso. (2006). Neuroscience : exploring the brain (3rd ed.). Oh, M. M., Simkin, D., & Disterhoft, J. F. (2016). Intrinsic Hippocampal Excitability Changes of Opposite Signs and Different Origins in CA1 and CA3 Pyramidal Neurons Underlie Aging-Related Cognitive Deficits. Front Syst Neurosci, 10, 52. doi:10.3389/fnsys.2016.00052 Platt, R. J., Chen, S., Zhou, Y., Yim, M. J., Swiech, L., Kempton, H. R., . . . Zhang, F. (2014). CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 159(2), 440-455. doi:10.1016/j.cell.2014.09.014 Rhodes, K. J., Carroll, K. I., Sung, M. A., Doliveira, L. C., Monaghan, M. M., Burke, S. L., . . . Trimmer, J. S. (2004). KChIPs and Kv4 alpha subunits as integral components of A-type potassium channels in mammalian brain. J Neurosci, 24(36), 7903-7915. doi:10.1523/JNEUROSCI.0776-04.2004 Sheng, M., Tsaur, M. L., Jan, Y. N., & Jan, L. Y. (1992). Subcellular segregation of two A-type K+ channel proteins in rat central neurons. Neuron, 9(2), 271-284. Simkin, D., Hattori, S., Ybarra, N., Musial, T. F., Buss, E. W., Richter, H., . . . Disterhoft, J. F. (2015). Aging-Related Hyperexcitability in CA3 Pyramidal Neurons Is Mediated by Enhanced A-Type K+ Channel Function and Expression. J Neurosci, 35(38), 13206-13218. doi:10.1523/JNEUROSCI.0193-15.2015 Spruston, N., Schiller, Y., Stuart, G., & Sakmann, B. (1995). Activity-dependent action potential invasion and calcium influx into hippocampal CA1 dendrites. Science, 268(5208), 297-300. doi:10.1126/science.7716524 Strassle, B. W., Menegola, M., Rhodes, K. J., & Trimmer, J. S. (2005). Light and electron microscopic analysis of KChIP and Kv4 localization in rat cerebellar granule cells. J Comp Neurol, 484(2), 144-155. doi:10.1002/cne.20443 Stuart, G. J., & Hausser, M. (2001). Dendritic coincidence detection of EPSPs and action potentials. Nat Neurosci, 4(1), 63-71. doi:10.1038/82910 Swiech, L., Heidenreich, M., Banerjee, A., Habib, N., Li, Y., Trombetta, J., . . . Zhang, F. (2015). In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol, 33(1), 102-106. doi:10.1038/nbt.3055 Watakabe, A., Ohtsuka, M., Kinoshita, M., Takaji, M., Isa, K., Mizukami, H., . . . Yamamori, T. (2015). Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex. Neurosci Res, 93, 144-157. doi:10.1016/j.neures.2014.09.002 Zhang, F., Wen, Y., & Guo, X. (2014). CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet, 23(R1), R40-46. doi:10.1093/hmg/ddu125 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73824 | - |
dc.description.abstract | A型鉀離子通道是一種電位閘型離子通道(Voltage-gated ion channel),包含了電位閘型鉀離子通道KCNA4以及KCND家族。在過去的研究中指出,這些A型鉀離子通道,尤其是第四之二亞型(KCND2),影響海馬迴CA1中的錐形細胞調控神經興奮性以及動作電位的向後傳導,而這些特性會促使海馬迴形成特別形式的神經可塑性。它們同樣在許多腦區中的記憶的穩固歷程、焦慮以及壓力行為上表現重要的腳色。常間回文重複序列叢集關聯蛋白(clustered, regularly interspaced, short palindromic repeats ,CRISPR/Cas9),可以造成基因的插入/刪除突變(indel mutations)而使得特定基因剔除。這裡,我們使用第九血清型的腺病毒運送含有嚮導RNA的質體感染cre重組酶轉殖的鏈球菌Cas酵素之活體老鼠中的海馬迴神經元以剔除特定的KCND2基因,並且以腺病毒質體中帶有的紅螢光蛋白(mcherry)以及cre重組酶轉殖老鼠中帶有的綠螢光蛋白(GFP)來標定感染之細胞。再經過免疫染色確認剔除KCND2通道蛋白後,我們能夠利用這個技術進一步探討KCND2通道蛋白缺乏後的細胞電生理變化。 | zh_TW |
dc.description.abstract | The A-type potassium channels, which involves the KCNA4 and the KCND family, are belongs to the voltage-gated ion channels. In previous studies, those A-type potassium channels, especially the KCND2, are important regulators of neuronal excitability and the action potential backpropagation which induce specific forms of synaptic plasticity in the CA1 pyramidal neurons of hippocampus. They also play essential roles in memory consolidation, anxiety and stress-related behaviors in many brain areas. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system can be used to knockout specific gene by inducing the insertion/deletion (indel) mutations. Herein, we delivered the single guide-RNAs (sgRNAs) using adeno-associated virus 9 (AAV9) vector to target the specific KCND2 (KV4.2) gene in the cre-dependent spCas9 mouse hippocampal neurons in vivo. I can identify the cells which were expressing sgRNAs and Cas9 proteins with reporter genes such as mcherry in AAV vectors as well as GFP in Cas9 heterozygous mice. After evaluating the knockout of KCND2 proteins by immunohistochemistry, we are going to use this techniques to investigate the protein depletion and electrophysiological changes of lacking of the KCND2 channels. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:11:09Z (GMT). No. of bitstreams: 1 ntu-108-R05b21007-1.pdf: 2927241 bytes, checksum: e437570c05089d9e5bfc43805565e2d5 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii Abstract iv Content v Chapter 1. Introduction 1 1.1 The A-type potassium channels 1 1.1.1 Overview 1 1.1.2 Distribution of KCND2 channels 2 1.1.3 Influence of deletion KCND2 in the brain 3 1.1.4 KCND2 in mouse hippocampus 4 1.1.4.1 The distribution of KCND2 among the hippocampus 4 1.1.4.2 The mechanism of memory consolidation in hippocampus 5 1.1.4.3 KCND2 regulates the signal propagation and synaptic plasticity in dendrites of pyramidal cells 6 1.1.4.4 Deletion of KV4.2 in mouse hippocampus 6 1.2 Genomic editing by Crispr/Cas9 Technique 8 1.2.1 The site-specific genomic editing tools 8 1.2.2 The Crispr/Cas9 system 9 1.2.3 In vivo genomic editing in mammalian brain 10 1.2.4 Utilizing Crispr/Cas9 technique in mouse hippocampus 12 1.3 Aim of this study 13 Chapter 2. Materials and Methods 14 2.1 Animals 14 2.2 Vectors design 14 2.3 The annealing and cloning of sgDNA 15 2.4 Transformation 16 2.5 Cell culture and transfection 17 2.6 Adeno-associated virus (AAV) Preparation 18 2.7 Stereotaxic surgery for AAV injection 18 2.8 Perfusion and section 19 2.9 Immunohistochemistry 19 2.10 Confocal imaging 21 Chapter 3. Results 22 3.1 AAV2-SaCas9-SaKCND2-2 22 3.1.1 Designing sgRNA / SaCas9 expressing vectors 22 3.1.2 Ex vivo validation for the efficiency of different Sa-gRNA sequences 23 3.3.3 The distribution of KV4.2 channels in mouse 24 3.3.4 The expression of sgRNA and SaCas9 in WT mice hippocampus 25 3.2 AAV9-SpKCND1_2-mcherry 26 3.2.1 Designing sgRNA-expressing vectors 26 3.2.2 Ex vivo validation for the efficiency of different Sp-gRNA sequences 27 3.2.3. Genomic editing of KCND2 by AAV9-SpKCND1_2-mcherry in Cas9 heterozygous mice 28 3.2.4 The morphology of KCND2 in the genomic editing area 29 Chapter 4. Discussion 31 4.1 The knockout efficiency was better in AAV9-SpKCND1_2-mcherry 31 4.2 The utility of Cas9 knock-in mice 32 4.3 The signals in stratum pyramidale within knockout area 32 Chapter 5. Reference 34 Figure 1. Flowchart of designing adeno-associated virus. 40 Figure 2. Designing sgRNA/SaCas9 expressing vectors. 41 Figure 3. Ex vivo validation for the efficiency of different Sa-gRNA sequences. 43 Figure 4. The distribution of KV4.2 channels in mouse. 46 Figure 5. The expression of sgRNA and SaCas9 in WT mice hippocampus. 47 Figure 6. Designing sgRNA-expressing vectors. 48 Figure 7. Ex vivo validation for the efficiency of different Sp-gRNA sequences. 50 Figure 8. Genomic editing of KCND2 by AAV9-SpKCND1_2-mcherry in Cas9 heterozygous mice by immunofluorescence staining. 53 Figure 9. Genomic editing of KCND2 by AAV9-SpKCND1_2-mcherry in Cas9 heterozygous mice by DAB staining. A, 55 Figure 10. The morphology of KCND2 in the genomic editing area. 56 Figure 11. The map of adeno-associated virus (AAV) vectors. 60 Table 1. Resources table 62 | |
dc.language.iso | en | |
dc.title | 運用CRISPR/Cas9技術剔除海馬迴神經元的A型鉀離子通道 | zh_TW |
dc.title | Employing CRISPR/Cas9 Techniques to Knockout A-type Potassium Channel in Hippocampus neurons | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳示國(Shih-Kuo Chen),蔡素宜(Su-Yi Tsai),楊琇雯(Hsiu-Wen Yang),陳志成(Chih-Cheng Chen) | |
dc.subject.keyword | A型鉀離子通道,常間回文重複序列叢集關聯蛋白,海馬迴,嚮導RNA,腺病毒, | zh_TW |
dc.subject.keyword | A-type potassium channels,the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system,hippocampus,single guide-RNAs (sgRNAs),adeno-associated virus, | en |
dc.relation.page | 62 | |
dc.identifier.doi | 10.6342/NTU201903709 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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