Please use this identifier to cite or link to this item:
DC FieldValueLanguage
dc.contributorChia-Che Changen_US
dc.contributorChia-Ning Shenen_US
dc.contributor.advisorHong-Lin Suen_US
dc.contributor.authorChang, Chia-Yuen_US
dc.identifier.citationAbremski, K. and S. Gottesman (1982). "Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination." J Biol Chem 257(16): 9658-62. Armstrong, L., O. Hughes, et al. (2006). "The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis." Hum Mol Genet 15(11): 1894-913. Aubert, J., M. P. Stavridis, et al. (2003). "Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice." Proc Natl Acad Sci U S A 100 Suppl 1: 11836-41. Barraud, P., L. Thompson, et al. (2005). "Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse." Eur J Neurosci 22(7): 1555-69. Bauer, C. E., J. F. Gardner, et al. (1985). "Extent of sequence homology required for bacteriophage lambda site-specific recombination." J Mol Biol 181(2): 187-97. Bushman, W., J. F. Thompson, et al. (1985). "Control of directionality in lambda site specific recombination." Science 230(4728): 906-11. Bushman, W., S. Yin, et al. (1984). "Determinants of directionality in lambda site-specific recombination." Cell 39(3 Pt 2): 699-706. Cai, L., Z. Ye, et al. (2007). "Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions." Cell Res 17(1): 62-72. Carpenter, M. K., E. S. Rosler, et al. (2004). "Properties of four human embryonic stem cell lines maintained in a feeder-free culture system." Dev Dyn 229(2): 243-58. Cartwright, P., C. McLean, et al. (2005). "LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism." Development 132(5): 885-96. Chung, S., B. S. Shin, et al. (2006). "Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation." J Neurochem 97(5): 1467-80. Ekonomou, A., I. Kazanis, et al. (2005). "Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1." PLoS Biol 3(6): e186. Elkabetz, Y., G. Panagiotakos, et al. (2008). "Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage." Genes Dev 22(2): 152-65. Fukuda, H., J. Takahashi, et al. (2006). "Fluorescence-activated cell sorting-based purification of embryonic stem cell-derived neural precursors averts tumor formation after transplantation." Stem Cells 24(3): 763-71. Gong, S., C. Zheng, et al. (2003). "A gene expression atlas of the central nervous system based on bacterial artificial chromosomes." Nature 425(6961): 917-25. Heng, B. C., H. Liu, et al. (2004). "Feeder cell density--a key parameter in human embryonic stem cell culture." In Vitro Cell Dev Biol Anim 40(8-9): 255-7. Hohenstein, K. A., A. D. Pyle, et al. (2008). "Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells." Stem Cells 26(6): 1436-43. Holliday, R. (1974). "Molecular aspects of genetic exchange and gene conversion." Genetics 78(1): 273-87. Humphrey, R. K., G. M. Beattie, et al. (2004). "Maintenance of pluripotency in human embryonic stem cells is STAT3 independent." Stem Cells 22(4): 522-30. Hsu, P. L. and A. Landy (1984). "Resolution of synthetic att-site Holliday structures by the integrase protein of bacteriophage lambda." Nature 311(5988): 721-6. Irion, S., H. Luche, et al. (2007). "Identification and targeting of the ROSA26 locus in human embryonic stem cells." Nat Biotechnol 25(12): 1477-82. James, D., A. J. Levine, et al. (2005). "TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells." Development 132(6): 1273-82. Kan, L., N. Israsena, et al. (2004). "Sox1 acts through multiple independent pathways to promote neurogenesis." Dev Biol 269(2): 580-94. Kikuchi, Y. and H. A. Nash (1979). "Nicking-closing activity associated with bacteriophage lambda int gene product." Proc Natl Acad Sci U S A 76(8): 3760-4. Landy, A. (1989). "Dynamic, structural, and regulatory aspects of lambda site-specific recombination." Annu Rev Biochem 58: 913-49. Lang, R. J., J. M. Haynes, et al. (2004). "Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation." Eur J Neurosci 20(12): 3209-21. Lee, E. C., D. Yu, et al. (2001). "A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA." Genomics 73(1): 56-65. Li, X. J., Z. W. Du, et al. (2005). "Specification of motoneurons from human embryonic stem cells." Nat Biotechnol 23(2): 215-21. Liu, P., N. A. Jenkins, et al. (2003). "A highly efficient recombineering-based method for generating conditional knockout mutations." Genome Res 13(3): 476-84. Ludwig, T. and A. T. J (2007). "Defined, feeder-independent medium for human embryonic stem cell culture." Curr Protoc Stem Cell Biol Chapter 1: Unit 1C 2. Malas, S., S. M. Duthie, et al. (1997). "Cloning and mapping of the human SOX1: a highly conserved gene expressed in the developing brain." Mamm Genome 8(11): 866-8. Malas, S., M. Postlethwaite, et al. (2003). "Sox1-deficient mice suffer from epilepsy associated with abnormal ventral forebrain development and olfactory cortex hyperexcitability." Neuroscience 119(2): 421-32. Malas, S., M. Sartor, et al. (1996). "Genetic and physical mapping of the murine Sox1 gene." Mamm Genome 7(8): 620-1. Marchenko, S. and L. Flanagan (2007). "Transfecting human neural stem cells with the Amaxa Nucleofector." J Vis Exp(6): 240. Moehle, E. A., J. M. Rock, et al. (2007). "Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases." Proc Natl Acad Sci U S A 104(9): 3055-60. Ogawa, K., R. Nishinakamura, et al. (2006). "Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells." Biochem Biophys Res Commun 343(1): 159-66. Parmar, M. and M. Li (2007). "Early specification of dopaminergic phenotype during ES cell differentiation." BMC Dev Biol 7: 86. Pevny, L. H., S. Sockanathan, et al. (1998). "A role for SOX1 in neural determination." Development 125(10): 1967-78. Placantonakis, D. G., M. J. Tomishima, et al. (2008). "Bac Transgenesis in Human Es Cells as a Novel Tool to Define the Human Neural Lineage." Stem Cells. Porteus, M. H. (2006). "Mammalian gene targeting with designed zinc finger nucleases." Mol Ther 13(2): 438-46. Sasaki, Y., T. Sone, et al. (2008). "Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter." J Biotechnol 136(3-4): 103-12. Sato, N., L. Meijer, et al. (2004). "Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor." Nat Med 10(1): 55-63. Siemen, H., M. Nix, et al. (2005). "Nucleofection of human embryonic stem cells." Stem Cells Dev 14(4): 378-83. Siemen, H., L. Nolden, et al. (2008). "Nucleofection of human embryonic stem cells." Methods Mol Biol 423: 131-8. Sjogren-Jansson, E., M. Zetterstrom, et al. (2005). "Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system." Dev Dyn 233(4): 1304-14. Sone, T., K. Yahata, et al. (2008). "Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning." J Biotechnol 136(3-4): 113-21. Sottile, V., M. Li, et al. (2006). "Stem cell marker expression in the Bergmann glia population of the adult mouse brain." Brain Res 1099(1): 8-17. Suter, D. M., D. Tirefort, et al. (2008). "A Sox1 to Pax6 switch drives neuroectoderm to radial glia progression during differentiation of mouse embryonic stem cells." Stem Cells. Tomishima, M. J., A. K. Hadjantonakis, et al. (2007). "Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library." Stem Cells 25(1): 39-45. Trouillas, M., C. Saucourt, et al. (2009). "Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives." BMC Genomics 10(1): 73. Urnov, F. D., J. C. Miller, et al. (2005). "Highly efficient endogenous human gene correction using designed zinc-finger nucleases." Nature 435(7042): 646-51. Watanabe, K., M. Ueno, et al. (2007). "A ROCK inhibitor permits survival of dissociated human embryonic stem cells." Nat Biotechnol 25(6): 681-6. Wood, H. B. and V. Episkopou (1999). "Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages." Mech Dev 86(1-2): 197-201. Xu, R. H., R. M. Peck, et al. (2005). "Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells." Nat Methods 2(3): 185-90. Xu, X., M. Zhan, et al. (2007). "Gene expression atlas of the mouse central nervous system: impact and interactions of age, energy intake and gender." Genome Biol 8(11): R234. Yahata, K., H. Kishine, et al. (2005). "Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: conditional gene expression at near physiological levels." J Biotechnol 118(2): 123-34. Yin, S., W. Bushman, et al. (1985). "Interaction of the lambda site-specific recombination protein Xis with attachment site DNA." Proc Natl Acad Sci U S A 82(4): 1040-4. Ying, Q. L., J. Nichols, et al. (2003). "BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3." Cell 115(3): 281-92. Zhang, S. C., M. Wernig, et al. (2001). "In vitro differentiation of transplantable neural precursors from human embryonic stem cells." Nat Biotechnol 19(12): 1129-33.en_US
dc.description.abstract在成人的神經系統損傷常會造成嚴重的後果,且因成人體中只有非常少量的神經幹細胞 (neural stem cell) 存在,使創傷後的治療相當困難。近年來胚幹細胞 (embryonic stem cell, ES cell) 的應用正蓬勃的發展, 胚幹細胞擁有可以分化 (differentiation) 為全身各種細胞型態的分化潛力 (pluripotency) 以及在體外培養時自我增生 (self-renew) 的能力,其優秀的自我增生及分化能力被認為是可使用於成體組織修補的最佳素材。藉由最早期神經幹細胞特定表現的細胞標誌 (marker) Sex determining region Y-box 1 (Sox-1) 為鑑定標準分離純化胚幹細胞分化成的神經幹細胞並植入體內修復神經創傷在小鼠模式已經確立其成效。本實驗主要目的以建立sox-1基因敲入 (knock-in) 以及轉基因的人類胚幹細胞為目的,因已經有研究指出人類胚幹細胞體外神經分化時表現的細胞標誌和小鼠胚幹細胞並不相同,其先表現 paired box protein 6 (Pax-6) 而後Pax-6與 Sox-1 同時表現,但其所代表的意義及此時期的細胞接受誘導 (patterning) 分化成為下游神經的能力尚未完全清楚,希望可以以此實驗建立之knock-in或轉基因人類胚幹細胞株在神經幹細胞表現sox-1時期引發的綠色螢光為標準,對此細胞時期更為了解。本實驗各自以不同方法構築了進行knock-in以及轉基因之載體,欲執行knock-in的載體,使用以位點特異性重組(site specific recombination) 原理的three-fragment multiGateway ,此系統只需要一次的重組反應中就能將knock-in所需要的兩段同源性片段 (homologous arm) 以及欲取代人類胚幹細胞染色體上 sox-1 位置的綠螢光及抗生素抗性篩選基因依順序接在knock-in所需要的質體 (plasmid) 上。而在轉基因載體的建立,則使用了細菌人工染色體 (bacterial artificial chromosome, BAC) 轉基因系統,以大腸桿菌株 EL350 中所含的短片段重組蛋白將 BAC上 sox-1 部分以綠螢光蛋白及抗生素抗性基因取代,此使用 BAC 轉基因的方法主要優點是其所包含的 sox-1 上下游的長片段有相當大的機會包含主要調控 sox-1 表現的區域,較將可能調控 sox-1 表現的區域一一確認並逐步建構轉基因載體更為方便。而後將構築好的knock-in及轉基因載體轉染 (transfection) 進入人類胚幹細胞後建立 sox-1 knock-in及轉基因之人類胚幹細胞株。後續則可以以此細胞株進行 sox-1+ 時期的神經幹細胞的特性確定、其他專一性細胞標誌的尋找以及在體外及體內的分化能力和神經修復能力等測試。zh_TW
dc.description.abstractThe injury of nervous system leads to serious damage, and still it is difficult to repair the damaged nervous system because of the lack of neural stem cell in adult . The embryonic stem cells (ES cells) have the characteristic of pluripotence and self-renewal , become one of the potentially suitable materials to repair the hurt adult tissue. In the mouse model, Sex determining region Y-box 1 (Sox-1) is one of the earliest neuronal specific cell marker, and the repair efficiency of sox-1 positive neural stem cells differentiated from ES cells in vitro has already been confirmed. The purpose of my experiment is to establish the sox-1 knock-in and transgene human ES cell line. There is remarkable difference of cell specific markers between mouse and human neural stem cells . Previous research suggests that one of the earliest human rosette neural stem cells markers is paired box protein 6 (Pax-6) and at later stage sox-1 express , but the distinguishing features between this two stage are still not really clear now. The sox-1 knock-in and transgene human ES cell line can help us to examine the differentiation potential and specific quality of sox-1+ cell stage of human neural stem cell. In my experience I use two systems to construct the knock-in and transgene vector . The three-fragment multiGateway system can put two homologous arm and the reporter gene needed of knock-in vector together only with two steps site-specific recombination without considering restriction enzyme cutting sites . I use another system to construct sox-1 transgene vector, the bacterial artificial chromosome (BAC) . I choose a human BAC clone that contain sox-1 and it's upstream and downstream sequence that may contain the sox-1 promoter and enhancer region , then we can construct the transgene vector without finding the exactly promoter region. I use a special E.coli strain EL350 that have the ability of short homologous region recombination. We can use this short homologous region recombination system to replace sox-1 region of BAC with fluorescence protein reporter gene . Finally we can transfect these two vectors into human ES cells and establish the sox-1 knock-in and transgene human ES cell line then identify it's characters, differentiation potential and nervous system repair ability.en_US
dc.description.tableofcontents中文摘要 ii 英文摘要 iii 目錄 iv 圖表目錄 v 第一章 前言 1 第二章 實驗材料與方法 8 第三章 結果 22 第四章 討論 26 第五章 參考文獻 30 第六章 圖表 36 圖1 Sox-1 K-I之載體構造及 K-I之同源性重組策略圖示 36 圖2 K-I時欲取代人類胚幹細胞染色體 sox-1 之 insert 片段圖示 37 圖3 Venus片段與 NLS 片段剪接示意圖 38 圖4 PGK promoter 與β-geo 片段剪接示意圖 39 圖5 sox-1 K-I 完整 insert 片段構築剪接示意圖 40 圖6 欲執行 sox-1 K-I之insert 片段構築與確認結果 41 圖7 sox-1 K-I 之 insert片段綠色螢光蛋白之作用及螢光位置確認 42 圖8 載體pDEST-R4 R3接入 sox-1 K-I 所需之負篩選基因剪接示意圖 43 圖9 載體pDEST-R4 R3接入 sox-1 K-I 所需之負篩選基因構築與確認 44 圖10 Sox-1 K-I BP recombination 所需片段進行 PCR 擴增反應 45 圖11 進行 BP 重組反應後確認 46 圖12 BAC重組質體重要片段及短片段重組策略圖示 47 圖13 BAC 轉基因實驗欲和 BAC 進行短序列重組之載體構築簡易圖示 48 圖14 BAC 轉基因實驗欲和 BAC 進行短序列重組之載體構築使用限制酵素剪切策略圖示 49 圖15 BAC 重組方式及轉染人類胚幹細胞之簡易圖示 50 圖16 欲和 BAC 進行重組所需片段 TA clone 及確認 51 圖17 BAC 轉基因insert 片段綠色螢光蛋白之作用及螢光位置確認 52 圖18 將sox-1同源短序列以及Venus-NLS接入質體PL452構築與確認 53 圖19 BAC 與質體重組後以PCR方式確認是否重組成功 54 圖20 PCR 確認後之重組 BAC 以限制酵素SalI 剪切確認是否成功重組 55 圖21 繼代中之人類胚幹細胞 56 圖22 人類胚幹細胞進行single cell dissociation 後之細胞型態 57 圖23 人類胚幹細胞進行神經分化後之 rosette like neural stem cell 型態 58 表1 K-I 載體構築中使用之引子列表 59 表2 BAC 轉基因載體構築及確認使用之引子列表 60 附錄圖1 . Gateway system 之完整流程圖 61zh_TW
dc.subjecthuman embryonic stem cellen_US
dc.subjectrosette neural stem cellen_US
dc.subjectrosette neural stem cellzh_TW
dc.title以 MultiGateway 系統以及 BAC 構築載體並建立 sox-1 knock-in及轉基因之人類胚幹細胞株zh_TW
dc.titleUse MultiGateway system and Bacterial Artificial Chromosome to construct vector and establish sox-1 knock-in and transgenic human embryonic stem cell lineen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
Appears in Collections:生命科學系所
Show simple item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.