臺灣博碩士論文加值系統

bHLH-PAS蛋白在脊椎動物的胚胎發育過程中扮演著重要的角色，其中低氧誘發蛋白HIF (Hypoxia induce factors)主要的功能在於調節生物體內的供氧平衡(oxygen homeostasis),在細胞遇到缺氧的情況下，會與ARNT結合而形成異偶合體結構(heterodimer)，進而影響到下游基因表現以調控細胞缺氧時的反應。
近來人類有很多視網膜方面的疾病，被指出與HIFα蛋白有密切的關係。此外，視網膜發育的表現基因：vsx1(visual system homeobox 1)產生突變，也會造成視網膜兩極細胞無法分化造成彩色視覺受到影響。
先前的研究發現，在斑馬魚hif-1α/2α/3α mix morpholino基因弱化(knockdown)實驗中，會造成視網膜發育中的兩極細胞(bipolar cell)發育受到影響，其中主要標示基因:Vsx1表現量與表現範圍明顯受到抑制。因此本實驗中主要分析斑馬魚Vsx1基因上游啟動子序列，發現含有多個HIF-α/ARNT偶合體辨識並結合的位置:HREs (Hypoxia response elements)。本實驗中以聚合?連鎖反應夾取包含HREs的不同長度片段，並接入螢光報導載體: T2KXIGDin中，以顯微注射至ㄧ細胞胚胎內觀察螢光表現的強度以及專一性。另外注射Hif-1α、2α、3α morpholino個別弱化其標的基因以觀察內生性的vsx1基因表現，以及含有vsx1啟動子的螢光報導基因表現為何。結果發現，Hif-2α可能透過辨識vsx1上游的HRE序列來調控vsx1基因的表現。這項實驗首次找出負責調控兩極細胞vsx1基因的低氧誘發因子，進而確認低氧誘發訊息傳遞系統在視網膜細胞分化中所扮演的角色。

bHLH-PAS is a protein family that play important functions in vertebrate development, cellular homeostasis, and metabolic balance. It includes the aryl hydrocarbon receptor nuclear translocator (ARNT) and its associate factor, hypoxia-inducible factor alpha subunit (HIF-α). The unstable HIF-α subunit act as intracellular oxygen sensor and it is stabilized by oxygen deficiency. Under hypoxia stress treatment, HIF-αsubunit associates with ARNT to form a heterodimeric complex and binds specifically to a 5’-RCGTG-3’ hypoxia-responsive element (HRE) in the promoter or enhancer of various hypoxia inducible genes to stimulate transcription of target genes.
Previously, it was revealed that blocking hif-1α/2α/3α translation caused small eye, incomplete retina development and defect bipolar and rod cell differentiation. Retinal markers vsx1 were all repressed in hif-1α/2α/3α morphant. In the upstream region of vsx1 contains several hypoxia response elements (HRE). It is speculated that vsx1 is probably requlated by HIFs.
To examine the role of HIF on the transcriptional activity of vsx1 upstream sequence, I have fused the upstream sequence of vsx1 with the codon sequence of enhanced green fluorescence protein (EGFP).The expression efficiency of reporter gene was examined individually in each hif-α knockdown morphant embryo. It appears that HIF-2α konckdown supressed the reporter expression and suggests that HIF-2α has important roles in vsx1 transcription and retina differentiation

摘要 i
Abstract iii
目錄 iv
壹.前言 1
ㄧ.低氧誘發蛋白(Hypoxia- induce-factor) Hifα簡介 1
二.細胞內氧濃度與低氧誘發蛋白結構穩定性 2
三.斑馬魚眼睛結構和發育過程: 3
四.Vsx1簡介： 6
五.Tol2 載體 system ： 7
A.跳躍子 7
B.Tol2簡介 7
六.實驗動機與目的 9
貳.實驗方法: 10
一、斑馬魚vsx1基因5’端上游啟動子之選殖 10
A. Zebrafish Genomic DNA (total DNA) 萃取 10
B. 斑馬魚vsx1基因5’端上游啟動子搜尋 11
C. 斑馬魚vsx1基因5’ 端上游啟動子DNA片段之選殖 13
D. 聚合?連鎖反應 (polymerase chain reaction ， PCR) 13
E. DNA洋菜膠電泳 14
F. 聚合?連鎖反應產物之純化 14
G.重組DNA接合反應 (Ligation) 15
H. 質體轉型 (Trsnsformation) 15
I. DNA分子之萃取 (DNA Extraction) 16
J. 選殖質體DNA之正確性判斷 18
K. 啟動子與綠色螢光蛋白載體(Tol2-EGFP vector)之構築 19
L. 單點突變實驗(site direct mutation) 20
二、 斑馬魚胚胎顯微注射 (Microinjection) 22
A. 斑馬魚之飼養 22
B. 班馬魚受精卵收集 22
C. 顯微注射針(Micropipette)的準備 23
D. 洋菜膠平台(蛋盤)的製備 23
E. 專一性寡核酸 ( antisense morpholino，MO)設計 24
F. 配製實驗注射hif-1α/hif-2α/ hif-3α MO之溶液 24
G. 配製實驗注射綠色光蛋白載體之溶液 24
H. 顯微注射 24
I. 螢光顯微鏡觀察及拍照 25
J. RNA全覆式原位雜交 (RNA whole-mount in situ hybridization) 25
?. 實驗結果 32
一. 弱化Hif-α基因對胚胎內生性vsx1表現的影響 32
二. vsx1上游啟動子序列分析 33
三. Tg (Vsx1:EGFP)轉基因魚報導基因的表現 34
四.弱化Hif-α基因對vsx1-3.5K-EGFP螢光表現的影響 35
五.單點突變3.5K上pax6序列螢光表現 35
肆.討論 37
伍. 圖片 42
圖1. 弱化Hif-α基因對內生性vsx1基因的影響: 42
圖2. vsx1啟動子報導質體在胚胎內表現情形: 43
圖3. 弱化Hif-α基因 對pVsx1-3.5K-EGFP質體報導基因的影響: 44
圖4.-3.2K上Pax6序列突變對vsx1啟動子的影響 45
圖5.斑馬魚vsx1基因5’端上游啟動子5000base pair序列: 48
圖6. Vsx1上游-4986bp序列低氧反應原件(HRE)分析及PCR反應引子設計: 49
圖7.vsx1上游啟動子上Pax6轉錄因子結合位置以及PCR引子設計 50
陸.附圖 51
附圖1.BHLH-PAS蛋白結構示意 51
附圖2.HIF-α蛋白結構相似性比較 51
附圖3. T2KXIGDin載體結構圖及限制?切位 52
附圖4.人類及老鼠vsx1上游-5000bp序列低氧反應原件(HRE)分析 53
柒.參考文獻 54
捌.實驗材料 58

朱國榮。2007。bHLH/PAS蛋白質在斑馬魚胚胎的眼睛發育過程中所扮演的角色。碩士論文。國立台灣海洋大學生物科技研究所。

劉宗麟。 2008。斑馬魚低氧誘發蛋白HIFα對於斑馬魚眼睛發育中rho與vsx1基因上游啟動子之基因調控。碩士論文。國立台灣海洋大學生物科技研究所。

Arjamaa, O. and M. Nikinmaa (2006). "Oxygen-dependent diseases in the retina: role of hypoxia-inducible factors." Exp Eye Res 83(3): 473-483.

Covello, K. L., J. Kehler, et al. (2006). "HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth." Genes Dev 20(5): 557-570.

Ding, K., M. Scortegagna, et al. (2005). "Retinal disease in mice lacking hypoxia-inducible transcription factor-2alpha." Invest Ophthalmol Vis Sci 46(3): 1010-1016.

Dorval, K. M., B. P. Bobechko, et al. (2005). "Transcriptional activity of the paired-like homeodomain proteins CHX10 and VSX1." J Biol Chem 280(11): 10100-10108.

Fedoroff, N., S. Wessler, et al. (1983). "Isolation of the transposable maize controlling elements Ac and Ds." Cell 35(1): 235-242.

Furlow, P. W., M. J. Percy, et al. (2009). "Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline." J Biol Chem 284(14): 9050-9058.

Grimm, C., A. Wenzel, et al. (2002). "HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration." Nat Med 8(7): 718-724.

Grindley, J. C., D. R. Davidson, et al. (1995). "The role of Pax-6 in eye and nasal development." Development 121(5): 1433-42.

Hewitson, K. S., L. A. McNeill, et al. (2002). "Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family." J Biol Chem 277(29): 26351-26355.

Kawakami, K. and A. Shima (1999). "Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio." Gene 240(1): 239-244.

Kawakami, K., A. Shima, et al. (2000). "Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage." Proc Natl Acad Sci U S A 97(21): 11403-11408.

Kawakami, K., H. Takeda, et al. (2004). "A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish." Dev Cell 7(1): 133-144.

Kimura, Y., C. Satou, et al. (2008). "V2a and V2b neurons are generated by the final divisions of pair-producing progenitors in the zebrafish spinal cord." Development 135(18): 3001-3005.

Knauer, S. K., G. Carra, et al. (2005). "Nuclear export is evolutionarily conserved in CVC paired-like homeobox proteins and influences protein stability, transcriptional activation, and extracellular secretion." Mol Cell Biol 25(7): 2573-2582.

Knauer, S. K., G. Carra, et al. (2005). "Nuclear export is evolutionarily conserved in CVC paired-like homeobox proteins and influences protein stability, transcriptional activation, and extracellular secretion." Mol Cell Biol 25(7): 2573-82.

Li, Z., S. Bao, et al. (2009). "Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells." Cancer Cell 15(6): 501-513.

Lisy, K. and D. J. Peet (2008). "Turn me on: regulating HIF transcriptional activity." Cell Death Differ 15(4): 642-649.

Loosli, F., S. Winkler, et al. (2001). "Medaka eyeless is the key factor linking retinal determination and eye growth." Development 128(20): 4035-4044.

Maxwell, P. H., C. W. Pugh, et al. (2001). "The pVHL-hIF-1 system. A key mediator of oxygen homeostasis." Adv Exp Med Biol 502: 365-376.

Maxwell, P. H., M. S. Wiesener, et al. (1999). "The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis." Nature 399(6733): 271-275.

Mintz-Hittner, H. A., E. V. Semina, et al. (2004). "VSX1 (RINX) mutation with craniofacial anomalies, empty sella, corneal endothelial changes, and abnormal retinal and auditory bipolar cells." Ophthalmology 111(4): 828-836.

Nangaku, M., R. Inagi, et al. (2008). "Hypoxia and hypoxia-inducible factor in renal disease." Nephron Exp Nephrol 110(1): e1-7.

Ohtoshi, A., S. W. Wang, et al. (2004). "Regulation of retinal cone bipolar cell differentiation and photopic vision by the CVC homeobox gene Vsx1." Curr Biol 14(6): 530-536.

Ozaki, H., A. Y. Yu, et al. (1999). "Hypoxia inducible factor-1alpha is increased in ischemic retina: temporal and spatial correlation with VEGF expression." Invest Ophthalmol Vis Sci 40(1): 182-189.

Passini, M. A., A. L. Kurtzman, et al. (1998). "Cloning of zebrafish vsx1: expression of a paired-like homeobox gene during CNS development." Dev Genet 23(2): 128-141.

Patel, S. A. and M. C. Simon (2008). "Biology of hypoxia-inducible factor-2alpha in development and disease." Cell Death Differ 15(4): 628-634.

Percy, M. J. (2008). "Familial erythrocytosis arising from a gain-of-function mutation in the HIF2A gene of the oxygen sensing pathway." Ulster Med J 77(2): 86-88.

Percy, M. J., P. W. Furlow, et al. (2008). "A gain-of-function mutation in the HIF2A gene in familial erythrocytosis." N Engl J Med 358(2): 162-168.

Pohlman, R. F., N. V. Fedoroff, et al. (1984). "The nucleotide sequence of the maize controlling element Activator." Cell 37(2): 635-643.

Scortegagna, M., M. A. Morris, et al. (2003). "The HIF family member EPAS1/HIF-2alpha is required for normal hematopoiesis in mice." Blood 102(5): 1634-1640.

Stephen, L. X., D. Basel, et al. (2002). "Developmental absence of the premolar teeth: dental management." Int J Paediatr Dent 12(3): 219-22.

Tam, B. Y., K. Wei, et al. (2006). "VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis." Nat Med 12(7): 793-800.

Urasaki, A., G. Morvan, et al. (2006). "Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition." Genetics 174(2): 639-649.

Yoon, D., Y. D. Pastore, et al. (2006). "Hypoxia-inducible factor-1 deficiency results in dysregulated erythropoiesis signaling and iron homeostasis in mouse development." J Biol Chem 281(35): 25703-25711.