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標題: | 藉由報導基因EGFP探討SP7基因促進子在斑馬魚骨頭之專一性表現 Functional Analysis of Bone-Specific Sp7 Gene Enhancer Elements by Transgenic Zebrafish with Green Fluorescent Protein |
作者: | Shi-Yi Kuo 郭士毅 |
指導教授: | 張百恩 |
關鍵字: | SP7,OSX,成骨細胞,Tol2,斑馬魚, SP7,OSX,Osteoblast,Tol2,Zebrafish, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | SP7是屬於SP家族中的C2H2-type zinc finger transcription factor,被認定為骨細胞分化時的主要調控子,是由C2C12老鼠的骨骼肌幹細胞鑑別出的,又稱之為osterix (Osx),Osx的cDNA含有428個胺基酸,分子量約為44.7KD,OSX會與NFAT共同調控骨細胞的骨形成作用。
我將斑馬魚的SP7基因序列和各物種間的SP7序列在UCSC網頁互相比對(如日本青鱂魚、老鼠、人),發現在此段基因上游有三段保守序列(p1、p2、p3),接著將這三段物種間保守序列的SP7近端啟動子在ATG上游(p0)鍵結至pEGFP-1載體,此結構體含有βB1-Crystallin加強子(enhancer)及啟動子(promoter)片段,此基因能讓斑馬魚眼睛表現綠色螢光,便於早期觀察或挑選轉殖魚。成功做出三個結構體後,利用Tol2轉位子系統轉殖到斑馬魚。將結構體與Tol2 RNA適當比例混合再用顯微注射的方式打入一個細胞期的斑馬魚受精卵中,藉由綠螢光報導基因來觀察這三個加強子對斑馬魚骨骼發育的專一性表現。除了上述三個結構體外,先做了一個只接入近端啟動子(p0)的結構體做對照。 在F0親代,對照組P0中可以發現只有近端啟動子的斑馬魚在一週大的時候眼睛都有表現綠色螢光,極少數的魚在脊索的地方也有綠色螢光表現。注射P1結構體(加強子及近端啟動子)的斑馬魚在一週時(7dpf)也是只有眼睛有綠色螢光的表現,而在84天時發現鰓蓋骨、胸鰭、肋骨等地方都有綠色螢光的表現。在F0親代,p2和p3結構體在螢光表現形態上幾乎一樣。不同於p1,剛孵化3天(3dpf)的結構體p2和p3小魚就能看到胸鰭和頭部有螢光表現,6天時靠近尾巴脊索下緣開始出現螢光的表現,13天後可以看到脊椎骨、背鰭、臀鰭、腹鰭根部的地方也有明顯的螢光,一個月後(30dpf)螢光逐漸減弱。我共篩選出p1兩個Stable line, p2一個Stable line和p3 兩個Stable lines。在stable lines,進一步的觀察,發現不論是p0、p1、p2、p3,其子代與親代螢光表現無異,僅異位表現如脊索、肌肉小亮點GFP消失。同樣地,p2和p3結構體的Stable line在螢光表現形態上幾乎一樣,所以p2和p3的加強子片段可能是由於DNA duplication的結果。在上述stable line中,並未觀察到鰭條骨有螢光表現。 最後我做了p3 stable line (No.1) F1斑馬魚截尾與鱗片再生的實驗,觀察鱗片與尾鰭再生時GFP的螢光表現,實驗結果發現再生的尾鰭與鱗片都無明顯螢光表現。另外,因為p2及p3 stable line,其螢光表現與shh基因表現位置在尾柄腹面重疊,因此懷疑Shh蛋白(signaling molecule)可能誘導SP7基因之表現;所以為了探討Shh蛋白與SP7之間的關係,我還做了p3 stable line (No.1) F1斑馬魚魚苗浸泡Cyclopamine (Shh抑制物)的實驗,結果顯示,p3 F1斑馬魚,在浸泡Cyclopamine一天後,尾柄腹側螢光GFP表現明顯減弱;意謂著骨骼SP7基因會受到Shh蛋白訊息之調控。 SP7 is a C2H2-type zinc finger transcription factor of the SP gene family and a putative master regulator of bone cell differentiation. It is identified from mouse C2C12 skeletal muscle progenitor cells, a novel zinc finger-containing transcription factor, called osterix (Osx), that is specifically expressed in all developing bones. The Osx cDNA encodes a 428-amino acid polypeptide with a predicted molecular mass of 44.7 kD. From UCSC genome browser web site, by alignment of the SP7 gene sequence of the zebrafish and other various species (eg, medaka, mice, humans), three conserved sequences upstream of this gene (p1, p2, p3) are located. These three inter-species conserved sequences were cloned by PCR and ligated with the SP7 proximal promoter (p0) upstream of ATG into the pEGFP1 vector, which harbors the βB1-crystallin enhancer and its cognate promoter sequence that drive the reporter gene GFP in the zebrafish eye lens. The construct of proximal promoter p0 serves as the basal promoter. These constructs of chimeric enhancers-promoters (βB1-crystallin and SP7) can facilitate the early observation of GFP in the lens and the screening of transgenic lines. After I have successfully made these constructs, they were microinjected into one-cell stage zebrafish embryos. The expression patterns of EGFP were observed. In transient transgenic assay, in F0 fish injected with p0 promoter construct, at 7 dpf (day-post-fertilization), expression of GFP was well observed in the regions of lens. Not any expression was observed in bone tissues and only some ectopic expression was observed in the notochord in few fish. In group of p1 construct, at 7 dpf, GFP expression was observed in the lens, but not in any bone tissues. However, until 84 dpf, GFP can since then be observed in the operculum, the base of pectoral fins, and ribs. In contrast, the expression patterns of p2 and p3 constructs are almost identical, but disparate from that of p1 construct. For p2 and p3 constructs, at the hatching stage (3 dpf), GFP was observed in the head mesenchyme and pectoral fins. Then at 6dpf, GFP was observed in the ventral part of notochord near caudal fins. At 13 dpf, GFP was observed in the vertebrae, in the bases of dorsal fin, anal fin, and pelvic fins, and in the ribs. The expression in these tissues continues until 1 month, and then degrades gradually. I have screened the F0 fish and got stable lines of each construct, including p0 construct (none), p1 construct (2 lines), p2 construct (1 line), p3 construct (2 lines). The progeny of these stable lines reveals about the same expression patterns as the F0 fish, except that some ectopic expression in notochord and muscle disappeared and were not observed. And in stable lines, the expression patterns of p2 and p3 constructs are identical, insinuating the DNA duplication of these enhancer fragments have been duplicated during evolution. Eventually, I inspect the expression of GFP during fin and scale regeneration by fin-amputation and scale-removing in p3 stable line (No.1). The experimental results revealed that no significant fluorescence of GFP was detectable in either manipulation, suggesting some enhancer-elements are still elusive in my constructs. In addition, I found the expression region in the ventral notochord near caudal fin of p2 and p3 constructs is tantamount to that of shh gene encoding a signaling molecule in previous publication. To investigate the relationship between Shh and SP7, the embryos of p3-construct F1 (No.1) zebrafish were treated with cyclopamine, an alkaloid of Shh inhibitors. The results displayed that when p3 F1 zebrafish were treated with cyclopamine at 9 dpf, the fluorescence of GFP in the caudal ventral part of notochord significantly diminished. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60011 |
DOI: | 10.6342/NTU201700122 |
全文授權: | 有償授權 |
顯示於系所單位: | 口腔生物科學研究所 |
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