以基因轉殖方法藉由報導基因 EGFP 辨識人類 DMP1 基因在牙齒專一表現之加強子

Identification of Tooth-Specific Enhancer Element of Human Dentin Matrix Protein 1 (DMP1) Gene by Transgenic Assays

陳文裕(Wen-Yu Chen)

指導教授 ： 張百恩

國立臺灣大學/牙醫專業學院/口腔生物科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00809

摘要

骨骼及牙齒為脊椎動物中兩個主要的礦化組織，而骨生成及齒質形成過程中，骨細胞與牙本質母細胞會分泌非膠原性蛋白，並在細胞外形成基質(ECM)，其中一類非膠原性的蛋白稱為SIBLING家族(SIBLING family；Small Integrin-Binding Ligand, N-linked Glycoprotein)。而DMP-1是這家族蛋白的一個成員。這些SIBLING家族的蛋白質具有許多共同的特性，例如，含有大量的唾液酸(sialic acid)、磷酸鹽成分，以及精氨酸-甘氨酸-天門冬氨酸(Arginine-Glycine-Aspartic acid；RGD sequence)細胞結合序列，此類酸性蛋白質在骨頭及牙齒組織的形成及礦化時會被分泌到細胞外基質(ECM)與羥基磷灰石(hydroxyapatite)結合，幫助羥基磷灰石晶體形成與組織礦化。
先前以綠色螢光蛋白EGFP為報導基因，在斑馬魚及小鼠的動物模式中分析人類DMP-1啟動子的活性可發現人類DMP-1啟動子的上游2.783 kb片段對基因的調控在斑馬魚和小鼠間具有保留性(余俐亭，2004)，另外也發現DMP-1的表現並非侷限在礦化的組織，在非礦化的組織中也有DMP-1的表現。然而，並未發現綠色螢光有在牙齒表現，因此DMP-1啟動子的上游2.783 kb片段可能並未包括促使DMP-1基因在牙齒表現的序列(林麗芬，2007)。
有鑑於此，我以斑馬魚及小鼠為模式動物試圖找出人類DMP-1基因上下游序列中調控DMP-1在牙齒表現的加強子區域。首先我先在基因庫(UCSC, University of California Santa Cruz, Genome Browser website)中搜尋各物種的DMP-1基因以及其上下游的基因體DNA片段。透過不同物種間DNA序列之相似性比對，在上下游共發現有七段高度保守DNA片段。
接著我以PCR的方法從人類子宮頸癌細胞基因組DNA中分別複製出此七段保守DNA片段，作為候選加強子(enhancer)，並以βB1-Crystallin 1.3 kb (Cr1.3)基因在眼睛水晶體表現之加強子(enhancer)片段(-1290 bp ~ -62 bp)作為動物過渡性轉殖實驗中的篩選標記(selection marker)，然後將Cr1.3及此七段保守序列分別接合2.783 kb之DMP-1啟動子上游，並且以綠色螢光蛋白(EGFP, enhanced green fluorescent protein)為報導基因。再將這些結構體中含E2(enhancer 2)以顯微注射的方式注入斑馬魚及小鼠之受精卵中，結果在過渡性轉殖實驗中，發現這些結構體能驅使綠色螢光蛋白表現在斑馬魚胚胎的水晶體及咽齒區域；另一方面，在小鼠頭部的切片觀察結果也發現，綠色螢光蛋白在小鼠的水晶體及牙本質母細胞表現。由以上結果推測所選殖的DMP-1上游約18.8 kb的659 bp之E2片段可能包含了調控該基因表現在牙齒之順式調控因子。

關鍵字

牙本質基質蛋白 ； 牙本質母細胞 ； 加強子 ； 斑馬魚 ； 咽齒 ； 綠色螢光蛋白

並列摘要

Bones and teeth are two major mineralized tissues in vertebrate. In the process of osteogenesis and dentinogenesis, osteoblasts and odontoblasts secrete non-collagenous proteins to form extracellular matrix (ECM). One group of the non-collagenous proteins is SIBLING family (SIBLING family; Small-Integrin-Binding Ligand, N-linked Glyco- protein). DMP-1 (Dentin Matrix Protein 1; DMP-1) is a member of the family. The members of SIBLING family share many common characteristics. For example, they have sialic acids, phosphates, and RGD sequence. Such acidic phosphorylated proteins will secrete to ECM to incorporate with hydroxyapatite, helping nucleation of hydroxyapatite crystal growth and mineralization.
Preliminarily, upstream 2,783 kb fragment of human DMP-1 promoter is functional by the expression of the reporter gene (EGFP, enhanced green fluorescent protein) in transgenic zabrafish and mice (Yu, 2004). The results suggest that location of the DMP-1 is not restricted to mineralized tissues and may be present in non-mineralized tissue (Lin, 2007). However, the reporter gene EGFP is not expressed in odontoblasts of teeth.
According to this finding, I attempt to indentify tooth-specific enhancer elements of human Dentin Matrix Protein 1 (DMP1) gene. By analyzing sequences which are located in upstream and downstream of DMP-1 gene between different species from UCSC bio-informatics, I found out seven highly conserved DNA sequences.
I cloned the DNA fragments of these regions from HeLa cell genome, as candidate enhancers, using βB1-Crystallin 1.3 kb (Cr1.3) enhancer elements (-1290 bp ~ -62 bp), which confer tissue-specificity in lens, as a selection marker in transient transgenic experiments. Then Cr1.3 and the seven DNA fragments of the highly conserved regions were ligated with 2.783 kb human DMP-1 promoter with EGFP as a reporter gene. They were analyzed in zebrafish and mouse by transgenic assay. The constructed plasmid including E2 (Enhancer 2) was injected into both zebrafish and mouse fertilized eggs. The results demonstrated that the reporter is expressed in lens and regions of pharyngeal teeth in zebrafish embryo with E2 enhancer-construct. In addition, the reporter is expressed in lens and odontoblasts in mouse by the same construct. Taken together, the evolutionarily conserved DNA sequence E2 might provide a cis-element for rendering tooth-specificity to human DMP-1 gene.

並列關鍵字

DMP-1 ； odontoblast ； enhancer ； zebrafish ； pharyngeal teeth ； EGFP

參考文獻

余俐亭, 人類DMP-1基因啟動子之選殖及分析. 國立臺灣大學醫學院口腔生物科學研究所口腔細胞生物學組碩士論文, 2004.

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