藉由綠色螢光蛋白報導基因與Tol2跳躍基因系統研究斑馬魚Six1基因啟動子之顱顏組織專一性表現

Functional Analysis of Six1 Gene Enhancer Elements During Craniofacial Development by Transgenic Zebrafish with Green Fluorescent Protein (GFP) Reporter Gene and Tol2 transposon system

楊坤翰(Kun-Han Yang)

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(2014年)

https://doi.org/10.6342/NTU.2014.01014

摘要

Six1同源基因對於脊椎動物的器官形成扮演著重要的角色，當老鼠缺乏Six1同源基因表現時，顱顏發育相關所衍生器官的例如骨骼肌、腎臟、甲狀腺、感覺器官與神經節等皆會產生嚴重的缺失。而人類的SIX1突變則會產生Branchio-Oto-Rena症候群 Branchio-Oto-Renal syndrome(BOR)，造成聽力喪失或是咽弓缺失。因此可得知其對於調控器官發育及細胞的分化上扮演著重要的角色。
根據前人的研究可以得知，在Six1基因的上下游有許多高度保守序列，可以調控其基因表現。包含有五段高度保守的序列(conserved sequence)，分別命名為UCR1 (upstream conserved region 1)、UCR2、UCR3、UCR4以及DCR (downstream conserved region)，其中UCR1、UCR2、UCR3以及UCR4位於six1上游，而且其中UCR1及UCR2位於斑馬魚six1近端2.6 kb的範圍內。然而，由於之前學長的研究(詳見呂智楷論文)所結合的是Six1近端130bp與280bp的啟動子片段，可能仍有其本身的促進子存在，並無法區分是否為這些高度保守片段的作用。
本實驗主要想了解這些高度保守序列是否會單獨影響Six1基因的表現，因此將斑馬魚six1基因近端111bp之啟動子片段與Tol2轉位子載體的質體結合，以顯微注射的方式送到斑馬魚胚胎中，確認此片段中不含任何促進子(enhancer)活性，GFP螢光蛋白不在任何組織表現。之後，再將UCR1、UCR2、UCR3、UCR4以及DCR2等各序列與此質體結合，以顯微注射的方式送到斑馬魚胚胎中，在此可觀察到含有各片段之結構體在體節(somite)、耳囊(otic vesicle)、神經丘(neuromast)以及咽弓區域(branchial arch)等不同的組織器官中有綠色螢光蛋白的表現，顯示這些片段所含之促進子(enhancer)，會影響Six1的基因表現。
綜合上述，本實驗於斑馬魚six1基因的上下游相關序列，發現這五個保守片段可能分別包含某些會影響Six1基因表現的促進子。

關鍵字

Six1 ； Tol2元素 ； 斑馬魚 ； 保守序列 ； 神經丘 ； 體節 ； 耳囊

並列摘要

he Six1 homeobox gene plays a critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes. Mutations in human SIX1 cause Branchio-Oto-Renal syndrome(BOR), characterized by hearing loss and branchial defects. Six1 genes are hypothesized to play important roles in organgenesis and cell differentiation.
Depending on the previous study, there are a lot of conserved sequences around the six1 gene downstream and upstream,including 5 conserved sequences, namly UCR1 (upstream conserved region 1), UCR2, UCR3, UCR4 and DCR (downstream conserved region), The previous study used six1 proximal 130bp and 280bp promoter fragments, which may contain enhancer elements, to delineate the UCRs and DCR activity. These fragments were inserted upstream of the zebrafish six1 proximal promoter(111bp) in Tol-2 transposon elements. The UCR1 and UCR2 are located within the six1 2.6 kb proximal region.
I analyzed the highly conserved sequences UCRs and DCR in conjugation with six1 111bp proximal fragment by transgenic zebrafish assay. The resulting reporter-EGFP expression showed spatial restrictions similar to those of six1.1 mRNA detected by in situ hybridization (i.e., mainly in somites, otic vesicle and neuromasts).These
results revealed enhancers which can influence six1 gene expression.
Taken together, these results suggested that these conserved sequences contain enhancers which can influence six1 gene expression.

並列關鍵字

Six1 ； zebrafish ； conserved sequence ； neuromast ； somite ； otic vesicle

參考文獻

呂智凱, 藉由綠色螢光蛋白報導基因研究斑馬魚Six1.1基因啟動子之顱顏組織專一性表現. 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, 2008.

Ando, Z., et al., 2005. Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4. FEBS J. 272, 3026-41.

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Boucher, C. A., et al., 1996. Cloning of the human SIX1 gene and its assignment to chromosome 14. Genomics. 33, 140-2.

Boucher, C. A., et al., 1995. A novel homeodomain-encoding gene is associated with a large CpG island interrupted by the myotonic dystrophy unstable (CTG)n repeat. Hum Mol Genet. 4, 1919-25.