Tissue-specific Analysis of CCN1/CYR61 Gene Promoter of Zebrafish by Transgenic Assay with Green Fluorescent Protein (GFP) Reporter Gene
Date Issued
2007
Date
2007
Author(s)
Chen, Chun-Yu
DOI
zh-TW
Abstract
Cysteine-rich 61(Cyr61/CCN1), one of CCN family members, is an secretory matricellular protein with multiple biological functions, such as regulating cell adhesion, inducing cell migration, enhancing growth-factor-induced cell division and prolonging cell survival. It’s function is tightly associated with embryonic development, angiogenesis, chondrogenesis, tumor forming and wound healing. Moreover, CCN1/ Cyr61 is expressed in various tissues. Based on the above mentions, CCN1/Cyr61 may play an important role in normal development of an organism.
The aim of this study is to identify the cis-regulatory elements of CCN1/Cyr61 gene. I cloned and constructed its promoter DNA fragments in EGFP1 vector. Those constructs were microinjected into zebrafish zygote for their functional assay. The transient transgenic assay reveals that the longest promoter fragment (-2902/+29) could induce EGFP expression in embryo’s notochord、somite、heart、hindbrain、fin、mandible (pharyngeal arches) and epithelium; on the other hand, the proximal region
(-140/+29) could only induce EGFP expression in notochord、somite、heart、hindbrain and epithelium.
Besides, I obtain a transgenic stable line (F1 generation) with longest promoter fragment (-2902/+29). The expression of green fluorescent protein can be observed in the previous described tissues, which corresponds with the endogenous one of Cyr61. Therefore, I demonstrated that the upstream region (-2902/+29) of zebrafish CCN1/Cyr61 gene harbors most of its cis-regulatory elements.
Comparing the upstream genomic DNA sequences of CCN1/Cyr61 from eight different animal species led to the identification of a highly conserved region (-2299~-2123) in the upstream promoter region, in which several putative transcription factor binding sites are located by TRANSFAC software analysis. Furthermore, I divided it into 4 smaller fragments and constructed each with HSV-thymidine kinase basal promoter. The transient transgenic experiments show that each different conserved sub-region has differential EGFP expression patterns. Together, the cis-regulatory elements in the upstream 2.9kb promoter region of zebrafish CCN1/Cyr61 gene is functionally conserved through vertebrates’ evolutionary process.
Subjects
CCN1/Cyr61
啟動子
斑馬魚
順勢調控因子
演化
promoter
zebrafish
cis-regulatory element
evolution
Type
other
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