Functional Analysis of Zebrafish CTGF gene promoter by Transgenic Assay with Green Fluorescent Protein (GFP) Reporter Gene
Date Issued
2007
Date
2007
Author(s)
Chien, Chia-Hung
DOI
zh-TW
Abstract
Connective tissue growth factor (CTGF), a member of CCN family, is a cysteine-rich, secreted, extracellular matrix-associated protein that regulates diverse cellular functions in different cell types. It modulates many cellular functions, including cell proliferation, migration, adhesion, and extracellular matrix production. Evidence suggests that there is a distinctive function of CTGF in the skeletal development. For instance, during Meckel's cartilage development, CTGF acts as a down-stream molecule of TGF-β to stimulate cell-cell interactions and the expression of condensation-associated genes. Actually, TGF-β response element is located on the CTGF gene, and CTGF can exert many functions by the induction of TGF-β.
The aim of this study was to analyze the zebrafish CTGF promoter. Its cognate genomic DNA fragments were amplified by polymerase chain reaction (PCR). Upstream promoter (enhancer) fragments were constructed with EGFP (enhanced green fluorescent protein) reporter gene or with HSV-tymidine kinase (TK) basal promoter and analyzed in vivo by transient transgenic assays using zebrafish embryos. Results demonstrate that the constructs of pZF-CTGF(-2893/
+105)-EGFP1 [pCTGF- EGFP1] and pZF-CTGF(-1593/-1462)-HSV-TK-EGFP1 [pCTGF-E2-TK EGFP1]can drive the specific expression of GFP in zebrafish embryo(5dpf). The expression
sites include mandible, cranium, cornea, heart, somite, notochord, floor plate, fin bud, and epidermis. It is identical to in situ hybridization result. Furthermore,the
proximal promoter constructs of the pZF-CTGF(-195/+22)-EGFP1 [pCTGF-P200- EGFP1], pZF-CTGF(-64/+22)-EGFP1 [pCTGF-vTATA-EGFP1] and pZF-CTGF(-195/-44)- HSV-TK-EGFP1 [pCTGF-195xTATA-TK-EGFP1] can also drive expression of GFP in zebrafish embryo(5dpf). The expression sites include notochord and somite. However, the expression area is fewer.
The zebrafish transgenic stable lines were not obtained from pZF-CTGF(-2893/+105)-EGFP1 [pCTGF-EGFP1] construct yet. The results suggest that the tissue-specific regulatory elements of the zebrafish CTGF reside within the upstream conserved region (-1593/-1462) and proximal promoter region (-195/ +22), and the regulatory mechanism of CTGF may be conserved among the vertebrate species.
Subjects
結締組織生長因子
CCN家族
梅克氏軟骨
啟動子
綠色螢光蛋白
CTGF
CCN family
promoter
GFP
Type
other
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