Using Tol2 System to Develop the Fluorescent Zebrafish Containing Antimicrobial Peptide of Epinecidin-1
Date Issued
2009
Date
2009
Author(s)
Peng, Kuan-Chieh
Abstract
For the purpose of understanding the application of antimicrobial peptides in aquaculture, transgenic zebrafish expressing epinecidin-1, was developed and reported here. First, we cloned zebrafish mylz2 promoter for this purpose. To characterize the activity of mylz2 promoter, various fragments of the mylz2 promoters were analyzed using firefly luciferase transient expression assay, in which maximum promoter activity was found in the 2.5 kb fragment. Besides, the 2.5 kb fragment also expressed considerable red fluorescent proteins in the skeletal muscle of transgenic zebrafish. econd, in order to improve the translation efficiency of the Tol2 transposase, we constructed the UTRs of zebrafish ba1 globin flanked by the transposase . TEEA and in vivo fluorescent observation, showed high transposition efficiency during embryonic development. fter the optimization of promoter and transgenic efficiency, Epi-1/DsRed transgenic zebrafish was developed and expression of Epi-1/DsRed in the muscle and blood were demonstrated by immunohistochemistry staining techniques. Moreover, we also found that Epi-1/DsRed gene was efficiently and significantly expressed in vivo against Vibrio vulnificus 204. Gene expression study in Q-PCR revealed that Epi-1/DsRed itself could induce endogenous MyD88 expression in vivo. After Epi-1/DsRed transgenic zebrafish were infected with Vibrio vulnificus 204, MyD88 and TLR4a were upregulated, but IL-1β and TNF-α were downregulated at 12 hours post infection. It suggest that epinecidin-1 may negatively regulate toll-like receptor signaling pathway.
Subjects
mylz2
epinecidin-1
transgenic fish
Tol2
Vibrio vulnificus
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