Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate d evelopmental and genetic analyses, iis use in transgenic studies still suff ers from the scarcity of homologous gene promoters, In the present study th ree different zebrafish cDNA clones were isolated and sequenced completely and their expression patterns were characterized by wholemount in situ hybr idization as well as by Northern blot hybridization. The first clone encode s a hype II cytokeratin (CK), which is specifically expressed in skin epith elia in early embryos and prominently expressed in the adult skin tissue. T he second clone is muscle specific and encodes a muscle creatine kinase (MC K). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fid elity of zebrafish embryos in transgenic expression, the promoters of the t hree genes were isolated using a rapid linker-mediated PCR approach and sub sequently ligated to a modified green fluorescent protein (gfp) reporter ge ne. When the three hybrid GFP constructs were introduced into zebrafish emb ryos by microinjection, the three promoters were activated faithfully in de veloping zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is c onsistent with the expression pattern of the endogenous cytokeratin gene. T he 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles an d not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tiss ues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, Further demonstrated that zebrafis h embryos can faithfully express exogenously introduced genes under the con trol of zebrafish promoters. Dev. Genet. 25:158-167, 1999. (C) 1999 Wiley-L iss, Inc.