Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp Haiyan Wan, Jiangyan He, Bensheng Ju, Tie Yan, Toong Jin Lam, and Zhiyuan Gong* Department of Biological Sciences, National University of Singapore, Singapore 119260 Abstract: Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluo- rescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F 1 transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism. Key words: dsRed, EGFP, cytokeratin, myosin light chain, skin, muscle. I NTRODUCTION The green fluorescent protein (gfp) gene, originally isolated from the jellyfish Aequorea victoria, is widely used as a re- porter gene for investigation of tissue-specific gene expres- sion and cellular localization of proteins because the fluo- rescence of its protein product, GFP, can be conveniently detected in living cells (Prasher et al., 1992; Chalfie et al., 1994; Tsien, 1998). Since the introduction of the wild-type GFP, many mutant forms have been created and screened for improvement of fluorescence brightness or for altered spectra of fluorescence shifted toward red or blue (Tsien and Prasher, 1998). So far, several mutant forms of GFP are commercially available, and they display different spectra of fluorescence from that of wild-type GFP. The wild-type GFP has a maximal spectrum at 508 nm, while several mu- tant forms have maximal spectra at 440 nm (blue), 477 nm (cyan), and 527 nm (yellow); these mutant forms are called blue fluorescent protein (BFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP), respectively (Clontech Catalog 2000, pp. 209–222). The availability of these different fluorescent proteins makes it feasible to carry out multiple labeling of different organelles or structures within the same cells or different tissues or cells in the same organism. Recently, a new fluorescent protein gene, red fluorescent protein (rfp), was isolated from a sea anemone relative (Dis- Received March 15, 2001; accepted September 18, 2001. *Corresponding author: telephone 65-8742860; fax 65-7792486; e-mail dbsgzy@nus.edu.sg Mar. Biotechnol. 4, 146–154, 2002 DOI: 10.1007/s10126-001-0085-3 © 2002 Springer-Verlag New York Inc.