Expression of Red Fluorescent Protein (RFP) in Transgenic Angelfish (Pterophyllum scalare Schultze, 1823)

Abstract

The freshwater angelfish (Pterophyllum scalare Schultze, 1823) is a common aquarium species. Similar to many other cichlids, angelfish provide parental care for their embryos and larvae. In contrast to zebrafish and medaka, it is difficult to raise angelfish eggs in the lab. Therefore, obtaining angelfish embryos for transgenic purposes will also be challenging. Our aim in this study was to employ microinjection techniques to develop transgenic angelfish expressing red fluorescent protein (RFP). Using the zebrafish myosin light chain 2 (mylz2) promoter, the expression patterns of the RFP founder transgenic angelfish were analyzed. An open loop of plasmid pDsred2-1 was cloned with the 1999-bp Mylz2 promoter fragment at the SacI and AgeI sites to generate the pMylz2-RFP transgenic construct. After angelfish laid their eggs on the substrate, the embryos were carefully collected with a plastic pipette in preparation for the microinjection procedure. A single-cell-stage angelfish embryo was microinjected with pMylz2-RFP. Sixteen of 524 pMylz2-RFP microinjected embryos survived to five days post fertilization (5 dpf), with twelve displaying red fluorescence. Only two RFP-positive larvae survived to adulthood. In adult angelfish, red LED illumination clearly displayed RFP expression in trunk muscles, indicating successful transmission of the transgene. The pMylz2-RFP transgene, however, was not passed on to offspring, indicating that it was not incorporated into the germline.