Recombinant constructs and transgenic fluorescent ornamental fish therefrom

Inventors

Blake, Alan • Crockett, Richard • Essner, Jeffrey • Hackett, Perry • Nasevicius, Aidas

Assignees

Glofish LLC

Abstract

The present invention relates to the method and use of reef coral fluorescent proteins in making transgenic red, green and yellow fluorescent zebrafish. Preferably, such fluorescent zebrafish are fertile and used to establish a population of transgenic zebrafish and to provide to the ornamental fish industry for the purpose of marketing. Thus, new varieties of ornamental fish of different fluorescence colors from a novel source are developed.

Core Innovation

The disclosed invention relates to recombinant constructs using coral fluorescent proteins, including DsRed2, ZsGreen1, and ZsYellow1, to create transgenic zebrafish that exhibit fluorescence. The constructs are integrated into transgenic zebrafish germ-line DNA so that fluorescent expression is present in the germ line, and the fluorescent expression cassettes comprise the coral fluorescent protein coding sequences together with transcriptional and processing elements.

The fluorescent expression cassettes include either a zebrafish fast skeletal muscle myosin light-chain promoter (zMLC promoter) or a carp ubiquitous β-actin enhancer/promoter. The constructs further include transcriptional/processing elements such as introns/exons and tandem SV40 polyadenylation and 3′UTR elements, and the invention also describes combining ubiquitous promoter and tissue-specific (muscle) promoter elements to achieve strong visible fluorescence, including dual-vector strategies.

The invention also defines transgenic events for Red zebrafish 1, Green zebrafish 1, and Yellow zebrafish 1, supported by ECACC-deposited sperm accession numbers. The described events are associated with specific integrated constructs, including examples designated as pZMLC-DsRed2-SV40×2, pZMLC-ZsGreen1-SV40×2, pZMLC-ZsYellow1-SV40×2, pCBAC-DsRed2-SV40×2, and pCBAC-ZsGreen1-SV40×2, and the zebrafish line establishment supports stable fluorescence selection criteria and fluorescence inheritance during breeding.

Claims Coverage

Independent claim clm-00001 defines a transgenic fluorescent zebrafish with germ-line integration of a construct defined by SEQ ID NO: 1 through SEQ ID NO: 5, and further restricts the zebrafish to one or more specified transformation events tied to ECACC-deposited sperm accession numbers. The inventive features focus on germ-line integration of SEQ ID NO: 1 through SEQ ID NO: 5 constructs and inclusion of Red zebrafish 1, Green zebrafish 1, and/or Yellow zebrafish 1 transformation events identified by specific ECACC sperm accession numbers.

Across the independent claim, the core claim coverage requires a transgenic fluorescent zebrafish with germ-line integration of SEQ ID NO: 1 through SEQ ID NO: 5 constructs and specifies which fluorescent transformation event or events are present by referencing ECACC-deposited sperm accession numbers for Red zebrafish 1, Green zebrafish 1, and Yellow zebrafish 1.

Stated Advantages

Documented Applications