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 invention provides a recombinant expression cassette and transgenic fluorescent fish progeny in which different fluorescent proteins are expressed in a visually detectable manner. The described system uses a first transgene encoding a first fluorescent protein under the control of a muscle specific promoter, and a second transgene encoding a second fluorescent protein under the control of a ubiquitous fish promoter. Fluorescent colors corresponding to the first and second fluorescent proteins yield visibly expressed fluorescence that differs from the color encoded by each transgene.

The disclosed constructs include coral fluorescent proteins such as DsRed2, ZsGreen1, and ZsYellow1 driven by fish promoters including ubiquitous carp β-actin enhancer/promoter and muscle-specific zebrafish myosin light chain promoter. The regulatory cassette components described include tandem SV40 poly(A) signals and poly(A)/3′UTR, with optional β-actin intron/exon elements. The description further defines construct sequence relationships mapped to SEQ ID NO:1 through SEQ ID NO:5.

The invention further includes producing stable, fertile fluorescent lines, including Golden zebrafish, that exhibit strong visible fluorescence, with the body coverage quantified as over 75% to 100% of the body, optionally excluding fins and eyes. Transgenic integration events are described for Red zebrafish 1, Green zebrafish 1, and Yellow zebrafish 1, including breeding and selection to generate transgenic fluorescent fish progeny. The described materials are stated to support visible fluorescent expression for ornamental use and also mention research and biosensor uses beyond ornamentals.

The background problem described in the provided partial content is the need for transgenic fluorescent fish progeny that visibly express strong fluorescent colors suitable for the intended use, including ornamental purposes. The described solution addresses this by combining muscle specific promoter control for one fluorescent protein transgene with ubiquitous fish promoter control for another fluorescent protein transgene, using defined nucleic acid sequence sets and producing progeny with strong whole-body fluorescence coverage.

Claims Coverage

The partial content includes two independent claims. Across these claims, the inventive features are centered on selecting a fluorescent fish with two different fluorescent protein transgenes and breeding to obtain progeny whose genome places one transgene under a muscle specific promoter and the other under a ubiquitous fish promoter, or both under muscle specific promoters, achieving strong visible whole-body fluorescence with defined SEQ ID NO sets.

Both independent claims require breeding to obtain transgenic fluorescent fish progeny with different fluorescent proteins and strong visible fluorescence over 75% to 100% of the body, optionally excluding fins and eyes. The claims differ in promoter arrangement: one positions the first transgene under a muscle specific promoter and the second transgene under a ubiquitous fish promoter, while the other positions both transgenes under muscle specific promoters. Each claim further limits nucleic acid sequences of at least one transgene to specified SEQ ID NO groups.

Stated Advantages

Documented Applications