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 relates to a transgenic fluorescent fish that visibly expresses fluorescent proteins encoded by chromosomally integrated transgenes. The fish comprises a first transgene under the control of a ubiquitous promoter and a second transgene under the control of a muscle specific promoter, and both transgenes encode the same fluorescent protein.

The transgenes are chromosomally integrated at the same, single locus, and the fish exhibits strong visible fluorescence over 75% to 100% of the body of the fish, with the option of excluding fins and eyes. The constructs use reef coral fluorescent proteins, including DsRed2, ZsGreen1, and ZsYellow1, to generate strong, visible fluorescence in the body-wide expression pattern.

The described expression cassettes include promoters and RNA-processing/translational elements such as tandem SV40 polyadenylation signals, and they can include intron and exon structure. The document also describes ubiquitous or housekeeping promoters such as carp β-actin and muscle-specific promoters such as a zebrafish myosin light chain promoter (zMLC), to provide ubiquitous expression and muscle-specific expression in a single fish genome at a shared chromosomal locus.

The problem addressed is producing transgenic fish with stable, strong visible fluorescence and appropriate widespread expression, including in ornamental zebrafish such as Golden zebrafish (slc24a5 mutant). The document emphasizes generating fluorescent fish lines with stable visible fluorescence and ubiquitous-like distribution across the body, using reef coral fluorescent proteins in chromosomally integrated constructs.

Claims Coverage

The independent claim recites 5 main inventive requirements, comprising a transgenic fluorescent fish with two chromosomally integrated transgenes at the same single locus, driven by distinct promoter types, encoding the same fluorescent protein, and producing strongly visible fluorescence across most of the body.

Overall, the claim coverage centers on a single-locus chromosomal integration of two transgenes encoding the same fluorescent protein, with one transgene driven by a ubiquitous promoter and the other by a muscle-specific promoter, producing strong visible fluorescence across 75% to 100% of the body, optionally excluding fins and eyes.

Stated Advantages

Documented Applications