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 patent describes transgenic fluorescent fish comprising, in the genome, two transgenes: a first transgene encoding a first fluorescent protein controlled by a ubiquitous promoter and a second transgene encoding a second fluorescent protein controlled by a tissue specific promoter. The first and second transgenes are integrated chromosomally at the same single locus, and the transgenic fluorescent fish are configured to exhibit a strong visible fluorescence over 75% to 100% of the body of the fish, optionally excluding fins and eyes.

The disclosed constructs and transgenic ornamental zebrafish use reef-coral fluorescent proteins, including DsRed2, ZsGreen1, and ZsYellow1. Expression is driven by zebrafish muscle-specific zMLC promoter and a ubiquitously active carp beta-actin promoter. The constructs also include RNA-processing/translational elements such as SV40 polyadenylation sequences, including tandem SV40x2, and an optional exon/intron arrangement associated with the carp beta-actin gene to support enhanced expression.

The document further describes generating and selecting transgenic lines exhibiting strong visible fluorescence and stable ubiquitous expression, including use of Golden zebrafish (slc24a5) to improve ornamental visibility. Transgenic events are reported as chromosomally integrated events deposited as “Red/Green/Yellow zebrafish 1” at ECACC, and the constructs are presented with schematics and fluorescent protein spectral properties.

Claims Coverage

The independent claim covers a transgenic fluorescent fish with two chromosomally integrated fluorescent-protein transgenes arranged at the same single locus, where one transgene is driven by a ubiquitous promoter and the other by a tissue specific promoter, producing strong visible fluorescence over most of the body (with an optional exclusion of fins and eyes).

Overall, the independent claim requires two fluorescent-protein transgenes integrated at the same single chromosomal locus, with ubiquitous versus tissue-specific promoter control, and a strong visible fluorescence phenotype spanning at least 75% of the body.

Stated Advantages

Documented Applications