Recombinant constructs and transgenic fluorescent ornamental fish therefrom

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.

1 .- 44 . (canceled) 
     
     
         45 . A method of producing a transgenic fluorescent, ornamental fish, comprising the steps of:
 (a) obtaining one or more cloning vectors, each comprising one or more fluorescent genes under the control of a fish promoter and one or more enhancing elements.   (b) producing a population of transgenic fluorescent, ornamental fish wherein one or more fish of said population comprise one or more fluorescent genes in its genome and express one or more fluorescent proteins.   
     
     
         46 . The method of  claim 45 , wherein said enhancing elements are introns and/or exons. 
     
     
         47 . The method of  claim 46 , wherein said intron and/exons are homologuous to the promoter wherein. 
     
     
         48 . The method of  claim 47 , wherein said intron and/or exons are in the 5′ untranslated region or the 3′ untranslated region of the transgene transcript. 
     
     
         49 . The method of  claim 48 , wherein the promoter is a carp β-actin promoter. 
     
     
         50 . The method of  claim 49 , wherein the enhancing elements are the first intron and exon of carp β-actin. 
     
     
         51 . The method of  claim 45 , wherein the transgenic fish further defined as a transgenic zebrafish. 
     
     
         52 . The method of  claim 51 , wherein the transgenic zebrafish has an integration event at a single locus after selection. 
     
     
         53 . A method of obtaining a population of the transgenic ornament fish with strong visible fluorescence, comprising the steps of:
 (a) producing a population of transgenic fluorescent, ornamental fish;   (b) selecting from the population at least one fish that exhibits ubiquitous fluorescent expression;   (c) monitoring the selected fish and continuing selecting for stable expression of strong visible fluorescence; and   (d) creating of a line of fish that exhibit strong visible fluorescence.   
     
     
         54 . The method of  claims 53 , wherein the selected at least one fish is selected by means of its strong visible fluorescence. 
     
     
         55 . The method of  claim 54 , wherein the at least one fish is selected using an ultraviolet or blue light of less than 50 lux. 
     
     
         56 . The method of  claim 54 , wherein the at least one fish is selected using an illuminance level of 50 to 1000 lux. 
     
     
         57 . A method of providing a fluorescent transgenic zebrafish to the ornamental fish market, comprising obtaining a transgenic zebrafish in accordance with claim  1  or  20 , or prepared by the method of claim  35 ,  39 ,  40  or  45 , and distributing said fish to the ornamental fish market. 
     
     
         58 . The method of  claim 57 , wherein the fish are distributed by a grower to a commercial distributor. 
     
     
         59 . The method of  claim 57 , wherein the fish are distributed by a grower or a commercial distributor to a retailer. 
     
     
         60 . The method of  claim 57 , wherein the retailer is a multi-product retailer having an ornamental fish department. 
     
     
         61 .- 71 . (canceled)