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 .- 39 . (canceled) 
     
     
         40 . 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 promoter, wherein said one or more cloning vectors does not have a gene encoding antibiotic resistance or a replication origin; and   (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.   
     
     
         41 . The method of  claim 40 , wherein obtaining the cloning vector comprises preparing a cloning vector and inserting therein one or more fluorescent genes under the control of a promoter and removing vector backbone components including gene(s) encoding antibiotic resistance gene and the replication of origin. 
     
     
         42 . The method of  claim 40 , wherein the transgenic fish is further defined as a transgenic zebrafish. 
     
     
         43 . The method of  claim 41 , wherein the transgenic zebrafish is defined as a transgenic Golden zebrafish. 
     
     
         44 . The method of  claim 40 , wherein the transgenic fish has an integration event at a single locus after selection. 
     
     
         45 .- 71 . (canceled) 
     
     
         72 . The method of  claim 40 , wherein said fish so produced do not comprise a transgenic antibiotic resistance gene or origin of replication. 
     
     
         73 . A transgenic fluorescent, ornamental fish produced by the method of  claim 40 . 
     
     
         74 . A transgenic fluorescent, ornamental fish having a genome comprising one or more fluorescent genes under the control of a promoter and expressing one or more fluorescent proteins encoded by said fluorescent genes, and wherein said fish do not comprises a transgenic antibiotic resistance gene or origin of replication. 
     
     
         75 . The transgenic fish of  claim 74 , wherein the one or more fluorescent proteins are selected from the group consisting of ZsGreen1, ZsYellow1, DsRed2, GFP, eGFP, YFP, eYFP, BFP, eBFP, CFP, eCFP, FP, AmCyan1, DsRed-Express, AsRed2, HcRed1, mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mOrange, mKO, MCitrine, Venus, Ypet, EYFP, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire.