Recombinant constructs and transgenic fluorescent ornamental fish therefrom
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. A method of producing a transgenic, fluorescent fish having improved fluorescence comprising:
(a) obtaining transgenic, fluorescent fish that exhibit a fluorescence by virtue of their comprising:
i) a first transgene encoding a fluorescent protein, wherein the first transgene is under the control of a ubiquitous fish promoter, and
ii) a second transgene encoding the same fluorescent protein as in the first transgene, wherein the second transgene is under the control of a muscle-specific fish promoter, wherein said transgenic, fluorescent fish were generated by chromosomal introduction of said first and second transgenes positioned on a single vector; and
(b) selecting a transgenic fish exhibiting strong visible fluorescence over 75% to 100% of the body of said fish, optionally excluding fins and eyes and
(c) obtaining transgenic progeny of said selected transgenic fish, wherein the progeny exhibits fluorescence.
2. The method of claim 1 , wherein the transgenic, fluorescent fish is further defined as a transgenic, fluorescent zebrafish, catfish, zebrafish, medaka, carp, tilapia, goldfish, tetras, barbs, sharks (family cyprinidae), angelfish, loach, koi, glassfish, catfish, angel fish, discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius.
3. The method of claim 2 , wherein the transgenic, fluorescent fish is further defined as a transgenic, fluorescent zebrafish, tetra or barb.
4. The method of claim 3 , wherein the transgenic, fluorescent fish is further defined as a transgenic zebrafish.
5. The method of claim 4 , wherein the transgenic zebrafish is further defined as a transgenic Golden zebrafish.
6. The method of claim 1 , wherein the fluorescent protein is selected from the group consisting of ZsGreen1, ZsYellow1, DsRed2, GFP, eGFP, YFP, eYFP, BFP, eBFP, CFP, eCFP, FP, AmCyan 1, 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.
7. The method of claim 6 , wherein the fluorescent protein is ZsGreen1.
8. The method of claim 6 , wherein the fluorescent protein is DsRed2.
9. The method of claim 1 , wherein the ubiquitous fish promoter is selected from the group consisting of those for housekeeping genes, including β-actin, EF-1, histone 2A, acidic ribosomal phosphoprotein, α-catenin, β-cateniny-catenin, ubiquitin CSrb7, creatine kinase (mitochondrial 1), ubiquitous Ca2+transporting ATPase, ancient ubiquitous protein, ubiquitin specific peptidase 4, acetyl-Coenzyme A acryltransferase 2, monoglyceride lipase, splicing factor 3b subunit 1 and tubulin β5.
10. The method of claim 9 , wherein the ubiquitous fish promoter is the carp-actin promoter.
11. The method of claim 1 , wherein said muscle specific promoter is a fish myosin light chain promoter.
12. The method of claim 1 , wherein the muscle-specific fish promoter is selected from the group consisting of myosin light chain, muscle creatine kinase, MyoD, myogenin, desmin, muscle enolase-3, β-sarkoglycan, dystrophin, serum response factor, a-tropomyosin, myosin heavy chain, mitochondrial creatine kinase 2, Ca2+transporting ATPase (fast twitch 1), skeletal troponin T1(slow), tropomodulin 4, four and a half LIM domains 1, fast-type binding protein C, calsequestrin 1, fast muscle troponin C2, phosphorylase kinase alpha 1, and skeletal Troponin I (fast 2).
13. The method of claim 12 , wherein the myosin light chain promoter is a zebrafish fast skeletal myosin light chain promoter.
14. A method of providing a fluorescent transgenic fish to the ornamental fish market, comprising obtaining a transgenic fish prepared by the method of claim 1 , and distributing said fish to the ornamental fish market.
15. The method of claim 14 , wherein the fish are distributed by a grower to a commercial distributor.
16. The method of claim 14 , wherein the fish are distributed by a grower or a commercial distributor to a retailer.
17. The method of claim 14 , wherein the retailer is a multi-product retailer having an ornamental fish department.