US 20090025645A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0025645 A1 Blake et al. (43) Pub. Date: Jan. 29, 2009 (54) RECOMBINANT CONSTRUCTS AND (22) Filed: Aug. 15, 2007 TRANSGENC FLUORESCENT ORNAMENTAL FISH THEREFROM Related U.S. Application Data (60) Provisional application No. 60/838,006, filed on Aug. 16, 2006, provisional application No. 60/842,721, (75) Inventors: Alan Blake, Austin, TX (US); filed on Sep. 7, 2006. Richard Crockett, New York, NY (US); Jeffrey Essner, Ames, IA Publication Classification (US); Perry Hackett, Saint Paul, (51) Int. Cl. MN (US); Aidas Nasevicius, Plant CI2N 15/85 (2006.01) City, FL (US) AOIK 67/027 (2006.01) AOIK 63/02 (2006.01) AOIK 6L/00 (2006.01) Correspondence Address: (52) U.S. Cl. .............. 119/203; 800/20: 800/21; 119/215 FULBRIGHT & UAWORSK L.L.P. (57) ABSTRACT 600 CONGRESSAVE., SUITE 2400 AUSTIN, TX 78701 (US) 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 (73) Assignee: Yorktown Technologies, L.C., Zebrafish are fertile and used to establish a population of Austin, TX (US) 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 (21) Appl. No.: 11/839,364 Source are developed. Xinyl (5728) Not I (671) ^ Spel (1111) pZMLC-SV40X2-Yellow puC ori Zsyellow 1 6043 bp, inserted DNA, Notl (671)-Xhol (3823) EcoRI (1829) PaC -- XhoIW . (3823)--- ZMLC-1934. Promoter Patent Application Publication Jan. 29, 2009 Sheet 1 of 6 US 2009/0025645 A1 Xinji I (5(4) -- Amp (bla) Y SV40(A) Spe (111) Construct 2: p2MLC-SV40x2-DsRed2 is: pUC ori DSRed2 6009 bp; inserted DNA, Not (671) - XhoI (3789 EcoRI (1795) Plac w - Xhol (3789) ZMLC-1934 POOter FIG.1 Patent Application Publication Jan. 29, 2009 Sheet 2 of 6 US 2009/0025645 A1 Xinji (57.26 Not (671) V40(A) SV40(A) Spel (11.11) Construct 1: p.ZMLC-SV40X2-ZsGreen 1 pUC ori 6041 bp; inserted DNA, Notl (671)-Xhol (3821) i? ZSGreen 1 EcoRI (1827) XhoI (382) ZMLC-1934 Promoter FG. 2 Patent Application Publication Jan. 29, 2009 Sheet 3 of 6 US 2009/0025645 A1 Xinful (5728) Not I (671) NV40A No. Spel (1111) pZMLC-SV40X2-Yellow ZSYellow 1 pUC ori 6043 bp, inserted DNA, Nott (671)-XhoI (3823) EcoRI (1829) PaC --- XhoI (3823) ZMLC-1934. Promoter FIG. 3 Patent Application Publication Jan. 29, 2009 Sheet 4 of 6 US 2009/0025645 A1 Xbr 25 COE Ori (s-actin promoter Attil (3870) SV40(A) S-actin intron 1 Spel (3427 K7I (2741) ECR 273 FG. 4 Patent Application Publication Jan. 29, 2009 Sheet 5 of 6 US 2009/0025645 A1 Xia (215) COE 1 Ori ?-actin promoter Amp ConstructE" 2: pCBAC-SV40X2-ZsGreenE:6'56-actin 1 exon 1 alcifll 3902) F-actin intron 1 so N. SV40(A) Zsgreen 1 Siel (3-459) Kpril (274 EcoRI (29.13 F.G. 5 Patent Application Publication Jan. 29, 2009 Sheet 6 of 6 US 2009/0025645 A1 Step 1. Using a restriction enzyme, the construct plasmid is opened, and the antibiotic resistance gene is separated from the desired DNA. Altibiotic Resistance Gene -> Step 2. The DNA fragments are collected and loaded into a gel. Step 3. Using an electric charge, the fragments of DNA are separated. Step 4. The desired DNA, in our case, the green fluorescent protein gene construct, is collected and microinjected into a recently fertilized zebrafish embryo. F.G. 6 US 2009/0025645 A1 Jan. 29, 2009 RECOMBINANT CONSTRUCTS AND direct injection of DNA into muscle tissue (Xu et al., 1999). TRANSGENC FLUORESCENT The first transgenic fish report was published by Zhu et al., ORNAMENTAL FISH THEREFROM (1985) using a chimeric gene construct consisting of a mouse metallothionein gene promoter and a human growth hormone gene. Most of the early transgenic fish studies have concen 0001. The present application claims the benefit of previ trated on growth hormone gene transfer with an aim of gen ously filed provisional application Ser. Nos. 60/838.006, filed erating fast growing “superfish'. While a majority of early Aug. 16, 2006, and 60/842,721, filed Sep. 7, 2006, the dis attempts used heterologous growth hormone genes and pro closures of which are incorporated by reference herein in their moters and failed to produce gigantic Superfish (e.g. Chour entirety. routet al., 1986; Penman et al., 1990; Brem et al., 1988: Gross et al., 1992), enhanced growth of transgenic fish has been BACKGROUND OF THE INVENTION demonstrated in several fish species including Atlantic 0002 1. Field of the Invention salmon, several species of Pacific salmons, and loach (e.g. Du 0003. This invention relates to transgenic gene constructs et al., 1992: Delvin et al., 1994, 1995: Tsai et al., 1995). with fish gene promoters and heterologous genes for genera 0008. The Zebrafish, Danio rerio, is a new model organism tion of transgenic fish, particularly fluorescent transgenic for vertebrate developmental biology. As an experimental fish. model, the Zebrafish offers several major advantages such as 0004 2. Description of Related Art easy availability of eggs and embryos, tissue clarity through 0005 Transgenic technology involves the transfer of a out embryogenesis, external development, short generation foreign gene into a host organism enabling the host to acquire time and easy maintenance of both the adult and the young. a new and inheritable trait. The technique was first developed Transgenic Zebrafish have been used as an experimental tool in mice by Gordon et al. (1980). They injected foreign DNA in Zebrafish developmental biology. However, for the orna into fertilized eggs and found that some of the mice developed mental fish industry the dark Striped pigmentation of the adult from the injected eggs retained the foreign DNA. Applying Zebrafish does not aid in the efficient display of the various the same technique, Palmiter et al. (1982) introduced a chi colors that are currently available in the market. More meric gene containing a rat growth hormone gene under a recently, Lamason et al. (2005) in their report showed that the mouse heavy metal-inducible gene promoter and generated Golden zebrafish carry a recessive mutation in the slo24.a5 the first batch of genetically engineered Supermice, which gene, a putative cation exchanger, and have diminished num were almost twice as large as non-transgenic siblings. This ber, size and density of melanosomes which are the pig work has opened a promising avenue in using the transgenic mented organelles of the melanocytes and hence are lightly approach to provide to animals new and beneficial traits for pigmented as compared to the wild type Zebrafish. The avail livestock husbandry and aquaculture. ability of the Golden Zebrafish for transgenesis with fluores 0006. In addition to the stimulation of somatic growth for cent proteins would result in better products for the ornamen increasing the gross production of animal husbandry and tal fish industry as it would allow for a better visualization of aquaculture, transgenic technology also has many other the various colors. potential applications. First, transgenic animals can be used 0009 Green fluorescent protein (GFP) is a useful tool in as bioreactors to produce commercially useful compounds by the investigation of various cellular processes. The GFP gene expression of a useful foreign gene in milk or in blood. Many was isolated from the jelly-fish Aqueous victoria. More pharmaceutically useful protein factors have been expressed recently, various other new fluorescent protein genes have in this way. For example, human 1-antitrypsin, which is com been isolated from the Anthozoa class of coral reefs (Matz et monly used to treat emphysema, has been expressed at a al., 1999) called DsRed, red fluorescent protein gene: concentration as high as 35 mg/ml (10% of milk proteins) in ZsGreen, green fluorescent protein gene and Zsyellow, yel the milk of transgenic sheep (Wright et al., 1991). Similarly, low fluorescent protein gene. The novel fluorescent proteins the transgenic technique can also be used to improve the encoded by these genes share 26-30% identity with GFP nutritional value of milk by selectively increasing the levels (Miyawaki, 2002). These are bright fluorescent proteins and of certain valuable proteins such as caseins and by Supple each emits a distinct wavelength. They are physico-chemi menting certain new and useful proteins such as lysozyme for cally very stable, extremely versatile, emitting strong visible antimicrobial activity (Maga and Murray, 1995). Second, fluorescence in a variety of cell types and display exceptional transgenic mice have been widely used in medical research, photostability and hence fluoresce over extended periods of particularly in the generation of transgenic animal models for time. Because of their distinct spectra, they can be used in human disease studies (Lathe and Mullins, 1993). More combination. The crystal structure of the Dsked protein sug recently, it has been proposed to use transgenic pigs as organ gests that the chromofore is located on a central a-helical donors for Xenotransplantation by expressing human regula segment embedded within a tightly folded f-barrel and that tors of complement activation to prevent hyperacute rejection the Dsked protein forms tetramers in vivo (Wall et al., 2000). during organ transplantation (Cozzi and White, 1995). The 0010 Coral reef fluorescent proteins have broad applica development of disease resistant animals has also been tested tion in research and development. Thered fluorescent protein, in transgenic mice (e.g. Chen et al., 1988). DsRed, has been used as a reporter in the transgenic studies 0007 Fish are also an intensive research subject of trans involving various animal model systems: for example, fila genic studies.