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. There are many ways of introducing a foreign mentous fungi (Eckert et al., 2005, Mikkelsen et al., 2003); gene into fish, including: microinjection (e.g., Zhu et al., ascidian (Zeller et al., 2006); Zebrafish (Zhu et al., 2005, Zhu 1985: Du et al., 1992), electroporation (Powers et al., 1992), et al., 2004, Gong et al., 2003, Finley et al., 2001); xenopus sperm-mediated gene transfer (Khoo et al., 1992: Sin et al., (Werdien et al., 2001); insect (Cho et al., 2006, Handler et al., 1993), gene bombardment or gene gun (Zelenin et al., 1991), 2001, Horn et al., 2002); drosophila (Barolo et al., 2004); liposome-mediated gene transfer (Szelei et al., 1994), and the silkworm (Royer et al., 2005); mouse (Schmid et al., 2006, US 2009/0025645 A1 Jan. 29, 2009

Vintersten et al., 2004); rat (Sato et al., 2003); and plants 0013. In another preferred embodiment, the fish for use (Wenek et al., 2003). It has also been used a marker in imag with the disclosed constructs and methods is the Golden ing studies in stem cells (Tolar et al., 2005, Long et al., 2005) Zebrafish. Zebrafish skin color is determined by pigment cells and mouse (Long et al., 2005, Hadjantonakis et al., 2003). in their skin, which contain pigment granules called melano Green fluorescent protein, ZsCreen, has been used as a trans Somes. The number, size and density of the melanosomes per formation marker in insects (Sarkar et al., 2006), knock-in pigment cell influence the color of the fish skin. Golden mouse model for the study of KIT expressing cells (Wouters Zebrafish have diminished number, size, and density of mel et al., 2005) and as reporters for plant transformation (Wenck anosomes and hence have lighter skin when compared to the et al., 2003). Yellow fluorescent protein, Zsyellow, has been wild type Zebrafish. Golden Zebrafish have a mutation in used a reporter for plant transformation (Wencket al., 2003) Slc24.a5 gene, rendering the fish skin lighter or less pigmented and for visualizing fungal pathogens (Bourett et al., 2002). (Lamason et al., 2005). All of these transgenic experiments have aimed at developing 0014. In another embodiment of the invention, a method newer markers and reporters for transgenesis; however, for making transgenic fluorescent fish is provided comprising progress in the field of ornamental fish industry has been at least the following steps: a) preparing a vector which has a limited. transgenic fluorescence expression cassette comprising one sequence from a group of SEQID NO: 1, SEQID NO:2, SEQ SUMMARY OF THE INVENTION ID NO:3, SEQ ID NO.4 and SEQ ID NO:5, two or more 0011. In certain embodiments, the present invention con sequences from a group of SEQIDNO:1, SEQIDNO:2, SEQ cerns making recombinant constructs and transgenic fluores ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 are used in cent fish and providing Such fish to the ornamental fish indus combination, specifically, SEQID NO: 1 and SEQID NO:2 try. The term recombinant construct is used to mean are used together and SEQ ID NO:3 and SEQ ID NO:5 are recombinant DNA constructs having sequences which do not used together; b) making the transgenic Zebrafish using the occur in nature or exist in a form that does not occur in nature vectors; and, c) selecting transgenic Zebrafish that fluoresce or exist in association with other materials that do not occur in by monitoring fluorescence under a light of appropriate wave nature. The term transgenic has historically been used in length. The transgenic expression cassette has a set of tran many contexts with various meanings. In the embodiments of Scriptional regulatory motifs, herein referred to as a promoter, this invention transgenic is understood to mean genetic mate which may be from the host species (herein referred to as a rial artificially introduced into the genome of an organism. An homologous promoter) or from another species (herein organism incorporating Such genetic material, or progeny to referred to as a heterologous promoter), heterologous fluo which this genetic material was passed, would be considered rescent gene, and appropriate RNA-processing and/or trans a transgenic organism. Such transgenic organisms may also, lational enhancing motif. The term promoter as used herein in certain embodiment, be referred to generally as a geneti refers to the DNA elements that direct and regulate transcrip cally modified organism (GMO), which is defined as an tion. For instance, the Zebrafish fast skeletal muscle myosin organism whose genetic material has been altered using the light chain promoter and carp B-actin promoter may be used genetic engineering techniques generally known as recombi according to the invention. nant DNA technology. This modified DNA is then transferred 0015. In certain specific embodiments there are provided into an organism preferably resulting in the expression of methods to use multiple vectors to express at least one fluo modified or novel traits. The term “GMO does not cover rescent protein in order to enhance expression. The preferred organisms whose genetic makeup has been altered by con mode is to make a transgenic fish comprising in its genome a ventional cross breeding or by “mutagenesis' breeding, as first fluorescent transgene under the control of a ubiquitous these methods predate the discovery of the recombinant DNA fish promoter, and a second fluorescent transgene under the techniques. Technically, however, such techniques are by control of a tissue specific fish promoter. The ubiquitous fish definition genetic modification. The term fluorescent is used promoter is selected from the group consisting of those tran to mean an entity that absorbs light of one wavelength and Scriptional motifs that direct gene expression in most cells, emits at a different wavelength. and more preferably in all cells; they are also preferably 0012 Specific embodiments of the present invention are promoters for housekeeping genes, such as tubulin, riboso directed to methods of making transgenic fluorescent fish mal protein, and actingenes. The tissue specific fish promoter having one sequence from a group of SEQID NO: 1, SEQID is selected from the group consisting of those transcriptional NO:2, SEQ ID NO:3, SEQ ID NO.4 and SEQ ID NO:5, as motifs that are active in specific cells of differentiated tissues well as transgenic fish developed by Such methods. Thus, a Such as muscle, brain, liver, blood and eyes. In a preferred transgenic Zebrafish having integrated into its germ line cell embodiment, the tissue specific fish promoter is muscle spe DNA a transgenic construct comprising one or more of SEQ cific. As used herein, a promoter drives expression “specifi ID NO:1 through SEQID NO:5 is also included as part of the cally' in a tissue if the level of expression is at least 5-fold, invention. Further more, the invention provides transgenic preferably at least 10-fold higher, more preferably at least Zebrafish egg and/or sperm cells comprising a sequence 50-fold higher in that tissue than in any other tissue. according to SEQID NO:1 through SEQID NO:5 integrated 0016. More than one construct can be injected into the fish in its/their genome(s). In certain aspects of the invention, two embryos simultaneously. For example, in the present inven or more sequences from a group of SEQID NO: 1, SEQID tion, both Red Zebrafish 1 and Green zebrafish 1 incorporate NO:2, SEQ ID NO:3, SEQID NO.4 and SEQID NO:5 are more than one transgenic expression cassettes, with one being used in one Zebrafish. In a specific aspect, SEQID NO: 1 and a ubiquitous promoter, and the other being a strong muscle SEQIDNO:2 are used in the same fish and SEQID NO:3 and promoter. In particular, Red Zebrafish 1 incorporates the cas SEQID NO:5 are used in the same fish. In preferred embodi settes represented by FIG. 1 and FIG. 4, and Green Zebrafish ments, it is contemplated that the transgenic fluorescent fish 1 incorporates the cassettes represented by FIG. 2 and FIG. 5. are fertile transgenic fluorescent fish. While the present invention incorporates only the transgenic US 2009/0025645 A1 Jan. 29, 2009

insert cassettes shown in the Figures, it is understood that promoters of these specific genes fused to fluorescent protein multiple transgenic insert cassettes of any type can be simul open reading frames (ORFs) can be used to visualize specific taneously injected into a fish embryo from any species. chromatophores. The specific genes can be roughly divided 0017. The steps involved in making the transgenic fish into two major groups: regulatory proteins and biosynthesis further involve isolation and separation of the transgenic enzymes, involved in specific pigment synthesis. Expression expression cassette from the vector backbone to remove any of regulatory proteins usually is at lower level than that of gene encoding antibiotic resistance (e.g., amplicillin or kana biosynthesis enzymes therefore use of promoters of biosyn mycin) and origin of replication. In a preferred mode, a Suit thesis enzymes are most preferred. able promoter would be expected to drive stable and consis 0021. The heterologous fluorescent gene may be, for tent expression throughout the life of the fish. To achieve such example, a gene encoding DSRed2, ZSGreen1 and Zsyel stable expression, it is necessary to choose a promoter that is low 1. The heterologous fluorescent gene may also be any known to drive stable and consistent expression throughout variation or mutation of these genes, encoding fluorescent the life of the fish. For example, a promoter that drives expres proteins including green fluorescent protein (GFP), enhanced sion only during the six months of the life of the fish would not green fluorescent protein (eGFP), yellow fluorescent protein be suitable for use. Examples of suitable promoters may be (YFP), enhanced yellow fluorescent protein (eYFP), blue selected from the group consisting of those for housekeeping fluorescent protein (BFP), enhanced blue fluorescent protein genes, such as tubulin, ribosomal protein, and actin gene (eBFP), cyan fluorescent protein (CFP) and enhanced cyan promoters. fluorescent protein (eCFP) or any of the proteins listed in 0018. It is also preferred to use regulatory elements, for Table 4, or any variation or mutation thereof, or any other example, RNA processing and translational enhancing ele fluorescence proteins. The steps involved in making the trans ments in the transgenic insert cassette to produce a transgenic genic fish also involve introduction of the transgenic expres fluorescent, ornamental fish. The RNA processing signals, sion cassette into the Zebrafish embryos or Zebrafish embry preferably, are one or more polyadenylation signals and/or onic stem cells. Such embryos and cells are allowed to grow one or more introns. Since introns are sequences between and mature into adult fish and then they are screened for the exons, the presence of an intron automatically indicates the presence of the transgenic expression cassette using the Vari presence of two exons. Accordingly, two introns indicate the ous molecular biology methods described in the detailed presence of three exons, and so on. The carp beta-actin intron description section and/or by functional biochemical assays used in SEQ ID 2 and SEQ ID 5 is an example of such an Such as assaying for the activity of the introduced fluorescent intron, and the untranslated carp beta-actin exonused in SEQ gene by exposing the said fish to light of appropriate wave ID 2 and SEQID 5 is an example of such an exon. Exons and length and/or by visibly inspecting the fish and observing the introns other than carp beta-actin can be used as well. The expression. Transgenic fluorescent fish are further bred to translational enhancing elements, preferably, are 5' untrans insure transmission of the transgenic expression cassette via lated leader sequences of 40-200 nucleotides, and more pref the germ cells of a fish as further described in this application. erably untranslated leader sequences of 40-70 nucleotides. It 0022. The present invention also provides a method to is known that the presence of introns in primary transcripts obtain a progenitor of a new line of fluorescent transgenic can increase expression, possibly by causing the transcript to fish, and a population therefrom, which exhibit strong visible enter the processing and transport system for mRNA. It is also fluorescence. Strong visible fluorescence means that a person preferred that the intron be homologous to the host species, with 20/20 vision (i.e., average vision) will be able to distin and more preferably homologous to the expression sequences guish between the fluorescent fish in question and a non used (that is, that the intron be from the same gene that some fluorescent fish of the same species at a distance of at least 5 or all of the expression sequences are from). feet in a lighted office, with a preferred distance of at least 10 0019. The disclosed transgene constructs preferably feet in a lighted office, and a more preferred distance of at include other sequences which improve expression from, or least 15 feet in a lighted office, and an even more preferred stability of the construct. For example, including a polyade distance of at least 20 feet in a lighted office, with the illumi nylation signal on the constructs encoding a protein ensures nation level defined in Table 5. One can observe all transgenic that transcripts from the transgene will be processed and fluorescent fish from a particular population that exhibit transported as mRNA. The identification and use of polyade strong visible fluorescence under the various lighting condi nylation signals in expression constructs is well established. tions and select the fish that exhibits the highest level of It is preferred that efficient polyadenylation signals, such as visible fluorescence of the fluorescent protein. Selected fish those derived from viruses, be used in the transgenic con with strong visible fluorescence are monitored and their prog structs, and more preferred to use at least two polyadenylation eny selected continuously to ensure stability of expression signals, which more preferably are two copies of SV40 poly and maintenance of strong visible fluorescence. Thus a new adenylation sequence. line of fish that exhibit strong visible fluorescence is created 0020. It is also a subject of this invention to disclose for further breeding. expression of the fluorescent protein gene only in chromato 0023 Transgenic fish made by the present disclosure will phores. There are several types of chromatophores found in emit red, yellow-green and yellow-orange fluorescence under animals: melanophores (black), Xanthophores (yellow), light of distinct wavelengths and hence will be unique and erythrophores (red), cyanophores (blue), leucophores (white) attractive to the ornamental fish industry. In yet another and iridophores (reflective). Different species of fish contain embodiment of the invention, a method of making the trans all types of chromatophores, usually a Subset of them in genic fish available to the consumer by a grower or a com different combinations. Zebrafish contain melanophores, mercial distributor through a retailer for sale to the public. In xantophores and iridophores. These different cell types such embodiment, the fish may also be sold by the grower or express specific genes, characteristic only for them or specific commercial distributor to a regional wholesale distributor, for a subset of chromatophores. In a preferred embodiment, who will then sell to a retailer for sale to the public. The US 2009/0025645 A1 Jan. 29, 2009 fluorescent transgenic fish are also useful for the development 0029. Throughout this application, the term “about is of a biosensor System and as research models for embryonic used to indicate that a value includes the inherent variation of studies such as cell lineage, cell migration, cell and nuclear error for the device, the method being employed to determine transplantation, cell-cell interaction in vivo, etc. the value, or the variation that exists among the study Sub 0024 Transgenic Zebrafish comprising an expression cas jects. sette according to the invention may be homozygous or het 0030. Other objects, features and advantages of the erozygous with respect to the expression cassette. In some present invention will become apparent from the following preferred aspects, fish for use in breeding of transgenic detailed description. It should be understood, however, that Zebrafish of the invention will be homozygous for an expres the detailed description and the specific examples, while indi sion cassette. Homozygous fish bred with fish lacking an cating preferred embodiments of the invention, are given by expression cassette (e.g., Golden Zebrafish) will in nearly all way of illustration only, since various changes and modifica cases produce 100% heterozygous offspring. Likewise, trans tions within the spirit and scope of the invention will become genic fish for commercial retail will preferably be heterozy apparent to those skilled in the art from this detailed descrip gous for an expression cassette. Furthermore in Some very tion. specific aspects a transgenic fish of the invention comprises the specific integration event of the Red fluorescent expres BRIEF DESCRIPTION OF THE DRAWINGS sion cassette described in Example 3. 0031. The following drawings are part of the present 0025. In certain specific embodiments there are provided specification and are included to further demonstrate certain transgenic fluorescent Zebrafish comprising specific trans aspects of the present invention. The invention may be better genic integration events. These fish are of particular interest understood by reference to the drawing in combination with since, for example, they embody an esthetically pleasing level the detailed description of specific embodiments presented of protein fluorescence. Thus, in some embodiments there is herein. provided a transgenic Zebrafish comprising a chromosomally 0032 FIG. 1: The figure shows a schematic map of the integrated expression cassette encoding a DSRed2 gene transgenic construct, pZMLC-DsRed2-SV40x2. The 2.1-kb wherein the zebrafish comprises the Red Zebrafish 1 transfor eukaryotic promoter sequence ZMLC-1934 promoter was mation event, sperm comprising said Red Zebrafish 1 trans amplified by PCR from pMLC vector and cloned into XhoI formation event having been deposited as ECACC accession and EcoRI restriction sites. The 684 bp DsRed2 fluorescent no. 06090403. In some other aspects, there is provided a protein CDS was amplified by PCR from posRed2-N1 transgenic Zebrafish comprising a chromosomally integrated (Clontech) and inserted into EcoRI and Spel sites. The 440 expression cassette encoding a ZsGreen1 gene wherein the bp 3'UTR/poly(A) sequence encoding tandem SV40 poly Zebrafish comprises the Green zebrafish 1 transformation adenylation signals was PCR amplified from pK-SV40(A)x2 event, sperm comprising said Green Zebrafish 1 transforma and cloned into Spel and NotI sites. XhoI, XmnI and NotI tion event having been deposited as ECACC accession no. restriction sites were used to isolate the expression construct 060904.01. In still other aspects, there is provided a transgenic from the vector backbone. Also shown is the amplicillin Zebrafish comprising a chromosomally integrated expression (Amp, formally known as beta-lactamase (bla)) resistance cassette encoding a Zsyellow 1 gene wherein the Zebrafish gene in the backbone of the p3luescript plasmid. The total comprises the Yellow zebrafish 1 transformation event, sperm length of the recombinant plasmid pzMLC-DsRed2-SV40x2 comprising said Yellow zebrafish 1 transformation event hav is 6009 bp. ing been deposited as ECACC accession no. 06090402. As 0033 FIG. 2: The figure shows a schematic map of the described above, transgenic fish comprising these specific transgenic construct, pZMLC-ZsGreen1-SV40x2. The 1.9- transgenic events may be homozygous or heterozygous for kb eukaryotic promoter sequence ZMLC-1934 promoter was transgene, and in Some cases may comprise more than one of amplified by PCR from pMLC vector and cloned into XhoI the transgenic events, although it is preferred to have only one and EcoRI restriction sites. The 716 bp ZsGreen1 fluorescent integration location for any given transgenic modification. protein CDS was amplified by PCR from pZsGreen1-N1 Eggs, sperm and embryos comprising these specific trans (Clontech) and inserted into EcoRI and Spel sites. The 440 genic events are also included as part of the instant invention. bp 3'UTR/poly(A) sequence encoding tandem SV40 poly 0026. Any of the fluorescence genes noted in this applica adenylation signals was PCR amplified from pK-SV40(A)x2 tion may be used in similar embodiments of this invention. and clone into Spel and NotI sites. XhoI, XmnI and NotI Embodiments discussed in the context of a method and/or restriction sites were used to isolate the expression construct composition of the invention may be employed with respect from the vector backbone. Also shown is the amplicillin to any other method or composition described herein. Thus, (Amp) resistance gene in the backbone of the pBluescript an embodiment pertaining to one method or composition may plasmid. The total length of the recombinant plasmid be applied to other methods and compositions of the inven pzMLC-ZsGreen1-SV40x2 is 6041 bp. tion as well. 0034 FIG. 3: The figure shows is a schematic map of the 0027. As used herein the specification, “a” or “an may transgenic construct, pZMLC-Zsyellow 1-SV40x2. The 1.9- mean one or more. As used herein in the claim(s), when used kb eukaryotic promoter sequence ZMLC-1934 promoter was in conjunction with the word “comprising, the words “a” or amplified by PCR from pMLC vector and cloned into XhoI 'an' may mean one or more than one. and EcoRI restriction sites. The 718 bp Zsyellow 1 fluores 0028. The use of the term “or” in the claims is used to cent protein CDS was amplified by PCR from pZsyellow 1 mean “and/or unless explicitly indicated to refer to alterna N1 (Clontech) and inserted into EcoRI and Spel sites. The tives only or the alternatives are mutually exclusive, although 440-bp 3'UTR/poly(A) sequence encoding tandem SV40 the disclosure supports a definition that refers to only alter polyadenylation signals was PCR amplified from pK-SV40 natives and “and/or.” As used herein “another may mean at (A)x2 and clone into Spel and NotI sites. XhoI, XmnI and least a second or more. NotI restriction sites were used to isolate the expression con US 2009/0025645 A1 Jan. 29, 2009

struct from the vector backbone. Also shown is the ampicillin in the art and can be found, for example, in Sambrook et al., (Amp) resistance gene in the backbone of the pBluescript 2001; Sambrook et al., 1989 and U.S. Pub No. 2004/0143864 plasmid. The total length of the recombinant plasmid A1, all of which are hereby incorporated by reference in their pzMLC-ZsYellow 1-SV40x2 is 6043 bp. entireties. 0035 FIG. 4: The figure shows a schematic map of the 0039. To develop successful transgenic fish with a predict transgenic construct, pCBAC-DsRed2-SV40x2. The 2.5-kb able pattern of transgenic expression, the first step is to make common carp beta-actin enhancer/promoter sequence, beta the appropriate genetic construct. The genetic construct gen actin exon-1 and beta-actin intron-1 was amplified by PCR erally comprises three portions: transcriptional regulators from pFV7b vector and cloned into Xbaland KpnI restriction comprising a promoter, a gene and appropriate RNA-process sites. The 684 bp DsRed2 fluorescent protein CDS was ampli ing and/or translational enhancing motif. The gene promoter fied by PCR from plDsRed2-N1 (Clontech) and inserted into would determine where, when and under what conditions the EcoRI and Spel sites. The 443-bp 3'UTR/poly(A) sequence gene is expressed. The gene contains protein coding portions encoding tandem SV40 polyadenylation signals sequence that determine the protein to be synthesized and thus the encoding tandem SV40 signal was PCR amplified from biological function. The gene might also contain intron pK-SV40(A)x2 and cloned into Spel and Aati I sites. Xbal sequences which can affect mRNA processing or which and Aat restriction sites were used to isolate the expression might contain transcription regulatory elements. The RNA construct from the vector backbone. Also shown is the ampi processing signals may include: one or more polyadenylation cillin (Amp) resistance gene in the backbone of the pBlue signals and one or more introns. Among the three portions, it Script plasmid. The total length of the recombinant plasmid is preferable to use a promoter that drives strong expression. pCBAC-DsRed2-SV40x2 is 5801 bp. The promoter may be a homologous promoter or it may be a 0036 FIG. 5: The figure shows a schematic map of the heterologous promoter. transgenic construct, pCBAC-ZsGreen 1-SV40x2. The 2.5- 0040. A promoter drives expression “predominantly' in a kb carp beta-actin enhancer/promoter sequence, beta-actin tissue if expression is at least 2-fold, preferably at least 5-fold exon 1 and beta-actin intron 1 was amplified by PCR from higher in that tissue compared to a reference tissue. A pro pFV7b vector and cloned into Xbaland KpnI restriction sites. moter drives expression “specifically' in a tissue if the level The 716 bp ZsGreen1 fluorescent protein CDS was amplified of expression is at least 5-fold, preferably at least 10-fold by PCR from pZsGreen1-N1 (Clontech) and inserted into higher, more preferably at least 50-fold higher in that tissue EcoRI and Spel sites. The 443 bp 3'UTR/poly(A) sequence than in any other tissue. Aubiquitous promoter drives expres encoding tandem SV40 polyadenylation signals sequence sion in most tissues, and preferably in all tissues. encoding tandem SV40 signal was PCR amplified from pK-SV40(A)x2 and cloned into Spel and Aati I sites. Xbal Recombinant DNA Constructs and Aat restriction sites were used to isolate the expression 0041 Recombinant DNA constructs comprising one or construct from the vector backbone. Also shown is the ampi more of the DNA sequences described herein and an addi cillin (Amp) resistance gene in the backbone of the pBlue tional DNA sequence are also included within the scope of Script plasmid. The total length of the recombinant plasmid this invention. These recombinant DNA constructs usually pCBAC-ZsGreen1-SV40x2 is 5833 bp. have sequences which do not occur in nature or existina form 0037 FIG. 6: Transgenic Construct purification and injec that does not occur in nature or exist in association with other tion process. The Figure depicts step by step the process of materials that do not occur in nature. The DNA sequences transgenic construct purification and injection. Step 1 illus described as constructs or in vectors above are “operably trates separation of the plasmid backbone sequence with the linked with other DNA sequences. DNA regions are oper antibiotic resistance gene and origins of replication (pUC ori ably linked when they are functionally related to each other. and fl(-)ori) (on left) and the expression construct (on right). Generally, operably linked means contiguous (or in close Step 2 and 3 show the method of purification of the expression proximity to). construct by loading and electrophoretic separation of the 0042. The disclosed transgenic constructs preferably DNA fragments on an agarose gel. The antibiotic resistance include other sequences that improve expression from, or gene and origins of replication (pUC ori and fl(-) ori) are stability of the construct. For example, including a polyade below the expression construct on the gel. Step 4 exemplifies nylation signal on the constructs encoding a protein ensures the process of microinjection of the gel-purified expression that mRNA transcripts from the transgene will be efficiently construct in to the fertilized Zebrafish embryos. translated as protein. The identification and use of polyade nylation signals in expression constructs is well established. DETAILED DESCRIPTION OF THE INVENTION It is preferred that defined and efficient polyadenylation sig nals, such as those derived from viruses, be used in the trans Transgenic Constructs genic constructs, and more preferred to use at least two poly 0038. The present invention encompasses transgenic con adenylation signals, which more preferably are two copies of structs which are genetic material artificially introduced into SV40 polyadenylation sequence. fish to produce a transgenic fish. The manner of introduction, 0043. In certain specific embodiments there are provided and, often, the structure of a transgenic construct, render Such methods to use multiple vectors to express at least one fluo a transgenic construct an exogenous construct. Although a rescent protein in order to enhance expression. The preferred transgenic construct can be made up of any assembly of mode is to make a transgenic fish comprising in its genome a nucleic acid sequences, for use in the disclosed transgenic fish first fluorescent transgene under the control of a ubiquitous it is preferred that the transgenic constructs combine regula fish promoter, and a second fluorescent transgene under the tory elements operably linked to a sequence encoding one or control of a tissue specific fish promoter. In a preferred more proteins. The methods and protocols for designing and embodiment, the tissue specific fish promoter is muscle spe making transgenic constructs are well known to those skilled cific. The ubiquitous fish promoter and the muscle specific US 2009/0025645 A1 Jan. 29, 2009 promoterare, for example, selected from Table 1 below. In the a search string. The search output will yield Ensembl gene ID Table 1, any promoter marked with an “X” is confirmed (e.g., ENSMUSG00000030730), gene homologues in other available at this time, with any unmarked promoter, or any organisms (e.g., Zebrafish—Danio rerio), as well as genomic other promoter of interest, available preferably through the information of the gene of interest, including genomic following steps: following the database searching instruc sequence of the coding region (introns and exons), as well as tions provided in detail below, conducting a literature search, genomic DNA sequence Surrounding the coding sequence and sequencing the gene and promoter of interest through (e.g., “Exon info' link directs a user to the sequence infor methods that are well know by artisans in the field. mation). Gene promoters are located upstream (5' flanking 0044) The provided Table 1 of muscle-specific and ubiq sequence) from the coding sequence, often within several uitous promoters constitutes only a small portion of publicly (e.g., five) kilobases. In addition, some regulatory sequences available promoters. An extensive list of genes with expres can be found in introns of the gene of interest—these regula sion of interest (e.g., muscle-specific expression) can be tory sequences are usually omitted from constructing tissue found using NCBI protein database server (www at ncbi.nlm. specific gene expression “drivers' due to complexity of locat nih.gov/sites/entrez?db=Protein). For example, in order to ing them. It is to be understood that the same approach can be find mouse genes expressed in skeletal muscles a search used starting with "zebrafish skeletal muscle' or “medaka string “mouse skeletal muscle' can be used. The search skeletal muscle' or any other species. The searcher may then results in a list of proteins including their accession number continue the search as Suggested above to find the genome (e.g., CAA47621) and their name (e.g., mouse fast skeletal and promoter information of interest. It is also to be under muscle SR calcium ATPase). In order to find genome infor stood that methods similar to the one described for searching mation (e.g., sequence) of the found proteins, Ensembl the database referenced above can be used to search other Genome Browser (www at ensembl.org/index.html) can be existing sequence databases, as well as databases that are employed, using the accession number ((e.g., CAA47621) as likely to be compiled in the future.

TABLE 1. Muscle specific and ubiquitous promoters for fish expression Gene promoter Zebrafish Fugu Tetraodon Medaka Xenopus (2) Rat Mouse Dog Bovine Muscle specific

Muscle creatine kinase X X X X X X X X MyoD X X X X X X X X Myogenin X X X X X X X X Desmin X X X X X Muscle enolase-3 X X X X beta-Sarkoglycan X X X X X X X X Dystrophin X X X X X X X Serum response factor X X X X X X X X a-tropomyosin X X X X X X X X Myosin heavy chain X X X X X X X Mitochondrial creatine kinase 2 X X X X X X Myosin light chain X X X X X X X X Ca2+ transporting ATPase (fast twitch 1) X skeletal Troponin T1 (slow) X Tropomodulin 4 X Four and a half LIM domains 1 X Fast-type myosin binding protein C X Calsequestrin 1 X Fast muscle troponin C2 X Phosphorylase kinase alpha 1 X Skeletal troponin I(fast 2) X Ubiquitous

EF-1 alpha X X X X X X X X Histone 2AZA X X X X X X Acidic ribosomal phosphoprotein PO (ARP) X X X X X alpha-catenin X X X X X X X X beta-catenin X X X X X X X gamma-catenin X X X X X X X X Srb7 X X X X X X Creatine kinase(mitochondrial 1) X Ubiquitous Ca2+ transporting ATPase X Ancient ubiquitous protein X Ubiquitin specific peptidase 4 X Acetyl-Coenzyme A acryltransferase 2 X Monoglyceride lipase X Splicing factor 3b subunit 1 X Tubulin B5 X Beta-Actin (2) indicates text missing or illegible when filed US 2009/0025645 A1 Jan. 29, 2009

0045 Table 2, below, is a partial list of Ensembl gene ID numbers of mouse and Zebrafish skeletal muscle specific and TABLE 2-continued ubiquitous genes found using this approach. Ensembl IDs of Muscle Specific Promoters TABLE 2 SERUMRESPONSE FACTOR PROMOTERS Ensembl IDs of Muscle Specific Promoters Zebrafish: (ENSDARG00000053918) Fugu: (SINFRUGOOOOO162928) MUSCLE CREATINE KINASE PROMOTERS Tetraodon: (GSTENG00025 109001) Medaka: (ENSORLGOOOOOO13036) Zebrafish: (ENSDARGO0000035327) Xenopus tropicalis: (ENSXETG00000018511) Fugu: (SINFRUGO0000143294) Rat: (ENSRNOG00000018232) Tetraodon: (GSTENGOOO12956001) Mouse: (ENSMUSG00000015605) Medaka: (ENSORLGOOOOOOOO449) Dog: (ENSCAFG00000001829) Xenopus tropicalis: (ENSXETG00000019108) Bovine: (ENSBTAGOOOOOO12777) Rat: (ENSRNOGOOOOOO16837) ALPHA-TROPOMYOSIN PROMOTERS Mouse: (ENSMUSG00000030399) Dog: (ENSCAFG00000004507) Zebrafish: (ENSDARG00000033683) Bovine: (ENSBTAG00000013921) Fugu: (SINFRUGOOOOO130484) MYOD PROMOTERS Tetraodon: (GSTENGOOO1595.0001) Medaka: (ENSORLGOOOOOO12326) Zebrafish: (ENSDARG000000301.10) Rat: (ENSRNOG00000018184) Fugu: (SINFRUGOOOOO154785) Mouse: (ENSMUSG00000032366) Tetraodon: (GSTENGOOOO3954.001) Dog: (ENSCAFGOOOOOO16966) Medaka: (ENSORLGOOOOOOOO694) Bovine: (ENSBTAGO0000005373) Xenopus tropicalis: (ENSXETGOOOOOOO1320) MYOSIN HEAVY CHAIN PROMOTERS Rat: (ENSRNOGOOOOOO11306) Mouse: (ENSMUSGOOOOOOO9471) Zebrafish: (ENSDARG00000035437) Dog: (ENSCAFGOOOOOOO9066) Fugu: (SINFRUGO0000135173) Bovine: (ENSBTAG00000002216) Medaka: (ENSORLGOOOOOOO1985) MYOGENIN PROMOTERS Xenopus tropicalis: (ENSXETGOOOOOO23939) Rat: (ENSRNOG00000031400) Zebrafish: (ENSDARGOOOOOOO9438) Mouse: (ENSMUSG00000033196) Fugu: (SINFRUGOOOOO121801) Dog: (ENSCAFG00000023926) Tetraodon: (GSTENG00013986001) Bovine: (ENSBTAG00000007090) Medaka: (ENSORLGOOOOOO15906) MITOCHONDRIAL CREATINE KINASE Xenopus tropicalis: (ENSXETG00000001704) (SARCOMERIC, CKMT2) PROMOTERS Rat: (ENSRNOG00000030743) Mouse: (ENSMUSG00000026459) Zebrafish: (ENSDARGOOOOOO35079) Dog: (ENSCAFGOOOOOO 10309) Fugu: (SINFRUGOOOOO160265) Bovine: (ENSBTAGOOOOOOO6030) Tetraodon: (GSTENG00028607001) DESMIN PROMOTERS Medaka: (ENSORLG00000000769) Mouse: (ENSMUSG00000021622) Zebrafish: (ENSDARGOOOOOO58656) Dog: (ENSCAFGOOOOOOO8707) Fugu: (SINFRUGOOOOO121939) Bovine: (ENSBTAG00000001003) Xenopus tropicalis: (ENSXETG00000019275) MYOSINLIGHT CHAIN PROMOTERS Rat: (ENSRNOGOOOOOO19810) Mouse: (ENSMUSG00000026208) Zebrafish: (ENSDARGOOOOOO17441) Dog: (ENSCAFGOOOOOO15475) Fugu: (SINFRUGOOOOO125026) Bovine: (ENSBTAGO0000005353) Tetraodon: (GSTENGOOO15855.001) MUSCLE ENOLASE3 BETA PROMOTERS Medaka: (ENSORLGOOOOOO15981) Xenopus tropicalis: (ENSXETGOOOOOOO6917) Zebrafish: (ENSDARGOOOOOO39007) Rat: (ENSRNOGOOOOOO13262) Tetraodon: (GSTENG00003809001) Mouse: (ENSMUSG00000061816) Rat: (ENSRNOG00000004078) Dog: (ENSCAFGOOOOOO13875) Mouse: (ENSMUSG00000060600) Bovine: (ENSBTAGOOOOOOO9707) Bovine: (ENSBTAGO0000005534) BETA-SARCOGLYCAN PROMOTERS 0046 While this approach will result in a great number of Zebrafish: (ENSDARG00000052341) Fugu: (SINFRUGOOOOO123612) sequences, additional points should be considered to generate Tetraodon: (GSTENGOOO32779001) a list of strong promoters. For example, abundant structural Medaka: (ENSORLG00000000171) genes (e.g., myosin) or abundant enzymes (e.g., SR calcium Xenopus tropicalis: (ENSXETG00000011676) ATPase) are likely to yield strong promoters. This screening Rat: (ENSRNOGOOOOOOO2135) Mouse: (ENSMUSG00000029156) can easily be performed by an artisan in the field. Dog: (ENSCAFG00000002001) 0047 Preferably more than one construct with different Bovine: (ENSBTAG00000014601) promoters can be injected into the fish embryos simulta DYSTROPHIN PROMOTERS neously. For example, in the present invention, both Red Zebrafish: (ENSDARGOOOOOOO8487) Zebrafish 1 and Green zebrafish 1 incorporate more than one Fugu: (SINFRUGOOOOO144815) transgenic expression cassette, with one being a ubiquitous Tetraodon: (GSTENG00024870001) promoter, and the other being a strong muscle promoter. In Medaka: (ENSORLG00000020638) particular, Red Zebrafish 1 incorporates the cassettes repre Xenopus tropicalis: (ENSXETGOOOOOO12391) Rat: (ENSRNOGOOOOOOO3667) sented by FIG. 1 and FIG. 4, and Green Zebrafish 1 incorpo Mouse: (ENSMUSG00000045103) rates the cassettes represented by FIG.2 and FIG.5. While the Bovine: (ENSBTAGOOOOOOO8254) present invention incorporates only the transgenic insert cas settes shown in the Figures, it is understood that multiple US 2009/0025645 A1 Jan. 29, 2009 transgenic insert cassettes of any type can be simultaneously groups: regulatory proteins (for example, kit a receptor injected into a fish embryo from any species. tyrosine kinase, specific to melanophores) and biosynthesis 0048. It is also a subject of this invention to disclose enzymes, involved in specific pigment synthesis (for expression of the fluorescent protein gene specifically in example, sepiapterin reductase, involved in yellow pigment chromatophores. Chromatophores are pigment-containing synthesis in Xanthophores). Expression of regulatory proteins and light-reflecting cells found in animals. There are several usually is at lower level than that of biosynthesis enzymes types of chromatophores: melanophores (black), Xantho therefore use of promoters of biosynthesis enzymes are most phores (yellow), erythrophores (red), cyanophores (blue), preferred. A chromatophore-specific gene expression is out leucophores (white) and iridophores (reflective). Of those, lined in Table 3 below. only melanophores, called melanocytes, are found in higher 0049. Of all chromatophores, melanophores have been Vertebrates, such as mammals. Different species of fish con studied most extensively (due to their relevance to human tain all types of chromatophores, usually a Subset of them in biology). Therefore, a lot is known about transcription factors different combinations. Zebrafish contain melanophores, specific to melanophores, as well as biosynthesis enzymes xantophores and iridophores. These different cell types involved in melanin synthesis in different classes of organ express specific genes, characteristic only for them or specific isms, ranging from lower vertebrates to humans. The next for a Subset of chromatophores. For example, tyrosinase best characterized chromatophores are the Xanthophores, for related protein 1 (tyrp1) is found only in melanophores; which a number of genes have been isolated, yielding, a ednrb1 is found in malenocytes and iridophores. Promoters of number of known promoters to choose from. With respect to these specific genes fused to fluorescent protein open reading iridophores, a few specific genes have been isolated (for frames (ORFs) can be used to visualize specific chromato example, endothelin receptor b1 Ednrb.1). The least known phores. For example, fugu tyrp 1 promoter can be used to drive chromatophores are the cyanophores—neither the nature of fluorescent protein expression in melanophores in Zebrafish. their pigment, nor specification pathway of the cells perse is The specific genes can be roughly divided into two major known.

TABLE 3 Chromatophore-specific expressed genes in fishes Chromatophore Protein Synth Reg Organism Reference Iridophore ednrb.1 Reg Zebrafish Parichy etal, Developmental (endothelin Biology 227, 294-306 receptor b1) (2000) Xanthophore Xanthine Synth Guppy Ben et al, Mar Biotechnol dehydrogenase (Poecilia (NY). 2003 Nov-Dec: reticulata); 5(6): 568-78. Epub 2003 Zebrafish Aug 21; Parichy etal, Developmental Biology 227, 294-306 (2000) sepiapterin Synth medaka Negishi etal, Pigment Cell reductase (Oryzias Res. 2003 Oct; 16(5): 501-3 latipes) Xanthine Synth oxidoreductase FmSCSf1 Reg zebrafish Ziegler, Pigment Cell Res. 2003 Jun; 16(3): 172-82; Ziegler et al., J Biol Chem. 2000 Jun 23; 275 (25): 18926-32: Parichy etal, Development 127, 3031-3044 (2000) Melanophores Mitf Reg Zebrafish kit Reg Zebrafish yrp1 Synth zebrafish, Zou et al., Pigment Cell Res. fugu 2006 Dec. 19(6): 615-27 yrosinase Synth 88 Miura et al., Jpn. J Genet. nigromaculata 1995 Feb; 70(1): 79-92 yrosinase Synth medaka Inagaki etal, Pigment Cell Res. 1998 Oct; 11(5): 283-90 yrosinase Synth Mouse in Matsumoto et al., Pigment medaka Cell Res. 1992 Nov; 5(5 Pt 2): 322-7 rp2 (tyrosinase- Synth OSC Zhao & Overbeek, Dev Biol. related protein 2) 1999 Dec 1: 216(1): 154-63 dopachrome Synth automerase US 2009/0025645 A1 Jan. 29, 2009

0050. It is also known that the presence of introns in pri mary transcripts can increase expression, possibly by causing TABLE 4-continued the transcript to enter the processing and transport system for mRNA. It is preferred that the intron be homologous to the Fluorescent Proteins (FP) with their Maximum host species, and more preferably homologous to the expres Excitation and Emission Wavelengths sion sequences used (that is, that the intron be from the same FP Excitation max (nm) Emission max (nm) gene that some or all of the expression sequences are from). to Tomato 554 581 The use and importance of these and other components useful mStrawberry 574 596 for transgenic constructs are discussed in Palmiter et al. J-Red S84 610 DsRed-monomer 556 S86 (1991); Sippeletal. (1992); Kollias and Grosveld (1992); and mOrange S48 S62 Clarket al. (1993). mKO S48 559 0051. The steps involved in making the transgenic fish MCitrine S16 529 Venus 515 528 further involve isolation and separation of the transgenic Ypet 517 530 expression cassette from the vector backbone to remove the EYFP S14 527 gene encoding antibiotic (e.g., amplicillin or kanamycin) Emerald 487 509 resistance and origin of replication. In a preferred mode, a EGFP 488 507 suitable promoter is chosen which is expected to drive stable CyPet 435 477 mCFPm 433 475 expression throughout the life of the fish. To achieve such Cerulean 433 475 stable expression, it is necessary to choose a promoter that is T-Sapphire 399 511 known to drive stable and consistent expression throughout the life of the fish. For example, a promoter that drives expres sion only during the six months of the life of the fish would not 0053. The sequences of the DNAs which are useful in the be suitable to achieve stable expression throughout the life of invention are set forth in the attached Sequence Listing. 0054) The sequence listed herein as SEQ ID NO:1 is the the fish. transgenic fluorescence expression cassette having Zebrafish 0052. The heterologous fluorescent gene may be, for fast skeletal muscle specific myosin light chain (ZMLC) pro example, a gene encoding DsRed2, ZSGreen1 and Zsyel moter, DsRed2 (a red fluorescent protein gene from Antho low 1. The heterologous fluorescent gene may also be any Zoa, a reefcoral), and two copies of the SV40 polyadenylation variation or mutation of these genes, encoding fluorescent sequence. The sequence listed in SEQ ID NO: 1 is the proteins including green fluorescent protein (GFP), enhanced complementary sequence to the coding DNA strand. green fluorescent protein (eGFP), yellow fluorescent protein 0055. The sequence listed herein as SEQ ID NO:2 is the (YFP), enhanced yellow fluorescent protein (eYFP), blue transgenic fluorescence expression cassettehaving carp ubiq fluorescent protein (BFP), enhanced blue fluorescent protein uitous B-actin enhancer/promoter, DsRed2 (a red fluorescent (eBFP), cyan fluorescent protein (CFP) and enhanced cyan protein gene from Anthozoa, a reef coral), and two copies of fluorescent protein (eCFP) or any of the proteins listed in the SV40 polyadenylation sequence. The first exon and intron Table 4, below, or any variation or mutation thereof, or any of B-actin has been incorporated in the SEQID NO:2 to allow other fluorescence proteins. The steps involved in making the for increased expression of the fluorescence protein gene. transgenic fish also involve introduction of the transgenic 0056. The sequence listed herein as SEQ ID NO:3 is the expression cassette into the Zebrafish embryos or Zebrafish transgenic fluorescence expression cassette having Zebrafish embryonic stem cells. Such embryos and cells are allowed to fast skeletal muscle specific myosin light chain (ZMLC) pro grow and mature into adult fish and then they are screened for moter, ZsCreenl (a green fluorescent protein gene from the presence of the transgenic expression cassette using the Anthozoa, a reef coral), and two copies of the SV40 polyade various molecular biology methods described in the detailed nylation sequence. The sequence listed in SEQID NO:3 is the description section and/or by functional biochemical assays complementary sequence to the coding DNA strand. Such as assaying for the activity of the introduced fluorescent 0057 The sequence listed herein as SEQ ID NO:4 is the gene by exposing the said fish to light of appropriate wave transgenic fluorescence protein expression cassette having length and/or by visibly inspecting the fish and observing the Zebrafish fast skeletal muscle specific myosin light chain expression. Transgenic fluorescent fish are further bred to (ZMLC) promoter, Zsyellow 1 (a yellow fluorescent protein insure transmission of the transgenic expression cassette to gene from Anthozoa, a reef coral), and two copies of SV40 the germ cells of a fish as further described in this application. polyadenylation sequence. The sequence listed in SEQ ID NO:4 is the complementary sequence to the coding DNA TABLE 4 Strand. Fluorescent Proteins (FP) with their Maximum 0058. The sequence listed herein as SEQ ID NO:5 is the Excitation and Emission Wavelengths transgenic fluorescence protein expression cassette having carp ubiquitous B-actin enhancer/promoter, ZSOreen 1 (a FP Excitation max (nm) Emission max (nm) green fluorescent protein gene from Anthozoa, a reef coral), AmCyan1 458 489 and two copies of SV40 polyadenylation sequence. The first ZsCreen1 493 505 exon and intron of B-actin has been incorporated in the SEQ ZSYellow 1 529 539 DSRed2 563 582 ID NO:5 to allow for increased expression of the fluorescence DsRed-Express 557 579 gene AsRed2 576 592 HcRed1 S88 618 Chimeric Genes mPlum 590 649 mCherry 587 610 0059. The present invention encompasses chimeric genes comprising a promoter described herein operatively linked to US 2009/0025645 A1 Jan. 29, 2009 a heterologous gene. Thus, a chimeric gene can comprise a guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius. A promoterofa Zebrafish operatively linked to a zebrafish struc more complete list of ornamental fish species can be found in tural gene other than that normally found linked to the pro Table 5 below: moter in the genome. Alternatively, the promoter can be operatively linked to a gene that is exogenous to a Zebrafish, TABLE 5 as exemplified by the DSRed2 and other genes specifically exemplified herein. Furthermore, a chimeric gene can com Ornamental Fish Species prise an exogenous promoter linked to any structural gene not Scientific Name Common Name normally linked to that promoter in the genome of an organ ism. Steatocrantis Castiaritis African Blockhead Apistograma agassizi Agassizi Hyphessobrycon h axelrodi, sp bino Black Neon Tetra Substitutions, Additions and Deletions Lamprophogtis brichardi bino Bricardi Cichld Paracheirodon innessi, sp. bino Brilliant Neon Tetra 0060. As possible variants of the above specifically exem Hemigrammits caudovitatus bino Buenos Aires Tetra plified polypeptides, the polypeptide may have additional Hemigrammus erythrozonus bino Glow Light Tetra Hemigrammus ocellifer bino Head Tail Light Tetra individual amino acids or amino acid sequences inserted into Pelvicachromis pulcher bino Kribensis Cichlid the polypeptide in the middle thereof and/or at the N-terminal Aplocheit is normani bino Lampeye and/or C-terminal ends thereof So long as the polypeptide HypheSSobrycon pulchripinnis sp bino Lemon Tetra possesses the desired physical and/or biological characteris Paracheirodon innessi Oilino O Neon Tetra Macropodus opercularis spp bino Paradise Fish tics. Likewise, some of the amino acids or amino acid Pterophyllum scalare bino Red Eye Angel sequences may be deleted from the polypeptide so long as the Epaizeorhynchos fienatus bino Redfin Shark polypeptide possesses the desired physical and/or biochemi Hem. Rhodostomus sp. bino Rummy Nose cal characteristics. Amino acid Substitutions may also be Capoeia tetrazona bino Tiger Barb Astronotus ocellatus bino Tiger Oscar made in the sequences so long as the polypeptide possesses Tanichty's albonubes sp. bino White Cloud the desired physical and biochemical characteristics. DNA Lepisosteus oculatus igator Gar coding for these variants can be used to prepare gene con Luciosoma spilopleura pollo Shark Toxotesiactiiatrix Archer Fish structs of the present invention. Xiphophorus variatus Assorted Variatus 0061. A nucleic acid sequence “encodes' or “codes for a Badis badis Badis Badis polypeptide if it directs the expression of the polypeptide Heliostoma temmincki Balloon Kissing Gourami referred to. The nucleic acid can be DNA or RNA. Unless Corydoras metae Bandit Corydoras Pangasius Stitchi Bangkok Catfish otherwise specified, a nucleic acid sequence that encodes a Ancistri is doichopterus Big-Fin Bristlenose Golden Longfin polypeptide includes the transcribed strand, the hnRNA and Peocilia latipinna Black Balloon Molly the spliced RNA or the DNA representative thereof. Cichlasoma macuicauda ack Belt Cichlid Carrasilisatiratiis ack Butterfly Tail Callochromis macrops ack Eared Callochromis Degenerate Sequences Leptosoma Kittinba ack Finned Slender Cichlid Apteronotus albifrons ack Ghost 0062. In accordance with degeneracy of genetic code, it is Acanthopthalmis myersi ack Kuhlii possible to substitute at least one base of the base sequence of Bogrichthys hypselopterus ack Lancer Hyphessobrycon h axelrodi ack Neon Tetra a gene by another kind of base without causing the amino acid Nematobrycon palmerispp ack Palmeri sequence of the polypeptide produced from the gene to be Megalamphodus megalopterus ack Phantom changed. Hence, the DNA of the present invention may also Rasbora trilineata ack Scissor Tail Rasbora have any base sequence that has been changed by Substitution Labeo chrysopekadion ack Shark in accordance with degeneracy of genetic code. Piinii is filamentosus ack Spot Barb Rasbora agilis ack Stripe Rasbora Gymnocorymbusternetzi ack Tetra DNA Modification Astyanax fas. mexicantis ind Cave Tetra Brachydanio kerri ue Danio 0063. The DNA is readily modified by substitution, dele Inpaichty's kerri ue Emperor Tetra tion or insertion of nucleotides, thereby resulting in novel Trichogaster trichopterus ue Gourami Boehlkea fiedcochui ue King Tetra DNA sequences encoding the polypeptide or its derivatives. Xiphophorus machiatus ue Platy These modified sequences are used to produce mutant Melanoiaenia lacustris ue Rainbow polypeptide and to directly express the polypeptide. Methods Poecilia reticulata ue Ribbon Guppy for Saturating a particular DNA sequence with random muta Pseudotropheus zebra Blue Zebra Melanoiaenia boesemani Boesemani Rainbow tions and also for making specific site-directed mutations are Gastromyzon punctitiatus Borneo Sucker known in the art; see e.g. Sambrook et al., (1989). Datnoides microlepis Borneo Tiger Fish Paracheirodon innesi Brilliant Diamond Head Neon Rasbora birtiani Brittan S Rasbora Transgenic Fish Brachygobius doriae Bumble Bee Goby Anomalochromis thomasi Butterfly Cichlid 0064. The disclosed constructs and methods can be used Notesthes robusta Butterfly Goby with any type of fish that is an egg-layer. It is preferred that Paracheirodon axelrodi Cardinal Tetra - M fish belonging to species and varieties of fish of commercial Nomorhampheus iemi Celebes Halfbeak Telmatherina ladigesi Celebes Rainbow value, particularly commercial value within the ornamental Chaca bankanensis Chaca-Chaca fish industry, be used. Such fish include but are not limited to Capoeia oligolepis Checkered Barb catfish, Zebrafish, medaka, carp, tilapia, goldfish, tetras, Capoeia iiiteya Cherry Barb barbs, sharks (family cyprinidae), angelfish, loach, koi, glass Sphaerichthys Osphromenoides Chocolate Gourami fish, catfish, angel fish, discus, eel, tetra, goby, gourami, US 2009/0025645 A1 Jan. 29, 2009 11

TABLE 5-continued TABLE 5-continued Ornamental Fish Species Ornamental Fish Species Scientific Name Common Name Scientific Name Common Name Clarias battracus Clarias-Spotted Leptosoma malasa Leptosoma Malasa Epiplatys annitiatus Clown Killie? Rocket Rasbora paviei Line Rasbora Botia macracantha Clown Loach Capoeia artilius Long Fin Barb Haplochromis sp Cobalt Ice Blue Cichlid Alliesthes longipinnis Long Fin Characin Apistograma Cacatioides Cockatoo Dwarf Rasbora einihoveni Long-Band Rasbora Hyphessobrycon colombianus Colombia Tetra Meianotaenia naccuiochi Macculloch'S Rainbow Phenacogrammals interruptus Congo Tetra Paretropheus menoramba Madagascar Cichlid Corydoras aenetis Corydoras Albino Bedotia gaeli Madagascar Rainbow Corydoras panda Corydoras Panda Haplochromis compressiceps Malawi Eye Biter Corydoras paleathis Corydoras Peppered Ompok sp. Malay Glass Catfish Corydora,Spigny Corydoras Pigmy Betta splendens Male Betta Corydoras rabauti Corydoras Rabauti Cichlason managiiense Managuense Cichlid Corydoras similis Corydoras Similis Polypterus palmas Marbled Bichir Corydoras sterbai Corydoras Sterbai Xiphophorus helieri Millenium Swordtail Synodontis multipunctatus Cuckoo Synodontis Monodactylus argentiis Mono Angel Polypterus Senegalus Cuvier’S Bichir Cyrtocara moorii Morrii Synodontis decorus Decorated Synodontis Sawbwa resplendens Naked Micro Rasbora Polypterus delhezi Delhezi Bichir Hyphessobrycon h.axelrodisp. Negro Brilliant Black Neon Moenkhausia pitteri Diamond Tetra Melanoiaenia praecox Neon Dwarf Rainbow HypheSSobrycon amandae Ember Tetra Aplocheilus panchax New Golden Wonder Nematobrycon palmeri Emperor Tetra Synodontis ocellifer Ocellated Synodontis Polypterus endlicheri Endlicheri Bichir Coisa iabiosa Orange Thick Lipped Gourami Aphyocharax alburntis False Flame Tetra Polypterus ornatipinnis Ornate Bichir Synodontis eupterus Feathered Fin Synodontis Botia Locahanta Pakistani Loach Cichlasoma festae FestaS Cichlid Piinii is fasciatus Panda Barb Cichlasoma meeki Firemouth Cichlid Apistogramma pandhirini Pandurini Dwarf Piinii is pentazona Five Banded Barb Macropodus opercularis Paradise Fish Epaizeorhynchus kalopterus Flying Fox Cichlasoma sp. Parrot Crossocheilus Siamensis Flying Fox Cichia Oceiiaris Peackock Bass Cichlid Popondetta firCata Forktail Rainbow Trichogaster leeri Pearl Gourami Cyphoiilapia fontosa Frontosa Cichlid Cichlasoma carpinte Pearl Scale Cichlid Cyathopharynx furcifer Furcifer Lamprologits calvus Pearly Lamprologus Sturisoma firsochi Fursochi Cat Fish Tropheus PEMBA Pemba River Tropheus AphyOSemion gardneri Gardneri Killifish Thayeria boelkea Penguin Tetra Pseudomugilgeririidae Gertrudae Chalceus macrolepidotits Pinktail Characin Danio mailabarinchus Giant Danio Mogurnda mogurnda Purple Striped Gudgeon Ambassis ranga Glass Angel Rasbora sp. Rasbora Red Fin Prionobrama filigera Glass Bloodfin Aphyocharax rathbuni Red Belly Tetra Hypostonis piecostomits Glass Cleaner Plecostomus Cichlasoma iabiatum Red Devil Hemigrammus rodwayi Gold Tetra Moenkhauisia Santaefilomenae Red Eye Tetra Puntius Sachsi Golden Barb Pseudotrophies sp. Red Eyed Tangarine Cichlid Nannacara anomalia Golden Dwarf Cichlid Mastacembelus erythrotaenia Red Fire Eel Nannostomus beckfordi Golden Pencil Tetra Copadichromis bordeyi Red Kadango Pristeia maxiiiaris Golden Pristella Rasbora paticiperforata Red Line Rasbora Meianotaenia herbri axelrodi Golden Rainbow Colossoma macropodiin Red Pacu Scleropages formosus Green Arowana Megalamphodus Swegiesi Red Phantom Brachydanio rerio Green Danio Glossoiepis incisus Red Rainbow Aequidens rivulatus Green Terror Cichlid Cichlasoma severum Red Severum Cichlid Macrognathus circumcinctus Half Banded Spiny Eel Notropis lutrensis Red Shiner Rasbora heteromorpha Harlequin Rasbora Megalamphodus rosetts Red Tail Yellow Phantom Gasteropelectis Sternicia Hatchet Fish Epaizeorhynchos fienatus Redfin Shark Rasbora dorsioceiiata High Spot Rasbora Epaizeorhynchos bicolor Redtail Black Shark Geophagus Steindachneri Hondae Humphead Puntius conchonius Rosy Barb Ctenoiticit is hittieta Hujeta Hyphessobrycon bentosi Rosy Tetra Scleropagesjardini ardini Arowana Puntius rhomboceiiatus Round Banded Clown Barb Hemichromis paynei ewel Cichlid Piinii is nigrofasciatus Ruby Barb Melanochromis johanni ohanni Cichlid Hemigrammus bleheri Rummy Nose Tetra Julidichromis dickfeldi Juidchirmis Dickfeldi Arius graeffei Salmon Catfish Juidichromis ornatus Juidochromis Ornatus HypheSSobrycon serpae Serpae Tetra Julidichromis transcriptus Julidochromis Transcriptus HypheSSobrycon serpae sp Serpae Tetra Veil tail Geophagusiurupari urupari Cichlid Osteoglossum bichirrhosum Silver Arowana Tropheus IKOLA Kaisar Tropheus Distichodus affinis Silver Distichodus HypheSSobrycon loweae Kitti Tetra Metynnis hypsauchen Silver Dollar Stigmatogobius Sadant indio Knight Goby Selenotoca multifasciata Silver Sca Cyprint is Carpio Koi. Hasemania nanna Silver Tipped Tetra Acanthopthalmus kuhli Kuhlii Loach Balantiocheilos melanopterus Silver Tricolor Shark Lamprologus Silindericits Lamprologus Silindericus Rasbora espei Slender Wedge Rasbora Lamprologits leieupi Lemon Cichlid Pseudomugil Signifer Southern Blue Eye Labidochromis caeruleus Lemon Mbuna Cichlid Chiodits punctatus Spotted Headstander Hyphessobrycon pulchripinnis Lemon Tetra Rasbora maculata Spotted Pygmy Rasbora Ctenopoma actitirostre Leopard Bushfish Metynnis machiatus Spotted Silver Dollar Brachydanio frankei Leopard Danio Puntius ineatus Striped/Lined Barb US 2009/0025645 A1 Jan. 29, 2009

0067 Embryos or embryonic cells can generally be TABLE 5-continued obtained by collecting eggs as soon as possible after they are laid by methods that are well known to those of ordinary Ornamental Fish Species experience in the ornamental fish production field. Depend Scientific Name Common Name ing on the type of fish, it is generally preferred that the eggs be fertilized prior to or at the time of collection. This is prefer Scleropages formosus Super Red Arowana ably accomplished by placing a male and female fish together Corynopoma risei Swordtail Characin Cichlasoma Synspilum Synspillum Cichlid in a tank that allows egg collection under conditions that Irianiherina werneri Threadfin Rainbow stimulate mating. A fertilized egg cell prior to the first cell Capoeia tetrazona Tiger Barb division is considered a one cell embryo, and the fertilized Pseudoplatystoma fasciatin Tiger Shovelnose Catfish egg cell is thus considered an embryonic cell. Tilapia buttikoferi Tiger Zebra Tilapia Petrochromistrewavasae Trewavas'S Petrochromis 0068. The transgene may randomly integrate into the Tropheus duboisi Tropheus Duboisi genome of the embryo in one or more copies (concatemers). Mystus micracanthus Two Spotted Catfish After introduction of the transgenic construct, the embryo is Uaru amphiacanthoides Uaru - Triangle Cichlid Sphaerichthys vallianti Valliant S Gourami allowed to develop into a fish. The fish that were injected as Thayeria boehlkea sp. Veil tail Penguin Tetra embryos are allowed to interbreed and the offspring are Opthalmotilapia ventralis Ventralis screened for the presence of the transgene. Fish harboring the Haplochromis venustus Venustus transgene may be identified by any suitable means. In the Synodontis Schoutedeni Vermiculated Synodntis Tanichty's albonubes White Cloud preferred case, one or more of the transgenic constructs will Tanichtyhs albonubes White Cloud Minnow have integrated into the cellular genome, which can be probed Osphronentis gotirami White Giant Gourami for the presence of construct sequences. To identify trans Symphysodon aequifasciata White Smoke genic fish actually expressing the transgene, the presence of Aphyocharax paraguayensis White Spot Tetra Crenicichia saxalitus White Spotted Pike Cichlid an expression product can be assayed. Several techniques for Mastacenbei is armatiis White Spotted Spiny Eel Such identification are known and used for transgenic animals Gymnocorymbusternetzi White Tetra and most can be applied to transgenic fish. Probing of poten Beita coccina Wine Red Betta tial or actual transgenic fish for nucleic acid sequences Meianochromis auratus Yellow Auratus Cichlid Hemmigrammopetensius caudalis Yellow Congo present in or characteristic of a transgenic construct can be Apistograma borelli Yellow Dwarf Cichlid accomplished by Southern or northern blotting, polymerase chain reaction (PCR) or other sequence-specific nucleic acid amplification techniques. 0065. The more preferred fish for use with the disclosed 0069. The simplest way to confirm the presence of a fluo constructs and methods is zebrafish, Danio rerio. Zebrafish rescent protein expressing transgene in a given fish is by are increasingly popular ornamental animals and would be of visual inspection, as the fish in question would be brightly added commercial value in various colors. Zebrafish embryos colored and immediately distinguishable from non-trans are easily accessible and nearly transparent. The most pre genic fish. Preferred techniques for identifying fluorescent ferred fish for use with the disclosed constructs and methods protein expressing transgenic Zebrafish are described in the is the Golden Zebrafish. Zebrafish skin coloris determined by examples. The present invention also provides a method to pigment cells in their skin, which contain pigment granules obtain a new population or the progenitor of a new line of called melanosomes. The number, size and density of the fluorescent transgenic fish exhibits strong visible fluores melanosomes per pigment cell influence the color of the fish cence, strong visible fluorescence means that a person with skin. Golden Zebrafish have diminished number, size, and 20/20 vision (i.e., average vision) will be able to distinguish density of melanosomes and hence have lighter skin when between the fluorescent fish in question and a non-fluorescent compared to the wild type Zebrafish. Golden zebrafish have a fish of the same species at a distance of at least 5 feet in a mutation in Slc24.a5 gene, Slc24.a5 codes for a putative cation lighted office, with a preferred distance of at least 10 feet in a exchanger localized to intracellular membrane, rendering the lighted office, and a more preferred distance of at least 15 feet fish skin lighter or less pigmented (Lamason et al., 2005). in a lighted office, and an even more preferred distance of at least 20 feet in a lighted office, with the illumination level 0066. The disclosed transgenic fish are produced by intro defined in Table 6. One can observe all transgenic fluorescent ducing a transgenic construct into the genomes of cells of a fish from a particular population that exhibit strong visible fish, preferably embryonic cells, and most preferably in a fluorescence under the various lighting conditions and select single cell embryo. Where the transgenic construct is intro the fish that exhibits the highest level of visible fluorescence duced into embryonic cells, the transgenic fish is obtained by of the fluorescent protein. Selected fish with strong visible allowing the embryonic cellor cells to develop into a fish. The fluorescence are monitored and selected continuously to disclosed transgenic constructs can be introduced into embry ensure stability of expression and maintenance of the strong onic fish cells using any suitable technique. Many techniques visible fluorescence trait. Thus a new line of fish exhibiting for Such introduction of exogenous genetic material have strong visible fluorescence is created for further breeding. been demonstrated in fish and other animals. These include 0070 The invention further encompasses progeny of a microinjection (Culpet al., (1991), electroporation (Inoue et transgenic fish containing a genomically integrated trans al., 1990; Muller et al., 1993; Murakami et al., 1994; Muller genic construct, as well as transgenic fish derived from a et al., 1992; and Symonds et al., 1994), particle gunbombard transgenic fish egg, sperm cell, embryo, or other cell contain ment (Zelenin et al., 1991), and the use of liposomes (Szelei ing a genomically integrated transgenic construct. “Progeny.” et al., 1994). The preferred method for introduction of trans as the term is used herein, can result from breeding two genic constructs into fish embryonic cells is by microinjec transgenic fish of the invention, or from breeding a first trans tion. genic fish of the invention to a second fish that is not a US 2009/0025645 A1 Jan. 29, 2009

transgenic fish of the invention. In the latter case, the second tions, parameters, reagents, or starting materials which must fish can, for example, be a wild-type fish, a specialized strain be utilized exclusively in order to practice the art of the of fish, a mutant fish, or another transgenic fish. The hybrid present invention. progeny of these matings have the benefits of the transgene for fluorescence combined with the benefits derived from Example 1 these other lineages. Design and Generation of the Construct Plasmids Fertilization from Frozen Sperm 0078. The promoter of the Zebrafish fast skeletal muscle 0071 Sperm freezing methods are well known in the art, myosin light chain (ZMLC2) (Ju et al., 2003) and the carp for example see Walker and Streisinger (1983). Frozen B-actin enhancer/promoter sequence (Lui et al., 1990) were Zebrafish sperm may be used to fertilize eggs also as cloned into pBluescript II SK (-) and puC18 respectively. described in Walker and Streisinger (1983), incorporated Red fluorescent protein gene, DSRed2; green fluorescent pro herein by references. Briefly, a droplet of ice-cold 100% tein gene, ZSGreen1 and yellow fluorescent protein gene, Hank's Saline is placed next to Zebrafish eggs in a petri dish. ZsYellow 1 were amplified by PCR from posRed2-N1, pZs Frozen sperm is thawed for a few seconds in air then expelled Green1-N1 and pZsyellow 1-N1 (Clontech Inc., Matz. et al., into the droplet of Hank's saline and the solution is mixed 1999) respectively and cloned into pBluescript II SK (-) with the eggs. The mixture is incubated for about ~1 minute ZMLC2 and pUC18-carp B-actin such that the promoter was and then fish water added. operably linked to the fluorescent gene. Tandem SV40(A) polyA/3'UTR sequence from pK-SV40(A)X2 plasmid were Vectors cloned 3' to the fluorescent protein gene coding region. It is preferred to use more than one copy of the selected polyaden 0072 The invention is further directed to a replicable vec lyation sequence, and more preferred to use a viral polyade tor containing cDNA that codes for the polypeptide and that is nylation sequence, as this will increase the efficiency of the capable of expressing the polypeptide. fluorescent protein gene expression. The resulting five con 0073. The present invention is also directed to a vector struct vector maps are provided as FIG. 1 through FIG. 5. comprising a replicable vector and a DNA sequence corre sponding to the above described gene inserted into said vec Example 2 tor. The vector may be an integrating or non-integrating vec Preparation of the Construct for Delivery tor depending on its intended use and is conveniently a plasmid. The present invention also encompasses the removal (0079. The vectors puC18-carp B-actin-DsRed2 and of the vector backbone from the plasmid before the transgenic pUC18-carp B-actin-ZsGreen1 were restriction double construct may be introduced into the Zebrafish. digested with Xbaland Aati Ienzymes for three hours (FIG. 6, Step 1) and then run on 0.8% agarose gel to separate the Transformed Cells transgenic insert cassette from the vector backbone (FIG. 6, Step 2 and 3). Transgenic insert cassette band (-3.5 kb) which 0.074 The invention further relates to a transformed cellor contained the promoter, the open reading frame and the microorganism containing cDNA or a vector which codes for 3'UTR was excised and purified using phenolicholoroform the polypeptide or a fragment or variant thereof and that is extraction. capable of expressing the polypeptide. 0080. The transgenic vectors pBluescript IISK(-)-ZMLC Dsked2-SV40x2, pBluescript II SK(-)-ZMLC-ZsGreen1 Expression Systems. Using Vertebrate Cells SV40x2, and pBluescript II SK(-)-ZMLC-ZsYellow 1 SV40x2 were restriction triple digested with XhoI, XmnI and 0075 Interest has been great invertebrate cells, and propa NotI enzymes for three hours and then run on 0.8% agarose gation of vertebrate cells in culture (tissue culture) has gel to separate the transgenic insert cassette from the vector become a routine procedure. Examples of vertebrate host cell backbone. The transgenic insert cassette band (-3.2 kb) lines useful in the present invention preferably include cells which contained the promoter, the open reading frame and the from any of the fish described herein. Expression vectors for 3'UTR was excised and gel purified. Such cells ordinarily include (if necessary) an origin of rep lication, a promoter located upstream from the gene to be Example 3 expressed, along with a ribosome-binding site, RNA splice site (if intron-containing genomic DNA is used or if an intron Making the Transgenic Fish is necessary to optimize expression of a cDNA), and a poly I0081. The purified transgenic insert cassette which con adenylation site. tained the promoter, the open reading frame and the 3'UTR 0076. In another aspect of the present invention, also was microinjected into the Zebrafish embryos (FIG. 6, Step included is the commercial marketability of the transgenic 4). fluorescent fish to the ornamental fish industry. I0082 While only one construct was injected into Yellow Zebrafish 1, to increase the chances of developing a fish with EXAMPLES strong visible fluorescence, more than one construct was injected simultaneously in Red Zebrafish 1 and Green 0077. The invention will now be further described with Zebrafish 1. For the purposes of this application, strong visible reference to the following examples. These examples are fluorescence means that a person with 20/20 vision (i.e., intended to be merely illustrative of the invention and are not average vision) will be able to distinguish between the fluo intended to limit or restrict the scope of the present invention rescent fish in question and a non-fluorescent fish of the same in any way and should not be construed as providing condi species at a distance of at least 5 feet in a lighted office, with US 2009/0025645 A1 Jan. 29, 2009

a preferred distance of at least 10 feet in a lighted office, and taneously injected into a fish embryo from any species. Once a more preferred distance of at least 15 feet in a lighted office, injected, the embryos were allowed to grow into adult fish. At and an even more preferred distance of at least 20 feet in a that point, they were spawned to determine if their offspring lighted office, with the illumination level defined in Table 6. carried the fluorescence trait. The preferred method of spawn 0083) Given the same illumination levels, distances, and ing is a single pair spawn between a Zebrafish that had been observer of average vision, another preferable quality of fish injected as an embryo and a wild-type Zebrafish. The off that exhibit strong visible fluorescence are those fish that also spring of the transgenic Zebrafish were raised to maturity and exhibit ubiquitous expression of the fluorescence, defined the fluorescent fish selected for further examination. In the herein to mean strong fluorescence that is not limited to a preferred method, the offspring should be screened for by particular tissue type or body location, with Such expression exposure to lights of specific wavelengths while they are still preferably including fins, eyes, Stripes or spots. Typically, embryos. For example, for green fluorescent protein an exci ubiquitous fluorescent expression will mean that the fluores tation max at 493 nm with emission max at 505 nm, for red cent expression is visible over 75% to 100% of the body of the fluorescent protein an excitation max at 563 nm and an emis fish (excluding fins and eyes). The inventors have discovered sion max at 582 nm and for yellow fluorescent protein an that the use of a ubiquitous promoter in combination with a excitation maxat 529 mm and an emission maxat 539 nm was tissue specific promoter (Such as a muscle promoter), particu used corresponding, for example, to ZSGreen1, DSRed2 and larly where such fish are prepared using at least two expres ZSYellow 1. sion vectors, will generally result in fish having the desirable I0087. The foregoing method was used to screen for the ubiquitous expression trait. In this more preferred example, most esthetically pleasing fish while still maintaining the the fluorescent pattern exhibited by the fish would also be free ability to efficiently breed. from any patches of non-expression or noticeably weak or dull expression, with the possible exception of non-expres Example 4 sion in fins, eyes, and stripes or spots. Expression in the fins, eyes, and stripes or spots is also preferred, but not required for Selecting the Transgenic Fish a fish to be considered as exhibiting ubiquitous fluorescent I0088 Any fish showing fluorescence as embryos or juve expression. niles were grown to maturity and examined for fluorescence 0084 Examples of fish exhibiting strong visible fluores as an adult to determine which specific fluorescent fish was to cence are the lines which are the subject of the present inven be used as a progenitor for a new line. In this endeavor, the tion. Color photographs of these fish are available through most valuable expression pattern is one that meets the defi World Wide Web at glofish.com/photos.asp. Color photo nition of a fish exhibiting strong visible expression as defined graphs of fish that are fluorescent, yet that do not exhibit herein, and even more preferred are those that also exhibit strong visible fluorescence are available through World Wide ubiquitous expression, as this strong expression would Web at glofish.com/old glofish.asp. increase both the aesthetic appeal and commercial value of 0085. To obtain strong visible fluorescence, it is preferred the fish. In particular, it is important to be sure the fish exhibits to use a promoter that expresses ubiquitously and co-inject strong visible fluorescence in all of the lighting conditions this promoter with a strong muscle promoter. It is also pre described in Table 6 below. ferred to use enhancing elements in the transgenic insert cassette. For example, in the present invention, both Red TABLE 6 Zebrafish 1 and Green zebrafish 1 incorporate more than one transgenic expression cassette, with one being a ubiquitous Common Light Levels - Indoors and Outdoors promoter, and the other being a strong muscle promoter. In Condition Illumination (lux) particular, Red Zebrafish 1 incorporates the cassettes repre Full Daylight 10,000 sented by FIG. 1 and FIG. 4, and Green Zebrafish 1 incorpo Overcast Day 1OOO rates the cassettes represented by FIG. 2 and FIG. 5. In the Lighted Home >150 cassette that includes the ubiquitous promoter, there is also an Lighted Office 500 intron and exon, which exemplifies the type of RNA process Dark Indoor Room SO ing element that is helpful in achieving strong visible fluo Moderately LitRoom 100-1SO CSCCC. I0086 To co-inject the embryos, multiple purified trans I0089. Accordingly, to ensure that a progenitor for a new genic insert cassettes can simply be loaded into the microin line of fluorescent fish exhibits strong visible fluorescence, jection needle simultaneously and then injected. Alterna one can observe all transgenic fluorescent fish from a particu tively, in the preferred method, the injection of constructs lar population that exhibit strong visible fluorescence under containing multiple (two or more) fluorescent protein expres the various lighting conditions noted above in Table 6, and sion cassettes can be made using common molecular biology select the fish that exhibits the highest level of visible fluo techniques, such as DNA digestion and ligation. In the most rescence of the fluorescent protein. Selection of this fish is preferred method, a plasmid can be made which contains based on visible observation only, as commercial appeal will several fluorescent protein expression cassettes in tandem, be based on visual appearance. When testing the fish in a and then treated in the same way as disclosed herein for a completely darkroom, it is preferred to use an ultraviolet light single fluorescent protein expressing plasmid (that is, made, to observe the level of the fish's fluorescent expression, as the isolated, purified, and linearized with the antibiotic resistance ambient light will typically be insufficient to observe even the marker gene and replication origin removed before injection). most strongly expressing fluorescent fish. While the present invention incorporates only the transgenic (0090. It is also preferred to provide fish that exhibit a insert cassettes shown in the Figures, it is understood that reasonably stable color over the entire life of the fish, varying multiple transgenic insert cassettes of any type can be simul no more than about 20% at any given age as compared to very US 2009/0025645 A1 Jan. 29, 2009

young fish of the same line. For example, the inventors have while the homozygous fish population was maintained noted that some fish, particularly those that are not prepared through traditional methods to ensure a future homozygous by the more preferred methods of the present invention, tend breeding population. to dramatically lose their color brilliance over time, and can become indistinguishable from non-transgenic fish of the Example 7 same species, even as young as one year old. Preferred trans Potential Application of the Transgenic Fish genic fish of the present invention can be selected for this trait 0094. The fluorescent transgenic fish have use as orna by monitoring the fish over its development cycle. It is also mental fish in the market. Stably expressing transgenic lines preferred to select fish that are stable without regard to the can be developed by breeding a transgenic individual with a ambient physical environment of the tank (e.g., color of wildtypefish, mutant fish or another transgenic fish. Multiple gravel, plants, etc.). This can be ensured by selecting fish that color fluorescent fish may be generated by the same technique do no lose their color brilliancy over time or in response to the as red fluorescent fish, yellow fluorescent fish and green fluo physical environment. rescent fish. By recombining different tissue specific promot 0.091 Mendelian inheritance of the fluorescent trait is con ers and fluorescent protein genes, more varieties of transgenic sistent with an integration event at a single locus in the fish of different fluorescent color patterns will be created. By selected fish. The progeny from the originally selected expression of two or more different fluorescent proteins in the Zebrafish comprising this particular transgenic event can be same tissue, an intermediate color may be created. For used for further breeding through traditional means with example, combing expression of both red fluorescent protein unmodified Zebrafish to establish a new line of fluorescent gene and yellow fluorescent protein gene under a muscle fish through methods that are well known to those of ordinary specific promoter, an orange fluorescent Zebrafish may be skill in the production of fish, wherein the vast majority of created. fluorescent fish derived from this progenitor exhibited a mate 0.095 The fluorescent transgenic fish should also be valu able in the market for scientific research tools because they rially similar fluorescence pattern and strength as the found can be used for embryonic studies Such as tracing cell lineage ing fish. It is also preferred that the selected fish be monitored and cell migration. Cells from transgenic fish expressing for stability and consistency of expression, as any life-cycle green fluorescent protein can also be used as cellular and variance from strong visible fluorescence that is seen in the genetic markers in cell transplantation and nuclear transplan selected fish may be passed along to the offspring. Addition tation experiments. Additionally these fish can be used to ally, to facilitate consistency of expression, it is also neces mark cells in genetic mosaic experiments and in fish cancer sary to remove from the breeding population of this line any models. fish that appear from time to time with an expression pattern 0096 All of the compositions and/or methods disclosed which is visibly weaker than the original founder. and claimed herein can be made and executed without undue 0092. The specific transgenic events embodied in these experimentation in light of the present disclosure. While the fish are designated Red Zebrafish 1, Green Zebrafish 1 and compositions and methods of this invention have been Yellow zebrafish 1 respectively. Sperm from these fish may be described interms of preferred embodiments, it will be appar used to fertilize Zebrafish eggs and thereby breed transgenic ent to those of skill in the art that variations may be applied to Zebrafish that comprise these specific transgenic integration the compositions and/or methods and in the steps or in the events. Sperm from each line is deposited at the European sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. Collection of Cell Cultures (ECACC) as “Red Zebrafish 1” More specifically, it will be apparent that certainagents which (provisional accession no. 06090403), “Green Zebrafish 1” are both chemically and physiologically related may be Sub (provisional accession no. 06090401) and “Yellow zebrafish stituted for the agents described herein while the same or 1’ (provisional accession no. 06090402). similar results would beachieved. All such similar substitutes and modifications apparent to those skilled in the art are Example 6 deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Breeding the Transgenic Fish REFERENCES 0093. Once the transgenic line had been established as (0097. The following references, to the extent that they described above, fish that were homozygous for the fluores provide exemplary procedural or other details Supplementary cence trait were obtained by crossing fish that were heterozy to those set forth herein, are specifically incorporated herein gous for the fluorescence trait, and then the progeny were by reference. screened to determine whether they were homozygous for the 0.098 U.S. Pub No. US 2004/0143864 fluorescence trait. The preferred method of screening the (0099 Barolo et al., Biotechniques, 36(3):436-440; 442, progeny is through a test cross with a wild-type Zebrafish, 2004. where any fluorescent fish that produces 100% fluorescent 0100 Bourett et al., Fungal Genet. Biol., 37(3):211-220, offspring would be homozygous for the fluorescent trait. 2002. Once enough homozygous fish were found to create a mini 0101 Brem et al., Aquaculture, 68:209-219, 1988. mal breeding population, they were crossed to produce addi 01.02 Carvan et al., Ann. NY Acad. Sci., 919:133-147, tional homozygous progeny. Upon adulthood, these progeny 2OOO. were crossed with wild-type fish to obtain progeny that were (0103 Chen et al., J. Virol., 62:3883-3887, 1988. heterozygous for the fluorescent trait. These heterozygous 0104 Cho et al., Insect. Biochem. Mol. Biol., 36(4):273 fish were then sold to the commercial ornamental fish market, 281, 2006. US 2009/0025645 A1 Jan. 29, 2009 16

0105 Chourrout et al., Aquaculture, 51:143-150, 1986. 0.134 Muller et al., Mol. Mar: Biol. Biotechnol., 1:276 01.06 Clarket al., Phil. Trans. R. Soc. Lond. B., 339:225 281, 1992. 232, 1993. 0.135 Murakami et al., J. Biotechnol., 34:35-42, 1994. 0107 Cozzi and White, Nat. Med., 1(9):964-966, 1995. (0.136 Navarro et al., J. Virol.., 78(9):4744-4752, 2004. 0108 Culpet al., Proc. Natl. Acad. Sci. USA, 88(18):7953 0.137 Palmiter et al., Nature, 300:611-615, 1982. 7957, 1991. 0.138 Palmiter et al., Proc. Natl. Acad. Sci. USA, 88:478 0109) Delvin et al., Can. J. Fisheries Aqua. Sci., 52:1376 482, 1991. 1384, 1995. I0139 Penman et al., Aquaculture, 85:35-50, 1990. 0140 Powers et al., Mol. Marine Biol. Biotechnol., 1:301 0110 Delvin et al., Nature, 371:209-210, 1994. 308, 1992. 0111 Du et al., Bio/Technology, 10:176-181, 1992. 0141 Royer et al., Transfenic Res., 14(4):463-472, 2005. 0112 Eckert et al., FEMS Microbiol. Lett., 253(1):67-74, 0.142 Sambrook and Russell, Molecular Cloning: A Labo 2005. ratory Manual 3" Ed., Cold Spring Harbor Laboratory 0113 Finley et al., Biotechniques, 31 (1):66-70; 72, 2001. Press, 2001. 0114 Gong et al., Biochem. Biophys. Res. Commun., 308 0.143 Sambrook et al., In: Molecular cloning: a labora (1):58-63, 2003. tory manual, 2" Ed., Cold Spring Harbor Laboratory 0115 Gordon et al., Proc. Natl. Acad. Sci. USA, 77:7380 Press, Cold Spring Harbor, N.Y., 1989. 7384, 1980. 0144 Sarkar et al., BMC Biotechnol. 6(1):27, 2006. 0116 Gross et al., Aquaculature, 103:253-273, 1992. 0145 Sato et al., Biochem. Biophys. Res. Commun., 311 0117 Hadjantonakis et al., Nat. Rev. Genet., 4(8):613 (2):478-481, 2003. 625, 2003. 0146 Schmid et al., Glia., 53(4):345-351, 2006. 0118 Hamada et al., Brain Res. Mol. Brain Res., 139(1): 0147 Sin et al., Aquaculature, 1 17:57-69, 1993. 42-51, 2005. 0148 Sippel et al., In: The Regulatory Domain Organiza 0119 Handler and Harrell, Biotechniques, 31(4):820: tion of Eukaryotic Genomes. Implications For Stable Gene 824-828, 2001. Transfer, Transgenic Animals, Grosveld and Kollias 0120 Horn et al., Insect. Biochem. Mol. Biol., 32(10): (Eds.), Academic Press, 1-26, 1992. 1221-1235, 2002. 0149 Symonds et al., Aquaculture, 119:313-327, 1994. 0121 Inoue et al., Cell. Differ. Develop., 29:123-128, 0150. Szelei et al., Transgenic Res., 3:116-119, 1994. 1990. 0151 Tolar et al., Mol. Ther, 12(1):42-48, 2005. 0122 Ju et al., Dev Dyn., 227(1):14-26, 2003. 0152 Tsai et al., Can. J Fish Aquat. Sci., 52:776-787, (0123 Khoo et al., Aquaculture, 107:1-19, 1992. 1995. 0.124 Kollias and Grosveld, In: The Study of Gene Regu O153 Vintersten et al., Genesis, 40(4):241-246, 2004. lation in Transgenic Mice, Transgenic Animals, Grosveld 0154) Walker and Streisinger, Genetics 103: 125-136, and Kollias (Eds.), Academic Press, 79-98, 1992. 1983. 0125 Lamason et al., Science, 310(5755):1782-1786, O155 Wall et al., Nat. Struct. Biol., 7(12): 1133-1138, 2005. 2OOO. 0126 Lathe and Mullins, Transgenic Res., 205):286-299, 0156 Wencket al., Plant Cell Rep., 22(4):244-251, 2003. 1993. O157 Werdien et al., Nucleic Acids Res., 29(11):E53-3, O127 Liu et al., Biotechnology, 8:1268-1272, 1990. 2001. 0128 Long et al., BMC Biotechnol., 5:20, 2005. 0158 Wouters et al., Physiol. Genomics, 203):412-421, 0129 Maga and Murray, Biotechnology, 13(13): 1452 2005. 1457, 1995. 0159) Wright et al., Biotechnology, 9:830-834, 1991. 0130 Matz et al., Nat. Biotechnol., 17:969-973, 1999. (0160 Xu et al., DNA Cell Biol., 18, 85-95, 1999. 0131 Mikkelsen et al., FEMS Microbiol. Lett., 223(1): (0161 Zelenin et al., FEBS Lett., 287(1-2):118-120, 1991. 135-139, 2003. (0162 Zeller et al., Dev. Dyn., 235(2):456-467, 2006. (0132) Miyawaki, Cell Struct. Funct. 27(5):343-347, (0163 Zhu and Zon, Methods Cell Biol., 76:3-12, 2004. 2002. (0164 Zhu et al., Dev. Biol., 28.1(2):256-269, 2005. 0133) Muller et al., FEBS Lett., 324:27-32, 1993. (0165 Zhu et al., Z. Angew. Ichthyol., 1:31-34, 1985.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 5

<210 SEQ ID NO 1 <211 LENGTH: 3119 &212> TYPE: DNA <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Cloned expression cassette <4 OO SEQUENCE: 1

ggcc.gcaa.ca gcggaatgac cacatttgta gaggttttac ttgctittaaa aaac ct coca 60

US 2009/0025645 A1 Jan. 29, 2009 18

- Continued tittgaaaatgaat atttittc. tccatctotc agtgaatata ggcaatgitat tittggtgcat 24 OO ttaaacaaaa cagatttatt aaa.ca.gctat atttattaaa ataat attitt agt cacaaaa 246 O ttgaaagata aaacaattta aataaagcta aatattgcaa caaaaaaatt acaacct aca 252O aattittgaac taattitttitt coattgttitt gcttctic titg attitt cocct gtttataaat 2580 ttgt atttaa tatttittcaa taa catataa atatgggtgt agtagtttitt ggaccgittat 264 O cataagtt at tttgttaaat aagct coaga tittagct tca citct cagaaa taaaggg.cgt. 27 OO gagctgtcac toggggtggta cctitt coaaa tdataaaaat ttgtacctta aaggit coat a 276 O ataaaacctic aagggtatat attagtacct aaaaagtaca aaagttgtttic ticittaaaatt 282O tittagg cact aatatatact tttgaggitat caatatggac cctittaagta caaatatgta 288O

CCttittgaaa agg taccacc gcagtgacag Cttgcggacc atttatttct gagagtgtac 294 O tgactaatct aatgtatatg cacaaatata acatagottc ct attaaaaa tattaattta 3 OOO aaagacagat ttgggtgttt CCC agtgtag ggttgcaact ggatggg cat CCCtgcata 3 O 6 O aaaaatacac tatgagtt ggtggctic at tcct ctgtgg catcgatac C9tcgacct 3119

<210 SEQ ID NO 2 <211 LENGTH: 3656 &212> TYPE: DNA <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Cloned expression cassette <4 OO SEQUENCE: 2 ctagaaccat gattacgc.ca agcttittaga cct tct tact tittggggatt atataagtat 6 O tittct caata aatat ct cat atct tact.gt gig tittaactg. citgaatctaa aattittaata 12 O caaaagtagt tatatttgtt gtacattgta aactata act taact tcagt ttcagagaaa 18O ct catgtgct caaaatgtaa aaaaagtttc ctdttaaata ttttgtaaat g tattgaaga 24 O caaaataaga aaaaaaaaaa tataa.gc.cac taaat cacac tdtcc ttggit atcagcaaga 3OO gattctgaca taatcagotg tttttgttta t tact.gc.cat tdaaggc.cat gtgcattagt 360 cc caagttac acattaaaaa gttcacatgta gcttaccaac at cagtgctg ttcaa.gcaca 42O gcct catcta ctatt caaac tdtggcacca totaaaatat gcc agaattt ttittatttaa 48O tgaatttgac cct gaaatat g tattaatat cactic ctdtg atttittttgt aat cagotta 54 O caattacagg aatgcaa.gcc tdatt catta caagttt cac tacactittct ctogacaacat 6OO caccitact ga act cagacca gctagttgct c cittaagtat acaat catgt cactaatcct 660 catttcaatgaaaaat acco ctattgtact togg tacttgg tagataacca cagagcagta 72 O titatgc catt attgttgaata caataagagg taaatgacct acagagctgc tigctgctgtt 78O gtgttagatt gtaalacacag cacaggat.ca aggaggtgtc. Cat Cactatg accalatact a 84 O gcactittgca Caggct Cttt gaaaggctga aaagagcctt attggcgitta t cacaacaaa 9 OO atacgcaaat acggaaaa.ca acg tattgaa citt cqcaaac aaaaaacagc gattittgatg 96.O aaaatcgctt aggccttgct cittcaaacaa to cagottct c cttctitt.ca citct caagtt 1 O2O gcaagaagca agtgtagcaa ttgcacgcg acagc.cgggt gtgtgacgct ggaccalatca 108 O gagggcagag Ctc.cgaaagt ttaccttitta tigctagagc C9gcatctgc cgt catataa 114 O alaga.gc.gc.gc C cagogtctic agcct cactt tagctic ct c cacacgcagc tagtgcggaa 12 OO

US 2009/0025645 A1 Jan. 29, 2009 24

- Continued gCaggcagtg Ctgcagcagg gtgttgaccta Ctttagctaa C9ttagc.cgg Ctalaccagda 1740 ttcatctgcc ggtaacttga gttctaatatt citctatotga tat cqaagtg atcaaagaca 18OO cgtctgttag ct cactittaa ccaactgtag tdaaaaatag cqcagtgtgc agc ccttcaa 1860 gtctitt catt taggctgatt attcaatcat titt attaact attaacg.cgt tactaaacgt. 1920 alaggtaacgt agt cagttitt taataactgg taaaagtac tigttgggitt taaatggtga 198O cittataattig togttggaggg ggaaacctitt ttgataaagg ctatataatc. tcaaatgaat 2O4. O gggctgagga tiggtgttcac aggtgctitta gtgaagt ccg Ctcgtgaaga gtc.gctgaag 21OO tgactgcaga t ctgtagcgc atgcgttittg gCagacggcc gttgaaattic ggttgagtaa 216 O ttgataccag gtgaggctag aggatgtaga aatt catttgttgtagaattt agggagtggc 222 O ctggcgtgat gaatgtcgaa atc.cgttcct ttt tactgaa ccc tatgtct c togctgagtg 228O ccacaccgcc ggcacaaag.c gttct caaacc attgccttitt atggtaataa tdagaatgca 234 O gagggactitc Ctttgttctgg cacatctgag gcgc.gcattg tca cact agc accCactago 24 OO ggtcagactg. Cagacaaaca ggaagctgac tocacatggit Cacatgctica citgaagttgtt 246 O gact tcc.ctg acagotgtgc actittctaaa ccggittittct catt cattta cagttcago c 252O gggt accgaa titcgacggtc gccaccatgg CCC agtic caa goacggcctg accaaggaga 2580 tgac catgaa gtaccgcatg gagggctg.cg tdgacggcca Caagttcgtg at Caccggcg 264 O agggcatcgg ct accc ctitc aagggcaa.gc aggcc at Caa cctgtgcgt.g. gtggagggcg 27 OO gccCCttgcc Ctt cqc.cgag gaCat Cttgt CCC.cgc.ctt catgitacggc aaccgcgtgt 276 O t caccgagta cccc.caggac atcgt.cgact acttcaagaa citcctg.cccc gcc.ggctaca 282O

Cctgggaccg CtcCtt Cotg titcgaggacg gcgc.cgtgtg Catctgcaac gcc.gacatca 288O cc.gtgagcgt ggaggagaac to atgtacc acgagtic caa gttctacggc gtgaact tcc 294 O cc.gc.cgacgg ccc.cgtgatg aagaagatga cc.gacaactg ggagc cct cc tecgaga aga 3 OOO t catcc.ccgt gcc caa.gcag ggcatcttga agggcgacgt gag catgitac Ctgctgctga 3 O 6 O aggacggtgg cc.gcttgcgc tigc.cagttcg acaccgtgta Caaggccaa.g. tcc.gtgc.ccc 312 O gcaagatgcc cactggcac ttcatc.ca.gc acaagctgac cc.gcgaggac cqcagcgacg 318O cCaagaacca gaagtggcac ctgaccgagc acgc.catcgc Ctcc.ggct cc gccttgc cct 324 O gagact agtg atccagagtt cattgatgag tittggacaaa C cacaac tag aatgcagtga 33 OO aaaaaatgct ttatttgttga aatttgttgat gctattgctt tatttgtaac cattataagc 3360 tgcaataaac aagttaacaa caacaattgc att catttta tottt caggit to agggggag 342O atgtgggagg ttttittaaag caagtaaaac Ctctacaaat gtggt cattg atgagtttgg 3480 acaaac caca act agaatgc agtgaaaaaa atgctittatt tdtgaaattt gtgatgctat 354 O tgctittattt gtaaccatta taagctgcaa taaacaagtt aacaacaa.ca attgcattca 36OO ttittatgttt Caggitt Cagg gggagatgtg ggaggtttitt taaagcaagt aaaacct ct a 366 O caaatgtggt cattcc.gctg ttgacgtc 3 688

1. A transgenic fluorescent Zebrafish having integrated into sperm comprising said Red Zebrafish 1 transformation event its germline cell DNA a transgenic construct comprising one having been deposited as ECACC accession no. 06090403. or more of SEQID NO: 1 through SEQID NO:5. the Green Zebrafish 1 transformation event, sperm compris 2-12. (canceled) ing said Green Zebrafish 1 transformation event having been 13. The transgenic Zebrafish of claim 1, wherein the deposited as ECACC accession no. 06090401, or the Yellow Zebrafish comprises the Red Zebrafish 1 transformation event, Zebrafish 1 transformation event, sperm comprising said Yel US 2009/0025645 A1 Jan. 29, 2009 25 low zebrafish 1 transformation event having been deposited ii) the second vector comprises a second gene encoding as ECACC accession no. 06090402. a second fluorescent protein, wherein said second 14-19. (canceled) gene is under the control of a tissue-specific fish pro 20. A transgenic fluorescent fish comprising in its genome: moter, and (b) producing a population of transgenic fluorescent, orna a) a first transgene encoding a first fluorescent protein, mental fish wherein one or more fish of said population wherein said first transgene is under the control of a comprise both said first and second genes and express at ubiquitous fish promoter, and least one fluorescent protein. (b) a second transgene encoding a second fluorescent pro 40-52. (canceled) tein, wherein said second transgene is under the control 53. A method of obtaining a population of the transgenic of a tissue specific fish promoter. ornament fish with strong visible fluorescence, comprising 21. The transgenic fish of claim 20, wherein the first and the steps of: second fluorescent proteins are selected from the group con (a) producing a population of transgenic fluorescent, orna sisting of ZsGreen1, ZsYellow 1, DsRed2, GFP, eGFP. YFP. mental fish; eYFP, BFP, eBFP, CFP, eCFP, FP, AmCyan1, DsRed-Ex (b) selecting from the population at least one fish that press, AsFed2, HcRed1, mPlum, mCherry, tdTomato, exhibits ubiquitous fluorescent expression; mStrawberry, J-Red, Dsked-monomer, mOrange, mKO, (c) monitoring the selected fish and continuing selecting MCitrine, Venus, Ypet, EYFP. Emerald, CyPet, mCFPm, Cer for stable expression of strong visible fluorescence; and ulean, and T-Sapphire. (d) creating of a line of fish that exhibit strong visible 22-23. (canceled) fluorescence. 24. The transgenic fish of claim 20, wherein the ubiquitous 54. The method of claim 53, wherein at least one fish is fish promoter is selected from the group consisting those for selected by means of its strong visible fluorescence. housekeeping genes, including B-actin, EF-1 C, histone 2A, 55. The method of claim 54, wherein at least one fish is acidic ribosomal phosphoprotein, C-catenin, 3-cateniny selected using an ultraviolet or blue light of less than 50 lux. catenin, ubiquitin CSrb7, creatine kinase(mitochondrial 1), 56. The method of claim 54, wherein the at least one fish is ubiquitous Ca2+ transporting ATPase, ancient ubiquitous selected using an illuminance level of 50 to 1000 lux. protein, ubiquitin specific peptidase 4, acetyl-Coenzyme A 57. A method of providing a fluorescent transgenic acryltransferase 2, monoglyceride lipase, splicing factor 3b Zebrafish to the ornamental fish market, comprising obtaining subunit 1 and tubulin B5. a transgenic Zebrafish in accordance with claim 1, and dis 25-26. (canceled) tributing said fish to the ornamental fish market. 27. The transgenic fish of claim 26, wherein said muscle 58-60. (canceled) specific fish promoter is selected from the group consisting 61. A method of producing a transgenic fluorescent, orna those from genes expressed in differentiated tissues, such as mental fish, comprising the steps of: promoters of myosin light chain, muscle creatine kinase, (a) obtaining at least two cloning vectors, each comprising MyoD, myogenin, desmin, muscle enolase-3, 3-Sarkoglycan, one or more fluorescent genes, a fish promoter and one dystrophin, serum response factor, a-tropomyosin, myosin or more polyadenylation signals. heavy chain, mitochondrial creatine kinase 2, Ca2+ transport (b) producing a population of transgenic fluorescent, orna ing ATPase (fast twitch 1), skeletal troponin T1 (slow), tro mental fish wherein one or more fish of said population pomodulin 4, four and a halfLIM domains 1, fast-type nyosin each comprise and express the fluorescence genes of binding protein C, calsequestrin 1, fast muscle troponin C2, said at least two cloning vectors in its genome and phosphorylase kinase alpha1, and skeletal Troponin I (fast 2). express one or more fluorescent proteins. 62-64. (canceled) 28. (canceled) 65. A method of producing a transgenic fluorescent, orna 29. The transgenic fish of claim 20, wherein said tissue mental fish, comprising the steps of: specific fish promoter is a skin specific fish promoter, an eye (a) obtaining one or more cloning vectors, each comprising specific promoter or a bone specific promoter. one or more fluorescent genes under the control a chro 30-34. (canceled) matophore-specific fish promoter. 35. A method of making a transgenic Zebrafish, comprising (b) producing a population of transgenic fluorescent, orna the steps of: mental fish wherein one or more fish of said population (a) preparing a vector which comprises a transgenic fluo comprise one or more fluorescent genes in its genome and express one or more fluorescent proteins in chro rescence expression cassette comprising one or more of matophores. SEQID NO: 1 through SEQID NO:5; 66. The method of claim 65, wherein said chromatophore is (b) preparing a transgenic Zebrafish using the vector, and a melanophore, a Xanthophore or an iridophore. (c) selecting transgenic Zebrafish that fluoresce. 67-68. (canceled) 36-37. (canceled) 69. The method of claim 65, wherein said chromatophore 38. The method of claim 35, wherein the vector has a gene specific fish promoter is a promoter of a biosynthesis enzyme encoding antibiotic resistance and origin of replication specific for one or a Subset of chromatophores or a promoter removed before its being introduced into the transgenic of a regulatory protein specific for one or a Subset of chro Zebrafish genome. matophore, or wherein said chromatophore-specific fish pro 39. A method of producing a transgenic fluorescent, orna moter is selected from the group consisting of promoters of mental fish, comprising the steps of: endothelin receptor b1, Xanthine dehydrogenase, sepiapterin reductase, Xanthine oxidoducatase, Fms/Csfl, Mitf, kit, tyro (a) obtaining at least a first and second cloning vector, sinase-related protein1, tyrosinase, tyrosinase-related protein wherein 2 and dopachrome tautomerase. i) the first vector comprises a first gene encoding a first 70-71. (canceled) fluorescent protein, wherein said first gene is under the control of a ubiquitous fish promoter, and