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 TRANSGENIC FLUORESCENT Related US. Application Data ORNAMENTAL FISH THEREFROM (60) Provisional application No. 60/838,006, ?led on Aug. 16, 2006, provisional application No. 60/842,721, (75) Inventors: Alan Blake, Austin, TX (US); ?led on Sep. 7, 2006. Richard Crockett, NeW York, NY (Us); Jeffrey Essner’ Ames’ IA Publication Classi?cation (US); Perry Hackett, Saint Paul, (51) IIlt- Cl MN (US); Aidas Nasevicius, Plant C12N 15/85 (2006-01) City, FL (Us) A01K 67/027 (2006.01) A01K 63/02 (2006.01) A01K 61/00 (2006.01) Correspondence Address: (52) US. Cl...... 119/203; 800/20; 800/21; 119/215 FULBRIGHT & JAWORSKI LLP (57) ABSTRACT 600 CONGRESS AVE., SUITE 2400 AUSTIN, TX 78701 (US) The present invention relates to the method and use of reef coral ?uorescent proteins in making transgenic red, green and yelloW ?uorescent Zebra?sh. Preferably, such ?uorescent (73) Assignee: Yorktown Technologies, L.C., Zebra?sh are fertile and used to establish a population of Austin, TX (US) transgenic Zebra?sh and to provide to the omamental ?sh industry for the purpose of marketing. Thus, neW varieties of ornamental ?sh of different ?uorescence colors from a novel (21) Appl. No.: 11/839,364 source are developed.

Xn'ml (5728) Mail (671.) /

swam} Spel' (11 11.)

pZMLC-SV4OX2-Yeliow ZsYe|l0w1 PUG a“ 5043 bp; Inserted DNA,NO1.| (671} - Xhol (3823)

Hawaii (18229} P lac f , . H" .Xho! (38:23) ZMLC~19B4 Promoter Patent Application Publication Jan. 29, 2009 Sheet 1 0f 6 US 2009/0025645 A1

Xmn Iv (5694} ‘H. Amp (ma) X“ \ -

SV40(A) SgwI (1.111}

pUC ori 13' 6009Construct bp; Inserted 2.: pZMLC-SV40x2-D5Red2 DNA, NotI(671) - Xho!(3789 DsRed2

Ecol-ll (1795}

ZMLC~1934 Promoier

FIG.1 Patent Application Publication Jan. 29, 2009 Sheet 2 0f 6 US 2009/0025645 A1

Spa-i ( in if)

Construct 1: pZMLC-SV4OX2-ZsGreen1 5,3} pUC 0" 6041 bp; Inserted DNA, Not! (671) _ Xhol (3821) ZsGreeni

EcuRi' {1827) P Eac

X1101 (3821) zMLC-1934 Promoter

FIG. 2 Patent Application Publication Jan. 29, 2009 Sheet 3 0f 6 US 2009/0025645 A1

Natl (671,)

W i M KMVYVALOW) swam) Spa! (1111.)

PUC 9" ‘@043 bp; Inserted DNA, Not] (6713 - Xhol (3823) f

“72mm (18229)

zMLC-1‘934 Promoter

FIG. 3 Patent Application Publication Jan. 29, 2009 Sheet 4 0f 6 US 2009/0025645 A1

Kim] (1122.51

iB-actin promoter

Amp

An r11‘ (3870)’ =SV4-0(A) _ B-actin intron 1 sv40(A)

Km! (34%}

Hamid {2343} FIG. 4 Patent Application Publication Jan. 29, 2009 Sheet 5 0f 6 US 2009/0025645 A1

Xbul (2115)

C01E1 Ori

f5~actin promoter

Amp Construct 2: pCBAC-SV40X2-ZsGnee-n1 5833 bp; Inserted DNAZXIJQI rzrs'g-Aam r3902) lg'ac'tm 9mm

fs-actin intron 1 swam)

ZsGreem ‘ “ SW1 (mass) Kprrl (2T4!

FIG. 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.

Antibiotic Resistance Gene "I

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 ?uorescent protein gene construct‘, is collected and microinjected into a recently fertilized zebra?sla embryo.

FIG. 6 US 2009/0025645 A1 Jan. 29, 2009

RECOMBINANT CONSTRUCTS AND direct injection of DNA into muscle tissue (Xu et al., 1999). TRANSGENIC FLUORESCENT The ?rst transgenic ?sh 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 ?sh studies have concen [0001] The present application claims the bene?t of previ trated on groWth hormone gene transfer With an aim of gen ously ?led provisional application Ser. Nos. 60/838,006, ?led erating fast groWing “super?sh”. While a majority of early Aug. 16, 2006, and 60/842,721, ?led 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 super?sh (e.g. Chour entirety. rout et al., 1986; Penman et al., 1990; Brem et al., 1988; Gross et al., 1992), enhanced groWth of transgenic ?sh has been BACKGROUND OF THE INVENTION demonstrated in several ?sh species including Atlantic [0002] 1. Field of the Invention salmon, several species of Paci?c 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 ?sh gene promoters and heterologous genes for genera [0008] The Zebra?sh, Danio rerio, is a neW model organism tion of transgenic ?sh, particularly ?uorescent transgenic for vertebrate developmental biology. As an experimental ?sh. model, the Zebra?sh offers several major advantages such as [0004] 2. Description of RelatedArt easy availability of eggs and embryos, tissue clarity through [0005] Transgenic technology involves the transfer of a out embryo genesis, 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 ?rst developed Transgenic Zebra?sh have been used as an experimental tool in mice by Gordon et al. (1980). They injected foreign DNA in Zebra?sh developmental biology. HoWever, for the oma into fertiliZed eggs and found that some of the mice developed mental ?sh industry the dark striped pigmentation of the adult from the injected eggs retained the foreign DNA. Applying Zebra?sh does not aid in the e?icient 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 Zebra?sh carry a recessive mutation in the slc24a5 the ?rst 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 bene?cial traits for pigmented as compared to the Wild type Zebra?sh. The avail livestock husbandry and aquaculture. ability of the Golden Zebra?sh for transgenesis With ?uores [0006] In addition to the stimulation of somatic groWth for cent proteins Would result in better products for the omamen increasing the gross production of animal husbandry and tal ?sh 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 ?uorescent 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-?sh Aqueous Victoria. More pharmaceutically useful protein factors have been expressed recently, various other neW ?uorescent 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 ?uorescent protein gene; concentration as high as 35 mg/ml (10% of milk proteins) in ZsGreen, green ?uorescent protein gene and ZsYelloW, yel the milk of transgenic sheep (Wright et al., 1991). Similarly, loW ?uorescent protein gene. The novel ?uorescent 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 ?uorescent 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, ?uorescence in a variety of cell types and display exceptional transgenic mice have been Widely used in medical research, photostability and hence ?uoresce 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 DsRed 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 [3-barrel and that tors of complement activation to prevent hyperacute rejection the DsRed protein forms tetramers in vivo (Wall et al., 2000). during organ transplantation (CoZZi and White, 1995). The [0010] Coral reef ?uorescent proteins have broad applica development of disease resistant animals has also been tested tion in research and development. The red ?uorescent 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, ?la genic studies. There are many Ways of introducing a foreign mentous fungi (Eckert et al., 2005, Mikkelsen et al., 2003); gene into ?sh, including: microinjection (e.g., Zhu et al., ascidian (Zeller et al., 2006); Zebra?sh (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 ?sh for use (Wenek et al., 2003). It has also been used 21 marker in imag With the disclosed constructs and methods is the Golden ing studies in stem cells (Tolar et 211., 2005, Long et al., 2005) Zebra?sh. Zebra?sh 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 ?uorescent protein, ZsGreen, has been used as 21 trans somes. The number, siZe and density of the melanosomes per formation marker in insects (Sarkar et al., 2006), knock-in pigment cell in?uence the color of the ?sh skin. Golden mouse model for the study of KIT expressing cells (Wouters Zebra?sh 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 ?uorescent protein, ZsYelloW, has been Wild type Zebra?sh. Golden Zebra?sh have a mutation in used a reporter for plant transformation (Wenck et al., 2003) slc24a5 gene, rendering the ?sh skin lighter or less pigmented and for visualiZing fungal pathogens (Bourett et al., 2002). (Lamason et al., 2005). All of these trans genic experiments have aimed at developing [0014] In another embodiment of the invention, a method neWer markers and reporters for transgenesis; hoWever, for making transgenic ?uorescent ?sh is provided comprising progress in the ?eld of ornamental ?sh industry has been at least the following steps: a) preparing a vector Which has a limited. transgenic ?uorescence expression cassette comprising one sequence from a group ofSEQ ID N01 1, SEQ ID N012, SEQ SUMMARY OF THE INVENTION ID N013, SEQ ID N014 and SEQ ID N015, tWo or more [0011] In certain embodiments, the present invention con sequences from a group of SEQ ID N011, SEQ ID N012, SEQ cerns making recombinant constructs and transgenic ?uores ID N013, SEQ ID N014 and SEQ ID N015 are used in cent ?sh and providing such ?sh to the ornamental ?sh indus combination, speci?cally, SEQ ID N01 1 and SEQ ID N012 try. The term recombinant construct is used to mean are used together and SEQ ID N013 and SEQ ID N015 are recombinant DNA constructs having sequences Which do not used together; b) making the transgenic Zebra?sh using the occur in nature or exist in a form that does not occur in nature vectors; and, c) selecting transgenic Zebra?sh that ?uoresce or exist in association With other materials that do not occur in by monitoring ?uorescence 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 arti?cially 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 ?uo 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 modi?ed organism (GMO), Which is de?ned as an tion. For instance, the Zebra?sh fast skeletal muscle myosin organism Whose genetic material has been altered using the light chain promoter and carp [3-actin promoter may be used genetic engineering techniques generally knoWn as recombi according to the invention. nant DNA technology. This modi?ed DNA is then transferred [0015] In certain speci?c embodiments there are provided into an organism preferably resulting in the expression of methods to use multiple vectors to express at least one ?uo modi?ed 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 ?sh comprising in its genome a ventional cross breeding or by “mutagenesis” breeding, as ?rst ?uorescent transgene under the control of a ubiquitous these methods predate the discovery of the recombinant DNA ?sh promoter, and a second ?uorescent transgene under the techniques. Technically, hoWever, such techniques are by control of a tissue speci?c ?sh promoter. The ubiquitous ?sh de?nition genetic modi?cation. The term ?uorescent 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] Speci?c embodiments of the present invention are promoters for ‘housekeeping genes’, such as tubulin, riboso directed to methods of making transgenic ?uorescent ?sh mal protein, and actin genes. The tissue speci?c ?sh promoter having one sequence from a group of SEQ ID N01 1, SEQ ID is selected from the group consisting of those transcriptional N012, SEQ ID N013, SEQ ID N014 and SEQ ID N015, as motifs that are active in speci?c cells of differentiated tissues Well as transgenic ?sh developed by such methods. Thus, a such as muscle, brain, liver, blood and eyes. In a preferred transgenic Zebra?sh having integrated into its germ line cell embodiment, the tissue speci?c ?sh promoter is muscle spe DNA a transgenic construct comprising one or more of SEQ ci?c. As used herein, a promoter drives expression “speci? ID N011 through SEQ ID N015 is also included as part ofthe 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 Zebra?sh egg and/ or sperm cells comprising a sequence 50-fold higher in that tissue than in any other tissue. according to SEQ ID N011 through SEQ ID N015 integrated [0016] More than one construct can be injected into the ?sh 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 SEQ ID N01 1, SEQ ID tion, both Red Zebra?sh 1 and Green Zebra?sh 1 incorporate N012, SEQ ID N013, SEQ ID N014 and SEQ ID N015 are more than one transgenic expression cassettes, With one being used in one Zebra?sh. In a speci?c aspect, SEQ ID N01 1 and a ubiquitous promoter, and the other being a strong muscle SEQ ID N012 are used in the same ?sh and SEQ ID N013 and promoter. In particular, Red Zebra?sh 1 incorporates the cas SEQ ID N015 are used in the same ?sh. In preferred embodi settes represented by FIG. 1 and FIG. 4, and Green Zebra?sh ments, it is contemplated that the transgenic ?uorescent ?sh 1 incorporates the cassettes represented by FIG. 2 and FIG. 5. are fertile transgenic ?uorescent ?sh. 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 speci?c genes fused to ?uorescent protein multiple transgenic insert cassettes of any type can be simul open reading frames (ORFs) can be used to visualiZe speci?c taneously injected into a ?sh embryo from any species. chromatophores. The speci?c genes can be roughly divided [0017] The steps involved in making the transgenic ?sh into tWo major groups: regulatory proteins and biosynthesis further involve isolation and separation of the transgenic enZymes, involved in speci?c 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., ampicillin 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 ?uorescent gene may be, for tent expression throughout the life of the ?sh. To achieve such example, a gene encoding DsRed2, ZsGreenl and ZsYel stable expression, it is necessary to choose a promoter that is loWl. The heterologous ?uorescent gene may also be any knoWn to drive stable and consistent expression throughout variation or mutation of these genes, encoding ?uorescent the life of the ?sh. For example, a promoter that drives expres proteins including green ?uorescent protein (GFP), enhanced sion only during the six months of the life of the ?sh Would not green ?uorescent protein (eGFP), yelloW ?uorescent protein be suitable for use. Examples of suitable promoters may be (Y FP), enhanced yelloW ?uorescent protein (eYFP), blue selected from the group consisting of those for housekeeping ?uorescent protein (BFP), enhanced blue ?uorescent protein genes, such as tubulin, ribosomal protein, and actin gene (eBFP), cyan ?uorescent protein (CFP) and enhanced cyan promoters. ?uorescent 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 ?uorescence proteins. The steps involved in making the trans ments in the transgenic insert cassette to produce a transgenic genic ?sh also involve introduction of the transgenic expres ?uorescent, ornamental ?sh. The RNA processing signals, sion cassette into the Zebra?sh embryos or Zebra?sh 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 ?sh 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 ?uorescent intron, and the untranslated carp beta-actin exon used in SEQ gene by exposing the said ?sh to light of appropriate Wave ID 2 and SEQ ID 5 is an example of such an exon. Exons and length and/ or by visibly inspecting the ?sh and observing the introns other than carp beta-actin can be used as Well. The expression. Transgenic ?uorescent ?sh 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 ?sh 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 ?uorescent transgenic can increase expression, possibly by causing the transcript to ?sh, and a population therefrom, Which exhibit strong visible enter the processing and transport system for mRNA. It is also ?uorescence. Strong visible ?uorescence 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 ?uorescent ?sh in question and a non used (that is, that the intron be from the same gene that some ?uorescent ?sh of the same species at a distance of at least 5 or all of the expression sequences are from). feet in a lighted o?ice, With a preferred distance of at least 10 [0019] The disclosed transgene constructs preferably feet in a lighted o?ice, and a more preferred distance of at include other sequences Which improve expression from, or least 15 feet in a lighted o?ice, and an even more preferred stability of, the construct. For example, including a polyade distance of at least 20 feet in a lighted o?ice, With the illumi nylation signal on the constructs encoding a protein ensures nation level de?ned in Table 5. One can observe all transgenic that transcripts from the transgene Will be processed and ?uorescent ?sh from a particular population that exhibit transported as mRNA. The identi?cation and use of polyade strong visible ?uorescence under the various lighting condi nylation signals in expression constructs is Well established. tions and select the ?sh that exhibits the highest level of It is preferred that e?icient polyadenylation signals, such as visible ?uorescence of the ?uorescent protein. Selected ?sh those derived from viruses, be used in the transgenic con With strong visible ?uorescence are monitored and their pro g 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 ?uorescence. Thus a neW adenylation sequence. line of ?sh that exhibit strong visible ?uorescence is created [0020] It is also a subject of this invention to disclose for further breeding. expression of the ?uorescent protein gene only in chromato [0023] Transgenic ?sh made by the present disclosure Will phores. There are several types of chromatophores found in emit red, yelloW-green and yelloW-orange ?uorescence 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 ?sh industry. In yet another and iridophores (re?ective). Different species of ?sh contain embodiment of the invention, a method of making the trans all types of chromatophores, usually a subset of them in genic ?sh available to the consumer by a groWer or a com different combinations. Zebra?sh contain melanophores, mercial distributor through a retailer for sale to the public. In xantophores and iridophores. These different cell types such embodiment, the ?sh may also be sold by the groWer or express speci?c genes, characteristic only for them or speci?c 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

?uorescent transgenic ?sh 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 zebra?sh 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, ?sh for use in breeding of transgenic detailed description. It should be understood, hoWever, that zebra?sh of the invention Will be homozygous for an expres the detailed description and the speci?c examples, While indi sion cassette. Homozygous ?sh bred With ?sh lacking an cating preferred embodiments of the invention, are given by expression cassette (e.g., Golden zebra?sh) Will in nearly all Way of illustration only, since various changes and modi?ca cases produce 100% heterozygous offspring. Likewise, trans tions Within the spirit and scope of the invention Will become genic ?sh 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. speci?c aspects a transgenic ?sh of the invention comprises the speci?c integration event of the Red ?uorescent expres BRIEF DESCRIPTION OF THE DRAWINGS sion cassette described in Example 3. [0031] The folloWing draWings are part of the present [0025] In certain speci?c embodiments there are provided speci?cation and are included to further demonstrate certain transgenic ?uorescent zebra?sh comprising speci?c trans aspects of the present invention. The invention may be better genic integration events. These ?sh 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 speci?c embodiments presented of protein ?uorescence. Thus, in some embodiments there is herein. provided a transgenic zebra?sh comprising a chromosomally [0032] FIG. 1: The ?gure shoWs a schematic map of the integrated expression cassette encoding a DsRed2 gene transgenic construct, pZMLC-DsRed2-SV40><2. The 2.1-kb Wherein the zebra?sh comprises the Red zebra?sh 1 transfor eukaryotic promoter sequence zMLC-1934 promoter Was mation event, sperm comprising said Red zebra?sh 1 trans ampli?ed 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 ?uorescent no. 06090403. In some other aspects, there is provided a protein CDS Was ampli?ed by PCR from pDsRed2-N1 transgenic zebra?sh comprising a chromosomally integrated (Clontech) and inserted into EcoRI and SpeI sites. The 440 expression cassette encoding a ZsGreen1 gene Wherein the bp 3'UTR/poly(A) sequence encoding tandem SV40 poly zebra?sh comprises the Green zebra?sh 1 transformation adenylation signals Was PCR ampli?ed from pK-SV40(A)x2 event, sperm comprising said Green zebra?sh 1 transforma and cloned into SpeI 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 06090401 . In still other aspects, there is provided a transgenic from the vector backbone. Also shoWn is the ampicillin zebra?sh comprising a chromosomally integrated expression (Amp, formally knoWn as beta-lactamase (bla)) resistance cassette encoding a ZsYelloW1 gene Wherein the zebra?sh gene in the backbone of the pBluescript plasmid. The total comprises theYelloW zebra?sh 1 transformation event, sperm length of the recombinant plasmid pzMLC-DsRed2-SV40x2 comprising saidYelloW zebra?sh 1 transformation event hav is 6009 hp. ing been deposited as ECACC accession no. 06090402. As [0033] FIG. 2: The ?gure shoWs a schematic map of the described above, transgenic ?sh comprising these speci?c transgenic construct, pZMLC-ZsGreen1-SV40><2. 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 ampli?ed 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 ?uorescent integration location for any given transgenic modi?cation. protein CDS Was ampli?ed by PCR from pZsGreen1-N1 Eggs, sperm and embryos comprising these speci?c trans (Clontech) and inserted into EcoRI and SpeI 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 ?uorescence genes noted in this applica adenylation signals Was PCR ampli?ed from pK-SV40(A)x2 tion may be used in similar embodiments of this invention. and clone into SpeI 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 ampicillin 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 hp. tion as Well. [0034] FIG. 3: The ?gure shoWs is a schematic map of the [0027] As used herein the speci?cation, “a” or “an” may transgenic construct, pZMLC-ZsYelloW1-SV40><2. 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 ampli?ed by PCR from pMLC vector and cloned into XhoI “an” may mean one or more than one. and EcoRI restriction sites. The 718 bp ZsYelloW1 ?uores [0028] The use of the term “or” in the claims is used to cent protein CDS Was ampli?ed by PCR from pZsYelloW1 mean “and/or” unless explicitly indicated to refer to alterna N1 (Clontech) and inserted into EcoRI and SpeI 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 de?nition that refers to only alter polyadenylation signals Was PCR ampli?ed from pK-SV40 natives and “and/or.” As used herein “another” may mean at (A)x2 and clone into SpeI 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 US. Pub No. 2004/0143864 plasmid. The total length of the recombinant plasmid A1, all of Which are hereby incorporated by reference in their pZMLC-ZsYelloW1-SV40x2 is 6043 bp. entireties. [0035] FIG. 4: The ?gure shoWs a schematic map of the [0039] To develop successful transgenic ?sh With a predict transgenic construct, pCBAC-DsRed2-SV40><2. The 2.5-kb able pattern of transgenic expression, the ?rst 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 ampli?ed by PCR erally comprises three portions: transcriptional regulators from pFV7b vector and cloned into XbaI and KpnI restriction comprising a promoter, a gene and appropriate RNA-process sites. The 684 bp DsRed2 ?uorescent protein CDS Was ampli ing and/ or translational enhancing motif. The gene promoter ?ed by PCR from pDsRed2-N1 (Clontech) and inserted into Would determine Where, When and under What conditions the EcoRI and SpeI 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 ampli?ed from biological function. The gene might also contain intron pK-SV40(A)x2 and cloned into SpeI and AatII sites. XbaI sequences Which can affect mRNA processing or Which and AatII 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 ?gure shoWs a schematic map of the heterologous promoter. transgenic construct, pCBAC-ZsGreen1-SV40><2. 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 ampli?ed by PCR from higher in that tissue compared to a reference tissue. A pro pFV7b vector and cloned into XbaI and KpnI restriction sites. moter drives expression “speci?cally” in a tissue if the level The 716 bp ZsGreenl ?uorescent protein CDS Was ampli?ed 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 SpeI sites. The 443 bp 3'UTR/poly(A) sequence than in any other tissue. A ubiquitous promoter drives expres encoding tandem SV40 polyadenylation signals sequence sion in most tissues, and preferably in all tissues. encoding tandem SV40 signal Was PCR ampli?ed from pK-SV40(A)x2 and cloned into SpeI and AatII sites. XbaI Recombinant DNA Constructs and AatII 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 exist in a form [0037] FIG. 6: Transgenic Construct puri?cation and inj ec 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 puri?cation 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 f 1 (—) 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 puri?cation 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 exempli?es nylation signal on the constructs encoding a protein ensures the process of microinjection of the gel-puri?ed expression that mRNA transcripts from the transgene Will be ef?ciently construct in to the fertilized Zebra?sh embryos. translated as protein. The identi?cation and use of polyade nylation signals in expression constructs is Well established. DETAILED DESCRIPTION OF THE INVENTION It is preferred that de?ned and e?icient 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 arti?cially introduced into SV40 polyadenylation sequence. ?sh to produce a transgenic ?sh. The manner of introduction, [0043] In certain speci?c embodiments there are provided and, often, the structure of a transgenic construct, render such methods to use multiple vectors to express at least one ?uo 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 ?sh comprising in its genome a nucleic acid sequences, foruse in the disclosed transgenic ?sh ?rst ?uorescent transgene under the control of a ubiquitous it is preferred that the transgenic constructs combine regula ?sh promoter, and a second ?uorescent transgene under the tory elements operably linked to a sequence encoding one or control of a tissue speci?c ?sh promoter. In a preferred more proteins. The methods and protocols for designing and embodiment, the tissue speci?c ?sh promoter is muscle spe making transgenic constructs are Well knoWn to those skilled ci?c. The ubiquitous ?sh promoter and the muscle speci?c US 2009/0025645 A1 Jan. 29, 2009

promoter are, 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 con?rmed (e.g., ENSMUSG00000030730), gene homologues in other available at this time, with any unmarked promoter, or any organisms (e. g., zebra?shiDanio 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 ?eld. mation). Gene promoters are located upstream (5' ?anking [0044] The provided Table l of muscle-speci?c and ubiq sequence) from the coding sequence, often within several uitous promoters constitutes only a small portion of publicly (e.g., ?ve) kilobases. In addition, some regulatory sequences available promoters. An extensive list of genes with expres can be found in introns of the gene of interestithese regula sion of interest (e.g., muscle-speci?c expression) can be tory sequences are usually omitted from constructing tissue found using NCBI protein database server (www at ncbi.nlm. speci?c 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 ?nd mouse genes expressed in skeletal muscles a search used starting with “Zebra?sh 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 ?nd 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 ?nd 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 speci?c and ubiquitous promoters for ?sh expression

Gene promoter Zebra?sh Fugu Tetraodon Medaka Xenopus @Rat Mouse Dog Bovine Muscle speci?c

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

EF-l alpha Histone 2A ZA Acidic ribosornal phosphoprotein PO (ARP) alpha-catenin beta-catenin gaInrna-catenin

Creatine kinase(rnitochondrial l) Ubiquitous Ca2+ transporting ATPase Ancient ubiquitous protein Ubiquitin speci?c peptidase 4 Acetyl-Coenzyrne A acryltransferase 2 Monoglyceride lipase Splicing factor 3b subunit 1 Tubulin [55 Beta-Actin

@ indicates text missing or illegible when ?led US 2009/0025645 A1 Jan. 29, 2009

[0045] Table 2, below, is a partial list of Ensembl gene ID numbers of mouse and Zebra?sh skeletal muscle speci?c and TABLE 2-continued ubiquitous genes found using this approach. Ensembl IDs of Muscle Speci?c Promoters TABLE 2 SERUM RESPONSE FACTOR PROMOTERS

Ensembl IDs of Muscle Speci?c Promoters Zebra?sh: (ENSDARG00000053918) Fugu: (S INFRUG000001 62928) MUSCLE CREATINE KINASE PROMOTERS Tetraodon: (GSTENG00025109001) Medaka: (ENSORLG0000001303 6) Zebra?sh: (ENSDARG00000035327) Xenopus lropicalis: (ENSXETG00000018511) Fugu: (SINFRUG00000143294) Rat: (ENSRNOG000000 1 823 2) Tetraodon: (GSTENG0001295 6001) Mouse: (ENSMUSG00000015605) Medaka: (ENSORLG00000000449) Dog: (ENS CAFG00000001 829) Xenopus lropicalis: (ENSXETG00000019108) Bovine: (ENSBTAG00000012777) Rat: (ENSRNOG00000016837) ALPHA-TROPOMYOSIN PROMOTERS Mouse: (ENSMUSG00000030399) Dog: (ENSCAFG00000004507) Zebra?sh: (ENSDARG00000033683) Bovine: (ENSBTAG00000013921) Fugu: (S1NFRUG00000130484) MYOD PROMOTERS Tetraodon: (GSTENG00015950001) Medaka: (ENSORLG00000012326) Zebra?sh: (ENSDARG00000030110) Rat: (ENSRNOG00000018184) Fugu: (SINFRUG00000154785) Mouse: (ENSMUSG00000032366) Tetraodon: (GSTENG00003954001) Dog: (ENSCAFG00000016966) Medaka: (ENSORLG00000000694) Bovine: (ENSBTAG00000005373) Xenopus lropicalis: (ENSXETG00000001320) MYOSIN HEAVY CHAIN PROMOTERS Rat: (ENSRNOG00000011306) Mouse: (ENSMUSG00000009471) Zebra?sh: (ENSDARG00000035437) Dog: (ENSCAFG00000009066) Fugu: (SINFRUG00000135173) Bovine: (ENSBTAG00000002216) Medaka: (ENSORLG00000001985) MYOGENIN PROMOTERS Xenopus lropicalis: (ENSXETG00000023939) Rat: (ENSRNOG00000031400) Zebra?sh: (ENSDARG00000009438) Mouse: (ENSMUSG00000033196) Fugu: (SINFRUG00000121801) Dog: (ENSCAFG00000023926) Tetraodon: (GSTENG00013986001) Bovine: (ENSBTAG00000007090) Medaka: (ENSORLG00000015906) MITOCHONDRIAL CREATINE KINASE Xenopus lropicalis: (ENSXETG00000001704) (SARCOMERIC, CKMTZ) PROMOTERS Rat: (ENSRNOG00000030743) Mouse: (ENSMUSG00000026459) Zebra?sh: (ENSDARG00000035079) Dog: (ENSCAFG00000010309) Fugu: (S1NFRUG00000160265) Bovine: (ENSBTAG00000006030) Tetraodon: (GSTENG00028607001) DESMIN PROMOTERS Medaka: (ENSORLG00000000769) Mouse: (ENSMUSG00000021622) Zebra?sh: (ENSDARG00000058656) Dog: (ENSCAFG00000008707) Fugu: (SINFRUG00000121939) Bovine: (ENSBTAG00000001003) Xenopus lropicalis: (ENSXETG00000019275) MYOSIN LIGHT CHAIN PROMOTERS Rat: (ENSRNOG00000019810) Mouse: (ENSMUSG00000026208) Zebra?sh: (ENSDARG00000017441) Dog: (ENSCAFG00000015475) Fugu: (SINFRUG00000125026) Bovine: (ENSBTAG00000005353) Tetraodon: (GSTENG00015 855001) MUSCLE ENOLASE 3 BETA PROMOTERS Medaka: (ENSORLG00000015981) Xenopus lropicalis: (ENSXETG00000006917) Zebra?sh: (ENSDARG00000039007) Rat: (ENSRNOG00000013262) Tetraodon: (GSTENG00003809001) Mouse: (ENSMUSG00000061816) Rat: (ENSRNOG00000004078) Dog: (ENSCAFG00000013 875) Mouse: (ENSMUSG00000060600) Bovine: (ENSBTAG00000009707) Bovine: (ENSBTAG00000005534) BETA-SARCOGLYCAN PROMOTERS [0046] While this approach Will result in a great number of Zebra?sh: (ENSDARG00000052341) sequences, additional points should be considered to generate Fugu: (SINFRUG00000123612) Tetraodon: (GSTENG00032779001) a list of strong promoters. For example, abundant structural Medaka: (ENSORLG00000000171) genes (e.g., myosin) or abundant enZymes (e.g., SR calcium Xenopus lropicalis: (ENSXETG00000011676) ATPase) are likely to yield strong promoters. This screening Rat: (ENSRNOG00000002135) can easily be performed by an artisan in the ?eld. Mouse: (ENSMUSG00000029156) Dog: (ENSCAFG00000002001) [0047] Preferably more than one construct With different Bovine: (ENSBTAG00000014601) promoters can be injected into the ?sh embryos simulta DYSTROPHIN PROMOTERS neously. For example, in the present invention, both Red Zebra?sh 1 and Green Zebra?sh 1 incorporate more than one Zebra?sh: (ENSDARG00000008487) Fugu: (S INFRUG00000 1448 15) transgenic expression cassette, With one being a ubiquitous Tetraodon: (GSTENG00024870001) promoter, and the other being a strong muscle promoter. In Medaka: (ENSORLG00000020638) particular, Red Zebra?sh 1 incorporates the cassettes repre Xenopus lropicalis: (ENSXETG00000012391) sented by FIG. 1 and FIG. 4, and Green Zebra?sh 1 incorpo Rat: (ENSRNOG00000003667) Mouse: (ENSMUSG00000045103) rates the cassettes represented by FIG. 2 and FIG. 5. While the Bovine: (ENSBTAG00000008254) 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 ?sh embryo from any species. tyrosine kinase, speci?c to melanophores) and biosynthesis [0048] It is also a subject of this invention to disclose enZymes, involved in speci?c pigment synthesis (for expression of the ?uorescent protein gene speci?cally in example, sepiapterin reductase, involved in yelloW pigment chromatophores. Chromatophores are pigment-containing synthesis in xanthophores). Expression of regulatory proteins and light-re?ecting 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-speci?c gene expression is out leucophores (White) and iridophores (re?ective). 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 ?sh 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. Zebra?sh contain melanophores, speci?c to melanophores, as Well as biosynthesis enZymes xantophores and iridophores. These different cell types involved in melanin synthesis in different classes of organ express speci?c genes, characteristic only for them or speci?c 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 (tyrpl) is found only in melanophores; Which a number of genes have been isolated, yielding, a ednrbl is found in malenocytes and iridophores. Promoters of number of known promoters to choose from. With respect to these speci?c genes fused to ?uorescent protein open reading iridophores, a feW speci?c genes have been isolated (for frames (ORFs) can be used to visualiZe speci?c chromato example, endothelin receptor bl Ednrbl). The least knoWn phores. For example, fugu tyrp 1 promoter can be used to drive chromatophores are the cyanophores-neither the nature of ?uorescent protein expression in melanophores in Zebra?sh. their pigment, nor speci?cation pathWay of the cells per se is The speci?c genes can be roughly divided into tWo major knoWn.

TABLE 3

Chromatophore-speci?c expressed genes in ?shes

Chromatophore Protein Synth/ Reg Organism Reference Iridophore ednrbl Reg Zebra?sh Parichy et al, Developmental (endothelin Biology 227, 294-306 receptor bl) (2000) Xanthophore xanthine Synth Guppy Ben et al, Mar Biotechnol dehydrogenase (Poecilia (NY). 2003 Nov-Dec; rericulala); 5(6): 568-78. Epub 2003 Zebra?sh Aug 21; Parichy et al, Developmental Biology 227, 294-306 (2000) sepiapterin Synth medaka Negishi et al, Pigment Cell reductase (Oryzias Res. 2003 Oct; 16(5): 501-3 Zalipes) Xanthine Synth oxidoreductase Fms/Csfl Reg Zebra?sh Ziegler, Pigment Cell Res. 2003 Jun; 16(3): 172-82; Ziegler et al, J Biol Chem. 2000 Jun 23; 275(25): 18926-32; Parichy et al, Development 127, 3031-3044 (2000) Melanophores Mitf Reg Zebra?sh kit Reg Zebra?sh tyrpl Synth Zebra?sh, Zou et al, Pigment Cell Res. fugu 2006 Dec; 19(6): 615-27 tyrosinase Synth rana Miura et al, Jpn J Genet. nigromaculata 1995 Feb; 70(1): 79-92 tyrosinase Synth medaka Inagaki et al, Pigment Cell Res. 1998 Oct; 11(5): 283-90 tyrosinase Synth Mouse in Matsumoto et al, Pigment medaka Cell Res. 1992 Nov; 5(5 Pt 2): 322-7 t1p2 (tyrosinase- Synth mouse Zhao & Over beek, Dev Biol. related protein 2) 1999 Dec 1; 216(1): 154-63 dopachrome Synth tautomerase 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 Fluorescent Proteins (“PP”) With their Maximum mRNA. It is preferred that the intron be homologous to the Excitation and Emission Wavelengths host species, and more preferably homologous to the expres 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). tdTomato 554 5 81 The use and importance of these and other components useful mStraWberry 574 596 for transgenic constructs are discussed in Palmiter et al. .I-Red 5 84 610 (1991); Sippel et al. (1992); Kollias and Grosveld (1992); and DsRed-monomer 556 5 86 mOrange 548 5 62 Clark et al. (1993). mKO 548 559 [0051] The steps involved in making the transgenic ?sh MCitrine 516 529 further involve isolation and separation of the transgenic Venus 515 528 Ypet 517 530 expression cassette from the vector backbone to remove the EYFP 514 527 gene encoding antibiotic (e.g., ampicillin 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 ?sh. 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 ?sh. For example, a promoter that drives expres sion only during the six months of the life of the ?sh 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. the ?sh. [0054] The sequence listed herein as SEQ ID N011 is the transgenic ?uorescence expression cassette having Zebra?sh [0052] The heterologous ?uorescent gene may be, for fast skeletal muscle speci?c myosin light chain (ZMLC) pro example, a gene encoding DsRed2, ZsGreenl and ZsYel moter, DsRed2 (a red ?uorescent protein gene from Antho loW1. The heterologous ?uorescent gene may also be any Zoa, a reef coral), and tWo copies of the SV40 polyadenylation variation or mutation of these genes, encoding ?uorescent sequence. The sequence listed in SEQ ID N01 1 is the proteins including green ?uorescent protein (GFP), enhanced complementary sequence to the coding DNA strand. green ?uorescent protein (eGFP), yelloW ?uorescent protein [0055] The sequence listed herein as SEQ ID N012 is the (YEP), enhanced yelloW ?uorescent protein (eYFP), blue transgenic ?uorescence expression cassette having carp ubiq ?uorescent protein (BFP), enhanced blue ?uorescent protein uitous [3-actin enhancer/promoter, DsRed2 (a red ?uorescent (eBFP), cyan ?uorescent protein (CFP) and enhanced cyan protein gene from AnthoZoa, a reef coral), and tWo copies of ?uorescent protein (eCFP) or any of the proteins listed in the SV40 polyadenylation sequence. The ?rst exon and intron Table 4, beloW, or any variation or mutation thereof, or any of [3-actin has been incorporated in the SEQ ID N012 to alloW other ?uorescence proteins. The steps involved in making the for increased expression of the ?uorescence protein gene. transgenic ?sh also involve introduction of the transgenic [0056] The sequence listed herein as SEQ ID N013 is the expression cassette into the Zebra?sh embryos or Zebra?sh transgenic ?uorescence expression cassette having Zebra?sh embryonic stem cells. Such embryos and cells are alloWed to fast skeletal muscle speci?c myosin light chain (ZMLC) pro groW and mature into adult ?sh and then they are screened for moter, ZsGreenl (a green ?uorescent 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 SEQ ID N013 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 ?uorescent [0057] The sequence listed herein as SEQ ID N014 is the gene by exposing the said ?sh to light of appropriate Wave transgenic ?uorescence protein expression cassette having length and/ or by visibly inspecting the ?sh and observing the Zebra?sh fast skeletal muscle speci?c myosin light chain expression. Transgenic ?uorescent ?sh are further bred to (ZMLC) promoter, ZsYelloW1 (a yelloW ?uorescent 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 ?sh as further described in this application. polyadenylation sequence. The sequence listed in SEQ ID N014 is the complementary sequence to the coding DNA TABLE 4 strand. Fluorescent Proteins (“PP”) With their Maximum [0058] The sequence listed herein as SEQ ID N015 is the Excitation and Emission Wavelengths transgenic ?uorescence protein expression cassette having carp ubiquitous [3-actin enhancer/promoter, ZsGreenl (a FP Excitation max (nm) Emission max (nm) green ?uorescent protein gene from AnthoZoa, a reef coral), AmCyanl 458 489 and tWo copies of SV40 polyadenylation sequence. The ?rst ZsGreenl 493 505 exon and intron of [3-actin has been incorporated in the SEQ ZsYelloWl 529 539 ID N015 to alloW for increased expression of the ?uorescence DsRed2 5 63 5 82 DsRed-Express 557 579 gene AsRedZ 576 592 HcRedl 5 88 618 Chimeric Genes mPlum 590 649 mCherry 5 87 610 [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 ?sh, Molly ?sh, or pangasius. A promoter of a Zebra?sh operatively linked to a Zebra?sh struc more complete list of ornamental ?sh 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 Zebra?sh, TABLE 5 as exempli?ed by the DsRed2 and other genes speci?cally exempli?ed herein. Furthermore, a chimeric gene can com Ornamental Fish Species prise an exogenous promoter linked to any structural gene not Scienti?c Name Common Name normally linked to that promoter in the genome of an organ ism. Slealocranus casuarius African Blockhead Apislograma agassizi Agassizi Hyphessobrycon h axelrodi, sp Albino Black Neon Tetra Substitutions, Additions and Deletions Lamprophogus brichardi Albino Bricardi Cichld Paracheirodon innessi, sp. Albino Brilliant Neon Tetra [0060] As possible variants of the above speci?cally exem Hemigrammus caudovilalus Albino Buenos Aires Tetra pli?ed polypeptides, the polypeptide may have additional Hemigrammus erylhrozonus Albino Glow Light Tetra individual amino acids or amino acid sequences inserted into Hemigrammus oceZZzfer Albino Head Tail Light Tetra Pelvicachromis pulcher Albino Kribensis Cichlid the polypeptide in the middle thereof and/ or at the N-terminal ApZocheZius normani Albino Lampeye and/or C-terminal ends thereof so long as the polypeptide Hyphessobrycon pulchripinnis sp Albino Lemon Tetra possesses the desired physical and/or biological characteris Paracheirodon innessi Albino Neon Tetra Macropodus opercularis spp Albino Paradise Fish tics. Likewise, some of the amino acids or amino acid PlerophyZZum scaZare Albino Red Eye Angel sequences may be deleted from the polypeptide so long as the Epalzeorhynchosfrenalus Albino Red?n Shark polypeptide possesses the desired physical and/ or biochemi Hem. Rhodoslomus sp. Albino Rummy Nose cal characteristics. Amino acid substitutions may also be Capoela lelrazona Albino Tiger Barb Aslronolus oceZZalus Albino Tiger Oscar made in the sequences so long as the polypeptide possesses Tanichlys albonubes sp. Albino White Cloud the desired physical and biochemical characteristics. DNA Lepisosleus oculalus Alligator Gar coding for these variants can be used to prepare gene con Luciosoma spiZopZeura Apollo Shark structs of the present invention. Toxolesjaculalrix Archer Fish Xiphophorus varialus 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 Helosloma lemmincki Balloon Kissing Gourami referred to. The nucleic acid can be DNA or RNA. Unless Corydoras melae Bandit Corydoras Pangasius sulchi Bangkok Cat?sh otherwise speci?ed, a nucleic acid sequence that encodes a Ancislrus dolichoplerus Big-Fin Bristlenose Golden Long?n polypeptide includes the transcribed strand, the hnRNA and PeociZia Zaripinna Black Balloon Molly the spliced RNA or the DNA representative thereof. CichZasoma maculicauda Black Belt Cichlid Carrasius auralus Black Butter?y Tail Callochromis macrops Black Eared Callochromis Degenerate Sequences Leplosoma Kilumba Black Finned Slender Cichlid Apleronolus albzfrons Black Ghost [0062] In accordance with degeneracy of genetic code, it is Acanlhoplhalmus myersi Black Kuhlii possible to substitute at least one base of the base sequence of Bogrichlhys hypseloplerus Black Lancer a gene by another kind of base without causing the amino acid Hyphessobrycon h axelrodi Black Neon Tetra Nemalobrycon palmeri spp Black Palmeri sequence of the polypeptide produced from the gene to be Megalamphodus megaloplerus Black Phantom changed. Hence, the DNA of the present invention may also Rasbora lrilineala Black Scissor Tail Rasbora have any base sequence that has been changed by substitution Labeo chrysopekadion Black Shark in accordance with degeneracy of genetic code. Punrius?lamenlosus Black Spot Barb Rasbora agiZis Black Stripe Rasbora Gymnocorymbus lemelzi Black Tetra DNA Modi?cation Aslyanaxfas. mexicanus Blind Cave Tetra Brachydanio kerri Blue Danio [0063] The DNA is readily modi?ed by substitution, dele Inpaichlys kerri Blue Emperor Tetra tion or insertion of nucleotides, thereby resulting in novel Trichogasler lrichoplerus Blue Gourami Boehlkeafredcochui Blue King Tetra DNA sequences encoding the polypeptide or its derivatives. Xiphophorus maculalus Blue Platy These modi?ed sequences are used to produce mutant Melanolaenia Zacuslris Blue Rainbow polypeptide and to directly express the polypeptide. Methods PoeciZia rericulala Blue Ribbon Guppy for saturating a particular DNA sequence with random muta Pseudolropheus zebra Blue Zebra tions and also for making speci?c site-directed mutations are Melanolaenia boesemani Boesemani Rainbow Gaslromyzon punclulalus Borneo Sucker known in the art; see eg Sambrook et al., (1989). Damoides microlepis Borneo Tiger Fish Paracheirodon innesi Brilliant Diamond Head Neon Transgenic Fish Rasbora birllani Brittan’S Rasbora Brachygobius doriae Bumble Bee Goby Anomalochromis lhomasi Butter?y Cichlid [0064] The disclosed constructs and methods can be used Noleslhes robusla with any type of ?sh that is an egg-layer. It is preferred that Paracheirodon axelrodi Cardinal Tetra — M ?sh belonging to species and varieties of ?sh of commercial Nomorhampheus Ziemi Celebes Halfbeak value, particularly commercial value within the ornamental Telmalherina Zadigesi Celebes Rainbow ?sh industry, be used. Such ?sh include but are not limited to Chaca bankanensis Chaca-Chaca Capoela oligolepis Checkered Barb cat?sh, Zebra?sh, medaka, carp, tilapia, gold?sh, tetras, Capoela rilleya Cherry Barb barbs, sharks (family cyprinidae), angel?sh, loach, koi, glass Sphaerichlhys osphromenoides Chocolate Gourami ?sh, cat?sh, angel ?sh, discus, eel, tetra, goby, gourami, US 2009/0025645 A1 Jan. 29, 2009

TABLE 5-c0ntinued TABLE 5-c0ntinued

Omamental Fish Species Omamental Fish Species

Scienti?c Name Common Name Scienti?c Name Common Name

CZarias balracus Clarias-Spotted Leplosoma maZasa Leptosoma Malasa Epiplalys annulalus Clown Killie/Rocket Rasborapaviei Line Rasbora Bolia macracanlha Clown Loach Capoela arulius Long Fin Barb Haplochromis sp Cobalt/Ice Blue Cichlid Aleslhes Zongz'pinnis Long Fin Characin Apislograma cacaluoides Cockatoo Dwarf Rasbora einlhovenii Long-Band Rasbora Hyphessobrycon colombianus Colombia Tetra Melanolaenia maccuZZochi Macculloch’S Rainbow Phenacogrammus inlerruplus Congo Tetra Parelropheus menoramba Madagascar Cichlid Corydoras aeneus Corydoras Albino Bedoria gaeyi Madagascar Rainbow Corydoraspanda Corydoras Panda Haplochromis compressiceps Malawi Eye Biter Corydoras palealus Corydoras Peppered Ompok sp. Malay Glass Cat?sh Corydoraspigmy Corydoras Pigmy Bella splendens Male Betta Corydoras rabauri Corydoras Rabauti CichZasom managuense Managuense Cichlid Corydoras simiZis Corydoras Similis Polypleruspalmas Marbled Bichir Corydoras slerbai Corydoras Sterbai Xiphophorus heZZeri Millenium Swordtail Synodontis mulripunclalus Cuckoo Synodontis Monodaclylus argenlus Mono Angel Polyplerus senegalus Cuvier’S Bichir Cyrlocara moorii Morrii Synodontis decorus Decorated Synodontis Sawbwa resplendens Naked Micro Rasbora Polyplerus deZhezi DelheZi Bichir Hyphessobrycon h.axeZr0di sp. Negro Brilliant Black Neon Moenkhausia pilleri Diamond Tetra Melanolaenia praecox Neon Dwarf Rainbow Hyphessobrycon amandae Ember Tetra Aplocheilluspanchax New Golden Wonder Nemalobrycon palmeri Emperor Tetra Synodontis oceZZzfer Ocellated Synodontis Polyplerus endlicheri Endlicheri Bichir Colisa Zabiosa Orange Thick Lipped Gourami Aphyocharax albumus False Flame Tetra Polyplerus omaripinnis Ornate Bichir Synodontis euplerus Feathered Fin Synodontis Boria Locahanla Pakistani Loach CichZasomafeslae Festa’S Cichlid Punriusfascialus Panda Barb CichZasoma meeki Firemouth Cichlid Apislogramma pandurini Pandzm'ni Dwarf Pzmrz'us penlazona Five Banded Barb Macropodus opercularis Paradise Fish Epalzeorhynchus kaloplerus Flying Fox CichZasoma sp. Parrot Crossocheilus siamensis Flying Fox CichZa OceZZaris Peackock Bass Cichlid Popondella furcala Forktail Rainbow Trichogasler Zeeri Pearl Gourami Cyphorilapiafronlosa Frontosa Cichlid CichZasoma carpinle Pearl Scale Cichlid Cyalhopharynxfurczfer Furcifer Lamprologus caZvus Pearly Lamprologus Slurisomafursochi Fursochi Cat Fish Tropheus PEMBA Pemba River Tropheus Aphyosemion gardneri Gardneri Killi?sh Thayeria boelkea Penguin Tetra Pseudomugil gerlrudae Gertrudae ChaZceus macrolepidolus Pinktail Characin Danio malabarinchus Giant Danio Mogumda mogurnda Purple Striped Gudgeon Ambassis ranga Glass Angel Rasbora sp. Rasbora Red Fin Prionobrama?ligera Glass Blood?n Aphyocharax ralhbzmi Red Belly Tetra Hyposlomus plecoslomus Glass Cleaner Plecostomus CichZasoma Zabialum Red Devil Hemigrammus rodwayi Gold Tetra Moenkhausia sanlae?lomenae Red Eye Tetra Pzmrz'us sachsi Golden Barb Pseudolrophues sp. Red Eyed Tangarine Cichlid Nannacara anomaZa Golden Dwarf Cichlid Maslacembelus erylhrolaenia Red Fire Eel Nannoslomus beckfordi Golden Pencil Tetra Copadichromis borleyi Red Kadango PrisleZZa maxiZZaris Golden Pristella Rasborapauczperforala Red Line Rasbora Melanolaenia herbrl axelrodi Golden Rainbow Colossoma macropodum Red Pacu Scleropagesformosus Green Arowana Megalamphodus sweglesi Red Phantom Brachydanio rerio Green Danio Glossolepis incisus Red Rainbow Aequidens rivulalus Green Terror Cichlid CichZasoma severum Red Severum Cichlid Macrognalhus circumcinclus Half Banded Spiny Eel Nolropis Zulrensis Red Shiner Rasbora heleromorpha Harlequin Rasbora Megalamphodus roseus Red TailYellow Phantom Gasleropelecus slernicla Hatchet Fish Epalzeorhynchosfrenalus Red?n Shark Rasbora dorsioceZZala High Spot Rasbora Epalzeorhynchos bicolor Redtail Black Shark Geophagus sleindachneri Hondae Humphead Pzmrz'us conchonius Rosy Barb Clenolucius hujela Hujeta Hyphessobrycon benlosi Rosy Tetra Scleropagesjardini Jardini Arowana Pzmrz'us rhombocellalus Round Banded Clown Barb Hemichromispaynei Jewel Cichlid Pzmrz'us nigrofascialus Ruby Barb Melanochromisjohanni Johanni Cichlid Hemigrammus bZeheri Rummy Nose Tetra Julidichromis dickfeldi Juldchrmis Dickfeldi Arias graefei Salmon Cat?sh Julidichromis ornalus Julidochromis Omalus Hyphessobrycon serpae Serpae Tetra Julidichromis lranscrzplus Julidochromis Transcnplus Hyphessobrycon serpae sp Serpae Tetra Veiltail Geophagusjurupari Jurupari Cichlid Osleoglossum bichirrhosum Silver Arowana Tropheus IKOLA Kaisar Tropheus Distichodus a?inis Silver Distichodus Hyphessobrycon Zoweae Kitti Tetra Melynnis hypsauchen Silver Dollar Srigmalogobius sadanzmdio Knight Goby Selenoloca mulrzfasciala Silver Scat Cyprinus Carpio Koi Hasemania nanna Silver Tipped Tetra Acanlhoplhalmus kuhZii Kuhlii Loach Balanliocheilos melanoplerus Silver Tricolor Shark Lamprologus Silindericus Lamprologus Silindericus Rasbora espei Slender Wedge Rasbora Lamprologus ZeZeupi Lemon Cichlid Pseudomugil signzfer Southern Blue Eye Labidochromis caeruleus Lemon Mbuna Cichlid ChiZodus punclalus Spotted Headstander Hyphessobrycon pulchn'pinnis Lemon Tetra Rasbora maculala Spotted Pygmy Rasbora Clenopoma acuriroslre Leopard Bush?sh Melynnis maculalus Spotted Silver Dollar Brachydaniofrankei Leopard Danio Pzmrz'us Zinealus 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 Omamental Fish Species experience in the ornamental ?sh production ?eld. Depend Scienti?c Name Common Name ing on the type of ?sh, it is generally preferred that the eggs be fertilized prior to or at the time of collection. This is prefer Scleropagesformosus Super Red Arowana ably accomplished by placing a male and female ?sh together Corynopoma riseii Swordtail Characin CichZasoma synspilum Synspillum Cichlid in a tank that allows egg collection under conditions that Irianlherina wemeri Thread?n Rainbow stimulate mating. A fertilized egg cell prior to the ?rst cell Capoela lelrazona Tiger Barb division is considered a one cell embryo, and the fertilized Pseudoplaglslomafascialum Tiger Shovelnose Cat?sh egg cell is thus considered an embryonic cell. TiZapia bullikoferi Tiger Zebra Tilapia Pelrochromis lrewavasae 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). Myslus micracanlhus Two Spotted Cat?sh After introduction of the transgenic construct, the embryo is Uaru amphiacanlhoides Uaru — Triangle Cichlid allowed to develop into a ?sh. The ?sh that were injected as Sphaerichlhys vaZZianri Valliant’S Gourami Thayeria boehlkea sp. Veiltail Penguin Tetra embryos are allowed to interbreed and the offspring are Oplhalmorilapia venlralis Venlralis screened for the presence of the trans gene. Fish harboring the Haplochromis venuslus Venuslus transgene may be identi?ed by any suitable means. In the Synodonl‘is schouledeni Vermiculated Synodntis Tanichlys albonubes White Cloud preferred case, one or more of the transgenic constructs will Tanichlyhs albonubes White Cloud Minnow have integrated into the cellular genome, which can be probed Osphronemus gourami White Giant Gourami for the presence of construct sequences. To identify trans Symphysodon aequzfasciala White Smoke genic ?sh actually expressing the transgene, the presence of Aphyocharax paraguayensis White Spot Tetra Crenicichla saxalilus White Spotted Pike Cichlid an expression product can be assayed. Several techniques for Maslacembelus armalus White Spotted Spiny Eel such identi?cation are known and used for transgenic animals Gymnocorymbus lemelzi White Tetra and most can be applied to transgenic ?sh. Probing of poten Bella coccina Wine Red Betta tial or actual transgenic ?sh for nucleic acid sequences Melanochromis auralus Yellow Auratus Cichlid Hemmigrammopelersius caudalis Yellow Congo present in or characteristic of a transgenic construct can be Apislograma boreZZi Yellow Dwarf Cichlid accomplished by Southern or northern blotting, polymerase chain reaction (PCR) or other sequence-speci?c nucleic acid ampli?cation techniques. [0065] The more preferred ?sh for use with the disclosed [0069] The simplest way to con?rm the presence of a ?uo constructs and methods is zebra?sh, Danio rerio. Zebra?sh rescent protein expressing transgene in a given ?sh is by are increasingly popular ornamental animals and would be of visual inspection, as the ?sh in question would be brightly added commercial value in various colors. Zebra?sh embryos colored and immediately distinguishable from non-trans are easily accessible and nearly transparent. The most pre genic ?sh. Preferred techniques for identifying ?uorescent ferred ?sh for use with the disclosed constructs and methods protein expressing transgenic zebra?sh are described in the is the Golden Zebra?sh. Zebra?sh skin color is 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 ?uorescent transgenic ?sh exhibits strong visible ?uores melanosomes per pigment cell in?uence the color of the ?sh cence, strong visible ?uorescence means that a person with skin. Golden zebra?sh 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 ?uorescent ?sh in question and a non-?uorescent compared to the wild type zebra?sh. Golden zebra?sh have a ?sh of the same species at a distance of at least 5 feet in a mutation in slc24a5 gene, slc24a5 codes for a putative cation lighted of?ce, with a preferred distance of at least 10 feet in a exchanger localized to intracellular membrane, rendering the lighted of?ce, and a more preferred distance of at least 15 feet ?sh skin lighter or less pigmented (Lamason et al., 2005). in a lighted o?ice, and an even more preferred distance of at least 20 feet in a lighted of?ce, with the illumination level [0066] The disclosed transgenic ?sh are produced by intro de?ned in Table 6. One can observe all transgenic ?uorescent ducing a transgenic construct into the genomes of cells of a ?sh from a particular population that exhibit strong visible ?sh, preferably embryonic cells, and most preferably in a ?uorescence under the various lighting conditions and select single cell embryo. Where the transgenic construct is intro the ?sh that exhibits the highest level of visible ?uorescence duced into embryonic cells, the transgenic ?sh is obtained by of the ?uorescent protein. Selected ?sh with strong visible allowing the embryonic cell or cells to develop into a ?sh. The ?uorescence are monitored and selected continuously to disclosed transgenic constructs can be introduced into embry ensure stability of expression and maintenance of the strong onic ?sh cells using any suitable technique. Many techniques visible ?uorescence trait. Thus a new line of ?sh exhibiting for such introduction of exogenous genetic material have strong visible ?uorescence is created for further breeding. been demonstrated in ?sh and other animals. These include [0070] The invention further encompasses progeny of a microinj ection (Culp et al., (1991), electroporation (Inoue et transgenic ?sh containing a genomically integrated trans al., 1990; Muller et al., 1993; Murakami et al., 1994; Muller genic construct, as well as transgenic ?sh derived from a et al., 1992; and Symonds et al., 1994), particle gun bombard transgenic ?sh 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 ?sh embryonic cells is by microinjec transgenic ?sh of the invention, or from breeding a ?rst trans tion. genic ?sh of the invention to a second ?sh that is not a US 2009/0025645 A1 Jan. 29, 2009

transgenic ?sh of the invention. In the latter case, the second tions, parameters, reagents, or starting materials Which must ?sh can, for example, be a Wild-type ?sh, a specialized strain be utilized exclusively in order to practice the art of the of ?sh, a mutant ?sh, or another transgenic ?sh. The hybrid present invention. progeny of these matings have the bene?ts of the transgene for ?uorescence combined With the bene?ts derived from Example 1 these other lineages. Design and Generation of the Construct Plasmids Fertilization from Frozen Sperm [0078] The promoter of the zebra?sh 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 [3-actin enhancer/promoter sequence (Lui et al., 1990) Were zebra?sh 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 ?uorescent protein gene, DsRed2; green ?uorescent pro herein by references. Brie?y, a droplet of ice-cold 100% tein gene, ZsGreen1 and yelloW ?uorescent protein gene, Hank’s saline is placed next to zebra?sh eggs in a petri dish. ZsYelloW1 Were ampli?ed by PCR from pDsRed2-N1, pZs Frozen sperm is thaWed for a feW seconds in air then expelled Green1-N1 and pZsYelloW1-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 [3-actin such that the promoter Was and then ?sh Water added. operably linked to the ?uorescent gene. Tandem SV40(A) polyA/3'UTR sequence from pK-SV40(A)X2 plasmid Were Vectors cloned 3' to the ?uorescent 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 e?iciency of the capable of expressing the polypeptide. ?uorescent protein gene expression. The resulting ?ve 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 [3-actin-DsRed2 and of the vector backbone from the plasmid before the transgenic pUC18-carp [3-actin-ZsGreen1 Were restriction double construct may be introduced into the zebra?sh. digested With XbaI andAatII enzymes 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 [0074] The invention further relates to a transformed cell or contained the promoter, the open reading frame and the microorganism containing cDNA or a vector Which codes for 3'UTR Was excised and puri?ed using phenolzcholoroform the polypeptide or a fragment or variant thereof and that is extraction. capable of expressing the polypeptide. [0080] The transgenic vectors pBluescript II SK(—)-zMLC DsRed2-SV40><2, pBluescript II SK(—)-zMLC-ZsGreen1 - Expression Systems Using Vertebrate Cells SV40><2, and pBluescript II SK(—)-zMLC-ZsYelloW1 - SV40x2 Were restriction triple digested With XhoI, XmnI and [0075] Interest has been great in vertebrate 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 ?sh described herein. Expression vectors for 3'UTR Was excised and gel puri?ed. 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 adenylation site. [0081] The puri?ed transgenic insert cassette Which con tained the promoter, the open reading frame and the 3'UTR [0076] In another aspect of the present invention, also Was microinjected into the zebra?sh embryos (FIG. 6, Step included is the commercial marketability of the transgenic 4). ?uorescent ?sh to the ornamental ?sh industry. [0082] While only one construct Was injected into YelloW zebra?sh 1, to increase the chances of developing a ?sh With EXAMPLES strong visible ?uorescence, more than one construct Was injected simultaneously in Red zebra?sh 1 and Green [0077] The invention Will noW be further described With zebra?sh 1. For the purposes of this application, strong visible reference to the folloWing examples. These examples are ?uorescence 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 ?uo intended to limit or restrict the scope of the present invention rescent ?sh in question and a non-?uorescent ?sh 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 of?ce, With