# Patent

## Title
Recombinant constructs and transgenic fluorescent ornamental fish therefrom

## Abstract

The present invention relates to the method and use of reef coral fluorescent proteins in making transgenic red, green and yellow fluorescent zebrafish. Preferably, such fluorescent zebrafish are fertile and used to establish a population of transgenic zebrafish and to provide to the ornamental fish industry for the purpose of marketing. Thus, new varieties of ornamental fish of different fluorescence colors from a novel source are developed.

## Background
The present application is a continuation of U.S. patent application Ser. No. 13/663,210, filed on Oct. 29, 2012, now U.S. Pat. No. 8,581,025, which was a continuation of U.S. patent application Ser. No. 12/577,467, filed on Oct. 12, 2009, which is a continuation of U.S. patent application Ser. No. 11/839,364 filed Aug. 15, 2007, now U.S. Pat. No. 7,700,825, which claims the benefit of previously filed provisional application Ser. Nos. 60/838,006, filed Aug. 16, 2006, and 60/842,721, filed Sep. 7, 2006, the disclosures of which are incorporated by reference herein in their entirety.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to transgenic gene constructs with fish gene promoters and heterologous genes for generation of transgenic fish, particularly fluorescent transgenic fish.
2. Description of Related Art
Transgenic technology involves the transfer of a foreign gene into a host organism enabling the host to acquire a new and inheritable trait. The technique was first developed in mice by Gordon et al. (1980). They injected foreign DNA into fertilized eggs and found that some of the mice developed from the injected eggs retained the foreign DNA. Applying the same technique, Palmiter et al. (1982) introduced a chimeric gene containing a rat growth hormone gene under a mouse heavy metal-inducible gene promoter and generated the first batch of genetically engineered supermice, which were almost twice as large as non-transgenic siblings. This work has opened a promising avenue in using the transgenic approach to provide to animals new and beneficial traits for livestock husbandry and aquaculture.
In addition to the stimulation of somatic growth for increasing the gross production of animal husbandry and aquaculture, transgenic technology also has many other potential applications. First, transgenic animals can be used as bioreactors to produce commercially useful compounds by expression of a useful foreign gene in milk or in blood. Many pharmaceutically useful protein factors have been expressed in this way. For example, human 1-antitrypsin, which is commonly used to treat emphysema, has been expressed at a concentration as high as 35 mg/ml (10% of milk proteins) in the milk of transgenic sheep (Wright et al., 1991). Similarly, the transgenic technique can also be used to improve the nutritional value of milk by selectively increasing the levels of certain valuable proteins such as caseins and by supplementing certain new and useful proteins such as lysozyme for antimicrobial activity (Maga and Murray, 1995). Second, transgenic mice have been widely used in medical research, particularly in the generation of transgenic animal models for human disease studies (Lathe and Mullins, 1993). More recently, it has been proposed to use transgenic pigs as organ donors for xenotransplantation by expressing human regulators of complement activation to prevent hyperacute rejection during organ transplantation (Cozzi and White, 1995). The development of disease resistant animals has also been tested in transgenic mice (e.g. Chen et al., 1988).
Fish are also an intensive research subject of transgenic studies. There are many ways of introducing a foreign gene into fish, including: microinjection (e.g., Zhu et al., 1985; Du et al., 1992), electroporation (Powers et al., 1992), sperm-mediated gene transfer (Khoo et al., 1992; Sin et al., 1993), gene bombardment or gene gun (Zelenin et al., 1991), liposome-mediated gene transfer (Szelei et al., 1994), and the direct injection of DNA into muscle tissue (Xu et al., 1999). The first transgenic fish report was published by Zhu et al., (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 concentrated on growth hormone gene transfer with an aim of generating fast growing “superfish”. While a majority of early attempts used heterologous growth hormone genes and promoters and failed to produce gigantic superfish (e.g. Chourrout et al., 1986; Penman et al., 1990; Brem et al., 1988; Gross et al., 1992), enhanced growth of transgenic fish has been demonstrated in several fish species including Atlantic salmon, several species of Pacific salmons, and loach (e.g. Du et al., 1992; Delvin et al., 1994, 1995; Tsai et al., 1995).
The zebrafish, Danio rerio, is a new model organism for vertebrate developmental biology. As an experimental model, the zebrafish offers several major advantages such as easy availability of eggs and embryos, tissue clarity throughout embryogenesis, external development, short generation time and easy maintenance of both the adult and the young. Transgenic zebrafish have been used as an experimental tool in zebrafish developmental biology. However, for the ornamental fish industry the dark striped pigmentation of the adult zebrafish does not aid in the efficient display of the various colors that are currently available in the market. More recently, Lamason et al. (2005) in their report showed that the Golden zebrafish carry a recessive mutation in the slc24a5 gene, a putative cation exchanger, and have diminished number, size and density of melanosomes which are the pigmented organelles of the melanocytes and hence are lightly pigmented as compared to the wild type zebrafish. The availability of the Golden zebrafish for transgenesis with fluorescent proteins would result in better products for the ornamental fish industry as it would allow for a better visualization of the various colors.
Green fluorescent protein (GFP) is a useful tool in the investigation of various cellular processes. The GFP gene was isolated from the jelly-fish Aqueous victoria. More recently, various other new fluorescent protein genes have been isolated from the Anthozoa class of coral reefs (Matz et al., 1999) called DsRed, red fluorescent protein gene; ZsGreen, green fluorescent protein gene and ZsYellow, yellow fluorescent protein gene. The novel fluorescent proteins encoded by these genes share 26-30% identity with GFP (Miyawaki, 2002). These are bright fluorescent proteins and each emits a distinct wavelength. They are physico-chemically very stable, extremely versatile, emitting strong visible fluorescence in a variety of cell types and display exceptional photostability and hence fluoresce over extended periods of time. Because of their distinct spectra, they can be used in combination. The crystal structure of the DsRed protein suggests that the chromofore is located on a central α-helical segment embedded within a tightly folded β-barrel and that the DsRed protein forms tetramers in vivo (Wall et al., 2000).
Coral reef fluorescent proteins have broad application in research and development. The red fluorescent protein, DsRed, has been used as a reporter in the transgenic studies involving various animal model systems: for example, filamentous fungi (Eckert et al., 2005, Mikkelsen et al., 2003); ascidian (Zeller et al., 2006); zebrafish (Zhu et al., 2005, Zhu et al., 2004, Gong et al., 2003, Finley et al., 2001); xenopus (Werdien et al., 2001); insect (Cho et al., 2006, Handler et al., 2001, Horn et al., 2002); drosophila (Barolo et al., 2004); silkworm (Royer et al., 2005); mouse (Schmid et al., 2006, Vintersten et al., 2004); rat (Sato et al., 2003); and plants (Wenek et al., 2003). It has also been used a marker in imaging studies in stem cells (Tolar et al., 2005, Long et al., 2005) and mouse (Long et al., 2005, Hadjantonakis et al., 2003). Green fluorescent protein, ZsGreen, has been used as a transformation marker in insects (Sarkar et al., 2006), knock-in mouse model for the study of KIT expressing cells (Wouters et al., 2005) and as reporters for plant transformation (Wenck et al., 2003). Yellow fluorescent protein, ZsYellow, has been used a reporter for plant transformation (Wenck et al., 2003) and for visualizing fungal pathogens (Bourett et al., 2002). All of these transgenic experiments have aimed at developing newer markers and reporters for transgenesis; however, progress in the field of ornamental fish industry has been limited.
SUMMARY OF THE INVENTION
In certain embodiments, the present invention concerns making recombinant constructs and transgenic fluorescent fish and providing such fish to the ornamental fish industry. The term recombinant construct is used to mean recombinant DNA constructs having sequences which do not occur in nature or exist in a form that does not occur in nature or exist in association with other materials that do not occur in nature. The term transgenic has historically been used in many contexts with various meanings. In the embodiments of this invention transgenic is understood to mean genetic material artificially introduced into the genome of an organism. An organism incorporating such genetic material, or progeny to which this genetic material was passed, would be considered a transgenic organism. Such transgenic organisms may also, in certain embodiment, be referred to generally as a genetically modified organism (GMO), which is defined as an organism whose genetic material has been altered using the genetic engineering techniques generally known as recombinant DNA technology. This modified DNA is then transferred into an organism preferably resulting in the expression of modified or novel traits. The term “GMO” does not cover organisms whose genetic makeup has been altered by conventional cross breeding or by “mutagenesis” breeding, as these methods predate the discovery of the recombinant DNA techniques. Technically, however, such techniques are by definition genetic modification. The term fluorescent is used to mean an entity that absorbs light of one wavelength and emits at a different wavelength.
Specific embodiments of the present invention are directed to methods of making transgenic fluorescent fish having one sequence from a group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, as well as transgenic fish developed by such methods. Thus, a transgenic zebrafish having integrated into its germ line cell DNA a transgenic construct comprising one or more of SEQ ID NO:1 through SEQ ID NO:5 is also included as part of the invention. Further more, the invention provides transgenic zebrafish egg and/or sperm cells comprising a sequence according to SEQ ID NO:1 through SEQ ID NO:5 integrated in its/their genome(s). In certain aspects of the invention, two or more sequences from a group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 are used in one zebrafish. In a specific aspect, SEQ ID NO:1 and SEQ ID NO:2 are used in the same fish and SEQ ID NO:3 and SEQ ID NO:5 are used in the same fish. In preferred embodiments, it is contemplated that the transgenic fluorescent fish are fertile transgenic fluorescent fish.
In another preferred embodiment, the fish for use with the disclosed constructs and methods is the Golden zebrafish. Zebrafish skin color is determined by pigment cells in their skin, which contain pigment granules called melanosomes. The number, size and density of the melanosomes per pigment cell influence the color of the fish skin. Golden zebrafish have diminished number, size, and density of melanosomes and hence have lighter skin when compared to the wild type zebrafish. Golden zebrafish have a mutation in slc24a5 gene, rendering the fish skin lighter or less pigmented (Lamason et al., 2005).
In another embodiment of the invention, a method for making transgenic fluorescent fish is provided comprising at least the following steps: a) preparing a vector which has a transgenic fluorescence expression cassette comprising one sequence from a group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, or two or more sequences from a group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 are used in combination, specifically, SEQ ID NO:1 and SEQ ID NO:2 are used together and SEQ ID NO:3 and SEQ ID NO:5 are used together; b) making the transgenic zebrafish using the vectors; and, c) selecting transgenic zebrafish that fluoresce by monitoring fluorescence under a light of appropriate wavelength. The transgenic expression cassette has a set of transcriptional regulatory motifs, herein referred to as a promoter, which may be from the host species (herein referred to as a homologous promoter) or from another species (herein referred to as a heterologous promoter), heterologous fluorescent gene, and appropriate RNA-processing and/or translational enhancing motif. The term promoter as used herein refers to the DNA elements that direct and regulate transcription. For instance, the zebrafish fast skeletal muscle myosin light chain promoter and carp β-actin promoter may be used according to the invention.
In certain specific embodiments there are provided methods to use multiple vectors to express at least one fluorescent protein in order to enhance expression. The preferred mode is to make a transgenic fish comprising in its genome a first fluorescent transgene under the control of a ubiquitous fish promoter, and a second fluorescent transgene under the control of a tissue specific fish promoter. The ubiquitous fish promoter is selected from the group consisting of those transcriptional motifs that direct gene expression in most cells, and more preferably in all cells; they are also preferably promoters for ‘housekeeping genes’, such as tubulin, ribosomal protein, and actin genes. The tissue specific fish promoter is selected from the group consisting of those transcriptional motifs that are active in specific cells of differentiated tissues such as muscle, brain, liver, blood and eyes. In a preferred embodiment, the tissue specific fish promoter is muscle specific. As used herein, a promoter drives expression “specifically” in a tissue if the level of expression is at least 5-fold, preferably at least 10-fold higher, more preferably at least 50-fold higher in that tissue than in any other tissue.
More than one construct can be injected into the fish embryos simultaneously. For example, in the present invention, both Red zebrafish 1 and Green zebrafish 1 incorporate more than one transgenic expression cassettes, with one being a ubiquitous promoter, and the other being a strong muscle promoter. In particular, Red zebrafish 1 incorporates the cassettes represented by FIG. 1 and FIG. 4, and Green zebrafish 1 incorporates the cassettes represented by FIG. 2 and FIG. 5. While the present invention incorporates only the transgenic insert cassettes shown in the Figures, it is understood that multiple transgenic insert cassettes of any type can be simultaneously injected into a fish embryo from any species.
The steps involved in making the transgenic fish further involve isolation and separation of the transgenic expression cassette from the vector backbone to remove any gene encoding antibiotic resistance (e.g., ampicillin or kanamycin) and origin of replication. In a preferred mode, a suitable promoter would be expected to drive stable and consistent expression throughout the life of the fish. To achieve such stable expression, it is necessary to choose a promoter that is known to drive stable and consistent expression throughout the life of the fish. For example, a promoter that drives expression only during the six months of the life of the fish would not be suitable for use. Examples of suitable promoters may be selected from the group consisting of those for housekeeping genes, such as tubulin, ribosomal protein, and actin gene promoters.
It is also preferred to use regulatory elements, for example, RNA processing and translational enhancing elements in the transgenic insert cassette to produce a transgenic fluorescent, ornamental fish. The RNA processing signals, preferably, are one or more polyadenylation signals and/or one or more introns. Since introns are sequences between exons, the presence of an intron automatically indicates the presence of two exons. Accordingly, two introns indicate the presence of three exons, and so on. The carp beta-actin intron used in SEQ ID 2 and SEQ ID 5 is an example of such an intron, and the untranslated carp beta-actin exon used in SEQ ID 2 and SEQ ID 5 is an example of such an exon. Exons and introns other than carp beta-actin can be used as well. The translational enhancing elements, preferably, are 5′ untranslated leader sequences of 40-200 nucleotides, and more preferably untranslated leader sequences of 40-70 nucleotides. It is known that the presence of introns in primary transcripts can increase expression, possibly by causing the transcript to enter the processing and transport system for mRNA. It is also preferred that the intron be homologous to the host species, and more preferably homologous to the expression sequences used (that is, that the intron be from the same gene that some or all of the expression sequences are from).
The disclosed transgene constructs preferably include other sequences which improve expression from, or stability of, the construct. For example, including a polyadenylation signal on the constructs encoding a protein ensures that transcripts from the transgene will be processed and transported as mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that efficient polyadenylation signals, such as those derived from viruses, be used in the transgenic constructs, and more preferred to use at least two polyadenylation signals, which more preferably are two copies of SV40 polyadenylation sequence.
It is also a subject of this invention to disclose expression of the fluorescent protein gene only in chromatophores. There are several types of chromatophores found in animals: melanophores (black), xanthophores (yellow), erythrophores (red), cyanophores (blue), leucophores (white) and iridophores (reflective). Different species of fish contain all types of chromatophores, usually a subset of them in different combinations. Zebrafish contain melanophores, xantophores and iridophores. These different cell types express specific genes, characteristic only for them or specific for a subset of chromatophores. In a preferred embodiment, promoters of these specific genes fused to fluorescent protein open reading frames (ORFs) can be used to visualize specific chromatophores. The specific genes can be roughly divided into two major groups: regulatory proteins and biosynthesis enzymes, involved in specific pigment synthesis. Expression of regulatory proteins usually is at lower level than that of biosynthesis enzymes therefore use of promoters of biosynthesis enzymes are most preferred.
The heterologous fluorescent gene may be, for example, a gene encoding DsRed2, ZsGreen1 and ZsYellow1. The heterologous fluorescent gene may also be any variation or mutation of these genes, encoding fluorescent proteins including green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), cyan fluorescent protein (CFP) and enhanced cyan fluorescent protein (eCFP) or any of the proteins listed in Table 4, or any variation or mutation thereof, or any other fluorescence proteins. The steps involved in making the transgenic fish also involve introduction of the transgenic expression cassette into the zebrafish embryos or zebrafish embryonic stem cells. Such embryos and cells are allowed to grow and mature into adult fish and then they are screened for the presence of the transgenic expression cassette using the various molecular biology methods described in the detailed description section and/or by functional biochemical assays such as assaying for the activity of the introduced fluorescent gene by exposing the said fish to light of appropriate wavelength and/or by visibly inspecting the fish and observing the expression. Transgenic fluorescent fish are further bred to insure transmission of the transgenic expression cassette via the germ cells of a fish as further described in this application.
The present invention also provides a method to obtain a progenitor of a new line of fluorescent transgenic fish, and a population therefrom, which exhibit strong visible fluorescence. Strong visible fluorescence means that a person with 20/20 vision (i.e., average vision) will be able to distinguish between the fluorescent fish in question and a non-fluorescent fish of the same species at a distance of at least 5 feet in a lighted office, with a preferred distance of at least 10 feet in a lighted office, and a more preferred distance of at least 15 feet in a lighted office, and an even more preferred distance of at least 20 feet in a lighted office, with the illumination level defined in Table 5. One can observe all transgenic fluorescent fish from a particular population that exhibit strong visible fluorescence under the various lighting conditions and select the fish that exhibits the highest level of visible fluorescence of the fluorescent protein. Selected fish with strong visible fluorescence are monitored and their progeny selected continuously to ensure stability of expression and maintenance of strong visible fluorescence. Thus a new line of fish that exhibit strong visible fluorescence is created for further breeding.
Transgenic fish made by the present disclosure will emit red, yellow-green and yellow-orange fluorescence under light of distinct wavelengths and hence will be unique and attractive to the ornamental fish industry. In yet another embodiment of the invention, a method of making the transgenic fish available to the consumer by a grower or a commercial distributor through a retailer for sale to the public. In such embodiment, the fish may also be sold by the grower or commercial distributor to a regional wholesale distributor, who will then sell to a retailer for sale to the public. The fluorescent transgenic fish are also useful for the development of a biosensor system and as research models for embryonic studies such as cell lineage, cell migration, cell and nuclear transplantation, cell-cell interaction in vivo, etc.
Transgenic zebrafish comprising an expression cassette according to the invention may be homozygous or heterozygous with respect to the expression cassette. In some preferred aspects, fish for use in breeding of transgenic zebrafish of the invention will be homozygous for an expression cassette. Homozygous fish bred with fish lacking an expression cassette (e.g., Golden zebrafish) will in nearly all cases produce 100% heterozygous offspring. Likewise, transgenic fish for commercial retail will preferably be heterozygous for an expression cassette. Furthermore in some very specific aspects a transgenic fish of the invention comprises the specific integration event of the Red fluorescent expression cassette described in Example 3.
In certain specific embodiments there are provided transgenic fluorescent zebrafish comprising specific transgenic integration events. These fish are of particular interest since, for example, they embody an esthetically pleasing level of protein fluorescence. Thus, in some embodiments there is provided a transgenic zebrafish comprising a chromosomally integrated expression cassette encoding a DsRed2 gene wherein the zebrafish comprises the Red zebrafish 1 transformation event, sperm comprising said Red zebrafish 1 transformation event having been deposited as ECACC accession no. 06090403. In some other aspects, there is provided a transgenic zebrafish comprising a chromosomally integrated expression cassette encoding a ZsGreen1 gene wherein the zebrafish comprises the Green zebrafish 1 transformation event, sperm comprising said Green zebrafish 1 transformation event having been deposited as ECACC accession no. 06090401. In still other aspects, there is provided a transgenic zebrafish comprising a chromosomally integrated expression cassette encoding a ZsYellow1 gene wherein the zebrafish comprises the Yellow zebrafish 1 transformation event, sperm comprising said Yellow zebrafish 1 transformation event having been deposited as ECACC accession no. 06090402. As described above, transgenic fish comprising these specific transgenic events may be homozygous or heterozygous for transgene, and in some cases may comprise more than one of the transgenic events, although it is preferred to have only one integration location for any given transgenic modification. Eggs, sperm and embryos comprising these specific transgenic events are also included as part of the instant invention.
Any of the fluorescence genes noted in this application may be used in similar embodiments of this invention. Embodiments discussed in the context of a method and/or composition of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.
FIG. 1: The figure shows a schematic map of the transgenic construct, pZMLC-DsRed2-SV40x2. The 2.1-kb eukaryotic promoter sequence zMLC-1934 promoter was amplified by PCR from pMLC vector and cloned into XhoI and EcoRI restriction sites. The 684 bp DsRed2 fluorescent protein CDS was amplified by PCR from pDsRed2-N1 (Clontech) and inserted into EcoRI and SpeI sites. The 440-bp 3′UTR/poly(A) sequence encoding tandem SV40 polyadenylation signals was PCR amplified from pK-SV40(A)x2 and cloned into SpeI and NotI sites. XhoI, XmnI and NotI restriction sites were used to isolate the expression construct from the vector backbone. Also shown is the ampicillin (Amp, formally known as beta-lactamase (bla)) resistance gene in the backbone of the pBluescript plasmid. The total length of the recombinant plasmid pzMLC-DsRed2-SV40x2 is 6009 bp.
FIG. 2: The figure shows a schematic map of the transgenic construct, pZMLC-ZsGreen1-SV40x2. The 1.9-kb eukaryotic promoter sequence zMLC-1934 promoter was amplified by PCR from pMLC vector and cloned into XhoI and EcoRI restriction sites. The 716 bp ZsGreen1 fluorescent protein CDS was amplified by PCR from pZsGreen1-N1 (Clontech) and inserted into EcoRI and SpeI sites. The 440-bp 3′UTR/poly(A) sequence encoding tandem SV40 polyadenylation signals was PCR amplified from pK-SV40(A)x2 and clone into SpeI and NotI sites. XhoI, XmnI and NotI restriction sites were used to isolate the expression construct from the vector backbone. Also shown is the ampicillin (Amp) resistance gene in the backbone of the pBluescript plasmid. The total length of the recombinant plasmid pzMLC-ZsGreen1-SV40x2 is 604 lbp.
FIG. 3: The figure shows is a schematic map of the transgenic construct, pZMLC-ZsYellow1-SV40x2. The 1.9-kb eukaryotic promoter sequence zMLC-1934 promoter was amplified by PCR from pMLC vector and cloned into XhoI and EcoRI restriction sites. The 718 bp ZsYellow1 fluorescent protein CDS was amplified by PCR from pZsYellow1-N1 (Clontech) and inserted into EcoRI and SpeI sites. The 440-bp 3′UTR/poly(A) sequence encoding tandem SV40 polyadenylation signals was PCR amplified from pK-SV40(A)x2 and clone into SpeI and NotI sites. XhoI, XmnI and NotI restriction sites were used to isolate the expression construct from the vector backbone. Also shown is the ampicillin (Amp) resistance gene in the backbone of the pBluescript plasmid. The total length of the recombinant plasmid pzMLC-ZsYellow 1-SV40x2 is 6043 bp.
FIG. 4: The figure shows a schematic map of the transgenic construct, pCBAC-DsRed2-SV40x2. The 2.5-kb common carp beta-actin enhancer/promoter sequence, beta-actin exon-1 and beta-actin intron-1 was amplified by PCR from pFV7b vector and cloned into XbaI and KpnI restriction sites. The 684 bp DsRed2 fluorescent protein CDS was amplified by PCR from pDsRed2-N1 (Clontech) and inserted into EcoRI and SpeI sites. The 443-bp 3′UTR/poly(A) sequence encoding tandem SV40 polyadenylation signals sequence encoding tandem SV40 signal was PCR amplified from pK-SV40(A)x2 and cloned into SpeI and AatII sites. XbaI and AatII restriction sites were used to isolate the expression construct from the vector backbone. Also shown is the ampicillin (Amp) resistance gene in the backbone of the pBluescript plasmid. The total length of the recombinant plasmid pCBAC-DsRed2-SV40x2 is 5801 bp.
FIG. 5: The figure shows a schematic map of the transgenic construct, pCBAC-ZsGreen1-SV40x2. The 2.5-kb carp beta-actin enhancer/promoter sequence, beta-actin exon 1 and beta-actin intron 1 was amplified by PCR from pFV7b vector and cloned into XbaI and KpnI restriction sites. The 716 bp ZsGreen1 fluorescent protein CDS was amplified by PCR from pZsGreenl-N1 (Clontech) and inserted into EcoRI and SpeI sites. The 443 bp 3′UTR/poly(A) sequence encoding tandem SV40 polyadenylation signals sequence encoding tandem SV40 signal was PCR amplified from pK-SV40(A)x2 and cloned into SpeI and AatII sites. XbaI and AatII restriction sites were used to isolate the expression construct from the vector backbone. Also shown is the ampicillin (Amp) resistance gene in the backbone of the pBluescript plasmid. The total length of the recombinant plasmid pCBAC-ZsGreen1-SV40x2 is 5833 bp.
FIG. 6: Transgenic Construct purification and injection process. The Figure depicts step by step the process of transgenic construct purification and injection. Step 1 illustrates separation of the plasmid backbone sequence with the antibiotic resistance gene and origins of replication (pUC on and fl(−) ori) (on left) and the expression construct (on right). Step 2 and 3 show the method of purification of the expression construct by loading and electrophoretic separation of the DNA fragments on an agarose gel. The antibiotic resistance gene and origins of replication (pUC on and fl(−) ori) are below the expression construct on the gel. Step 4 exemplifies the process of microinjection of the gel-purified expression construct in to the fertilized zebrafish embryos.



DETAILED DESCRIPTION OF THE INVENTION
Transgenic Constructs
The present invention encompasses transgenic constructs which are genetic material artificially introduced into fish to produce a transgenic fish. The manner of introduction, and, often, the structure of a transgenic construct, render such a transgenic construct an exogenous construct. Although a transgenic construct can be made up of any assembly of nucleic acid sequences, for use in the disclosed transgenic fish it is preferred that the transgenic constructs combine regulatory elements operably linked to a sequence encoding one or more proteins. The methods and protocols for designing and making transgenic constructs are well known to those skilled in the art and can be found, for example, in Sambrook et al., 2001; Sambrook et al., 1989 and U.S. Pub No. 2004/0143864 A1, all of which are hereby incorporated by reference in their entireties.
To develop successful transgenic fish with a predictable pattern of transgenic expression, the first step is to make the appropriate genetic construct. The genetic construct generally comprises three portions: transcriptional regulators comprising a promoter, a gene and appropriate RNA-processing and/or translational enhancing motif. The gene promoter would determine where, when and under what conditions the gene is expressed. The gene contains protein coding portions that determine the protein to be synthesized and thus the biological function. The gene might also contain intron sequences which can affect mRNA processing or which might contain transcription regulatory elements. The RNA processing signals may include: one or more polyadenylation signals and one or more introns. Among the three portions, it is preferable to use a promoter that drives strong expression. The promoter may be a homologous promoter or it may be a heterologous promoter.
A promoter drives expression “predominantly” in a tissue if expression is at least 2-fold, preferably at least 5-fold higher in that tissue compared to a reference tissue. A promoter drives expression “specifically” in a tissue if the level of expression is at least 5-fold, preferably at least 10-fold higher, more preferably at least 50-fold higher in that tissue than in any other tissue. A ubiquitous promoter drives expression in most tissues, and preferably in all tissues.
Recombinant DNA Constructs
Recombinant DNA constructs comprising one or more of the DNA sequences described herein and an additional DNA sequence are also included within the scope of this invention. These recombinant DNA constructs usually have sequences which do not occur in nature or exist in a form that does not occur in nature or exist in association with other materials that do not occur in nature. The DNA sequences described as constructs or in vectors above are “operably linked” with other DNA sequences. DNA regions are operably linked when they are functionally related to each other. Generally, operably linked means contiguous (or in close proximity to).
The disclosed transgenic constructs preferably include other sequences that improve expression from, or stability of, the construct. For example, including a polyadenylation signal on the constructs encoding a protein ensures that mRNA transcripts from the transgene will be efficiently translated as protein. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that defined and efficient polyadenylation signals, such as those derived from viruses, be used in the transgenic constructs, and more preferred to use at least two polyadenylation signals, which more preferably are two copies of SV40 polyadenylation sequence.
In certain specific embodiments there are provided methods to use multiple vectors to express at least one fluorescent protein in order to enhance expression. The preferred mode is to make a transgenic fish comprising in its genome a first fluorescent transgene under the control of a ubiquitous fish promoter, and a second fluorescent transgene under the control of a tissue specific fish promoter. In a preferred embodiment, the tissue specific fish promoter is muscle specific. The ubiquitous fish promoter and the muscle specific promoter are, for example, selected from Table 1 below. In the Table 1, any promoter marked with an “X” is confirmed available at this time, with any unmarked promoter, or any other promoter of interest, available preferably through the following steps: following the database searching instructions provided in detail below, conducting a literature search, and sequencing the gene and promoter of interest through methods that are well know by artisans in the field.
The provided Table 1 of muscle-specific and ubiquitous promoters constitutes only a small portion of publicly available promoters. An extensive list of genes with expression of interest (e.g., muscle-specific expression) can be found using NCBI protein database server (www at ncbi.nlm.nih.gov/sites/entrez?db=Protein). For example, in order to find mouse genes expressed in skeletal muscles a search string “mouse skeletal muscle” can be used. The search results in a list of proteins including their accession number (e.g., CAA47621) and their name (e.g., mouse fast skeletal muscle SR calcium ATPase). In order to find genome information (e.g., sequence) of the found proteins, Ensembl Genome Browser (www at ensembl.org/index.html) can be employed, using the accession number ((e.g., CAA47621) as a search string. The search output will yield Ensembl gene ID (e.g., ENSMUSG00000030730), gene homologues in other organisms (e.g., zebrafish—Danio rerio), as well as genomic information of the gene of interest, including genomic sequence of the coding region (introns and exons), as well as genomic DNA sequence surrounding the coding sequence (e.g., “[Exon info]” link directs a user to the sequence information). Gene promoters are located upstream (5′ flanking sequence) from the coding sequence, often within several (e.g., five) kilobases. In addition, some regulatory sequences can be found in introns of the gene of interest—these regulatory sequences are usually omitted from constructing tissue-specific gene expression “drivers” due to complexity of locating them. It is to be understood that the same approach can be used starting with “zebrafish skeletal muscle” or “medaka skeletal muscle” or any other species. The searcher may then continue the search as suggested above to find the genome and promoter information of interest. It is also to be understood that methods similar to the one described for searching the database referenced above can be used to search other existing sequence databases, as well as databases that are likely to be compiled in the future.







TABLE 1







Muscle specific and ubiquitous promoters for fish expression
















Gene promoter
Zebrafish
Fugu
Tetraodon
Medaka
Xenopus
Rat
Mouse
Dog
Bovine





Muscle specific











Muscle creatine kinase
X
X
X
X
X
X
X
X
X


MyoD
X
X
X
X
X
X
X
X
X


Myogenin
X
X
X
X
X
X
X
X
X


Desmin
X
X



X
X
X
X


Muscle enolase-3
X

X


X
X

X


beta-sarkoglycan
X
X
X
X
X
X
X
X
X


Dystrophin
X
X
X
X
X
X
X

X


Serum response factor
X
X
X
X
X
X
X
X
X


a-tropomyosin
X
X
X
X
X
X
X
X
X


Myosin heavy chain
X
X

X
X
X
X
X
X


Mitochondrial creatine kinase 2
X
X
X
X


X
X
X


Myosin light chain
X
X
X
X
X
X
X
X
X


Ca2+ transporting ATPase (fast twitch 1)
X





X


skeletal Troponin T1 (slow)
X





X


Tropomodulin 4
X





X


Four and a half LIM domains 1
X





X


Fast-type myosin binding protein C
X





X


Calsequestrin 1
X





X


Fast muscle troponin C2
X





X


Phosphorylase kinase alpha 1
X





X


Skeletal troponin I (fast 2)
X





X


Ubiquitous


EF-1 alpha
X
X
X
X
X
X
X
X
X


Histone 2A ZA
X
X
X
X


X
X
X


Acidic ribosomal phosphoprotein PO (ARP)
X
X
X
X
X

X


alpha-catenin
X
X
X
X
X
X
X
X
X


beta-catenin

X
X
X
X
X
X
X
X


gamma-catenin
X
X
X
X
X
X
X
X
X


Srb7
X
X

X

X
X
X
X


Creatine kinase (mitochondrial 1)
X





X


Ubiquitous Ca2+ transporting ATPase
X





X


Ancient ubiquitous protein
X





X


Ubiquitin specific peptidase 4
X





X


Acetyl-Coenzyme A acryltransferase 2
X





X


Monoglyceride lipase
X





X


Splicing factor 3b subunit 1
X





X


Tubulin β5
X





X


Beta-Actin









Table 2, below, is a partial list of Ensembl gene ID numbers of mouse and zebrafish skeletal muscle specific and ubiquitous genes found using this approach.







TABLE 2





Ensembl IDs of Muscle Specific Promoters

















MUSCLE CREATINE KINASE PROMOTERS



Zebrafish: (ENSDARG00000035327)



Fugu: (SINFRUG00000143294)



Tetraodon: (GSTENG00012956001)



Medaka: (ENSORLG00000000449)



Xenopus tropicalis: (ENSXETG00000019108)



Rat: (ENSRNOG00000016837)



Mouse: (ENSMUSG00000030399)



Dog: (ENSCAFG00000004507)



Bovine: (ENSBTAG00000013921)



MYOD PROMOTERS



Zebrafish: (ENSDARG00000030110)



Fugu: (SINFRUG00000154785)



Tetraodon: (GSTENG00003954001)



Medaka: (ENSORLG00000000694)



Xenopus tropicalis: (ENSXETG00000001320)



Rat: (ENSRNOG00000011306)



Mouse: (ENSMUSG00000009471)



Dog: (ENSCAFG00000009066)



Bovine: (ENSBTAG00000002216)



MYOGENIN PROMOTERS



Zebrafish: (ENSDARG00000009438)



Fugu: (SINFRUG00000121801)



Tetraodon: (GSTENG00013986001)



Medaka: (ENSORLG00000015906)



Xenopus tropicalis: (ENSXETG00000001704)



Rat: (ENSRNOG00000030743)



Mouse: (ENSMUSG00000026459)



Dog: (ENSCAFG00000010309)



Bovine: (ENSBTAG00000006030)



DESMIN PROMOTERS



Zebrafish: (ENSDARG00000058656)



Fugu: (SINFRUG00000121939)



Xenopus tropicalis: (ENSXETG00000019275)



Rat: (ENSRNOG00000019810)



Mouse: (ENSMUSG00000026208)



Dog: (ENSCAFG00000015475)



Bovine: (ENSBTAG00000005353)



MUSCLE ENOLASE 3 BETA PROMOTERS



Zebrafish: (ENSDARG00000039007)



Tetraodon: (GSTENG00003809001)



Rat: (ENSRNOG00000004078)



Mouse: (ENSMUSG00000060600)



Bovine: (ENSBTAG00000005534)



BETA-SARCOGLYCAN PROMOTERS



Zebrafish: (ENSDARG00000052341)



Fugu: (SINFRUG00000123612)



Tetraodon: (GSTENG00032779001)



Medaka: (ENSORLG00000000171)



Xenopus tropicalis: (ENSXETG00000011676)



Rat: (ENSRNOG00000002135)



Mouse: (ENSMUSG00000029156)



Dog: (ENSCAFG00000002001)



Bovine: (ENSBTAG00000014601)



DYSTROPHIN PROMOTERS



Zebrafish: (ENSDARG00000008487)



Fugu: (SINFRUG00000144815)



Tetraodon: (GSTENG00024870001)



Medaka: (ENSORLG00000020638)



Xenopus tropicalis: (ENSXETG00000012391)



Rat: (ENSRNOG00000003667)



Mouse: (ENSMUSG00000045103)



Bovine: (ENSBTAG00000008254)



SERUM RESPONSE FACTOR PROMOTERS



Zebrafish: (ENSDARG00000053918)



Fugu: (SINFRUG00000162928)



Tetraodon: (GSTENG00025109001)



Medaka: (ENSORLG00000013036)



Xenopus tropicalis: (ENSXETG00000018511)



Rat: (ENSRNOG00000018232)



Mouse: (ENSMUSG00000015605)



Dog: (ENSCAFG00000001829)



Bovine: (ENSBTAG00000012777)



ALPHA-TROPOMYOSIN PROMOTERS



Zebrafish: (ENSDARG00000033683)



Fugu: (SINFRUG00000130484)



Tetraodon: (GSTENG00015950001)



Medaka: (ENSORLG00000012326)



Rat: (ENSRNOG00000018184)



Mouse: (ENSMUSG00000032366)



Dog: (ENSCAFG00000016966)



Bovine: (ENSBTAG00000005373)



MYOSIN HEAVY CHAIN PROMOTERS



Zebrafish: (ENSDARG00000035437)



Fugu: (SINFRUG00000135173)



Medaka: (ENSORLG00000001985)



Xenopus tropicalis: (ENSXETG00000023939)



Rat: (ENSRNOG00000031400)



Mouse: (ENSMUSG00000033196)



Dog: (ENSCAFG00000023926)



Bovine: (ENSBTAG00000007090)



MITOCHONDRIAL CREATINE KINASE



(SARCOMERIC, CKMT2) PROMOTERS



Zebrafish: (ENSDARG00000035079)



Fugu: (SINFRUG00000160265)



Tetraodon: (GSTENG00028607001)



Medaka: (ENSORLG00000000769)



Mouse: (ENSMUSG00000021622)



Dog: (ENSCAFG00000008707)



Bovine: (ENSBTAG00000001003)



MYOSIN LIGHT CHAIN PROMOTERS



Zebrafish: (ENSDARG00000017441)



Fugu: (SINFRUG00000125026)



Tetraodon: (GSTENG00015855001)



Medaka: (ENSORLG00000015981)



Xenopus tropicalis: (ENSXETG00000006917)



Rat: (ENSRNOG00000013262)



Mouse: (ENSMUSG00000061816)



Dog: (ENSCAFG00000013875)



Bovine: (ENSBTAG00000009707)









While this approach will result in a great number of sequences, additional points should be considered to generate a list of strong promoters. For example, abundant structural genes (e.g., myosin) or abundant enzymes (e.g., SR calcium ATPase) are likely to yield strong promoters. This screening can easily be performed by an artisan in the field.
Preferably more than one construct with different promoters can be injected into the fish embryos simultaneously. For example, in the present invention, both Red zebrafish 1 and Green zebrafish 1 incorporate more than one transgenic expression cassette, with one being a ubiquitous promoter, and the other being a strong muscle promoter. In particular, Red zebrafish 1 incorporates the cassettes represented by FIG. 1 and FIG. 4, and Green zebrafish 1 incorporates the cassettes represented by FIG. 2 and FIG. 5. While the present invention incorporates only the transgenic insert cassettes shown in the Figures, it is understood that multiple transgenic insert cassettes of any type can be simultaneously injected into a fish embryo from any species.
It is also a subject of this invention to disclose expression of the fluorescent protein gene specifically in chromatophores. Chromatophores are pigment-containing and light-reflecting cells found in animals. There are several types of chromatophores: melanophores (black), xanthophores (yellow), erythrophores (red), cyanophores (blue), leucophores (white) and iridophores (reflective). Of those, only melanophores, called melanocytes, are found in higher vertebrates, such as mammals. Different species of fish contain all types of chromatophores, usually a subset of them in different combinations. Zebrafish contain melanophores, xantophores and iridophores. These different cell types express specific genes, characteristic only for them or specific for a subset of chromatophores. For example, tyrosinase-related protein 1 (tyrp1) is found only in melanophores; ednrbl is found in malenocytes and iridophores. Promoters of these specific genes fused to fluorescent protein open reading frames (ORFs) can be used to visualize specific chromatophores. For example, fugu tyrp1 promoter can be used to drive fluorescent protein expression in melanophores in zebrafish. The specific genes can be roughly divided into two major groups: regulatory proteins (for example, kit—a receptor tyrosine kinase, specific to melanophores) and biosynthesis enzymes, involved in specific pigment synthesis (for example, sepiapterin reductase, involved in yellow pigment synthesis in xanthophores). Expression of regulatory proteins usually is at lower level than that of biosynthesis enzymes therefore use of promoters of biosynthesis enzymes are most preferred. A chromatophore-specific gene expression is outlined in Table 3 below.
Of all chromatophores, melanophores have been studied most extensively (due to their relevance to human biology). Therefore, a lot is known about transcription factors specific to melanophores, as well as biosynthesis enzymes involved in melanin synthesis in different classes of organisms, ranging from lower vertebrates to humans. The next best characterized chromatophores are the Xanthophores, for which a number of genes have been isolated, yielding, a number of known promoters to choose from. With respect to iridophores, a few specific genes have been isolated (for example, endothelin receptor b1 Ednrb1). The least known chromatophores are the cyanophores—neither the nature of their pigment, nor specification pathway of the cells per se is known.







TABLE 3







Chromatophore-specific expressed genes in fishes











Chromatophore
Protein
Synth/Reg
Organism
Reference





Iridophore
ednrb1
Reg
Zebrafish
Parichy et al, 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 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





latipes)




Xanthine
Synth





oxidoreductase






Fms/Csf1
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 et al, Development






127, 3031-3044 (2000)


Melanophores
Mitf
Reg
Zebrafish




kit
Reg
Zebrafish




tyrp1
Synth
zebrafish,
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



trp2 (tyrosinase-
Synth
mouse
Zhao & Over beek, Dev Biol.



related protein 2)


1999 Dec 1; 216(1): 154-63



dopachrome
Synth





tautomerase









It is also known that the presence of introns in primary transcripts can increase expression, possibly by causing the transcript to enter the processing and transport system for mRNA. It is preferred that the intron be homologous to the host species, and more preferably homologous to the expression sequences used (that is, that the intron be from the same gene that some or all of the expression sequences are from). The use and importance of these and other components useful for transgenic constructs are discussed in Palmiter et al. (1991); Sippel et al. (1992); Kollias and Grosveld (1992); and Clark et al. (1993).
The steps involved in making the transgenic fish further involve isolation and separation of the transgenic expression cassette from the vector backbone to remove the gene encoding antibiotic (e.g., ampicillin or kanamycin) resistance and origin of replication. In a preferred mode, a suitable promoter is chosen which is expected to drive stable expression throughout the life of the fish. To achieve such stable expression, it is necessary to choose a promoter that is known to drive stable and consistent expression throughout the life of the fish. For example, a promoter that drives expression only during the six months of the life of the fish would not be suitable to achieve stable expression throughout the life of the fish.
The heterologous fluorescent gene may be, for example, a gene encoding DsRed2, ZsGreen1 and ZsYellow1. The heterologous fluorescent gene may also be any variation or mutation of these genes, encoding fluorescent proteins including green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), cyan fluorescent protein (CFP) and enhanced cyan fluorescent protein (eCFP) or any of the proteins listed in Table 4, below, or any variation or mutation thereof, or any other fluorescence proteins. The steps involved in making the transgenic fish also involve introduction of the transgenic expression cassette into the zebrafish embryos or zebrafish embryonic stem cells. Such embryos and cells are allowed to grow and mature into adult fish and then they are screened for the presence of the transgenic expression cassette using the various molecular biology methods described in the detailed description section and/or by functional biochemical assays such as assaying for the activity of the introduced fluorescent gene by exposing the said fish to light of appropriate wavelength and/or by visibly inspecting the fish and observing the expression. Transgenic fluorescent fish are further bred to insure transmission of the transgenic expression cassette to the germ cells of a fish as further described in this application.







TABLE 4







Fluorescent Proteins (“FP”) with their Maximum Excitation and Emission


Wavelengths











FP
Excitation max (nm)
Emission max (nm)






AmCyan1
458
489



ZsGreen1
493
505



ZsYellow1
529
539



DsRed2
563
582



DsRed-Express
557
579



AsRed2
576
592



HcRed1
588
618



mPlum
590
649



mCherry
587
610



tdTomato
554
581



mStrawberry
574
596



J-Red
584
610



DsRed-monomer
556
586



mOrange
548
562



mKO
548
559



MCitrine
516
529



Venus
515
528



Ypet
517
530



EYFP
514
527



Emerald
487
509



EGFP
488
507



CyPet
435
477



mCFPm
433
475



Cerulean
433
475



T-Sapphire
399
511









The sequences of the DNAs which are useful in the invention are set forth in the attached Sequence Listing.
The sequence listed herein as SEQ ID NO:1 is the transgenic fluorescence expression cassette having zebrafish fast skeletal muscle specific myosin light chain (zMLC) promoter, DsRed2 (a red fluorescent protein gene from Anthozoa, a reef coral), and two copies of the SV40 polyadenylation sequence. The sequence listed in SEQ ID NO:1 is the complementary sequence to the coding DNA strand.
The sequence listed herein as SEQ ID NO:2 is the transgenic fluorescence expression cassette having carp ubiquitous β-actin enhancer/promoter, DsRed2 (a red fluorescent protein gene from Anthozoa, a reef coral), and two copies of the SV40 polyadenylation sequence. The first exon and intron of β-actin has been incorporated in the SEQ ID NO:2 to allow for increased expression of the fluorescence protein gene.
The sequence listed herein as SEQ ID NO:3 is the transgenic fluorescence expression cassette having zebrafish fast skeletal muscle specific myosin light chain (zMLC) promoter, ZsGreen1 (a green fluorescent protein gene from Anthozoa, a reef coral), and two copies of the SV40 polyadenylation sequence. The sequence listed in SEQ ID NO:3 is the complementary sequence to the coding DNA strand.
The sequence listed herein as SEQ ID NO:4 is the transgenic fluorescence protein expression cassette having zebrafish fast skeletal muscle specific myosin light chain (zMLC) promoter, ZsYellow1 (a yellow fluorescent protein gene from Anthozoa, a reef coral), and two copies of SV40 polyadenylation sequence. The sequence listed in SEQ ID NO:4 is the complementary sequence to the coding DNA strand.
The sequence listed herein as SEQ ID NO:5 is the transgenic fluorescence protein expression cassette having carp ubiquitous β-actin enhancer/promoter, ZsGreen1 (a green fluorescent protein gene from Anthozoa, a reef coral), and two copies of SV40 polyadenylation sequence. The first exon and intron of β-actin has been incorporated in the SEQ ID NO:5 to allow for increased expression of the fluorescence gene
Chimeric Genes
The present invention encompasses chimeric genes comprising a promoter described herein operatively linked to a heterologous gene. Thus, a chimeric gene can comprise a promoter of a zebrafish operatively linked to a zebrafish structural gene other than that normally found linked to the promoter in the genome. Alternatively, the promoter can be operatively linked to a gene that is exogenous to a zebrafish, as exemplified by the DsRed2 and other genes specifically exemplified herein. Furthermore, a chimeric gene can comprise an exogenous promoter linked to any structural gene not normally linked to that promoter in the genome of an organism.
Substitutions, Additions and Deletions
As possible variants of the above specifically exemplified polypeptides, the polypeptide may have additional individual amino acids or amino acid sequences inserted into the polypeptide in the middle thereof and/or at the N-terminal and/or C-terminal ends thereof so long as the polypeptide possesses the desired physical and/or biological characteristics. Likewise, some of the amino acids or amino acid sequences may be deleted from the polypeptide so long as the polypeptide possesses the desired physical and/or biochemical characteristics. Amino acid substitutions may also be made in the sequences so long as the polypeptide possesses the desired physical and biochemical characteristics. DNA coding for these variants can be used to prepare gene constructs of the present invention.
A nucleic acid sequence “encodes” or “codes for” a polypeptide if it directs the expression of the polypeptide referred to. The nucleic acid can be DNA or RNA. Unless otherwise specified, a nucleic acid sequence that encodes a polypeptide includes the transcribed strand, the hnRNA and the spliced RNA or the DNA representative thereof.
Degenerate Sequences
In accordance with degeneracy of genetic code, it is possible to substitute at least one base of the base sequence of a gene by another kind of base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, the DNA of the present invention may also have any base sequence that has been changed by substitution in accordance with degeneracy of genetic code.
DNA Modification
The DNA is readily modified by substitution, deletion or insertion of nucleotides, thereby resulting in novel DNA sequences encoding the polypeptide or its derivatives. These modified sequences are used to produce mutant polypeptide and to directly express the polypeptide. Methods for saturating a particular DNA sequence with random mutations and also for making specific site-directed mutations are known in the art; see e.g. Sambrook et al., (1989).
Transgenic Fish
The disclosed constructs and methods can be used with any type of fish that is an egg-layer. It is preferred that fish belonging to species and varieties of fish of commercial value, particularly commercial value within the ornamental fish industry, be used. Such fish include but are not limited to catfish, zebrafish, medaka, carp, tilapia, goldfish, tetras, barbs, sharks (family cyprinidae), angelfish, loach, koi, glassfish, catfish, angel fish, discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius. A more complete list of ornamental fish species can be found in Table 5 below:







TABLE 5







Ornamental Fish Species








Scientific Name
Common Name





Steatocranus casuarius
African Blockhead


Apistograma agassizi
Agassizi


Hyphessobrycon h axelrodi, sp
Albino Black Neon Tetra


Lamprophogus brichardi
Albino Bricardi Cichld


Paracheirodon innessi, sp.
Albino Brilliant Neon Tetra


Hemigrammus caudovitatus
Albino Buenos Aires Tetra


Hemigrammus erythrozonus
Albino Glow Light Tetra


Hemigrammus ocellifer
Albino Head Tail Light Tetra


Pelvicachromis pulcher
Albino Kribensis Cichlid


Aplochelius normani
Albino Lampeye


Hyphessobrycon pulchripinnis sp
Albino Lemon Tetra


Paracheirodon innessi
Albino Neon Tetra


Macropodus opercularis spp
Albino Paradise Fish


Pterophyllum scalare
Albino Red Eye Angel


Epalzeorhynchos frenatus
Albino Redfin Shark


Hem. Rhodostomus sp.
Albino Rummy Nose


Capoeta tetrazona
Albino Tiger Barb


Astronotus ocellatus
Albino Tiger Oscar


Tanichtys albonubes sp.
Albino White Cloud


Lepisosteus oculatus
Alligator Gar


Luciosoma spilopleura
Apollo Shark


Toxotes jaculatrix
Archer Fish


Xiphophorus variatus
Assorted Variatus


Badis badis
Badis Badis


Helostoma temmincki
Balloon Kissing Gourami


Corydoras metae
Bandit Corydoras


Pangasius sutchi
Bangkok Catfish


Ancistrus dolichopterus
Big-Fin Bristlenose Golden Longfin


Peocilia latipinna
Black Balloon Molly


Cichlasoma maculicauda
Black Belt Cichlid


Carrasius auratus
Black Butterfly Tail


Callochromis macrops
Black Eared Callochromis


Leptosoma Kitumba
Black Finned Slender Cichlid


Apteronotus albifrons
Black Ghost


Acanthopthalmus myersi
Black Kuhlii


Bogrichthys hypselopterus
Black Lancer


Hyphessobrycon h axelrodi
Black Neon Tetra


Nematobrycon palmeri spp
Black Palmeri


Megalamphodus megalopterus
Black Phantom


Rasbora trilineata
Black Scissor Tail Rasbora


Labeo chrysopekadion
Black Shark


Puntius filamentosus
Black Spot Barb


Rasbora agilis
Black Stripe Rasbora


Gymnocorymbus ternetzi
Black Tetra


Astyanax fas. mexicanus
Blind Cave Tetra


Brachydanio kerri
Blue Danio


Inpaichtys kerri
Blue Emperor Tetra


Trichogaster trichopterus
Blue Gourami


Boehlkea fredcochui
Blue King Tetra


Xiphophorus maculatus
Blue Platy


Melanotaenia lacustris
Blue Rainbow


Poecilia reticulata
Blue Ribbon Guppy


Pseudotropheus zebra
Blue Zebra


Melanotaenia boesemani
Boesemani Rainbow


Gastromyzon punctulatus
Borneo Sucker


Datnoides microlepis
Borneo Tiger Fish


Paracheirodon innesi
Brilliant Diamond Head Neon


Rasbora birttani
Brittan'S Rasbora


Brachygobius doriae
Bumble Bee Goby


Anomalochromis thomasi
Butterfly Cichlid


Notesthes robusta
Butterfly Goby


Paracheirodon axelrodi
Cardinal Tetra - M


Nomorhampheus liemi
Celebes Halfbeak


Telmatherina ladigesi
Celebes Rainbow


Chaca bankanensis
Chaca - Chaca


Capoeta oligolepis
Checkered Barb


Capoeta titteya
Cherry Barb


Sphaerichthys osphromenoides
Chocolate Gourami


Clarias batracus
Clarias - Spotted


Epiplatys annulatus
Clown Killie/Rocket


Botia macracantha
Clown Loach


Haplochromis sp
Cobalt/Ice Blue Cichlid


Apistograma cacatuoides
Cockatoo Dwarf


Hyphessobrycon colombianus
Colombia Tetra


Phenacogrammus interruptus
Congo Tetra


Corydoras aeneus
Corydoras Albino


Corydoras panda
Corydoras Panda


Corydoras paleatus
Corydoras Peppered


Corydoras pigmy
Corydoras Pigmy


Corydoras rabauti
Corydoras Rabauti


Corydoras similis
Corydoras Similis


Corydoras sterbai
Corydoras Sterbai


Synodontis multipunctatus
Cuckoo Synodontis


Polypterus senegalus
Cuvier'S Bichir


Synodontis decorus
Decorated Synodontis


Polypterus delhezi
Delhezi Bichir


Moenkhausia pitteri
Diamond Tetra


Hyphessobrycon amandae
Ember Tetra


Nematobrycon palmeri
Emperor Tetra


Polypterus endlicheri
Endlicheri Bichir


Aphyocharax alburnus
False Flame Tetra


Synodontis eupterus
Feathered Fin Synodontis


Cichlasoma festae
Festa'S Cichlid


Cichlasoma meeki
Firemouth Cichlid


Puntius pentazona
Five Banded Barb


Epalzeorhynchus kalopterus
Flying Fox


Crossocheilus siamensis
Flying Fox


Popondetta furcata
Forktail Rainbow


Cyphotilapia frontosa
Frontosa Cichlid


Cyathopharynx furcifer
Furcifer


Sturisoma fursochi
Fursochi Cat Fish


Aphyosemion gardneri
Gardneri Killifish


Pseudomugil gertrudae
Gertrudae


Danio malabarinchus
Giant Danio


Ambassis ranga
Glass Angel


Prionobrama filigera
Glass Bloodfin


Hypostomus plecostomus
Glass Cleaner Plecostomus


Hemigrammus rodwayi
Gold Tetra


Puntius sachsi
Golden Barb


Nannacara anomala
Golden Dwarf Cichlid


Nannostomus beckfordi
Golden Pencil Tetra


Pristella maxillaris
Golden Pristella


Melanotaenia herbrt axelrodi
Golden Rainbow


Scleropages formosus
Green Arowana


Brachydanio rerio
Green Danio


Aequidens rivulatus
Green Terror Cichlid


Macrognathus circumcinctus
Half Banded Spiny Eel


Rasbora heteromorpha
Harlequin Rasbora


Gasteropelecus sternicla
Hatchet Fish


Rasbora dorsiocellata
High Spot Rasbora


Geophagus steindachneri
Hondae Humphead


Ctenolucius hujeta
Hujeta


Scleropages jardini
Jardini Arowana


Hemichromis paynei
Jewel Cichlid


Melanochromis johanni
Johanni Cichlid


Julidichromis dickfeldi
Juldchrmis Dickfeldi


Julidichromis ornatus
Julidochromis Ornatus


Julidichromis transcriptus
Julidochromis Transcriptus


Geophagus jurupari
Jurupari Cichlid


Tropheus IKOLA
Kaisar Tropheus


Hyphessobrycon loweae
Kitti Tetra


Stigmatogobius sadanundio
Knight Goby


Cyprinus Carpio
Koi


Acanthopthalmus kuhlii
Kuhlii Loach


Lamprologus silindericus
Lamprologus Silindericus


Lamprologus leleupi
Lemon Cichlid


Labidochromis caeruleus
Lemon Mbuna Cichlid


Hyphessobrycon pulchripinnis
Lemon Tetra


Ctenopoma acutirostre
Leopard Bushfish


Brachydanio frankei
Leopard Danio


Leptosoma malasa
Leptosoma Malasa


Rasbora paviei
Line Rasbora


Capoeta arulius
Long Fin Barb


Alesthes longipinnis
Long Fin Characin


Rasbora einthovenii
Long-Band Rasbora


Melanotaenia maccullochi
Macculloch'S Rainbow


Paretropheus menoramba
Madagascar Cichlid


Bedotia gaeyi
Madagascar Rainbow


Haplochromis compressiceps
Malawi Eye Biter


Ompok sp.
Malay Glass Catfish


Betta splendens
Male Betta


Cichlasom managuense
Managuense Cichlid


Polypterus palmas
Marbled Bichir


Xiphophorus helleri
Millenium Swordtail


Monodactylus argentus
Mono Angel


Cyrtocara moorii
Morrii


Sawbwa resplendens
Naked Micro Rasbora


Hyphessobrycon h.axelrodi sp.
Negro Brilliant Black Neon


Melanotaenia praecox
Neon Dwarf Rainbow


Aplocheillus panchax
New Golden Wonder


Synodontis ocellifer
Ocellated Synodontis


Colisa labiosa
Orange Thick Lipped Gourami


Polypterus ornatipinnis
Ornate Bichir


Botia Locahanta
Pakistani Loach


Puntius fasciatus
Panda Barb


Apistogramma pandurini
Pandurini Dwarf


Macropodus opercularis
Paradise Fish


Cichlasoma sp.
Parrot


Cichla Ocellaris
Peackock Bass Cichlid


Trichogaster leeri
Pearl Gourami


Cichlasoma carpinte
Pearl Scale Cichlid


Lamprologus calvus
Pearly Lamprologus


Tropheus PEMBA
Pemba River Tropheus


Thayeria boelkea
Penguin Tetra


Chalceus macrolepidotus
Pinktail Characin


Mogurnda mogurnda
Purple Striped Gudgeon


Rasbora sp.
Rasbora Red Fin


Aphyocharax rathbuni
Red Belly Tetra


Cichlasoma labiatum
Red Devil


Moenkhausia santaefilomenae
Red Eye Tetra


Pseudotrophues sp.
Red Eyed Tangarine Cichlid


Mastacembelus erythrotaenia
Red Fire Eel


Copadichromis borleyi
Red Kadango


Rasbora pauciperforata
Red Line Rasbora


Colossoma macropodum
Red Pacu


Megalamphodus sweglesi
Red Phantom


Glossolepis incisus
Red Rainbow


Cichlasoma severum
Red Severum Cichlid


Notropis lutrensis
Red Shiner


Megalamphodus roseus
Red Tail Yellow Phantom


Epalzeorhynchos frenatus
Redfin Shark


Epalzeorhynchos bicolor
Redtail Black Shark


Puntius conchonius
Rosy Barb


Hyphessobrycon bentosi
Rosy Tetra


Puntius rhombocellatus
Round Banded Clown Barb


Puntius nigrofasciatus
Ruby Barb


Hemigrammus bleheri
Rummy Nose Tetra


Arius graeffei
Salmon Catfish


Hyphessobrycon serpae
Serpae Tetra


Hyphessobrycon serpae sp
Serpae Tetra Veiltail


Osteoglossum bichirrhosum
Silver Arowana


Distichodus affinis
Silver Distichodus


Metynnis hypsauchen
Silver Dollar


Selenotoca multifasciata
Silver Scat


Hasemania nanna
Silver Tipped Tetra


Balantiocheilos melanopterus
Silver Tricolor Shark


Rasbora espei
Slender Wedge Rasbora


Pseudomugil signifer
Southern Blue Eye


Chilodus punctatus
Spotted Headstander


Rasbora maculata
Spotted Pygmy Rasbora


Metynnis maculatus
Spotted Silver Dollar


Puntius lineatus
Striped/Lined Barb


Scleropages formosus
Super Red Arowana


Corynopoma riseii
Swordtail Characin


Cichlasoma synspilum
Synspillum Cichlid


Iriantherina werneri
Threadfin Rainbow


Capoeta tetrazona
Tiger Barb


Pseudoplatystoma fasciatum
Tiger Shovelnose Catfish


Tilapia buttikoferi
Tiger Zebra Tilapia


Petrochromis trewavasae
Trewavas'S Petrochromis


Tropheus duboisi
Tropheus Duboisi


Mystus micracanthus
Two Spotted Catfish


Uaru amphiacanthoides
Uaru -Triangle Cichlid


Sphaerichthys vallianti
Valliant'S Gourami


Thayeria boehlkea sp.
Veiltail Penguin Tetra


Opthalmotilapia ventralis
Ventralis


Haplochromis venustus
Venustus


Synodontis schoutedeni
Vermiculated Synodntis


Tanichtys albonubes
White Cloud


Tanichtyhs albonubes
White Cloud Minnow


Osphronemus gourami
White Giant Gourami


Symphysodon aequifasciata
White Smoke


Aphyocharax paraguayensis
White Spot Tetra


Crenicichla saxalitus
White Spotted Pike Cichlid


Mastacembelus armatus
White Spotted Spiny Eel


Gymnocorymbus ternetzi
White Tetra


Betta coccina
Wine Red Betta


Melanochromis auratus
Yellow Auratus Cichlid


Hemmigrammopetersius caudalis
Yellow Congo


Apistograma borelli
Yellow Dwarf Cichlid









The more preferred fish for use with the disclosed constructs and methods is zebrafish, Danio rerio. Zebrafish are increasingly popular ornamental animals and would be of added commercial value in various colors. Zebrafish embryos are easily accessible and nearly transparent. The most preferred fish for use with the disclosed constructs and methods is the Golden Zebrafish. Zebrafish skin color is determined by pigment cells in their skin, which contain pigment granules called melanosomes. The number, size and density of the melanosomes per pigment cell influence the color of the fish skin. Golden zebrafish have diminished number, size, and density of melanosomes and hence have lighter skin when compared to the wild type zebrafish. Golden zebrafish have a mutation in slc24a5 gene, slc24a5 codes for a putative cation exchanger localized to intracellular membrane, rendering the fish skin lighter or less pigmented (Lamason et al., 2005).
The disclosed transgenic fish are produced by introducing a transgenic construct into the genomes of cells of a fish, preferably embryonic cells, and most preferably in a single cell embryo. Where the transgenic construct is introduced into embryonic cells, the transgenic fish is obtained by allowing the embryonic cell or cells to develop into a fish. The disclosed transgenic constructs can be introduced into embryonic fish cells using any suitable technique. Many techniques for such introduction of exogenous genetic material have been demonstrated in fish and other animals. These include microinjection (Culp et al., (1991), electroporation (Inoue et al., 1990; Muller et al., 1993; Murakami et al., 1994; Muller et al., 1992; and Symonds et al., 1994), particle gun bombardment (Zelenin et al., 1991), and the use of liposomes (Szelei et al., 1994). The preferred method for introduction of transgenic constructs into fish embryonic cells is by microinj ection.
Embryos or embryonic cells can generally be obtained by collecting eggs as soon as possible after they are laid by methods that are well known to those of ordinary experience in the ornamental fish production field. Depending on the type of fish, it is generally preferred that the eggs be fertilized prior to or at the time of collection. This is preferably accomplished by placing a male and female fish together in a tank that allows egg collection under conditions that stimulate mating. A fertilized egg cell prior to the first cell division is considered a one cell embryo, and the fertilized egg cell is thus considered an embryonic cell.
The transgene may randomly integrate into the genome of the embryo in one or more copies (concatemers). After introduction of the transgenic construct, the embryo is allowed to develop into a fish. The fish that were injected as embryos are allowed to interbreed and the offspring are screened for the presence of the transgene. Fish harboring the transgene may be identified by any suitable means. In the preferred case, one or more of the transgenic constructs will have integrated into the cellular genome, which can be probed for the presence of construct sequences. To identify transgenic fish actually expressing the transgene, the presence of an expression product can be assayed. Several techniques for such identification are known and used for transgenic animals and most can be applied to transgenic fish. Probing of potential or actual transgenic fish for nucleic acid sequences present in or characteristic of a transgenic construct can be accomplished by Southern or northern blotting, polymerase chain reaction (PCR) or other sequence-specific nucleic acid amplification techniques.
The simplest way to confirm the presence of a fluorescent protein expressing transgene in a given fish is by visual inspection, as the fish in question would be brightly colored and immediately distinguishable from non-transgenic fish. Preferred techniques for identifying fluorescent protein expressing transgenic zebrafish are described in the examples. The present invention also provides a method to obtain a new population or the progenitor of a new line of fluorescent transgenic fish exhibits strong visible fluorescence, strong visible fluorescence means that a person with 20/20 vision (i.e., average vision) will be able to distinguish between the fluorescent fish in question and a non-fluorescent fish of the same species at a distance of at least 5 feet in a lighted office, with a preferred distance of at least 10 feet in a lighted office, and a more preferred distance of at least 15 feet in a lighted office, and an even more preferred distance of at least 20 feet in a lighted office, with the illumination level defined in Table 6. One can observe all transgenic fluorescent fish from a particular population that exhibit strong visible fluorescence under the various lighting conditions and select the fish that exhibits the highest level of visible fluorescence of the fluorescent protein. Selected fish with strong visible fluorescence are monitored and selected continuously to ensure stability of expression and maintenance of the strong visible fluorescence trait. Thus a new line of fish exhibiting strong visible fluorescence is created for further breeding.
The invention further encompasses progeny of a transgenic fish containing a genomically integrated transgenic construct, as well as transgenic fish derived from a transgenic fish egg, sperm cell, embryo, or other cell containing a genomically integrated transgenic construct. “Progeny,” as the term is used herein, can result from breeding two transgenic fish of the invention, or from breeding a first transgenic fish of the invention to a second fish that is not a transgenic fish of the invention. In the latter case, the second fish can, for example, be a wild-type fish, a specialized strain of fish, a mutant fish, or another transgenic fish. The hybrid progeny of these matings have the benefits of the transgene for fluorescence combined with the benefits derived from these other lineages.
Fertilization from Frozen Sperm
Sperm freezing methods are well known in the art, for example see Walker and Streisinger (1983). Frozen zebrafish sperm may be used to fertilize eggs also as described in Walker and Streisinger (1983), incorporated herein by references. Briefly, a droplet of ice-cold 100% Hank's saline is placed next to zebrafish eggs in a petri dish. Frozen sperm is thawed for a few seconds in air then expelled into the droplet of Hank's saline and the solution is mixed with the eggs. The mixture is incubated for about  ̃1 minute and then fish water added.
Vectors
The invention is further directed to a replicable vector containing cDNA that codes for the polypeptide and that is capable of expressing the polypeptide.
The present invention is also directed to a vector comprising a replicable vector and a DNA sequence corresponding to the above described gene inserted into said vector. The vector may be an integrating or non-integrating vector depending on its intended use and is conveniently a plasmid. The present invention also encompasses the removal of the vector backbone from the plasmid before the transgenic construct may be introduced into the zebrafish.
Transformed Cells
The invention further relates to a transformed cell or microorganism containing cDNA or a vector which codes for the polypeptide or a fragment or variant thereof and that is capable of expressing the polypeptide.
Expression Systems Using Vertebrate Cells
Interest has been great in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of vertebrate host cell lines useful in the present invention preferably include cells from any of the fish described herein. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome-binding site, RNA splice site (if intron-containing genomic DNA is used or if an intron is necessary to optimize expression of a cDNA), and a polyadenylation site.
In another aspect of the present invention, also included is the commercial marketability of the transgenic fluorescent fish to the ornamental fish industry.
EXAMPLES
The invention will now be further described with reference to the following examples. These examples are intended to be merely illustrative of the invention and are not intended to limit or restrict the scope of the present invention in any way and should not be construed as providing conditions, parameters, reagents, or starting materials which must be utilized exclusively in order to practice the art of the present invention.
Example 1
Design and Generation of the Construct Plasmids
The promoter of the zebrafish fast skeletal muscle myosin light chain (zMLC2) (Ju et al., 2003) and the carp β-actin enhancer/promoter sequence (Lui et al., 1990) were cloned into pBluescript II SK (−) and pUC18 respectively. Red fluorescent protein gene, DsRed2; green fluorescent protein gene, ZsGreen1 and yellow fluorescent protein gene, ZsYellow1 were amplified by PCR from pDsRed2-N1, pZsGreen1-N1 and pZsYellow1-N1 (Clontech Inc., Matz. et al., 1999) respectively and cloned into pBluescript II SK (−) zMLC2 and pUC18-carp β-actin such that the promoter was operably linked to the fluorescent gene. Tandem SV40(A) polyA/3′UTR sequence from pK-SV40(A)x2 plasmid were cloned 3′ to the fluorescent protein gene coding region. It is preferred to use more than one copy of the selected polyadenlyation sequence, and more preferred to use a viral polyadenylation sequence, as this will increase the efficiency of the fluorescent protein gene expression. The resulting five construct vector maps are provided as FIG. 1 through FIG. 5.
Example 2
Preparation of the Construct for Delivery
The vectors pUC18-carp β-actin-DsRed2 and pUC18-carp β-actin-ZsGreen1 were restriction double digested with XbaI and AatII enzymes for three hours (FIG. 6, Step 1) and then run on 0.8% agarose gel to separate the transgenic insert cassette from the vector backbone (FIG. 6, Step 2 and 3). Transgenic insert cassette band ( ̃3.5 kb) which contained the promoter, the open reading frame and the 3′UTR was excised and purified using phenol:choloroform extraction.
The transgenic vectors pBluescript II SK(−)-zMLC-DsRed2-SV40x2, pBluescript II SK(−)-zMLC-ZsGreen1-SV40x2, and pBluescript II SK(−)-zMLC-ZsYellow1-SV40x2 were restriction triple digested with XhoI, XmnI and NotI enzymes for three hours and then run on 0.8% agarose gel to separate the transgenic insert cassette from the vector backbone. The transgenic insert cassette band ( ̃3.2 kb) which contained the promoter, the open reading frame and the 3′UTR was excised and gel purified.
Example 3
Making the Transgenic Fish
The purified transgenic insert cassette which contained the promoter, the open reading frame and the 3′UTR was microinjected into the zebrafish embryos (FIG. 6, Step 4).
While only one construct was injected into Yellow zebrafish 1, to increase the chances of developing a fish with strong visible fluorescence, more than one construct was injected simultaneously in Red zebrafish 1 and Green zebrafish 1. For the purposes of this application, strong visible fluorescence means that a person with 20/20 vision (i.e., average vision) will be able to distinguish between the fluorescent fish in question and a non-fluorescent fish of the same species at a distance of at least 5 feet in a lighted office, with a preferred distance of at least 10 feet in a lighted office, and a more preferred distance of at least 15 feet in a lighted office, and an even more preferred distance of at least 20 feet in a lighted office, with the illumination level defined in Table 6.
Given the same illumination levels, distances, and observer of average vision, another preferable quality of fish that exhibit strong visible fluorescence are those fish that also exhibit ubiquitous expression of the fluorescence, defined herein to mean strong fluorescence that is not limited to a particular tissue type or body location, with such expression preferably including fins, eyes, stripes or spots. Typically, ubiquitous fluorescent expression will mean that the fluorescent expression is visible over 75% to 100% of the body of the fish (excluding fins and eyes). The inventors have discovered that the use of a ubiquitous promoter in combination with a tissue specific promoter (such as a muscle promoter), particularly where such fish are prepared using at least two expression vectors, will generally result in fish having the desirable ubiquitous expression trait. In this more preferred example, the fluorescent pattern exhibited by the fish would also be free from any patches of non-expression or noticeably weak or dull expression, with the possible exception of non-expression in fins, eyes, and stripes or spots. Expression in the fins, eyes, and stripes or spots is also preferred, but not required for a fish to be considered as exhibiting ubiquitous fluorescent expression.
Examples of fish exhibiting strong visible fluorescence are the lines which are the subject of the present invention. Color photographs of these fish are available through World Wide Web at glofish.com/photos.asp. Color photographs of fish that are fluorescent, yet that do not exhibit strong visible fluorescence are available through World Wide Web at glofish.com/old_glofish.asp.
To obtain strong visible fluorescence, it is preferred to use a promoter that expresses ubiquitously and co-inject this promoter with a strong muscle promoter. It is also preferred to use enhancing elements in the transgenic insert cassette. For example, in the present invention, both Red zebrafish 1 and Green zebrafish 1 incorporate more than one transgenic expression cassette, with one being a ubiquitous promoter, and the other being a strong muscle promoter. In particular, Red zebrafish 1 incorporates the cassettes represented by FIG. 1 and FIG. 4, and Green zebrafish 1 incorporates the cassettes represented by FIG. 2 and FIG. 5. In the cassette that includes the ubiquitous promoter, there is also an intron and exon, which exemplifies the type of RNA processing element that is helpful in achieving strong visible fluorescence.
To co-inject the embryos, multiple purified transgenic insert cassettes can simply be loaded into the microinjection needle simultaneously and then injected. Alternatively, in the preferred method, the injection of constructs containing multiple (two or more) fluorescent protein expression cassettes can be made using common molecular biology techniques, such as DNA digestion and ligation. In the most preferred method, a plasmid can be made which contains several fluorescent protein expression cassettes in tandem, and then treated in the same way as disclosed herein for a single fluorescent protein expressing plasmid (that is, made, isolated, purified, and linearized with the antibiotic resistance marker gene and replication origin removed before injection). While the present invention incorporates only the transgenic insert cassettes shown in the Figures, it is understood that multiple transgenic insert cassettes of any type can be simultaneously injected into a fish embryo from any species. Once injected, the embryos were allowed to grow into adult fish. At that point, they were spawned to determine if their offspring carried the fluorescence trait. The preferred method of spawning is a single pair spawn between a zebrafish that had been injected as an embryo and a wild-type zebrafish. The offspring of the transgenic zebrafish were raised to maturity and the fluorescent fish selected for further examination. In the preferred method, the offspring should be screened for by exposure to lights of specific wavelengths while they are still embryos. For example, for green fluorescent protein an excitation max at 493 nm with emission max at 505 nm, for red fluorescent protein an excitation max at 563 nm and an emission max at 582 nm and for yellow fluorescent protein an excitation max at 529 nm and an emission max at 539 nm was used corresponding, for example, to ZsGreen1, DsRed2 and ZsYellow1.
The foregoing method was used to screen for the most esthetically pleasing fish while still maintaining the ability to efficiently breed.
Example 4
Selecting the Transgenic Fish
Any fish showing fluorescence as embryos or juveniles were grown to maturity and examined for fluorescence as an adult to determine which specific fluorescent fish was to be used as a progenitor for a new line. In this endeavor, the most valuable expression pattern is one that meets the definition of a fish exhibiting strong visible expression as defined herein, and even more preferred are those that also exhibit ubiquitous expression, as this strong expression would increase both the aesthetic appeal and commercial value of the fish. In particular, it is important to be sure the fish exhibits strong visible fluorescence in all of the lighting conditions described in Table 6 below.







TABLE 6







Common Light Levels - Indoors and Outdoors










Condition
Illumination (lux)













Full Daylight
10,000



Overcast Day
1000



Lighted Home
>150



Lighted Office
500



Dark Indoor Room
<50



Moderately Lit Room
100-150









Accordingly, to ensure that a progenitor for a new line of fluorescent fish exhibits strong visible fluorescence, one can observe all transgenic fluorescent fish from a particular population that exhibit strong visible fluorescence under the various lighting conditions noted above in Table 6, and select the fish that exhibits the highest level of visible fluorescence of the fluorescent protein. Selection of this fish is based on visible observation only, as commercial appeal will be based on visual appearance. When testing the fish in a completely dark room, it is preferred to use an ultraviolet light to observe the level of the fish's fluorescent expression, as the ambient light will typically be insufficient to observe even the most strongly expressing fluorescent fish.
It is also preferred to provide fish that exhibit a reasonably stable color over the entire life of the fish, varying no more than about 20% at any given age as compared to very young fish of the same line. For example, the inventors have noted that some fish, particularly those that are not prepared by the more preferred methods of the present invention, tend to dramatically lose their color brilliance over time, and can become indistinguishable from non-transgenic fish of the same species, even as young as one year old. Preferred transgenic fish of the present invention can be selected for this trait by monitoring the fish over its development cycle. It is also preferred to select fish that are stable without regard to the ambient physical environment of the tank (e.g., color of gravel, plants, etc.). This can be ensured by selecting fish that do no lose their color brilliancy over time or in response to the physical environment.
Mendelian inheritance of the fluorescent trait is consistent with an integration event at a single locus in the selected fish. The progeny from the originally selected zebrafish comprising this particular transgenic event can be used for further breeding through traditional means with unmodified zebrafish to establish a new line of fluorescent fish through methods that are well known to those of ordinary skill in the production of fish, wherein the vast majority of fluorescent fish derived from this progenitor exhibited a materially similar fluorescence pattern and strength as the founding fish. It is also preferred that the selected fish be monitored for stability and consistency of expression, as any life-cycle variance from strong visible fluorescence that is seen in the selected fish may be passed along to the offspring. Additionally, to facilitate consistency of expression, it is also necessary to remove from the breeding population of this line any fish that appear from time to time with an expression pattern which is visibly weaker than the original founder.
The specific transgenic events embodied in these fish are designated Red zebrafish 1, Green zebrafish 1 and Yellow zebrafish 1 respectively. Sperm from these fish may be used to fertilize zebrafish eggs and thereby breed transgenic zebrafish that comprise these specific transgenic integration events. Sperm from each line is deposited at the European Collection of Cell Cultures (ECACC) as “Red zebrafish 1” (provisional accession no. 06090403), “Green zebrafish 1” (provisional accession no. 06090401) and “Yellow zebrafish 1” (provisional accession no. 06090402).
Example 6
Breeding the Transgenic Fish
Once the transgenic line had been established as described above, fish that were homozygous for the fluorescence trait were obtained by crossing fish that were heterozygous for the fluorescence trait, and then the progeny were screened to determine whether they were homozygous for the fluorescence trait. The preferred method of screening the progeny is through a test cross with a wild-type zebrafish, where any fluorescent fish that produces 100% fluorescent offspring would be homozygous for the fluorescent trait. Once enough homozygous fish were found to create a minimal breeding population, they were crossed to produce additional homozygous progeny. Upon adulthood, these progeny were crossed with wild-type fish to obtain progeny that were heterozygous for the fluorescent trait. These heterozygous fish were then sold to the commercial ornamental fish market, while the homozygous fish population was maintained through traditional methods to ensure a future homozygous breeding population.
Example 7
Potential Application of the Transgenic Fish
The fluorescent transgenic fish have use as ornamental fish in the market. Stably expressing transgenic lines can be developed by breeding a transgenic individual with a wild type fish, mutant fish or another transgenic fish. Multiple color fluorescent fish may be generated by the same technique as red fluorescent fish, yellow fluorescent fish and green fluorescent fish. By recombining different tissue specific promoters and fluorescent protein genes, more varieties of transgenic fish of different fluorescent color patterns will be created. By expression of two or more different fluorescent proteins in the same tissue, an intermediate color may be created. For example, combing expression of both red fluorescent protein gene and yellow fluorescent protein gene under a muscle-specific promoter, an orange fluorescent zebrafish may be created.
The fluorescent transgenic fish should also be valuable in the market for scientific research tools because they can be used for embryonic studies such as tracing cell lineage and cell migration. Cells from transgenic fish expressing green fluorescent protein can also be used as cellular and genetic markers in cell transplantation and nuclear transplantation experiments. Additionally these fish can be used to mark cells in genetic mosaic experiments and in fish cancer models.
* * *
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A method of producing a transgenic, fluorescent fish having improved fluorescence comprising:
(a) obtaining transgenic, fluorescent fish that exhibit a fluorescence by virtue of their comprising:
i) a first transgene encoding a fluorescent protein, wherein the first transgene is under the control of a ubiquitous fish promoter, and
ii) a second transgene encoding the same fluorescent protein as in the first transgene, wherein the second transgene is under the control of a tissue-specific fish promoter,
wherein said transgenic, fluorescent fish were generated by chromosomal introduction of said first and second transgenes positioned on a single vector; and
(b) selecting a transgenic fish exhibiting strong visible fluorescence over 75% to 100% of the body of said fish, optionally excluding fins and eyes.

2. The method of claim 1, further comprising obtaining transgenic progeny of said selected transgenic fish.

3. The method of claim 1, wherein the transgenic, fluorescent fish is further defined as a transgenic, fluorescent zebrafish, catfish, zebrafish, medaka, carp, tilapia, goldfish, tetras, barbs, sharks (family cyprinidae), angelfish, loach, koi, glassfish, catfish, angel fish, discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius.

4. The method of claim 3, wherein the transgenic, fluorescent fish is further defined as a transgenic, fluorescent zebrafish, tetra or barb.

5. The method of claim 4, wherein the transgenic, fluorescent fish is further defined as a, transgenic zebrafish.

6. The method of claim 5, wherein the transgenic zebrafish is further defined as a transgenic Golden zebrafish.

7. The method of claim 1, wherein the fluorescent protein is selected from the group consisting of ZsGreen1, ZsYellow1, DsRed2, GFP, eGFP, YFP, eYFP, BFP, eBFP, CFP, eCFP, FP, AmCyan 1, DsRed-Express, AsRed2, HcRed1,mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mOrange, mKO, MCitrine, Venus, Ypet, EYFP, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire.

8. The method of claim 7, wherein the fluorescent protein is ZsGreen1.

9. The method of claim 7, wherein the fluorescent protein is DsRed2.

10. The method of claim 1, wherein the ubiquitous fish promoter is selected from the group consisting of those for housekeeping genes, including β-actin, EF-1 α, histone 2A, acidic ribosomal phosphoprotein, α-catenin, β-cateniny-catenin, ubiquitin CSrb7, creatine kinase(mitochondrial 1), ubiquitous Ca2+transporting ATPase, ancient ubiquitous protein, ubiquitin specific peptidase 4, acetyl-Coenzyme A acryltransferase 2, monoglyceride lipase, splicing factor 3b subunit 1 and tubulin β5.

11. The method of claim 10, wherein the ubiquitous fish promoter is the carp β -actin promoter.

12. The method of claim 1, wherein the tissue-specific fish promoter is a muscle-specific fish promoter.

13. The method of claim 12, wherein the muscle-specific fish promoter is selected from the group consisting of myosin light chain, muscle creatine kinase, MyoD, myogenin, desmin, muscle enolase-3, β-sarkoglycan, dystrophin, serum response factor, a-tropomyosin, myosin heavy chain, mitochondrial creatine kinase 2, Ca2+ transporting ATPase(fast twitch 1), skeletal troponin T1(slow), tropomodulin 4, four and a half LIM domains 1, fast-type nyosin binding protein C, calsequestrin 1, fast muscle troponin C2, phosphorylase kinase alpha 1, and skeletal Troponin I (fast 2).

14. The method of claim 12, wherein said muscle specific promoter is a fish myosin light chain promoter.

15. The method of claim 12, wherein said ubiquitous promoter is a fish β-actin promoter.

16. The method of claim 13, wherein the myosin light chain promoter is a zebrafish fast skeletal myosin light chain promoter.

17. A method of providing a fluorescent transgenic fish to the ornamental fish market, comprising obtaining a transgenic fish prepared by the method of claim 1, and distributing said fish to the ornamental fish market.

18. The method of claim 17, wherein the fish are distributed by a grower to a commercial distributor.

19. The method of claim 17, wherein the fish are distributed by a grower or a commercial distributor to a retailer.

20. The method of claim 17, wherein the retailer is a multi-product retailer having an ornamental fish department.