Transgenic Cory

Abstract

The present invention relates to transgenic ornamental fish and methods for producing such fish by germ cell transfer techniques. Also disclosed are methods for establishing populations of such transgenic fish and for providing them to the ornamental fish industry for marketing purposes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as a PCT international patent application on January 11, 2022, and claims priority to and the benefit of U.S. Provisional Patent Applications Nos. 63 / 136,454, 63 / 136,460, and 63 / 136,464, all filed on January 12, 2021, the entire contents of each of which are incorporated herein by reference.

[0002] The present invention relates to transgenic fish, in particular red, pink, orange, green and yellow transgenic Cory fish.

[0003] introduction Transgenic technology involves the transfer of foreign genes into a host organism, allowing the host to acquire new genetic traits. Transgenic technology has many potential applications. For example, it can be used to introduce transgenes into fish to create new varieties of fish. There are many methods to introduce foreign genes 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 direct injection of DNA into muscle tissue (Xu et al., 1999).

[0004] 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 focused on growth hormone gene transfer with the aim of generating rapidly growing fish. Although most of the early attempts used heterologous growth hormone genes and promoters and failed to produce these fish (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 shown in several fish species, including Atlantic salmon, some species of Pacific salmon, and loach (e.g., Du et al., 1992; Delvin et al., 1994, 1995; Tsai et al., 1995).

[0005] Corydoras (Cory) is a genus of freshwater catfish in the family Callichthyidae and subfamily Corydoradinae. Species assigned to Corydoras exhibit a wide diversity of body colors. The genus is well known among aquarists for its many species. The species are well suited for tropical freshwater community aquariums, as they get along well with other species and are not aggressive at all. However, for the ornamental fish industry, bronze, albino, or black bodies are not conducive to efficient display of the various colors. Availability of such Cory with modified pigmentation for transgenesis with fluorescent proteins would result in better products for the ornamental fish industry due to better visualization of the various colors.

[0006] Many fluorescent proteins are known in the art and have been used to investigate various cellular processes, including fluorescent proteins that exhibit various green, red, pink, yellow, orange, blue, or purple colors. Transgenic experiments involving fluorescent proteins have provided new markers and reporters for gene transfer, but progress in the field of developing and producing cells that express such proteins has been limited.

[0007] Transgenic Cory In certain embodiments, the present disclosure relates to producing transgenic fluorescent fish and providing such fish to the ornamental fish industry.

[0008] In some embodiments, a transgenic fish or a method for making a transgenic fish is provided. In certain aspects, the transgenic fish is a fertile transgenic fluorescent fish. In certain embodiments, the fish for use with the disclosed constructs and methods is Cory. Cory's skin color is determined by pigment cells in the skin, which contain pigment granules called melanosomes (black or brown), xanthosomes (yellow), erythrosomes (orange or red), or iridosomes (rainbow colors, including white). The number, size, and density of pigment granules per pigment cell affect the skin color of the fish.

[0009] In certain specific embodiments, transgenic Cory or its progeny are provided that contain specific transgenic integration events, referred to herein as transformation events. These fish are of particular interest, for example, because they embody an aesthetically pleasing red color. Transgenic fish that contain these specific transgenic events can be homozygous or heterozygous (including, for example, hemizygous) for the transformation event. Homozygous fish bred with fish lacking the transformation event will produce 100% heterozygous offspring in nearly all cases. Germ cells, eggs, sperm, and embryos that contain these specific transgenic events are also included as part of the present invention.

[0010] In one such embodiment relating to a particular transgenic integration event, a red transgenic Cory or progeny thereof is provided that comprises a chromosomally integrated transgene, the Cory comprising a "Red Cory 1 transformation event", and a cryopreserved adult germline stem cell comprising a Red Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127094. The chromosomally integrated transgene may be present on one integrated expression cassette or on two or more integrated expression cassettes. In certain aspects, such a transgenic Cory is a fertile transgenic Cory. Such a transgenic Cory may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0011] Also disclosed are methods of providing transgenic Cory comprising a Red Cory 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Cory or progeny thereof comprising a chromosomally integrated transgene, the Cory comprising a "Red Cory 1 transformation event," and cryopreserved adult germline stem cells comprising a Red Cory 1 transformation event have been deposited under ATCC Accession No. PTA-127094, and distributing the fish to the ornamental fish market. Such fish may be distributed by the farmer to a commercial distributor, or such fish may be distributed by the farmer or commercial distributor to a retailer, such as, for example, a multi-product retailer having an ornamental fish department.

[0012] In some aspects, methods are provided for producing a transgenic Cory, comprising: (a) obtaining a Cory that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Cory comprising a "Red Cory 1 transformation event," and a cryopreserved adult germline stem cell comprising the Red Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127094; and (b) breeding the obtained Cory with a second Cory to provide a transgenic Cory comprising the Red Cory 1 transformation event. The second Cory can be a transgenic or non-transgenic Cory.

[0013] In further embodiments, methods of producing transgenic organisms are also provided, the methods comprising using germ cell transplantation to generate Red Cory 1 transformants to produce transgenic progeny, such cryopreserved adult germline stem cells being deposited under ATCC Accession No. PTA-127094. Such progeny can be, for example, Cory, a species of the Corydoras family, a fish species or genus related to Cory, or another fish species or genus.

[0014] In another embodiment relating to a particular transgenic integration event, an orange transgenic Cory or progeny thereof is provided that comprises a chromosomally integrated transgene, the Cory comprising an "Orange Cory 1 transformation event", and a cryopreserved adult germline stem cell comprising an Orange Cory 1 transformation event is deposited under ATCC Accession No. PTA-127095. The chromosomally integrated transgene may be present on one integrated expression cassette or on two or more integrated expression cassettes. In certain aspects, such a transgenic Cory is a fertile transgenic Cory. Such a transgenic Cory may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0015] Also disclosed are methods of providing transgenic Corys comprising an Orange Cory 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Cory or progeny thereof comprising a chromosomally integrated transgene, the Cory comprising an "Orange Cory 1 transformation event," and cryopreserved adult germline stem cells comprising an Orange Cory 1 transformation event have been deposited under ATCC Accession No. PTA-127095, and distributing the fish to the ornamental fish market. Such fish may be distributed by the farmer to a commercial distributor, or such fish may be distributed by the farmer or commercial distributor to a retailer, such as, for example, a multi-product retailer having an ornamental fish department.

[0016] In some aspects, methods are provided for producing a transgenic Cory, comprising: (a) obtaining a Cory that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Cory comprising an "Orange Cory 1 transformation event," and a cryopreserved adult germline stem cell comprising the Orange Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127095; and (b) breeding the obtained Cory with a second Cory to provide a transgenic Cory comprising the Orange Cory 1 transformation event. The second Cory can be a transgenic or non-transgenic Cory.

[0017] In further embodiments, methods of producing transgenic organisms are also provided, the methods comprising using germ cell transplantation to generate Orange Cory 1 transformation to produce transgenic progeny, such cryopreserved adult germline stem cells being deposited under ATCC Accession No. PTA-127095. Such progeny can be, for example, Cory, a species of the family Corydoras, a fish species or genus related to Cory, or another fish species or genus.

[0018] In another embodiment relating to a particular transgenic integration event, a green transgenic Cory or progeny thereof is provided that comprises a chromosomally integrated transgene, the Cory comprising a "Green Cory 1 transformation event", and a cryopreserved adult germline stem cell comprising a Green Cory 1 transformation event is deposited under ATCC Accession No. PTA-127096. The chromosomally integrated transgene may be present on one integrated expression cassette or on two or more integrated expression cassettes. In certain aspects, such a transgenic Cory is a fertile transgenic Cory. Such a transgenic Cory may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0019] Also disclosed are methods of providing transgenic Corys comprising a Green Cory 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Cory or progeny thereof comprising a chromosomally integrated transgene, the Cory comprising a "Green Cory 1 transformation event," and cryopreserved adult germline stem cells comprising the Green Cory 1 transformation event have been deposited under ATCC Accession No. PTA-127096, and distributing the fish to the ornamental fish market. Such fish may be distributed by the farmer to a commercial distributor, or such fish may be distributed by the farmer or commercial distributor to a retailer, such as, for example, a multi-product retailer having an ornamental fish department.

[0020] In some aspects, methods are provided for producing a transgenic Cory, comprising: (a) obtaining a Cory that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Cory comprising a "Green Cory 1 transformation event," a cryopreserved adult germline stem cell comprising the Green Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127096; and (b) breeding the obtained Cory with a second Cory to provide a transgenic Cory comprising the Green Cory 1 transformation event. The second Cory can be a transgenic or non-transgenic Cory.

[0021] In further embodiments, methods of producing transgenic organisms are also provided, the methods comprising using germ cell transplantation to generate Green Cory 1 transformation to produce transgenic progeny, such cryopreserved adult germline stem cells being deposited under ATCC Accession No. PTA-127096. Such progeny can be, for example, Cory, a species of the Corydoras family, a fish species or genus related to Cory, or another fish species or genus.

[0022] In a further embodiment, the yellow cory comprises two expression cassettes. Both cassettes may be homozygous. In other embodiments, each of the cassettes may be heterozygous. In alternative embodiments, one expression cassette may be homozygous and the other expression cassette may be heterozygous. It is understood that fish of different zygosity will produce different ratios of offspring when bred.

[0023] In at least one exemplary embodiment, a cory comprises a particular transgenic integration event, providing a yellow transgenic cory or progeny thereof comprising a chromosomally integrated transgene comprising a chromosomally integrated expression cassette encoding a green fluorescent protein and a chromosomally integrated expression cassette encoding an orange fluorescent protein.

[0024] In some exemplary embodiments, the yellow cory comprises a green fluorescent protein, or a portion thereof, and / or an orange fluorescent protein, or a portion thereof. In at least these exemplary embodiments, the transgenic cory is a fertile transgenic cory. Such transgenic cory may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0025] The chromosomally integrated transgene may be present on one integrated expression cassette or on more than one integrated expression cassette. In some exemplary embodiments, the yellow cory comprises a green fluorescent protein, or a portion thereof, and an orange fluorescent protein, or a portion thereof. In at least these exemplary embodiments, the transgenic green cory is a fertile transgenic cory and is crossed with a transgenic orange cory that is a fertile transgenic cory. In more specific aspects, the progeny of such a cross is a transgenic yellow cory. In at least these exemplary embodiments, such a transgenic cory may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0026] Also disclosed are methods of providing transgenic cories, including yellow cories, to the ornamental fish market. In some embodiments, the methods include obtaining a transgenic cory or progeny thereof that includes a chromosomally integrated transgene and distributing the fish to the ornamental fish market. Such fish may be distributed by a fish farmer to a commercial distributor, or such fish may be distributed by a fish farmer or commercial distributor to a retailer, such as, for example, a multi-product retailer with an ornamental fish department.

[0027] In some aspects, methods of producing a transgenic cory are provided that include (a) obtaining a cory that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, and (b) breeding the obtained cory with a second cory to provide a transgenic cory. The second cory can be a transgenic or non-transgenic cory. In further embodiments, methods of producing a transgenic organism are also provided, the methods including using a yellow cory to produce transgenic progeny. Such progeny can be, for example, cory, a fish species or genus related to cory, or another fish species or genus.

[0028] As used herein, "a" or "an" can mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" can mean one or more than one.

[0029] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer only to alternatives or where the alternatives are mutually exclusive, but the present disclosure also supports a definition that refers only to alternatives and only to "and / or." As used herein, "another" can mean at least a second, or more.

[0030] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device for the method being used to determine the value, or the variation that exists among study subjects.

[0031] Any embodiment of any of the methods, kits, and compositions can consist of, or consist essentially of, the recited features and / or steps, rather than comprise / include / contain / have. Thus, in any of the claims, the terms "consisting of" or "consisting essentially of" can be substituted with any of the above open-ended linking verbs to modify a given claim from what it would otherwise be using the open-ended linking verb.

[0032] Other objects, features and advantages of the present invention will become apparent from the following detailed description, however, it should be understood that the detailed description and preferred examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Transgenic fish In some aspects, the present disclosure relates to transgenic fish. Methods for producing transgenic fish are described in, for example, U.S. Patent Nos. 7,135,613, 7,700,825, and 7,834,239, each of which is incorporated by reference in its entirety. For example, transgenic red Cory can be produced using expression cassettes encoding red fluorescent proteins (RFPs), such as TurboRFP, DsRed2, tdTomato, dTomato, eqFP578, DsRed-Express, DsRed-Express2, TagRFP, TagRFP-T, RRvT, cgfTagRFP, mApple, mRuby3, mNectarine, meffRFP, mScarlet-I, and amilFP593. Transgenic orange Cory can be generated using expression cassettes encoding yellow fluorescent proteins (YFPs), such as ZsYellow1, TurboYFP, E2-Orange, phiYFP, zFP538, Venus, yPet, ccalYFP1, mGold, Citrine, mCitrine, Topaz, mPapaya, and mBanana. Transgenic green Cory can be generated using expression cassettes encoding green fluorescent proteins (GFPs), such as zsGreen1, eYGFPdp, AausFP1, ​​mNeonGreen, maxNeonGreen, mGreenLantern, Clover, mClover3, PlamGFP, aeurGFP, afraGFP, WasCFP, NowGFP, cerFP505, pporGFP, Kohinoor, efasGFP, eechGFP1, bfloGFPa1, LanFP1, and LanFP2. Transgenic yellow Cory can be generated using expression cassettes encoding a green fluorescent protein (GFP), such as zsGreen1, and a yellow fluorescent protein (YFP), such as ZsYellow1.

[0034] It is preferred to use fish belonging to species and varieties of fish that have commercial value, especially within the ornamental fish industry. Such fish include, but are not limited to, cory, catfish, zebrafish, and other danio, killifish, carp, tilapia, goldfish, tetras, barbs, sharks (family cyprinidae, such as rainbow sharks), angelfish, loach, koi, glassfish, discus, eels, gobies, gouramis, guppies, Xiphophorus, hatchetfish, mollyfish, or pangasius. A particular fish for use in the context of this disclosure is the Cory. Cory is an increasingly popular ornamental animal, and a variety of colors would add commercial value. Cory embryos are readily available. Cory skin color is determined by pigment cells in the skin, which contain pigment granules called melanosomes. The number, size, and density of melanosomes per pigment cell affect the skin color of the fish.

[0035] In commercial aquaculture, cories spawn naturally. In one exemplary embodiment, groups of females and males are placed in an aquarium where PVC tiles are suspended from the side of the aquarium as spawning medium. The tiles with the eggs deposited are collected and transferred to an aquarium and incubated at 74-82 degrees Fahrenheit until the fry hatch (i.e., approximately 3-5 days), at which point the tiles are removed. The fry are fed newly hatched Artemia nauplii for approximately two weeks. At this point, the fry are safe to handle and can be transferred outside to a grow-out tank or earthen pond. It takes approximately six months for cories to reach maturity. In a related embodiment, line breeding is maintained by cryopreserved testes.

[0036] embryo cell transplant Methods for freezing fish gonads are well known in the art, see, for example, Lee et al. (2013) and Seki et al. (2017), both of which are incorporated by reference in their entireties. Germ cells obtained from isolated frozen Cory gonads may be used to obtain the transgenic fish disclosed herein.

[0037] In an exemplary embodiment, less mature young female Cory are euthanized using an overdose of a sedative such as Tricaine, followed by brain disruption using scissors. The fish are blotted dry, dissected, and their ovaries are removed and transferred to ice-cold L-15 medium. Ideally, the collected tissue should be free of or have very few mature oocytes. Once the desired amount of tissue is obtained, the ovaries are macerated and cut into approximately 1-2 mm fragments using scissors. The tissue fragments are allowed to settle, the L-15 medium is removed, and 0.25% trypsin in 1x PBS containing 2U / ml DNaseI is added to the tissue, approximately 1 ml of solution for tissue collected from 4-5 fish. The tissue is incubated on ice for 30 minutes, mixing every 10 minutes. The tissue is then transferred to room temperature and incubated for 1 hour, gently triturating every 5 minutes. After incubation, the resulting cell suspension along with the remaining tissue fragments is filtered through a 40 micron sieve. The retained tissue is rinsed with L-15 medium, filtered and combined with the previously obtained filtrate. The filtrate is layered on top of 40% Percoll made in Opti-MEM medium and centrifuged in a swing rotor at 1500 RPM for 15 minutes at 10°C. The interface phase is collected and 2 volumes of L-15 medium are added. The suspension is centrifuged in a swing rotor at 1000 RPM for 5 minutes at 10°C. The supernatant is removed and discarded and the pellet is suspended in cold Opti-MEM medium. The suspension is kept on ice. To freeze the cells, 100 μL of ice-cold 1.8 M DMSO in 1×HBSS is transferred to a cryovial. 33 μL of cell suspension is added, mixed and transferred to an iCellBox or equivalent cell freezing container for a freezing rate of 1°C per minute. Once the iCellBox is full, it is transferred to a cooler containing dry ice and frozen overnight or for at least 12 hours. After freezing, the cryovials are transferred from the iCellBox to liquid nitrogen for long-term storage.

[0038] In an example method for recovering red Cory lineages, cryopreserved cells are thawed by transferring the cryovial from liquid nitrogen to a 10-15°C water bath and incubating for approximately 1 minute until the cell suspension is thawed. 1 ml of ice-cold Opti-MEM medium is added to the suspension, mixed, and centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 50 μL of the supernatant is removed and discarded, and 0.5 ml of Opti-MEM medium is added to the pellet. The pellet is suspended and the suspension is incubated at 30°C for 30 minutes. After incubation, the suspension is centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 15 μL of the supernatant is removed and discarded, and the cells are resuspended in the remaining supernatant. The cell suspension is then injected into 5-6 day old, tricaine anesthetized Cory fry that have been treated with Primordazin B, approximately 100-200 nl of suspension per fry. The injected fry are raised to adulthood and spawned using the usual protocol to obtain red Cory progeny. To treat the cory embryos with Primordazin B, approximately 200-300 less than 1 hour old cory eggs are removed from the tile and transferred to a Petri dish containing 40 ml of water suitable for the fish. A solution of Primordazin B in DMSO is added to a final concentration of 5.5 μg / ml and mixed. The eggs are incubated in the solution for 24 hours at room temperature and then transferred to a tank for hatching.

[0039] The present disclosure further encompasses transgenic fish containing the Red Cory 1 transformation event, as well as progeny of such transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the transgenic transgenic construct. "Progeny", as the term is used herein, may result from breeding two transgenic fish of the present invention, or from breeding a first transgenic fish of the present invention to a second fish that is not a transgenic fish of the present invention. In the latter case, the second fish may be, for example, a wild-type fish, a specialized fish strain, a mutant fish, or another transgenic fish. The second fish may be of the same species or may be of a different species or genus. The hybrid progeny of these crosses have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0040] The simplest way to identify fish containing the Red Cory 1 transformation event is by visual inspection, as the fish in question are red and immediately distinguishable from non-transgenic fish. It should be understood that depending on the insertion location of the particular RFP and / or expression cassette used, the transgenic red Cory may have a color that is maintained throughout the transgenic red Cory's lifetime and / or over multiple generations. It should also be understood that depending on the insertion location of the particular RFP and / or expression cassette used, the transgenic red Cory may have a color that fades throughout the transgenic red Cory's lifetime. For example, a red transgenic Cory may change color from red to pale red, or from red to pink. Furthermore, depending on the insertion location of the particular RFP and / or expression cassette used, the transgenic red Cory may have a color that fades over generations. For example, a red transgenic Cory may change color from one generation to the next, such that older generations may exhibit red, but younger generations may appear pale red, or pink.

[0041] In an example method for recovering orange Cory lines, cryopreserved cells are thawed by transferring the cryovial from liquid nitrogen to a 10-15°C water bath and incubating until the cell suspension is thawed, approximately 1 minute. 1 ml of ice-cold Opti-MEM medium is added to the suspension, mixed, and centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 50 μL of the supernatant is removed and discarded, and 0.5 ml of Opti-MEM medium is added to the pellet. The pellet is suspended and the suspension is incubated at 30°C for 30 minutes. After incubation, the suspension is centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 15 μL of the supernatant is removed and discarded, and the cells are resuspended in the remaining supernatant. The cell suspension is then injected into primordazin B-treated, tricaine-anesthetized 5-6 day old Cory fry, approximately 100-200 nl of suspension per fry. Injected fry are raised to adulthood and spawned using the usual protocol to obtain orange Cory progeny. To treat cory embryos with Primordazin B, approximately 200-300 less than 1 hour old cory eggs are removed from the tile and transferred to a Petri dish containing 40 ml of water suitable for fish. Primordazin B solution in DMSO is added to a final concentration of 5.5 μg / ml and mixed. The eggs are incubated in the solution for 24 hours at room temperature and then transferred to a tank for hatching.

[0042] The present disclosure further encompasses transgenic fish containing the Orange Cory 1 transformation event, as well as progeny of such transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the transgenic transgenic construct. "Progeny", as the term is used herein, may result from breeding two transgenic fish of the present invention, or from breeding a first transgenic fish of the present invention to a second fish that is not a transgenic fish of the present invention. In the latter case, the second fish may be, for example, a wild-type fish, a specialized fish strain, a mutant fish, or another transgenic fish. The second fish may be of the same species or may be of a different species or genus. The hybrid progeny of these crosses have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0043] The simplest way to identify fish that contain the Orange Cory 1 transformation event is by visual inspection, as the fish in question are orange in color and immediately distinguishable from non-transgenic fish.

[0044] In an example method for recovering green Cory lineages, cryopreserved cells are thawed by transferring the cryovial from liquid nitrogen to a 10-15°C water bath and incubating for approximately 1 minute until the cell suspension is thawed. 1 ml of ice-cold Opti-MEM medium is added to the suspension, mixed, and centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 50 μL of the supernatant is removed and discarded, and 0.5 ml of Opti-MEM medium is added to the pellet. The pellet is suspended and the suspension is incubated at 30°C for 30 minutes. After incubation, the suspension is centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 15 μL of the supernatant is removed and discarded, and the cells are resuspended in the remaining supernatant. The cell suspension is then injected into 5-6 day old, tricaine anesthetized Cory fry that have been treated with Primordazin B, approximately 100-200 nl of suspension per fry. The injected fry are raised to adulthood and spawned using the usual protocol to obtain green Cory progeny. To treat the cory embryos with Primordazin B, approximately 200-300 less than 1 hour old cory eggs are removed from the tile and transferred to a Petri dish containing 40 ml of water suitable for the fish. A solution of Primordazin B in DMSO is added to a final concentration of 5.5 μg / ml and mixed. The eggs are incubated in the solution for 24 hours at room temperature and then transferred to a tank for hatching.

[0045] The present disclosure further encompasses transgenic fish containing the Green Cory 1 transformation event, as well as progeny of such transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the transgenic transgenic construct. "Progeny", as the term is used herein, may result from breeding two transgenic fish of the present invention, or from breeding a first transgenic fish of the present invention to a second fish that is not a transgenic fish of the present invention. In the latter case, the second fish may be, for example, a wild-type fish, a specialized fish strain, a mutant fish, or another transgenic fish. The second fish may be of the same species or may be of a different species or genus. The hybrid progeny of these crosses have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0046] The simplest way to identify fish that contain a Green Cory 1 transformation event is by visual inspection, as the fish in question are green in color and immediately distinguishable from non-transgenic fish.

[0047] In an example method for recovering yellow Cory lines, cryopreserved cells are thawed by transferring the cryovial from liquid nitrogen to a 10-15°C water bath and incubating until the cell suspension is thawed, approximately 1 minute. 1 ml of ice-cold Opti-MEM medium is added to the suspension, mixed, and centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 50 μL of the supernatant is removed and discarded, and 0.5 ml of Opti-MEM medium is added to the pellet. The pellet is suspended and the suspension is incubated at 30°C for 30 minutes. After incubation, the suspension is centrifuged at 10°C, 1000 RPM for 5 minutes in a swing rotor. All but 15 μL of the supernatant is removed and discarded, and the cells are resuspended in the remaining supernatant. The cell suspension is then injected into 5-6 day old, tricaine-anesthetized Cory fry that have been treated with primoldazine B, approximately 100-200 nl of suspension per fry. Injected fry are raised to adulthood and spawned using normal protocols to obtain yellow Cory progeny. To treat cory embryos with Primordazin B, approximately 200-300 less than 1 hour old cory eggs are removed from the tile and transferred to a Petri dish containing 40 ml of water suitable for fish. Primordazin B solution in DMSO is added to a final concentration of 5.5 μg / ml and mixed. The eggs are incubated in the solution for 24 hours at room temperature and then transferred to a tank for hatching.

[0048] The present disclosure further encompasses transgenic fish, i.e., yellow transgenic cories, containing the Green Cory 1 transformation event and the Orange Cory 1 transformation event, as well as progeny of such transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the transgenic transgenic construct. "Progeny," as the term is used herein, may result from breeding two transgenic fish of the present invention, or from breeding a first transgenic fish of the present invention to a second fish that is not a transgenic fish of the present invention. In the latter case, the second fish may be, for example, a wild-type fish, a specialized fish strain, a mutant fish, or another transgenic fish. The second fish may be of the same species or may be of a different species or genus. The hybrid progeny of these crosses have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0049] The simplest way to identify yellow transgenic cories is by visual inspection, as the fish in question are yellow in color and immediately distinguishable from non-transgenic fish. EXAMPLES

[0050] Certain embodiments of the invention are further described with reference to the following examples, which are intended to be merely illustrative of the invention and are not intended to limit or restrict the scope of the invention in any manner whatsoever, nor should they be construed as providing conditions, parameters, reagents, or starting materials which must be utilized solely to practice the techniques of the invention.

[0051] Example 1 - Red Transgenic Cory Transgenic fish are provided that exhibit a red color. The particular transgenic event embodied in these fish is designated the "Red Cory 1 transformation event." Germ cells from these fish can be transplanted into Cory eggs, thereby obtaining transgenic Corys that contain the particular transgenic integration event. Cryopreserved adult germline stem cells from this line have been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Red Cory 1" (the deposit was designated accession number PTA-127094).

[0052] Fluorescent transgenic fish are used as ornamental fish in the market. Stably expressing transgenic lines can be developed by breeding transgenic individuals with wild-type fish, mutant fish, or other transgenic fish. The desired transgenic fish can be distinguished from non-transgenic fish by observing the fish under white light, sunlight, ultraviolet light, blue light, or any other useful light condition that allows visualization of the red color of the transgenic fish.

[0053] Example 2 - Orange Transgenic Cory Transgenic fish are provided that exhibit an orange color. The particular transgenic event embodied in these fish is designated the "Orange Cory 1 transformation event." Germ cells from these fish can be transplanted into Cory eggs, thereby obtaining transgenic Corys that contain the particular transgenic integration event. Cryopreserved adult germline stem cells from this line have been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Orange Cory 1" (the deposit was designated accession number PTA-127095).

[0054] Fluorescent transgenic fish are used as ornamental fish in the market. Stably expressing transgenic lines can be developed by breeding transgenic individuals with wild-type fish, mutant fish, or other transgenic fish. The desired transgenic fish can be distinguished from non-transgenic fish by observing the fish under white light, sunlight, ultraviolet light, blue light, or any other useful light condition that allows visualization of the orange color of the transgenic fish.

[0055] Example 3 - Green Transgenic Cory Transgenic fish are provided that exhibit a green color. The particular transgenic event embodied in these fish is designated the "Green Cory 1 transformation event." Germ cells from these fish can be transplanted into Cory eggs, thereby obtaining transgenic Corys that contain the particular transgenic integration event. Cryopreserved adult germline stem cells from this line have been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Green Cory 1" (the deposit was designated accession number PTA-127096).

[0056] Fluorescent transgenic fish are used as ornamental fish in the market. Stably expressing transgenic lines can be developed by breeding transgenic individuals with wild-type fish, mutant fish, or other transgenic fish. The desired transgenic fish can be distinguished from non-transgenic fish by observing the fish under white light, sunlight, ultraviolet light, blue light, or any other useful light condition that allows visualization of the green color of the transgenic fish.

[0057] Example 4 - Yellow Transgenic Cory Transgenic fish are provided that exhibit a yellow color. The particular transgenic event embodied in these fish is designated the "Yellow Cory 1 transformation event." Transgenic fish are provided that exhibit a yellow color. The particular transgenic event embodied in these fish is designated the Yellow Cory 1 transformation event. Adult germline stem cells from these fish can be obtained, for example, from the progeny of crossing a green fluorescent cory with an orange fluorescent cory. It should be understood that the ability to provide transgenic fish that display colors, including "color crossed" combinations, is not readily apparent to one of skill in the art, nor is the ability to obtain novel colors readily obtainable without undue experimentation. This is particularly evident when consistent visualization of the Yellow Cory 1 transformation event is desired over multiple generations. The following examples provide various relevant examples of yellow cories that can be obtained.

[0058] In some exemplary embodiments, expression cassettes encoding green fluorescent proteins (GFPs), such as zsGreen1, eYGFPdp, AausFP1, ​​mNeonGreen, maxNeonGreen, mGreenLantern, Clover, mClover3, PlamGFP, aeurGFP, afraGFP, WasCFP, NowGFP, cerFP505, pporGFP, Kohinoor, efasGFP, eechGFP1, bfloGFPa1, LanFP1, and LanFP2, and expression cassettes encoding yellow fluorescent proteins (YFPs), such as ZsYellow1, TurboYFP, E2-Orange, phiYFP, zFP538, Venus, yPet, ccalYFP1, mGold, Citrine, mCitrine, Topaz, mPapaya, and mBanana, are injected, thereby encoding both green and orange, but Cory includes a yellow visualization effect.

[0059] In other embodiments, a particular transgenic integration event is provided that includes a chromosomally integrated transgene that may be present on one integrated expression cassette for each fluorescent protein or on more than one integrated expression cassette for each fluorescent protein, GFP and YFP Cory or its progeny. In certain aspects, such a transgenic Cory is a fertile transgenic Cory. Such a transgenic Cory may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette. In a related embodiment, a yellow cory is formed from breeding a cory that includes a chromosomally integrated expression cassette encoding a green fluorescent protein with a cory that includes a chromosomally integrated expression cassette encoding a yellow fluorescent protein. The progeny of such breeding results in a fertile next generation line of fish. In some instances, a non-fertile progeny line is desired, allowing yellow cories to be sourced from a particular generation line, such as, for example, (F1, F2, F3, etc.).

[0060] Fluorescent transgenic fish are used as ornamental fish in the market. Stably expressing transgenic lines can be developed by breeding transgenic individuals with wild-type fish, mutant fish, or other transgenic fish. The desired transgenic fish can be distinguished from non-transgenic fish by observing the fish under white light, sunlight, ultraviolet light, blue light, or any other useful light condition that allows visualization of the yellow color of the transgenic fish.

[0061] Fluorescent transgenic fish should also be valuable in the scientific research tool market, as they can be used in embryonic studies such as tracing cell lineages and cell migration, and can be used to mark cells in genetic mosaic experiments and fish cancer models.

[0062] All compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to one skilled in the art that modifications may be applied to the compositions and / or methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that 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.

[0063] The above specification, examples, and data provide a complete description of the manufacture and use of the composition of the invention. Since many embodiments of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended.

Claims

1. 1. A transgenic Cory comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Cory comprising a transformation event selected from the group consisting of a "Red Cory 1 transformation event," an "Orange Cory 1 transformation event," and a "Green Cory 1 transformation event."

2. 2. The transgenic Cory of claim 1, further defined as a fertile transgenic Cory, wherein the cryopreserved adult germline stem cell comprising the Red Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127094, the cryopreserved adult germline stem cell comprising the Orange Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127095, or the cryopreserved adult germline stem cell comprising the Green Cory 1 transformation event has been deposited under ATCC Accession No. PTA-127096.

3. The transgenic Cory of claim 1 , wherein the fish is homozygous for the integrated expression cassette.

4. The transgenic Cory of claim 1 , wherein the fish is heterozygous for the integrated expression cassette.

5. 13. A method of providing transgenic Cory to the ornamental fish market, comprising obtaining a transgenic Cory according to claim 1 and distributing said fish to the ornamental fish market.

6. 6. The method of claim 5, wherein the fish are distributed by a fish farmer to a commercial distributor.

7. The method of claim 5 , wherein the fish are distributed to retailers by fish farmers or commercial distributors.

8. The method of claim 7 , wherein the retailer is a multi-item retailer having an ornamental fish department.

9. 1. A method for producing a transgenic Cory comprising: (a) obtaining a Cory comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Cory comprising a transformation event selected from the group consisting of "Red Cory 1 transformation event", "Orange Cory 1 transformation event", and "Green Cory 1 transformation event"; (b) breeding the resulting Cory with a second Cory to provide a transgenic Cory comprising the transformation event.

10. The method of claim 9 , wherein the second Cory is a non-transgenic Cory.

11. A transgenic cory comprising a chromosomally integrated expression cassette encoding a green fluorescent protein and a chromosomally integrated expression cassette encoding an orange fluorescent protein.

12. The transgenic cory of claim 11, further defined as a fertile transgenic cory.

13. 12. The transgenic cory of claim 11, further defined as a sterile transgenic Yellow Cory.

14. The transgenic cory of claim 11 , wherein the fish is homozygous for at least one of the integrated expression cassettes.

15. The transgenic cory of claim 11 or 14, wherein the fish is heterozygous for at least one of the integrated expression cassettes.

16. 12. A method of providing transgenic cory to the ornamental fish market, comprising obtaining the transgenic cory of claim 11 and distributing said fish to the ornamental fish market.

17. 17. The method of claim 16, wherein the fish are distributed by a fish farmer to a commercial distributor.

18. 17. The method of claim 16, wherein the fish are distributed to retailers by fish farmers or commercial distributors.

19. 20. The method of claim 18, wherein the retailer is a multi-item retailer having an ornamental fish department.

20. 1. A method for producing transgenic cory comprising: (a) obtaining a cory comprising a chromosomally integrated expression cassette encoding a green fluorescent protein and a chromosomally integrated expression cassette encoding an orange fluorescent protein; (b) breeding the resulting cory with a second cory.

21. 21. The method of claim 20, wherein the second cory is a non-transgenic cory.