Transgenic betta

Abstract

To provide a transgenic aquarium fish and a method for producing such a fish by germ cell transplantation technology.SOLUTION: A method for establishing a population of such transgenic aquarium fish and the method for supplying such fish to an aquarium fish industry for the purpose of transaction at a marketplace are also disclosed.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as a PCT international patent application on November 13, 2019, and claims priority to U.S. Patent Application No. 62 / 760,445, filed November 13, 2018, and entitled Transgenic Green Betta, U.S. Patent Application No. 62 / 760,464, filed November 13, 2018, and entitled Transgenic Orange Betta, U.S. Patent Application No. 62 / 760,480, filed November 13, 2018, and entitled Transgenic Red Betta, U.S. Patent Application No. 62 / 760,489, filed November 13, 2018, and entitled Transgenic Purple Betta, and U.S. Patent Application No. 62 / 760,498, filed November 13, 2018, and entitled Transgenic Blue Betta, the disclosures of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE PRESENT APPLICATION The present invention relates to transgenic fish. In particular, the present invention relates to an orange transgenic betta. In particular, the present invention relates to a purple transgenic betta. In particular, the present invention relates to a blue transgenic betta. In particular, the present invention relates to a green transgenic betta. In particular, the present invention relates to a red transgenic betta. In particular, the present invention relates to a pink transgenic betta. [Background technology]

[0003] Introduction Transgenic technology involves the introduction 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 species of fish. There are many methods for introducing 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). 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 focused on growth hormone gene transfer with the goal 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 demonstrated in several fish species, including Atlantic salmon, several species of Pacific salmon, and loach (e.g., Du et al., 1992; Delvin et al., 1994, 1995; Tsai et al., 1995).

[0004] The Siamese fighting fish (Betta splendens), sometimes colloquially known as betta, is a species of the gourami family, and it is popular as an ornamental fish. Species assigned to Betta splendens exhibit a wide diversity in terms of body shape and color. The genus is well known among aquatic life enthusiasts. Bettas are highly territorial fish and tend to be highly aggressive towards each other. Two males that come close to each other will most likely attack each other, and this usually results in the death of one or both fish if they are unable to escape. Female bettas can also become territorial towards each other if they are kept in tanks that are too small.

[0005] In the wild, fish only develop strong coloration when excited. Breeders can make this color permanent and breed purebreds of various hues. Colors available to the aquatic hobbyist include red, orange, yellow, blue, blue-gray, turquoise / green, black, pastel colors, white ("opaque" white, not to be confused with albinos) and multicolored fish.

[0006] Many different colors can be found in bettas, resulting from different pigment layers within their cuticle. The layers (from innermost to outermost) consist of red, yellow, black, iridescent (blue and green), and metallic (not a color in itself, but reacts with other colors to change how they are seen). Any combination of these layers can exist, leading to a wide variety of colors.

[0007] However, in the ornamental fish industry, flashy colors may be enhanced or new colors may be introduced to provide a variety of colors more efficiently. The availability of such bettas with modified pigmentation by genetic modification with fluorescent proteins would bring better products to the ornamental fish industry by better displaying the variety of colors.

[0008] 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 provide new markers and reporters of genetic modification, but progress in the field of developing and producing betta fish that express such proteins has been limited. Summary of the Invention [Problem to be solved by the invention]

[0009] Transgenic Betta In certain embodiments, the present disclosure relates to producing transgenic fluorescent fish and supplying such fish to the ornamental fish industry. [Means for solving the problem]

[0010] In some embodiments, a transgenic fish or a method for producing a transgenic fish is provided. In certain embodiments, the transgenic fish is a fertile, transgenic, fluorescent fish. In certain embodiments, the fish used in the disclosed compositions and methods is a betta. The color of the betta's skin is determined by the pigment cells in the skin, which contain pigment granules called melanosomes (black or brown), xanthosomes (yellow), erythrosomes (orange or red), or iridosomes (iridescent, including white). The number, size, and density of pigment granules per pigment cell affect the color of the fish's skin.

[0011] In certain specific embodiments, transgenic betta fish or their progeny are provided that contain specific transgenic integration events, referred to herein as transgenic events. These fish are particularly interesting, for example, because they embody an aesthetically pleasing green color. Transgenic fish that contain these specific transgenic events can be homozygous or heterozygous (including, for example, hemizygous) for the transgenic events. Homozygous fish mated with fish lacking the transgenic events will, in almost all cases, produce 100% heterozygous offspring. Germ cells, eggs, sperm, and embryos that contain these specific transgenic events are also included as part of the present invention.

[0012] In one embodiment of such a particular transgenic integration event, a green transgenic beta or its progeny is provided that includes a chromosomally integrated transgene, including a "green beta 1 transformation event" cryopreserved testis that includes the green beta 1 transformation event deposited at ECACC. The chromosomally integrated transgene may be present in one integrated expression cassette or in two or more integrated expression cassettes. In a particular embodiment, such a transgenic beta is a fertile transgenic beta. Such a transgenic beta may be homozygous or heterozygous (including, for example, hemizygous) with respect to the transgene or integrated expression cassette(s).

[0013] Also disclosed is a method of supplying a transgenic beta fish containing the green beta 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic beta fish or its progeny containing a chromosomally integrated transgene, wherein the beta fish contains a "green beta 1 transformation event" cryopreserved testis containing the green beta 1 transformation event deposited with the ECACC, and distributing the fish to the ornamental fish industry. Such fish may be distributed by breeders to wholesalers, or such fish may be distributed by breeders or wholesalers to retailers, such as, for example, multi-product retailers with ornamental fish departments.

[0014] In some embodiments, the method of producing a transgenic betta comprises: (a) obtaining a beta that fluoresces and contains one or more chromosomally integrated transgenes or expression cassettes, wherein the beta comprises a "Green Beta 1 transformation event" cryopreserved testis that contains the Green Beta 1 transformation event deposited at ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta that contains the green beta 1 transformation event. The second beta can be a transgenic or non-transgenic beta.

[0015] In further embodiments, methods of producing transgenic organisms are also provided, comprising using cryopreserved testes containing the green betta 1 transformation and deposited at ECACC to produce transgenic progeny. Such progeny may be, for example, bettas, species of the Betta splendida family, species or genera related to bettas, or other fish species or genera. In some embodiments, the fish may be produced using in vitro fertilization techniques.

[0016] In another such embodiment relating to a particular transgenic integration event, an orange betta or its progeny is provided that comprises a chromosomally integrated transgene, wherein the betta comprises an "orange betta 1 transformation event" cryopreserved testis that comprises the orange betta 1 transformation event deposited at ECACC. The chromosomally integrated transgene may be present in one integrated expression cassette or in two or more integrated expression cassettes. In a particular embodiment, the transgenic betta is a fertile transgenic betta. The transgenic betta may be homozygous or heterozygous (e.g., including hemizygous) for the transgene or integrated expression cassette(s).

[0017] Also disclosed is a method of supplying transgenic betta fish containing orange betta transformation events to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic betta fish or its progeny containing a chromosomally integrated transgene, wherein the betta fish contains "orange betta 1 transformation event" cryopreserved testes containing the orange betta 1 transformation event deposited with ECACC, and distributing the fish to the ornamental fish market. Such fish may be distributed by breeders to wholesalers, or such fish may be distributed by breeders or wholesalers to retailers, such as multi-product retailers with ornamental fish departments.

[0018] In some embodiments, the method of producing a transgenic betta comprises: (a) obtaining a beta that fluoresces and contains one or more chromosomally integrated transgenes or expression cassettes, wherein the beta includes an "Orange Beta 1 transformation event" cryopreserved testis that contains the Orange Beta 1 transformation event deposited at ECACC; and (b) mating the resulting betta with a second betta to provide a transgenic betta containing the Orange Betta 1 transformation event. The second betta can be a transgenic or non-transgenic betta.

[0019] In further embodiments, methods of producing transgenic organisms are also provided, comprising using cryopreserved testes containing the orange betta 1 transformation and deposited at ECACC to produce transgenic progeny. Such progeny may be, for example, bettas, species of the Betta splendida family, species or genera related to bettas, or other fish species or genera. In some embodiments, the fish may be produced using in vitro fertilization techniques.

[0020] In another embodiment of such a particular transgenic integration event, a red transgenic beta or its progeny is provided that includes a chromosomally integrated transgene, including a "red beta 1 transformation event" cryopreserved testis that includes the red beta 1 transformation event deposited at ECACC. The chromosomally integrated transgene may be present in one integrated expression cassette or in two or more integrated expression cassettes. In a particular embodiment, such a transgenic beta is a fertile transgenic beta. Such a transgenic beta may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0021] Also disclosed is a method of supplying a transgenic beta fish containing a red beta 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic beta fish or its progeny containing a chromosomally integrated transgene, wherein the beta fish contains a "red beta 1 transformation event" cryopreserved testis containing the red beta 1 transformation event deposited with ECACC, and distributing the fish to the ornamental fish industry. Such fish can be distributed by breeders to wholesalers, or such fish can be distributed by breeders or wholesalers to retailers, such as multi-product retailers with ornamental fish departments.

[0022] In some embodiments, the method of producing a transgenic betta comprises: (a) obtaining a beta that fluoresces and contains one or more chromosomally integrated transgenes or expression cassettes, wherein the beta includes a "Red Beta 1 transformation event" cryopreserved testis that contains the Red Beta 1 transformation event deposited at ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta that includes the Red Beta 1 transformation event. The second beta can be a transgenic or non-transgenic beta.

[0023] In further embodiments, methods of producing transgenic organisms are also provided, comprising using cryopreserved testes containing the red betta 1 transformation and deposited at ECACC to produce transgenic progeny. Such progeny may be, for example, bettas, species of the Betta splendida family, species or genera related to bettas, or other fish species or genera. In some embodiments, the fish may be produced using in vitro fertilization techniques.

[0024] In another embodiment of such a particular transgenic integration event, a purple transgenic beta or its progeny is provided that includes a chromosomally integrated transgene, including a "purple beta 1 transformation event" cryopreserved testis that includes the purple beta 1 transformation event deposited at ECACC. The chromosomally integrated transgene may be present in one integrated expression cassette or in two or more integrated expression cassettes. In a particular embodiment, such a transgenic beta is a fertile transgenic beta. Such a transgenic beta may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0025] Also disclosed is a method of supplying a transgenic beta fish containing a purple beta 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic beta fish or its progeny containing a chromosomally integrated transgene, wherein the beta fish contains a "purple beta 1 transformation event" cryopreserved testis containing the purple beta 1 transformation event deposited with ECACC, and distributing the fish to the ornamental fish industry. Such fish can be distributed by breeders to wholesalers, or such fish can be distributed by breeders or wholesalers to retailers, such as, for example, multi-product retailers with ornamental fish departments.

[0026] In some embodiments, the method of producing a transgenic betta comprises: (a) obtaining a beta that fluoresces and contains one or more chromosomally integrated transgenes or expression cassettes, wherein the beta includes a "purple beta 1 transformation event" cryopreserved testis that contains the purple beta 1 transformation event deposited at ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta that includes the Purple Beta 1 transformation event. The second beta can be a transgenic or non-transgenic beta.

[0027] In further embodiments, methods of producing transgenic organisms are also provided, comprising using cryopreserved testes containing the purple betta 1 transformation and deposited at ECACC to produce transgenic progeny. Such progeny may be, for example, bettas, species of the Betta splendida family, species or genera related to bettas, or other fish species or genera. In some embodiments, the fish may be produced using in vitro fertilization techniques.

[0028] In another embodiment of such a particular transgenic integration event, a blue transgenic beta or its progeny is provided that includes a chromosomally integrated transgene, including a "blue beta 1 transformation event" cryopreserved testis that includes the blue beta 1 transformation event deposited at ECACC. The chromosomally integrated transgene may be present in one integrated expression cassette or in two or more integrated expression cassettes. In a particular embodiment, such a transgenic beta is a fertile transgenic beta. Such a transgenic beta may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0029] Also disclosed is a method of supplying a transgenic beta fish containing a blue beta 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic beta fish or its progeny containing a chromosomally integrated transgene, wherein the beta fish contains a "blue beta 1 transformation event" cryopreserved testis that contains the blue beta 1 transformation event deposited with ECACC, and distributing the fish to the ornamental fish industry. Such fish can be distributed by breeders to wholesalers, or such fish can be distributed by breeders or wholesalers to retailers, such as multi-product retailers with ornamental fish departments.

[0030] In some embodiments, the method of producing a transgenic betta comprises: (a) obtaining a beta that fluoresces and contains one or more chromosomally integrated transgenes or expression cassettes, wherein the beta includes a "blue beta 1 transformation event" cryopreserved testis that contains the blue beta 1 transformation event deposited at ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta that contains the blue beta 1 transformation event. The second beta can be a transgenic or non-transgenic beta.

[0031] In further embodiments, methods of producing transgenic organisms are also provided, the methods comprising using cryopreserved testes containing the blue betta 1 transformation and deposited at ECACC to produce transgenic progeny. Such progeny can be, for example, bettas, species of the Betta splendida family, species or genera related to bettas, or other fish species or genera. In some embodiments, the fish can be produced using in vitro fertilization techniques.

[0032] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0033] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to only alternatives or the alternatives are not mutually exclusive, but the disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "other" may mean at least two or more.

[0034] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method employed to determine the value, or the variation that exists among study subjects.

[0035] Any embodiment of any of the methods, kits, and compositions can consist essentially of or consist 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 achieved by substituting any of the open-ended conjugated verbs listed above for the purpose of modifying the scope of a given claim from the use of an otherwise open-ended conjugated verb.

[0036] Other objects, features and advantages of the present invention will become apparent from the following detailed description. 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, but it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention Transgenic fish In some embodiments, the present disclosure relates to transgenic fish. Methods for producing transgenic fish are described, for example, in U.S. Patent Application Nos. 7,135,613; 7,700,825; and 7,834,239, each of which is incorporated herein by reference in its entirety. For example, a transgenic green betta can be produced using an expression cassette encoding a green fluorescent protein (GFP), such as zsGreen1. In another example, a transgenic orange betta can be produced using an expression cassette encoding an orange fluorescent protein (YFP), such as ZsYellow1. In another example, a transgenic red betta can be produced using an expression cassette encoding a red fluorescent protein (RFP), such as DsRed2. In another example, a transgenic purple betta can be produced using an expression cassette encoding a purple fluorescent protein (PFP), such as FP635. In another example, a transgenic blue betta can be produced using an expression cassette encoding a blue fluorescent protein (BFP), such as TagBFP, or TagBFP in combination with aeCP597.

[0038] 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, betta, catfish, zebrafish and other danios, killifish, carp, tilapia, goldfish, tetras, barbs, sharks (family cyprinidae, such as rainbow sharks), angelfish, loach, carp, glassfish, discus, eels, gobies, gouramis, guppies, xiphophorus, hatchetfish, mollyfish, or pangasius. A particular fish for use in the present disclosure is the betta. Bettas are ornamental animals that are becoming increasingly popular, and there would be commercial value in adding a variety of colors. Betta embryos are easily available and are nearly transparent. The color of the betta's skin is determined by the pigment cells in the skin, which contain pigment granules called melanosomes. The number, size, and density of melanosomes per pigment cell affect the color of the fish's skin.

[0039] In commercial farming, green bettas are produced in natural pairs. A mature male is placed in a 1-3 gallon tank and left overnight. The male builds a bubble nest and the female is added and left overnight. After spawning, the female is removed and the male is left in the tank to care for the developing eggs. After 3 days, the male is removed and the fry are fed newly hatched Artemia nauplii for 3-4 weeks until they are large enough to be safely transferred to a growout vat. It takes about 3 months for a betta to reach maturity. Regeneration of lineages from cryopreserved testes

[0040] Methods for freezing gonads are well known in the art; see, for example, Lee et al. (2013), and Seki et al. (2017), both of which are incorporated herein by reference in their entirety. Germ cells obtained from isolated frozen betta gonads can be used to obtain the transgenic fish disclosed herein.

[0041] In an exemplary embodiment, a young adult male green betta is euthanized using an overdose of a sedative such as Tricaine. The fish is decapitated, blotted dry, sprayed with 70% ethanol, dissected, and the testes are removed and transferred into ice-cold L-15 medium. Once the desired amount of tissue is obtained, the testes are transferred into ice-cold vitrification solution (L-15 medium containing 30% (v / v) ethylene glycol, 21% (w / v) Ficoll PM-70, and 0.35 M sucrose). After 20 minutes of incubation, the testes are blotted to remove the vitrification solution and immersed in liquid nitrogen. For long-term storage, samples are kept in liquid nitrogen.

[0042] To recover the green betta lineage, cryopreserved testes were thawed in 0.2M sucrose solution in L-15 medium at 25°C for 5 seconds and incubated in the same solution at 0°C for 5 minutes. The thawed testes were transferred into L-15 medium containing 10% fetal bovine serum (FBS), minced, transferred into L-15 medium containing 0.2% collagenase H, 0.15% dispase II, and 900U / ml DNase I, and incubated at 25°C for 1 hour with constant gentle agitation to disaggregate the tissue into cells. The cell suspension was rinsed once with L-15 containing 10% FBS and filtered through a 60 micron mesh. The cell suspension was then injected into 7-day-old betta fry anesthetized with Tricaine. The injected fry were raised to adulthood and allowed to spawn using standard protocols to obtain green betta progeny.

[0043] The present disclosure further encompasses the progeny of transgenic fish containing the green betta transformation event, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the genome-integrated transgenic construct. "Progeny" as used in this disclosure may result from mating two transgenic fish of the present invention, or mating 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 particular fish line, a mutant fish, or another transgenic fish. The second fish may be of the same species or a different species or genus. The hybrid progeny of these matings have the advantages of the transgene for fluorescence combined with the advantages derived from these other lines.

[0044] The easiest way to identify fish containing the Green Betta 1 transformation event is by visual inspection, as the fish are green in color and immediately distinguishable from non-transgenic fish.

[0045] In an exemplary embodiment, a young adult male orange betta is euthanized using an overdose of a sedative such as Tricaine. The fish is decapitated, blotted dry, sprayed with 70% ethanol, dissected, and the testes are removed and transferred into ice-cold L-15 medium. Once the desired amount of tissue is obtained, the testes are transferred into ice-cold vitrification solution (L-15 medium containing 30% (v / v) ethylene glycol, 21% (w / v) Ficoll PM-70, and 0.35 M sucrose). After 20 minutes of incubation, the testes are blotted to remove the vitrification solution and immersed in liquid nitrogen. For long-term storage, samples are kept in liquid nitrogen.

[0046] To recover the orange betta lineage, cryopreserved testes were thawed in 0.2M sucrose solution in L-15 medium at 25°C for 5 seconds and incubated in the same solution at 0°C for 5 minutes. The thawed testes were transferred into L-15 medium containing 10% fetal bovine serum (FBS), minced, transferred into L-15 medium containing 0.2% collagenase H, 0.15% dispase II, and 900U / ml DNase I, and incubated at 25°C for 1 hour with constant gentle agitation to disaggregate the tissue into cells. The cell suspension was rinsed once with L-15 containing 10% FBS and filtered through a 60 micron mesh. The cell suspension was then injected into 7-day-old betta fry anesthetized with Tricaine. The injected fry were raised to adulthood and allowed to spawn using standard protocols to obtain orange betta progeny.

[0047] The present disclosure further encompasses the progeny of transgenic fish containing the orange betta transformation event, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the genome-integrated transgenic construct. "Progeny" as used in this disclosure may result from mating two transgenic fish of the present invention, or from mating 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 particular fish line, a mutant fish, or another transgenic fish. The second fish may be of the same species or a different species or genus. The hybrid progeny of these matings have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0048] The easiest way to identify fish containing the Orange Betta 1 transformation event is by visual inspection, as the fish are orange in color and immediately distinguishable from non-transgenic fish.

[0049] In an exemplary embodiment, a young adult male red betta is euthanized using an overdose of a sedative such as Tricaine. The fish is decapitated, blotted dry, sprayed with 70% ethanol, dissected, and the testes are removed and transferred into ice-cold L-15 medium. Once the desired amount of tissue is obtained, the testes are transferred into ice-cold vitrification solution (L-15 medium containing 30% (v / v) ethylene glycol, 21% (w / v) Ficoll PM-70, and 0.35 M sucrose). After 20 minutes of incubation, the testes are blotted to remove the vitrification solution and immersed in liquid nitrogen. For long-term storage, samples are kept in liquid nitrogen.

[0050] To recover the red betta lineage, cryopreserved testes were thawed in 0.2M sucrose solution in L-15 medium at 25°C for 5 seconds and incubated in the same solution at 0°C for 5 minutes. The thawed testes were transferred into L-15 medium containing 10% fetal bovine serum (FBS), minced, transferred into L-15 medium containing 0.2% collagenase H, 0.15% dispase II, and 900U / ml DNase I, and incubated at 25°C for 1 hour with constant gentle stirring to disaggregate the tissue into cells. The cell suspension was rinsed once with L-15 containing 10% FBS and filtered through a 60 micron mesh. The cell suspension was then injected into 7-day-old betta fry anesthetized with Tricaine. The injected fry were raised to adulthood and allowed to spawn using standard protocols to obtain red betta progeny.

[0051] The present disclosure further encompasses the progeny of transgenic fish containing the red betta transformation event, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the genome-integrated transgenic construct. "Progeny" as used in this disclosure may result from mating two transgenic fish of the present invention, or from mating 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 particular fish line, a mutant fish, or another transgenic fish. The second fish may be of the same species or a different species or genus. The hybrid progeny of these matings have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0052] The easiest way to identify fish containing the red betta 1 transformation event is by visual inspection, as the fish are red and readily distinguishable from non-transgenic fish. It should also be understood that transgenic red bettas may have a fading color throughout the life of the transgenic red betta, depending on the insertion location of the particular RFP and / or expression cassette used. For example, a red transgenic betta may change color from red to pale red, or from red to pink. In addition, depending on the insertion location of the particular RFP and / or expression cassette used, a transgenic red betta may have a fading color over many generations. For example, a red transgenic betta may The color may vary from one generation to the next, such that older generations may exhibit a red color while younger generations may appear pale red or pink.

[0053] In an exemplary embodiment, a young adult male purple betta is euthanized using an overdose of a sedative such as Tricaine. The fish is decapitated, blotted dry, sprayed with 70% ethanol, dissected, and the testes are removed and transferred into ice-cold L-15 medium. Once the desired amount of tissue is obtained, the testes are transferred into ice-cold vitrification solution (L-15 medium containing 30% (v / v) ethylene glycol, 21% (w / v) Ficoll PM-70, and 0.35 M sucrose). After 20 minutes of incubation, the testes are blotted to remove the vitrification solution and immersed in liquid nitrogen. For long-term storage, samples are kept in liquid nitrogen.

[0054] To recover the purple betta lineage, cryopreserved testes were thawed in 0.2M sucrose solution in L-15 medium at 25°C for 5 seconds and incubated in the same solution at 0°C for 5 minutes. The thawed testes were transferred into L-15 medium containing 10% fetal bovine serum (FBS), minced, transferred into L-15 medium containing 0.2% collagenase H, 0.15% dispase II, and 900U / ml DNase I, and incubated at 25°C for 1 hour with constant gentle agitation to disaggregate the tissue into cells. The cell suspension was rinsed once with L-15 containing 10% FBS and filtered through a 60 micron mesh. The cell suspension was then injected into 7-day-old betta fry anesthetized with Tricaine. The injected fry were raised to adulthood and allowed to spawn using standard protocols to obtain purple betta progeny.

[0055] The present disclosure further encompasses the progeny of transgenic fish containing the purple betta transformation event, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing the genome-integrated transgenic construct. "Progeny" as used in this disclosure may result from mating two transgenic fish of the present invention, or from mating 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 particular fish line, a mutant fish, or another transgenic fish. The second fish may be of the same species or a different species or genus. The hybrid progeny of these matings have the advantages of the transgene for fluorescence combined with the advantages of being derived from these other lines.

[0056] The easiest way to identify fish containing the Purple Betta 1 transformation event is by visual inspection, as the fish are purple in color and immediately distinguishable from non-transgenic fish.

[0057] In an exemplary embodiment, a young adult male blue betta is euthanized using an overdose of a sedative such as Tricaine. The fish is decapitated, blotted dry, sprayed with 70% ethanol, dissected, and the testes are removed and transferred into ice-cold L-15 medium. Once the desired amount of tissue is obtained, the testes are transferred into ice-cold vitrification solution (L-15 medium containing 30% (v / v) ethylene glycol, 21% (w / v) Ficoll PM-70, and 0.35 M sucrose). After 20 minutes of incubation, the testes are blotted to remove the vitrification solution and immersed in liquid nitrogen. For long-term storage, samples are kept in liquid nitrogen.

[0058] To recover the blue betta lineage, cryopreserved testes were thawed in 0.2M sucrose solution in L-15 medium at 25°C for 5 seconds and incubated in the same solution at 0°C for 5 minutes. The thawed testes were transferred into L-15 medium containing 10% fetal bovine serum (FBS), minced, transferred into L-15 medium containing 0.2% collagenase H, 0.15% dispase II, and 900U / ml DNase I, and incubated at 25°C for 1 hour with constant gentle stirring to disaggregate the tissue into cells. The cell suspension was rinsed once with L-15 containing 10% FBS and filtered through a 60 micron mesh. The cell suspension was then injected into 7-day-old betta fry anesthetized with Tricaine. The injected fry were raised to adulthood and allowed to spawn using standard protocols to obtain blue betta progeny.

[0059] The present disclosure further encompasses the progeny of transgenic fish containing blue betta transformation events, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing genome-integrated transgenic constructs. "Progeny" as used in this disclosure may result from mating two transgenic fish of the present invention, or mating 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 particular fish line, a mutant fish, or another transgenic fish. The second fish may be of the same species or a different species or genus. The hybrid progeny of these matings have the advantages of the transgene for fluorescence combined with the advantages derived from these other lines.

[0060] The easiest way to identify fish containing the Blue Betta 1 transformation event is by visual inspection, as the fish are blue in color and immediately distinguishable from non-transgenic fish. EXAMPLES

[0061] 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 in no way intended to limit or restrict the scope of the invention, and should not be construed as providing conditions, parameters, reagents, or starting materials which must be exclusively utilized in practicing the techniques of the invention. Example 1 - Green transgenic betta

[0062] Transgenic fish exhibiting a green color are provided. The specific transgenic events embodied in these fish are designated "Green Betta 1 transformation events." Germ cells from these fish can be transplanted into betta juveniles, thereby breeding transgenic bettas containing these specific transgenic integration events. Cryopreserved testes from this line have been deposited under the provisions of the Budapest Treaty at the European Collection of Cell Cultures (ECACC), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, as "Green Betta 1." Example 2 - Orange transgenic betta

[0063] Transgenic fish exhibiting the color orange are provided. The particular transgenic event embodied in these fish is designated as the "Orange Betta 1 transformation event." Germ cells from these fish can be transplanted into betta juveniles, thereby breeding transgenic bettas containing these particular transgenic integration events. Cryopreserved testes from this line have been deposited under the provisions of the Budapest Treaty at the European Collection of Cell Cultures (ECACC), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, as "Orange Betta 1." Example 3 - Red and / or Pink Transgenic Bettas

[0064] Transgenic fish that exhibit the color red are provided. The specific transgenic events embodied in these fish are designated "Red Betta 1 transformation events." Germ cells from these fish can be transplanted into betta juveniles, thereby breeding transgenic bettas containing these specific transgenic integration events. Cryopreserved testes from this line have been deposited under the provisions of the Budapest Treaty at the European Collection of Cell Cultures (ECACC), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, as "Red Betta 1." Example 4 - Purple transgenic betta

[0065] Transgenic fish exhibiting the color purple are provided. The specific transgenic events embodied in these fish are designated "purple betta 1 transformation events." Germ cells from these fish can be transplanted into betta juveniles, thereby breeding transgenic bettas containing these specific transgenic integration events. Cryopreserved testes from this line have been deposited under the provisions of the Budapest Treaty at the European Collection of Cell Cultures (ECACC), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, as "purple betta 1." Example 5 - Blue transgenic betta

[0066] Transgenic fish that exhibit a blue color are provided. The specific transgenic events embodied in these fish are designated "Blue Betta 1 transformation events." Germ cells from these fish can be transplanted into betta larvae, thereby breeding transgenic bettas containing these specific transgenic integration events. Cryopreserved testes from this line have been deposited under the provisions of the Budapest Treaty at the European Collection of Cell Cultures (ECACC), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, as "Blue Betta 1."

[0067] Fluorescent transgenic fish are used as ornamental fish in the market. Stably expressing transgenic lines can be developed by crossing transgenic individuals with wild-type fish, mutant fish, or other transgenic fish. The transgenic fish of interest can be distinguished from non-transgenic fish by observing the fish under white light, sunlight, ultraviolet light, blue light, or other useful lighting conditions that allow the green, orange, red, pink, purple, or blue color of the transgenic fish to be visualized.

[0068] Fluorescent transgenic fish should also be valuable in the market of scientific research tools, as they can be used for embryonic studies such as tracking cell lineages and cell migration. In addition, these fish can be used to mark cells in genetic mosaic experiments and fish cancer models.

[0069] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with respect to preferred embodiments, it will be apparent that modifications can be applied in the compositions and / or methods, and in the steps or sequence of steps of the methods described herein, without departing from the concept, nature and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein to achieve the same or similar results. All such similar substitutes and modifications that are 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.

[0070] 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. A transgenic beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "green beta 1 transformation event" cryopreserved testis comprising the green beta 1 transformation event deposited with ECACC.

2. 2. The transgenic betta of claim 1, further defined as a fertile transgenic betta.

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

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

5. 13. A method for supplying a transgenic betta to the ornamental fish market, comprising obtaining a transgenic betta as described in claim 1 and distributing said fish to the ornamental fish market.

6. 6. The method of claim 5, wherein the fish are distributed to wholesalers by breeders.

7. The method of claim 5, wherein the fish are distributed to retailers by breeders or wholesalers.

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

9. 1. A method for producing a transgenic betta fish, comprising: (a) obtaining a beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "green beta 1 transformation event" cryopreserved testis comprising the green beta 1 transformation event deposited with ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta containing the green beta 1 transformation event.

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

11. 2. Progeny of the transgenic beta of claim 1, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said beta and progeny are fluorescent and include "Green Beta 1 Transformation Event" cryopreserved testes comprising the Green Beta 1 transformation event deposited with ECACC.

12. 12. The offspring fish of claim 11, further defined as a fertile transgenic betta.

13. The progeny fish of claim 11, wherein the fish is homozygous for the integrated expression cassette.

14. The progeny fish of claim 11, wherein the fish is heterozygous for the integrated expression cassette.

15. 12. A method of supplying transgenic fish to the ornamental fish market, comprising obtaining an offspring fish according to claim 11 and distributing said fish to the ornamental fish market.

16. 16. The method of claim 15, wherein the fish are distributed to wholesalers by breeders.

17. 17. The method of claim 16, wherein the fish are distributed to retailers by breeders or wholesalers.

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

19. 1. A method for producing a transgenic fish, comprising: (a) obtaining the transgenic fish of claim 11; and (b) mating the resulting fish with a second fish to provide a transgenic fish comprising said green betta 1 transformation event.

20. 20. The method of claim 19, wherein the second fish is a non-transgenic fish.

21. A transgenic beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises an "Orange beta 1 transformation event" cryopreserved testis comprising the Orange beta 1 transformation event deposited with ECACC.

22. 22. The transgenic betta of claim 21, further defined as a fertile transgenic betta.

23. 22. The transgenic betta of claim 21, wherein the fish is homozygous for the integrated expression cassette.

24. The transgenic betta of claim 21, wherein the fish is heterozygous for the integrated expression cassette.

25. 22. A method of supplying a transgenic betta to the ornamental fish market, comprising obtaining a transgenic betta as described in claim 21 and distributing said fish to the ornamental fish market.

26. 26. The method of claim 25, wherein the fish are distributed to wholesalers by breeders.

27. 26. The method of claim 25, wherein the fish are distributed to retailers by breeders or wholesalers.

28. 28. The method of claim 27, wherein the retailer is a multi-product retailer having an ornamental fish department.

29. 1. A method for producing a transgenic betta fish, comprising: (a) obtaining a beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, the beta comprising the "Orange Beta 1 transformation event" cryopreserved testis comprising the Orange Beta 1 transformation event deposited with ECACC; and (b) mating the resulting betta with a second betta to provide a transgenic betta containing the orange betta 1 transformation event.

30. 30. The method of claim 29, wherein the second beta is a non-transgenic beta.

31. 31. Progeny of the transgenic betta of claim 30, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said betta and progeny are fluorescent and include "Orange Betta 1 Transformation Event" cryopreserved testes comprising the Orange Betta 1 transformation event deposited with ECACC.

32. 32. The offspring fish of claim 31, further defined as a fertile transgenic betta.

33. 32. The progeny fish of claim 31 , wherein the fish is homozygous for the integrated expression cassette.

34. 32. The progeny fish of claim 31 , wherein the fish is heterozygous for the integrated expression cassette.

35. 32. A method of supplying transgenic fish to the ornamental fish market, comprising obtaining an offspring fish according to claim 31 and distributing said fish to the ornamental fish market.

36. 36. The method of claim 35, wherein the fish are distributed to wholesalers by breeders.

37. 37. The method of claim 36, wherein the fish are distributed to retailers by breeders or wholesalers.

38. 38. The method of claim 37, wherein the retailer is a multi-product retailer having an ornamental fish department.

39. 1. A method for producing a transgenic fish, comprising: (a) obtaining the transgenic fish of claim 31; and (b) mating the resulting fish with a second fish to provide a transgenic fish comprising said orange betta 1 transformation event.

40. 40. The method of claim 39, wherein the second fish is a non-transgenic fish.

41. A transgenic beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "Red beta 1 transformation event" cryopreserved testis comprising the Red beta 1 transformation event deposited with ECACC.

42. 42. The transgenic betta of claim 41, further defined as a fertile transgenic betta.

43. 42. The transgenic betta of claim 41, wherein the fish is homozygous for the integrated expression cassette.

44. 42. The transgenic betta of claim 41, wherein the fish is heterozygous for the integrated expression cassette.

45. 42. A method of supplying a transgenic betta to the ornamental fish market, comprising obtaining a transgenic betta of claim 41 and distributing said fish to the ornamental fish market.

46. 46. ​​The method of claim 45, wherein the fish are distributed to wholesalers by breeders.

47. 46. ​​The method of claim 45, wherein the fish are distributed to retailers by breeders or wholesalers.

48. 48. The method of claim 47, wherein the retailer is a multi-product retailer having an ornamental fish department.

49. 1. A method for producing a transgenic betta fish, comprising: (a) obtaining a beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "Red beta 1 transformation event" cryopreserved testis comprising the Red beta 1 transformation event deposited with ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta containing the Red Beta 1 transformation event.

50. 50. The method of claim 49, wherein the second beta is a non-transgenic beta.

51. 42. Progeny of the transgenic betta of claim 41, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said betta and progeny are fluorescent and include "Red Betta 1 Transformation Event" cryopreserved testes comprising the Red Betta 1 transformation event deposited with ECACC.

52. 52. The progeny fish of claim 51, further defined as a fertile transgenic betta.

53. 52. The progeny fish of claim 51 , wherein the fish is homozygous for the integrated expression cassette.

54. 52. The progeny fish of claim 51 , wherein the fish is heterozygous for the integrated expression cassette.

55. 52. A method of supplying transgenic fish to the ornamental fish market, comprising obtaining an offspring fish as described in claim 51 and distributing said fish to the ornamental fish market.

56. 56. The method of claim 55, wherein the fish are distributed to wholesalers by breeders.

57. 57. The method of claim 56, wherein the fish are distributed to retailers by breeders or wholesalers.

58. 58. The method of claim 57, wherein the retailer is a multi-product retailer having an ornamental fish department.

59. 1. A method for producing a transgenic fish, comprising: (a) obtaining the transgenic fish of claim 51; and (b) mating the resulting fish with a second fish to provide a transgenic fish comprising said red betta 1 transformation event.

60. 60. The method of claim 59, wherein the second fish is a non-transgenic fish.

61. A transgenic beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "purple beta 1 transformation event" cryopreserved testis comprising the purple beta 1 transformation event deposited with ECACC.

62. 62. The transgenic betta of claim 61, further defined as a fertile transgenic betta.

63. 62. The transgenic betta of claim 61, wherein the fish is homozygous for the integrated expression cassette.

64. The transgenic betta of claim 61, wherein the fish is heterozygous for the integrated expression cassette.

65. 62. A method of supplying a transgenic betta to the ornamental fish market, comprising obtaining a transgenic betta of claim 61 and distributing said fish to the ornamental fish market.

66. 66. The method of claim 65, wherein the fish are distributed to wholesalers by breeders.

67. 66. The method of claim 65, wherein the fish are distributed to retailers by breeders or wholesalers.

68. 68. The method of claim 67, wherein the retailer is a multi-product retailer having an ornamental fish department.

69. 1. A method for producing a transgenic betta fish, comprising: (a) obtaining a beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "purple beta 1 transformation event" cryopreserved testis comprising the purple beta 1 transformation event deposited with ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta containing the Purple Beta 1 transformation event.

70. 70. The method of claim 69, wherein the second beta is a non-transgenic beta.

71. 62. Progeny of the transgenic beta of claim 61, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said beta and progeny are fluorescent and include "Purple Beta 1 Transformation Event" cryopreserved testes comprising the Purple Beta 1 transformation event deposited with ECACC.

72. 72. The offspring fish of claim 71, further defined as a fertile transgenic betta.

73. 72. The offspring fish of claim 71, wherein the fish is homozygous for the integrated expression cassette.

74. 72. The offspring fish of claim 71, wherein the fish is heterozygous for the integrated expression cassette.

75. 72. A method of supplying transgenic fish to the ornamental fish market, comprising obtaining an offspring fish of claim 71 and distributing said fish to the ornamental fish market.

76. 76. The method of claim 75, wherein the fish are distributed to wholesalers by breeders.

77. 77. The method of claim 76, wherein the fish are distributed to retailers by breeders or wholesalers.

78. 78. The method of claim 77, wherein the retailer is a multi-product retailer having an ornamental fish department.

79. 1. A method for producing a transgenic fish, comprising: (a) obtaining the transgenic fish of claim 71; and (b) mating the resulting fish with a second fish to provide a transgenic fish comprising said purple betta 1 transformation event.

80. 80. The method of claim 79, wherein the second fish is a non-transgenic fish.

81. A transgenic beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "blue beta 1 transformation event" cryopreserved testis comprising the blue beta 1 transformation event deposited with ECACC.

82. 82. The transgenic betta of claim 81, further defined as a fertile transgenic betta.

83. 82. The transgenic betta of claim 81, wherein the fish is homozygous for the integrated expression cassette.

84. 82. The transgenic betta of claim 81, wherein the fish is heterozygous for the integrated expression cassette.

85. 82. A method of supplying a transgenic betta to the ornamental fish market, comprising obtaining a transgenic betta of claim 81 and distributing said fish to the ornamental fish market.

86. 86. The method of claim 85, wherein the fish are distributed to wholesalers by breeders.

87. 86. The method of claim 85, wherein the fish are distributed to retailers by breeders or wholesalers.

88. 88. The method of claim 87, wherein the retailer is a multi-product retailer having an ornamental fish department.

89. 1. A method for producing a transgenic betta fish, comprising: (a) obtaining a beta comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the beta comprises a "blue beta 1 transformation event" cryopreserved testis comprising the blue beta 1 transformation event deposited with ECACC; and (b) mating the resulting beta with a second beta to provide a transgenic beta containing the blue beta 1 transformation event.

90. 90. The method of claim 89, wherein the second beta is a non-transgenic beta.

91. 82. Progeny of the transgenic betta of claim 81, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said betta and progeny are fluorescent and include "blue betta 1 transformation event" cryopreserved testes comprising the blue betta 1 transformation event deposited with ECACC.

92. 92. The progeny fish of claim 91, further defined as a fertile transgenic betta.

93. 92. The offspring fish of claim 91, wherein the fish is homozygous for the integrated expression cassette.

94. 92. The offspring fish of claim 91, wherein the fish is heterozygous for the integrated expression cassette.

95. 92. A method of supplying transgenic fish to the ornamental fish market, comprising obtaining an offspring fish of claim 91 and distributing said fish to the ornamental fish market.

96. 96. The method of claim 95, wherein the fish are distributed to wholesalers by breeders.

97. 97. The method of claim 96, wherein the fish are distributed to retailers by breeders or wholesalers.

98. 98. The method of claim 97, wherein the retailer is a multi-product retailer having an ornamental fish department.

99. 1. A method for producing a transgenic fish, comprising: (a) obtaining the transgenic fish of claim 91; and (b) mating the resulting fish with a second fish to provide a transgenic fish comprising said blue betta 1 transformation event.

100. 100. The method of claim 99, wherein the second fish is a non-transgenic fish.