Transgenic barb

Abstract

To provide transgenic ornamental barbs, and methods of making such fish by in vitro fertilization techniques.SOLUTION: Also disclosed are methods of establishing a population of such transgenic barbs, and methods of providing them to the ornamental fish industry for the purpose of marketing.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as a PCT international patent application on January 10, 2020, and claims priority to U.S. Provisional Patent Application No. 62 / 790,683, entitled Transgenic Orange Barb, filed January 10, 2019, and U.S. Provisional Patent Application No. 62 / 790,687, entitled Transgenic Purple Barb, filed January 10, 2019, the disclosures of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates to transgenic fish. In particular, the present invention relates to transgenic orange bulbs. In particular, the present invention relates to transgenic purple bulbs. [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. Methods for introducing foreign genes into fish include 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 report of transgenic fish 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 early transgenic fish research focused on growth hormone gene transfer with the goal of generating rapidly growing fish. While most early attempts used heterologous growth hormone genes and promoters and failed to produce these fish (e.g., Chourlout et al., 1986; Penman et al., 1990; Brem et al., 1988; Gross et al., 1992), enhanced growth in 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).

[0005] Barbels are a non-phylogenetic group of ray-finned fish species, including members of the family Cyprinidae, particularly the genera Barbus and Puntius, but also many other fish species. They were previously classified as barbels within the subfamily Barbinae, but this group is paraphyletic to Cyprininae. They typically inhabit rough, rocky, slow-flowing waters with high dissolved oxygen levels. A typical adult barbel measures 25–100 cm in length and weighs 200–10 kg, with the most common being around 200 g. This genus is well-known among hobbyists. Because they are friendly and non-aggressive, they are well suited to mixed-species tropical freshwater aquaria. However, in the ornamental fish industry, their natural coloration does not compensate for the effective display of various colors. The availability of such barbels with modified pigmentation through genetic modification using fluorescent proteins would improve the ornamental fish industry by enhancing the display of 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 provide new markers and reporters of genetic modification, but progress in the development and production of valves that express such proteins has been limited. Summary of the Invention [Problem to be solved by the invention]

[0007] Transgenic Valves 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]

[0008] In some embodiments, transgenic fish or methods for producing transgenic fish are provided. In certain embodiments, the transgenic fish are fertile, transgenic, fluorescent fish. In certain embodiments, the fish used in the disclosed compositions and methods are bulbs. The skin color of bulbs is determined by skin pigment cells, which contain pigment granules called melanosomes (black or brown), xanthosomes (yellow), erythrosomes (orange or red), or iridosomes (iridescent colors, including white). The number, size, and density of pigment granules per pigment cell affect the skin color of the fish.

[0009] 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.

[0010] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer only to 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.

[0011] 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.

[0012] Any embodiment of any of the present methods, kits, and compositions can consist essentially of or consist of, rather than comprise / include / contain / have the recited features and / or steps. Thus, in any of the claims, the terms "consisting of" or "consisting essentially of" can be achieved by substituting any of the above-listed open-ended conjugated verbs for the purpose of modifying the scope of a given claim from otherwise using the open-ended conjugated verb.

[0013] 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 INVENTION

[0014] Detailed Description of the Invention Transgenic fish In some embodiments, the present disclosure relates to transgenic fish.The method for producing transgenic fish is described in, for example, US Patent Application Nos. 7,135,613; 7,700,825; 7,834,239, each of which is incorporated herein by reference in its entirety.For example, transgenic orange bulb can be produced using an expression cassette that encodes yellow fluorescent protein (YFP), such as ZsYellow1.In another example, transgenic purple bulb can be produced using an expression cassette that encodes purple fluorescent protein (PFP), such as FP635.

[0015] It is preferred to use fish belonging to species and varieties of fish that have commercial value, particularly within the ornamental fish industry. Such fish include, but are not limited to, corydoras, catfish, zebrafish and other danios, killifish, carp, tilapia, goldfish, tetras, barbs, sharks (family Cyprinidae, including 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 barb. Barb is an increasingly popular ornamental animal, and commercial value exists in the addition of various colors. Barb embryos are readily available and nearly transparent. Barb skin color is determined by skin pigment cells containing pigment granules called melanosomes. The number, size, and density of melanosomes per pigment cell affect the fish's skin color.

[0016] In some embodiments, transgenic bulbs or their progeny are provided that contain transgenic integration events, (herein referred to as transgenic events). These fish are particularly interesting because, for example, they exhibit an aesthetically pleasing orange color. Transgenic fish containing these specific transgenic bulbs may be homozygous or heterozygous (including, for example, hemizygous) for the transgenic event. Homozygous fish bred with fish lacking the transgenic event will almost always produce 100% heterozygous offspring. Sperm and embryos containing these specific transgenic events are also included as part of the present invention.

[0017] In at least one embodiment regarding a particular transgenic integration event, an orange transgenic bulb or its progeny has a chromosomally integrated transgene, the bulb comprising an "Orange Barb 1 transformation event," and sperm comprising the Orange Barb 1 transformation event have been deposited with the American Type Culture Collection (ATCC) under accession number PTA-126571. The chromosomally integrated transgene can be represented on one integrated expression cassette or on two or more integrated expression cassettes. In some aspects, such transgenic bulbs are fertile transgenic bulbs. Such transgenic bulbs can be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0018] Also provided are methods for providing transgenic valves containing an Orange Barb 1 transformation event to the ornamental fish market. In some embodiments, the methods include obtaining a transgenic valve or progeny thereof that contains a transgene integrated into a chromosome, wherein the valve contains an "Orange Barb 1 transformation event," and sperm containing the Orange Barb 1 transformation event has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-126571, and selling the fish to the ornamental fish market. Such fish can be sold by breeders to commercial distributors, or such fish are sold by breeders or commercial distributors to retail outlets, such as, for example, multi-species retailers with ornamental fish departments.

[0019] In some aspects, a method of producing a transgenic valve is provided, the method comprising: (a) obtaining a bulb that fluoresces and contains a transgene or expression cassette integrated into one or more chromosomes, the bulb comprising an "Orange Barb 1 transformation event," and sperm containing the Orange Barb 1 transformation event have been deposited with the American Type Culture Collection (ATCC) under accession number PTA-126571; and (b) crossing the resulting bulb with a second bulb to provide a transgenic bulb containing the Orange Barb 1 transformation event; The second valve can be a transgenic or non-transgenic valve.

[0020] In a further embodiment, a method of producing a transgenic organism is provided, the method comprising producing transgenic offspring using sperm containing the Orange Barb 1 transformation event deposited under accession number PTA-126571. Such offspring can be, for example, a barb, a species of the family Cyprinidae, a fish species or genera related to the barb, or another species or genera. In some embodiments, the fish can be produced using in vitro fertilization techniques known in the art or described herein.

[0021] In some embodiments relating to a particular transgenic integration event, purple transgenic bulbs or their progeny are provided that contain a chromosomally integrated transgene, the bulb comprising a "Purple Barb 1 transformation event," and sperm containing the Purple Barb 1 transformation event have been deposited with the American Type Culture Collection (ATCC) under accession number PTA-126570. The chromosomally integrated transgene can be present on one integrated expression cassette or on two or more integrated expression cassettes. In some aspects, such transgenic bulbs are fertile transgenic bulbs. Such transgenic bulbs can be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette.

[0022] Also provided are methods for providing transgenic bulbs containing a Purple Barb 1 transformation event to the ornamental fish market. In some embodiments, the methods include obtaining a transgenic bulb or progeny thereof that contains a transgene integrated into a chromosome, wherein the bulb contains a "Purple Barb 1 transformation event," and sperm containing the Purple Barb 1 transformation event has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-126570, and selling the fish to the ornamental fish market. Such fish can be sold by breeders to commercial distributors, or such fish are sold by breeders or commercial distributors to retail outlets, such as, for example, multi-species retailers with ornamental fish departments.

[0023] In some aspects, a method of producing a transgenic valve is provided, the method comprising: (a) obtaining a bulb that fluoresces and contains a transgene or expression cassette integrated into one or more chromosomes, the bulb comprising a "Purple Barb 1 transformation event," and sperm containing the Purple Barb 1 transformation event have been deposited with the American Type Culture Collection (ATCC) under accession number PTA-126570; and (b) crossing the resulting bulb with a second bulb to provide a transgenic bulb containing the Purple Barb 1 transformation event; The second valve can be a transgenic or non-transgenic valve.

[0024] In a further embodiment, a method of producing a transgenic organism is provided, the method comprising producing transgenic offspring using sperm containing the Purple Barb 1 transformation event deposited under Accession No. PTA-126570. Such offspring can be, for example, a barb, a species of the family Cyprinidae, a fish species or genera related to the barb, or another species or genera. In some embodiments, the fish can be produced using in vitro fertilization techniques known in the art or described herein.

[0025] In commercial aquaculture, orange and purple bulbs are naturally produced in pairs. One mated pair of bulbs should be placed in a small tank with artificial spawning mats and aeration. The water level in the tank should be 2-3 inches and 75-85°F. Low salinity (conductivity 100-200 uS / cm) and slightly acidic (pH ~6.9) promote spawning but are not required. Fish may be exposed to natural or artificial lighting cycles beginning at 8:00 AM and ending at 10:00 PM. The next day, the fish are removed around noon and the eggs in the tank are collected. The next day, fry hatch and begin swimming within 2-3 days. Once they are swimming, they can be transferred to tanks or outdoor ponds for growth and maturation.

[0026] Barb breeding stocks may be maintained indoors in aquaria, preferably 300 gallons or larger, but are typically conditioned outdoors in earthen ponds. Conditioned barbs are transferred from the outdoor earthen ponds to large indoor holding tanks. Barbs can spawn weekly; spawning every two or three weeks is preferred.

[0027] Fertilization from frozen sperm Methods for freezing fish sperm are well known in the art, see, for example, Walker and Streisinger (1983) and Draper and Moens (2007), which are incorporated by reference in their entireties. To obtain the transgenic fish disclosed herein, eggs can be fertilized using frozen bulb sperm.

[0028] Briefly, one or two mating pairs of barbs should be placed in a small aquarium equipped with an artificial spawning mat. The water level in the aquarium should be 2-3 inches and 75-85°F. Low salinity (conductivity 100-200 uS / cm) and slightly acidic (pH ~6.9) promote spawning but are not required. Fish may be exposed to natural or artificial light cycles beginning at 8:00 AM and ending at 10:00 PM. The following morning, eggs are removed and discarded. Barbs can be anesthetized by submersion in a 16 mg / 100 mL solution of tricaine. When gill movements become sluggish, the female is removed, washed underwater, and the belly gently wiped with a semi-dry paper towel. Eggs should not come into contact with water, as this will prevent fertilization. Gently squeeze eggs into a slightly concave surface by gently pressing the sides of the flanks with your thumb and index finger and moving your fingers toward the genital opening. Females immediately prior to spawning release their eggs very easily, so care should be taken not to squeeze the eggs when wiping the fish. Good eggs are yellowish and transparent. Eggs that have remained in the female's abdomen for too long are white and opaque. Females release eggs for approximately an hour. Eggs from several females can be pooled together and kept unfertilized for a few minutes. Sperm are thawed in a 33°C water bath for 18-20 seconds. 70 μl of room-temperature Hanks' solution is added to the vial and mixed. Sperm are then immediately added to the eggs and mixed gently. Activation is performed by adding 750 μl of fish water to the sperm and eggs and mixing. This mixture is incubated at room temperature for 5 minutes. The dish is then filled with fish water and incubated at 28°C. After 2-3 hours, the fertilized embryos are transferred to a smaller dish where they are further cultured.

[0029] Parichy and Johnson, 2001, which is incorporated by reference in its entirety, provides additional examples regarding in vitro fertilization.

[0030] The present disclosure further encompasses the progeny of transgenic fish containing the orange bulb transformation event, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing a genomically integrated transgenic construct. As used herein, "progeny" can result from mating two transgenic fish of the present invention, or from mating a first transgenic fish of the present invention with a second fish that is not a transgenic fish of the present invention. In the latter case, the second fish can be, for example, a wild-type fish, a particular fish strain, a mutant fish, or another transgenic fish. The second fish can be of the same species or a different species or genus. Hybrid progeny of these matings have the advantages of the fluorescence transgene combined with the advantages of being derived from these other strains.

[0031] The easiest way to identify fish containing the Orange Bulb1 transformation event is by visual inspection, as the fish are orange in color and readily distinguishable from non-transgenic fish.

[0032] The present disclosure further encompasses the progeny of transgenic fish containing the purple bulb transformation event, as well as transgenic fish derived from transgenic fish eggs, sperm cells, embryos, or other cells containing a genomically integrated transgenic construct. As used herein, "progeny" can result from mating two transgenic fish of the present invention, or from mating a first transgenic fish of the present invention with a second fish that is not a transgenic fish of the present invention. In the latter case, the second fish can be, for example, a wild-type fish, a particular fish strain, a mutant fish, or another transgenic fish. The second fish can be of the same species or a different species or genus. Hybrid progeny of these matings have the advantages of the fluorescence transgene combined with the advantages of being derived from these other strains.

[0033] The easiest way to identify fish containing the Purple Bulb1 transformation event is by visual inspection, as the fish are purple in color and readily distinguishable from non-transgenic fish. [Example]

[0034] Specific embodiments of the present 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, nor should they be construed as providing conditions, parameters, reagents, or starting materials that must be exclusively utilized in practicing the techniques of this invention. Example 1 - Orange Transgenic Bulb

[0035] Transgenic fish that exhibit an orange color are provided. The specific transgenic event embodied in these fish is designated the "Orange Valve 1 transformation event." Sperm from these fish can be used to fertilize valve eggs, thereby propagating transgenic valves containing these specific transgenic integration events. Sperm from this line have been deposited under the provisions of the Budapest Treaty as "Orange Valve 1" with ATCC, 10801 University Boulevard, Manassas, VA 20110 USA. (This deposit has been designated accession number PTA-126571.) Example 2 - Purple Transgenic Bulb

[0036] Transgenic fish that exhibit a purple color are provided. The specific transgenic event embodied in these fish is designated the "Purple Bulb 1 transformation event." Sperm from these fish can be used to fertilize bulb eggs, thereby propagating transgenic bulbs containing these specific transgenic integration events. Sperm from this line have been deposited under the provisions of the Budapest Treaty as "Purple Bulb 1" with ATCC, 10801 University Boulevard, Manassas, VA 20110 USA. (This deposit has been designated accession number PTA-126570.)

[0037] Fluorescent transgenic fish are commercially used as ornamental fish. 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 visualization of the orange or purple color of the transgenic fish.

[0038] 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. Additionally, these fish can be used to mark cells in genetic mosaic experiments and fish cancer models.

[0039] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent that variations can be applied to the compositions and / or methods, and to the steps or sequence of steps described herein, without departing from the concept, spirit, 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 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.

[0040] 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 bulb comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the bulb comprises "Orange Bulb 1 transformation event" sperm, the Orange Bulb 1 transformation event being deposited with the ATCC under deposit number PTA-126571.

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

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

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

5. 1. A method for producing a transgenic valve, comprising: (a) obtaining a bulb containing a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the bulb comprises "Orange Bulb 1 transformation event" sperm, which includes the Orange Bulb 1 transformation event deposited with the ATCC under accession number PTA-126571; and (b) crossing the resulting bulb with a second bulb to provide a transgenic bulb comprising said orange bulb 1 transformation event.

6. 6. The method of claim 5, wherein the second valve is not a transgenic valve.

7. 2. A transgenic bulb progeny fish as described in claim 1, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the transgenic bulb and progeny fish are fluorescent and comprise "Orange Bulb 1 Transformation Event" sperm comprising the Orange Bulb 1 Transformation Event deposited with the ATCC under deposit number PTA-126571.

8. 8. The progeny fish of claim 7, further defined as a fertile transgenic bulb.

9. The progeny fish of claim 7 , wherein the progeny fish are homozygous for the integrated expression cassette.

10. The progeny fish of claim 7 , wherein the progeny fish are heterozygous for the integrated expression cassette.

11. 1. A method for producing a transgenic valve, comprising: (a) obtaining offspring fish of the transgenic valve of claim 7; and (b) mating the resulting progeny fish with a second bulb to provide a transgenic bulb comprising said orange bulb 1 transformation event.

12. 12. The method of claim 11, wherein the second valve is not a transgenic valve.

13. A transgenic valve comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the transgenic valve comprises "Purple Valve 1 transformation event" sperm, which comprises the Purple Valve 1 transformation event deposited with the ATCC under deposit number PTA-126570.

14. 14. The transgenic valve of claim 13, further defined as a fertile transgenic valve.

15. 14. The transgenic valve of claim 13, wherein the transgenic valve is homozygous for the integrated expression cassette.

16. 14. The transgenic valve of claim 13, wherein the transgenic valve is heterozygous for the integrated expression cassette.

17. 1. A method for producing a transgenic valve, comprising: (a) obtaining a bulb containing a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the bulb comprises "purple bulb 1 transformation event" sperm, which includes the purple bulb 1 transformation event deposited with the ATCC under deposit number PTA-126570; and (b) crossing the resulting bulb with a second bulb to provide a transgenic bulb comprising said Purple Bulb 1 transformation event.

18. 18. The method of claim 17, wherein the second valve is not a transgenic valve.

19. 14. A transgenic bulb progeny fish as described in claim 13, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the transgenic bulb and progeny fish are fluorescent and comprise "Purple Bulb 1 Transformation Event" sperm comprising the Purple Bulb 1 Transformation Event deposited with the ATCC under deposit number PTA-126570.

20. 20. The progeny fish of claim 19, further defined as a fertile transgenic bulb.

21. 20. The progeny fish of claim 19, wherein the progeny fish are homozygous for the integrated expression cassette.

22. 20. The progeny fish of claim 19, wherein the progeny fish are heterozygous for the integrated expression cassette.

23. 1. A method for producing a transgenic valve, comprising: (a) obtaining offspring fish of the transgenic valve of claim 19; and (b) mating the resulting progeny fish with a second bulb to provide a transgenic bulb comprising the purple bulb 1 transformation event.

24. 24. The method of claim 23, wherein the second valve is not a transgenic valve.