Transgenic Pristella

Abstract

The present invention relates to transgenic ornamental fish and methods for producing such fish by in vitro fertilization 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 November 21, 2022, and claims priority to and benefit of U.S. Provisional Application No. 63 / 287,153, filed on December 8, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to transgenic fish, in particular green, orange, purple and red transgenic Pristella. [Background technology]

[0003] 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 generate 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] Pristella maxillaris, one of two species in the Pristella genus, is commonly known as the X-ray fish or X-ray tetra, due to its translucent body. It is a widespread and adaptable fish found in both acidic and alkaline waters of the Amazon and Orinoco river basins, as well as the coastal rivers of Guiana. Unlike most other characins, it tolerates (and is sometimes found in) slightly brackish waters. It is small (up to about 5 cm or 2.0 in long) and lives in large groups, with males being smaller and thinner than females, which makes them distinguishable from females. Like most other tetras, it feeds mainly on small insects and planktonic animals.

[0006] In the residential fish market, Pristella maxillaris is an adaptable little fish. It tolerates a range of water chemistry values ​​(i.e., pH 6-8). As a schooling species, it is usually kept in groups of at least six specimens, away from aggressive or predatory tank mates, but is otherwise easily kept in community tanks.

[0007] However, the availability of such Pristella 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.

[0008] Many fluorescent proteins are known in the art and have been used to investigate various cellular processes, including fluorescent proteins that exhibit a variety of 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 Pristella expressing such proteins has been limited.

[0009] Transgenic Pristella In certain embodiments, the present disclosure relates to producing transgenic fluorescent fish and providing such fish to the ornamental fish industry.

[0010] 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 Pristella. Pristella 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 (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 Pristella or progeny thereof 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 green color. Transgenic fish that contain these specific transgenic events can be homozygous or heterozygous (including, for example, hemizygous) for the transformation events. 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.

[0012] In one such embodiment relating to a particular transgenic integration event, a green transgenic Pristella or progeny thereof is provided that includes a chromosomally integrated transgene, the Pristella including a "Green Pristella 1 transformation event", and cryopreserved sperm including the Green Pristella 1 transformation event is deposited with the American Type Culture Collections (ATCC), Historic District, 10801 University Blvd, Manassas, VA 20110, under accession number (PTA-127162). 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 Pristella is a fertile transgenic Pristella. Such a transgenic Pristella may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0013] Also disclosed is a method of providing a transgenic Pristella comprising a Green Pristella 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Pristella comprising a chromosomally integrated transgene or a progeny thereof, the Pristella comprising a "Green Pristella 1 transformation event", cryopreserved sperm comprising the Green Pristella 1 transformation event being deposited with ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127162), and distributing the fish to the ornamental fish market. Such fish can be distributed by the farmer to a commercial distributor, or such fish can be distributed by the farmer or the commercial distributor to a retailer, such as a multi-product retailer with an ornamental fish section.

[0014] In some embodiments, a method for producing a transgenic Pristella is provided, comprising: (a) obtaining a Pristella that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Pristella comprising a "Green Pristella 1 transformation event", and cryopreserved sperm comprising the Green Pristella 1 transformation event has been deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127162); and (b) breeding the obtained Pristella with a second Pristella to provide a transgenic Pristella comprising the Green Pristella 1 transformation event. The second Pristella may be a transgenic or non-transgenic Pristella.

[0015] In further embodiments, methods of producing transgenic organisms are also provided, comprising using germ cell transplantation to generate Green Pristella 1 transformation to produce transgenic progeny, such cryopreserved sperm being deposited with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, under Accession Number (PTA-127162). Such progeny may be, for example, Pristella, a species of the Characidae family, a fish species or genus related to Pristella, or another fish species or genus.

[0016] In other embodiments, transgenic Pristella or progeny thereof 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 orange color. Transgenic fish that contain these specific transgenic events can be homozygous or heterozygous (including, for example, hemizygous) for the transformation events. Homozygous fish bred with fish lacking the transformation event 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.

[0017] In one such embodiment relating to a particular transgenic integration event, an orange transgenic Pristella or progeny thereof is provided that includes a chromosomally integrated transgene, the Pristella including an "Orange Pristella 1 transformation event", and cryopreserved sperm including the Orange Pristella 1 transformation event is deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127164). 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 Pristella is a fertile transgenic Pristella. Such a transgenic Pristella may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0018] Also disclosed is a method of providing a transgenic Pristella comprising an Orange Pristella 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Pristella comprising a chromosomally integrated transgene or a progeny thereof, the Pristella comprising an "Orange Pristella 1 transformation event", cryopreserved sperm comprising the Orange Pristella 1 transformation event has been deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127164), and distributing the fish to the ornamental fish market. Such fish can be distributed by the farmer to a commercial distributor, or such fish can be distributed by the farmer or the commercial distributor to a retailer, such as a multi-product retailer with an ornamental fish department.

[0019] In some embodiments, a method for producing a transgenic Pristella is provided, comprising: (a) obtaining a Pristella that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Pristella comprising an "Orange Pristella 1 transformation event", and cryopreserved sperm comprising the Orange Pristella 1 transformation event has been deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127164); and (b) breeding the obtained Pristella with a second Pristella to provide a transgenic Pristella comprising the Orange Pristella 1 transformation event. The second Pristella can be a transgenic or non-transgenic Pristella.

[0020] In further embodiments, methods of producing transgenic organisms are also provided, comprising using germ cell transplantation to generate Orange Pristella 1 transformants to produce transgenic progeny, such cryopreserved sperm being deposited with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, under accession number (PTA-127164). Such progeny may be, for example, Pristella, a species of the Characidae family, a fish species or genus related to Pristella, or another fish species or genus.

[0021] In alternative embodiments, transgenic Pristella or progeny thereof 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 purple 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.

[0022] In one such embodiment of a particular transgenic integration event, a purple transgenic Pristella or its progeny is provided that includes a chromosomally integrated transgene, the Pristella including a "Purple Pristella 1 transformation event", and cryopreserved sperm including the Purple Pristella 1 transformation event is deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127163). 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 Pristella is a fertile transgenic Pristella. Such a transgenic Pristella may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0023] Also disclosed is a method of providing a transgenic Pristella comprising a Purple Pristella 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Pristella comprising a chromosomally integrated transgene or a progeny thereof, the Pristella comprising a "Purple Pristella 1 transformation event", cryopreserved sperm comprising the Purple Pristella 1 transformation event being deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127163), 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 the commercial distributor to a retailer, such as a multi-product retailer with an ornamental fish department.

[0024] In some embodiments, a method for producing a transgenic Pristella is provided, comprising: (a) obtaining a Pristella that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Pristella comprising a "Purple Pristella 1 transformation event", and cryopreserved sperm comprising the Purple Pristella 1 transformation event has been deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127163); and (b) breeding the obtained Pristella with a second Pristella to provide a transgenic Pristella comprising the Purple Pristella 1 transformation event. The second Pristella may be a transgenic or non-transgenic Pristella.

[0025] In further embodiments, methods of producing transgenic organisms are also provided, comprising using germ cell transplantation to generate Purple Pristella 1 transformants to produce transgenic progeny, such cryopreserved sperm being deposited with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, under Accession Number (PTA-127163). Such progeny may be, for example, Pristella, a species of the Characidae family, a fish species or genus related to Pristella, or another fish species or genus.

[0026] In other exemplary embodiments, transgenic Pristella or progeny thereof 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 events. 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.

[0027] In one such embodiment relating to a particular transgenic integration event, a red transgenic Pristella or progeny thereof is provided that includes a chromosomally integrated transgene, the Pristella including a "Red Pristella 1 transformation event", and cryopreserved sperm including the Red Pristella 1 transformation event has been deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127165). 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 Pristella is a fertile transgenic Pristella. Such a transgenic Pristella may be homozygous or heterozygous (including, for example, hemizygous) for the transgene or integrated expression cassette(s).

[0028] Also disclosed is a method of providing a transgenic Pristella comprising a Red Pristella 1 transformation event to the ornamental fish market. In some embodiments, the method includes obtaining a transgenic Pristella comprising a chromosomally integrated transgene or a progeny thereof, the Pristella comprising a "Red Pristella 1 transformation event", cryopreserved sperm comprising the Red Pristella 1 transformation event being deposited with ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127165), and distributing the fish to the ornamental fish market. Such fish can be distributed by the farmer to a commercial distributor, or such fish can be distributed by the farmer or the commercial distributor to a retailer, such as a multi-product retailer with an ornamental fish department.

[0029] In some embodiments, a method for producing a transgenic Pristella is provided, comprising: (a) obtaining a Pristella that exhibits fluorescence and contains one or more chromosomally integrated transgenes or expression cassettes, the Pristella comprising a "Red Pristella 1 transformation event", and cryopreserved sperm comprising the Red Pristella 1 transformation event has been deposited with the ATCC (Historic District, 10801 University Blvd, Manassas, VA 20110) under accession number (PTA-127165); and (b) breeding the obtained Pristella with a second Pristella to provide a transgenic Pristella comprising the Red Pristella 1 transformation event. The second Pristella can be a transgenic or non-transgenic Pristella.

[0030] In further embodiments, methods of producing transgenic organisms are also provided, comprising using germ cell transplantation to generate Red Pristella 1 transformants to produce transgenic progeny, such cryopreserved sperm being deposited with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, under Accession Number (PTA-127165). Such progeny may be, for example, Pristella, species of the Characidae family, fish species or genus related to Pristella, or another fish species or genus.

[0031] 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.

[0032] 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.

[0033] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error in the device, the method being used to determine the value, or the variation that exists among testing subjects.

[0034] Any embodiment of any of the methods, kits, and compositions may 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" may be substituted with any of the above open-ended linking verbs in order to modify a given claim from other uses of the open-ended linking verb.

[0035] 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

[0036] 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 green Pristella can be produced using at least one expression cassette encoding green fluorescent protein (GFP), such as zsGreen1, afraGFP, WasCFP, NowGFP, cerFP505, pporGFP, Kohinoor, efasGFP, eechGFP1, UnaG, bfloGFPa1, LanFP1, and LanFP2. Alternatively, transgenic orange Pristella can be produced using at least one expression cassette encoding yellow fluorescent protein (YFP), such as ZsYellow1, phiYFP, zFP538, mPapaya, and mBanana. Alternatively, transgenic purple Pristella can be generated using at least one expression cassette encoding a purple fluorescent protein (PFP), such as FP635, Katushka2S, mKate2, mCherry2, mCherry, mKate-S158C, eqFP650, mPlum, jRed, and mRFP1. Alternatively, transgenic red Pristella can be generated using at least one expression cassette encoding a red fluorescent protein (RFP), such as TurboRFP, DsRed2, tdTomato, dTomato, eqFP578, DsRed-Express, DsRed-Express2, TagRFP, TagRFP-T, RRvT, cgfTagRFP, mRuby3, mNectarine, meffRFP, and amilFP593.

[0037] Preferably, fish belonging to species and varieties of fish that have commercial value, especially within the ornamental fish industry, are used. Such fish include, but are not limited to, Pristella, catfish, zebrafish, and other danio, killifish, carp, tilapia, goldfish, tetra, barb, sharks (family cyprinidae, such as rainbow sharks), angelfish, loach, koi, glassfish, discus, eels, gobies, gourami, guppies, Xiphophorus, hatchetfish, mollyfish, or pangasius. A particular fish for use in the context of this disclosure is Pristella. Pristella is an increasingly popular ornamental animal, and a variety of colors would add to its commercial value. Pristella embryos are readily available and nearly transparent. The color of Pristella's skin is determined by pigment cells within the skin, which contain pigment granules called melanosomes. The number, size, and density of melanosomes per pigment cell influence the color of fish skin.

[0038] In commercial aquaculture, Pristella spawn naturally. Briefly, one or two breeding pairs of tetras should be placed in a 2.5-5 gallon tank with an artificial spawning mat. The tank water level should be at least about 2-3 inches and maintained at 75-85°F. Low salinity (conductivity 100-200uS / cm) and slight acidity (pH about 6.9) promote spawning. Fish can be exposed to a natural or artificial light cycle, with the light period beginning at 8am and ending at 10pm. The next day, adults are removed and incubated at 75-85°F until the fry hatch and become free swimming (i.e., about 4-6 days). At this point, the fry can be safely handled and transferred outside to a grow-out tank or earthen pond. It takes about 4-6 months for Pristella to reach maturity. In a related embodiment, lineage breeding is maintained by cryopreserved sperm.

[0039] Fertilization from frozen sperm Methods for freezing fish sperm are well known in the art, see, e.g., Walker and Streisinger (1983) and Draper and Moens (2007), both of which are incorporated by reference in their entireties. Frozen tetrazoa sperm can be used to fertilize eggs to obtain the transgenic fish disclosed herein.

[0040] In an exemplary embodiment, a young adult male green Pristella is selected. It should be held with the ventral side up on its back. The ventral side should be wiped with a dry paper towel. The ventral fin can be pushed away from the anal opening. Once dry, the male's side should be gently pushed out and the released sperm should be collected and transferred to cold L-15 cell culture medium. The pipette should be pipetted in and out several times to rinse and flush the sperm. This process should be repeated with several males and, in good condition, about 10 males should be used per 50 μl of L-15. The goal is to collect at least 5-7 μl of sperm per 50 μl of L-15. After collection, the sperm should be frozen as soon as possible. They should be remixed immediately before freezing. For freezing, 30 μl of cryoprotectant solution (10% nonfat dry milk, 15% methanol, and 15% 2-methoxyethanol in L-15) should be transferred to a cryovial. Add 15 μl of sperm suspension and mix by pipetting 2-3 times. The cryovials should be closed, transferred to a 15 ml centrifuge tube and embedded vertically in dry ice. After 20 min, transfer the cryovials to liquid nitrogen. When convenient (when all cryovials have been transferred or there are long gaps between transfers), transfer the frozen cryotubes to a Dewar with liquid nitrogen for long-term storage.

[0041] In an exemplary method for recovering green Pristella strains, one or two breeding pairs of tetras should be placed in a 2.5-5 gallon tank with an artificial spawning mat. The tank water level should be at least about 2-3 inches and maintained at 75-85°F. Low salinity (conductivity 100-200 uS / cm) and slight acidity (about pH 6.9) promote spawning. The fish can be exposed to a natural or artificial light cycle, with the light period beginning at 8 am and ending at 10 pm. The following morning, ovulating females should be collected (they release eggs easily when gently pushed out). The female's abdomen should be wiped dry with paper towels. The eggs should not be exposed to water, as this will prevent fertilization. The eggs should be pushed out onto a slightly concave surface by applying light pressure with thumb and index finger on the side of the abdomen and sliding the fingers into the genital opening. Females that are ready to spawn will release the eggs quite easily and care should be taken to ensure that the eggs are not pushed out while wiping the fish. Good quality eggs are yellowish translucent and eggs that have remained longer inside the female will appear white and opaque. Females will only release eggs for an hour or so. Eggs from several females can be pooled and eggs can remain unfertilized for a few minutes. Sperm need to be thawed in a water bath at 30-33°C for 30 seconds. 70 μl of room temperature L-15 medium solution should be added to the vial and mixed. Sperm are then immediately added to the eggs and mixed gently. Sperm and eggs are activated by adding 750 μl of fish water and mixing. The mixture needs to be incubated at room temperature for 5 minutes. The eggs are then transferred to a small tank where they are further cultured using the usual protocol to obtain green Pristella progeny.

[0042] In an exemplary embodiment, a young adult male orange Pristella is selected. It should be held with the ventral side up on its back. The ventral side should be wiped with a dry paper towel. The ventral fin can be pushed away from the anal opening. Once dry, the male's side should be gently pushed out and the released sperm should be collected and transferred to cold L-15 cell culture medium. The pipette should be pipetted in and out several times to rinse and flush the sperm. This process should be repeated with several males and, in good condition, about 10 males should be used per 50 μl of L-15. The goal is to collect at least 5-7 μl of sperm per 50 μl of L-15. After collection, the sperm should be frozen as soon as possible. It should be remixed immediately before freezing. For freezing, 30 μl of cryoprotectant solution (10% nonfat dry milk, 15% methanol, and 15% 2-methoxyethanol in L-15) should be transferred to a cryovial. Add 15 μl of sperm suspension and mix by pipetting 2-3 times. The cryovials should be closed, transferred to a 15 ml centrifuge tube and embedded vertically in dry ice. After 20 min, transfer the cryovials to liquid nitrogen. When convenient (when all cryovials have been transferred or there are long gaps between transfers), transfer the frozen cryotubes to a Dewar with liquid nitrogen for long-term storage.

[0043] In an exemplary method for recovering orange Pristella strains, one or two breeding pairs of tetras should be placed in a 2.5-5 gallon tank with an artificial spawning mat. The tank water level should be at least about 2-3 inches and maintained at 75-85°F. Low salinity (conductivity 100-200 uS / cm) and slight acidity (about pH 6.9) promote spawning. The fish can be exposed to a natural or artificial light cycle, with the light period beginning at 8 am and ending at 10 pm. The following morning, ovulating females should be collected (they release eggs easily when gently pushed out). The female's abdomen should be wiped dry with paper towels. The eggs should not be exposed to water, as this will prevent fertilization. The eggs should be pushed out onto a slightly concave surface by applying light pressure with thumb and index finger on the side of the abdomen and sliding the fingers into the genital opening. Females that are ready to spawn will release the eggs quite easily and care should be taken to ensure that the eggs are not pushed out while wiping the fish. Good quality eggs are yellowish translucent and eggs that have remained longer inside the female will appear white and opaque. Females will only release eggs for an hour or so. Eggs from several females can be pooled and eggs can remain unfertilized for a few minutes. Sperm need to be thawed in a water bath at 30-33°C for 30 seconds. 70 μl of room temperature L-15 medium solution should be added to the vial and mixed. Sperm are then immediately added to the eggs and mixed gently. Sperm and eggs are activated by adding 750 μl of fish water and mixing. The mixture needs to be incubated at room temperature for 5 minutes. The eggs are then transferred to a small tank where they are further cultured using the usual protocol to obtain orange Pristella progeny.

[0044] In an exemplary embodiment, a young adult male purple Pristella is selected. It should be held with the ventral side up on its back. The ventral side should be wiped with a dry paper towel. The ventral fin can be pushed away from the anal opening. Once dry, the male's side should be gently squeezed and the released sperm should be collected and transferred to cold L-15 cell culture medium. The pipette should be pipetted in and out several times to rinse and flush the sperm. This process should be repeated with several males, and in good condition, about 10 males should be used per 50 μl of L-15. The goal is to collect at least 5-7 μl of sperm per 50 μl of L-15. After collection, the sperm should be frozen as soon as possible. It should be remixed immediately before freezing. For freezing, 30 μl of cryoprotectant solution (10% nonfat dry milk, 15% methanol, and 15% 2-methoxyethanol in L-15) should be transferred to a cryovial. Add 15 μl of sperm suspension and mix by pipetting 2-3 times. The cryovials should be closed, transferred to a 15 ml centrifuge tube and embedded vertically in dry ice. After 20 min, transfer the cryovials to liquid nitrogen. When convenient (when all cryovials have been transferred or there are long gaps between transfers), transfer the frozen cryotubes to a Dewar with liquid nitrogen for long-term storage.

[0045] In an exemplary method for recovering purple Pristella strains, one or two breeding pairs of tetras should be placed in a 2.5-5 gallon tank with an artificial spawning mat. The tank water level should be at least about 2-3 inches and maintained at 75-85°F. Low salinity (conductivity 100-200 uS / cm) and slight acidity (about pH 6.9) promote spawning. The fish can be exposed to a natural or artificial light cycle, with the light period beginning at 8 am and ending at 10 pm. The following morning, ovulating females should be collected (they release eggs easily when gently pushed out). The female's abdomen should be wiped dry with paper towels. The eggs should not be exposed to water, as this will prevent fertilization. The eggs should be pushed out onto a slightly concave surface by applying light pressure with thumb and index finger on the side of the abdomen and sliding the fingers into the genital opening. Females that are ready to spawn will release the eggs quite easily and care should be taken to ensure that the eggs are not pushed out while wiping the fish. Good quality eggs are yellowish translucent and eggs that have remained longer inside the female will appear white and opaque. Females will only release eggs for an hour or so. Eggs from several females can be pooled and eggs can remain unfertilized for a few minutes. Sperm need to be thawed in a water bath at 30-33°C for 30 seconds. 70 μl of room temperature L-15 medium solution should be added to the vial and mixed. Sperm are then immediately added to the eggs and mixed gently. Sperm and eggs are activated by adding 750 μl of fish water and mixing. The mixture needs to be incubated at room temperature for 5 minutes. The eggs are then transferred to a small tank where they are further cultured using the usual protocol to obtain purple Pristella progeny.

[0046] In an exemplary embodiment, a young adult male red Pristella is selected. It should be held with the ventral side up on its back. The ventral side should be wiped with a dry paper towel. The ventral fin can be pushed away from the anal opening. Once dry, the male's side should be gently pushed out and the released sperm should be collected and transferred to cold L-15 cell culture medium. The pipette should be pipetted in and out several times to rinse and flush the sperm. This process should be repeated with several males and, in good condition, about 10 males should be used per 50 μl of L-15. The goal is to collect at least 5-7 μl of sperm per 50 μl of L-15. After collection, the sperm should be frozen as soon as possible. It should be remixed immediately before freezing. For freezing, 30 μl of cryoprotectant solution (10% nonfat dry milk, 15% methanol, and 15% 2-methoxyethanol in L-15) should be transferred to a cryovial. Add 15 μl of sperm suspension and mix by pipetting 2-3 times. The cryovials should be closed, transferred to a 15 ml centrifuge tube and embedded vertically in dry ice. After 20 min, transfer the cryovials to liquid nitrogen. When convenient (when all cryovials have been transferred or there are long gaps between transfers), transfer the frozen cryotubes to a Dewar with liquid nitrogen for long-term storage.

[0047] In an exemplary method for recovering red Pristella strains, one or two breeding pairs of tetras should be placed in a 2.5-5 gallon tank with an artificial spawning mat. The tank water level should be at least about 2-3 inches and maintained at 75-85°F. Low salinity (conductivity 100-200 uS / cm) and slight acidity (about pH 6.9) promote spawning. The fish can be exposed to a natural or artificial light cycle, with the light period beginning at 8 am and ending at 10 pm. The following morning, ovulating females should be collected (they release eggs easily when gently pushed out). The abdomen of the female should be wiped dry with paper towels. The eggs should not be exposed to water, as this will prevent fertilization. The eggs should be pushed out onto a slightly concave surface by applying light pressure with thumb and index finger on the side of the abdomen and sliding the fingers into the genital opening. Females that are ready to spawn will release the eggs quite easily and care should be taken to ensure that the eggs are not pushed out while wiping the fish. Good quality eggs are yellowish translucent and eggs that have remained longer inside the female will appear white and opaque. Females will only release eggs for an hour or so. Eggs from several females can be pooled and eggs can remain unfertilized for a few minutes. Sperm need to be thawed in a water bath at 30-33°C for 30 seconds. 70 μl of room temperature L-15 medium solution should be added to the vial and mixed. Sperm are then immediately added to the eggs and mixed gently. Sperm and eggs are activated by adding 750 μl of fish water and mixing. The mixture needs to be incubated at room temperature for 5 minutes. The eggs are then transferred to a small tank where they are further cultured using the usual protocol to obtain red Pristella progeny.

[0048] Parichy and Johnson, 2001, which is incorporated by reference in its entirety, provides further examples regarding in vitro fertilization.

[0049] The present disclosure further encompasses transgenic fish containing the Green Pristella 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.

[0050] The simplest way to identify fish that contain the Green Pristella 1 transformation event is by visual inspection, as the fish in question are green in color and immediately distinguishable from non-transgenic fish.

[0051] The present disclosure further encompasses transgenic fish containing the Orange Pristella 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 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 lineages.

[0052] The simplest way to identify fish containing the Orange Pristella 1 transformation event is by visual inspection, as the fish in question are orange in color and immediately distinguishable from non-transgenic fish.

[0053] The present disclosure further encompasses transgenic fish containing the Purple Pristella 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.

[0054] The simplest way to identify fish that contain the Purple Pristella 1 transformation event is by visual inspection, as the fish in question are purple in color and immediately distinguishable from non-transgenic fish.

[0055] The present disclosure further encompasses transgenic fish containing the Red Pristella 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 lineages.

[0056] The simplest way to identify fish containing the Red Pristella 1 transformation event is by visual inspection, since 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 Pristella may have a color that is maintained throughout the transgenic red Pristella's life 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 Pristella may have a color that fades throughout the transgenic red Pristella's life. For example, a red transgenic Pristella may change color from red to light red, or from red to pink. Furthermore, depending on the insertion location of the particular RFP and / or expression cassette used, the transgenic red Pristella may have a color that fades over generations. For example, red transgenic Pristella may change color from one generation to the next such that the older generation may appear red, while the younger generation may appear pale red or pink. EXAMPLES

[0057] 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.

[0058] Example 1 - Green Transgenic Pristella Transgenic fish are provided that exhibit a green color. The particular transgenic event embodied in these fish is designated as the "Green Pristella 1 transformation event." Sperm from these fish can be used to fertilize tetra eggs, thereby breeding transgenic tetras that contain these particular transgenic integration events. Sperm from this line has been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Green Pristella 1" (the deposit was designated as Accession Number (PTA-127162)).

[0059] 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.

[0060] Example 2 - Orange Transgenic Pristella Transgenic fish are provided that exhibit an orange color. The particular transgenic event embodied in these fish is designated the "Orange Pristella 1 transformation event." Sperm from these fish can be used to fertilize tetra eggs, thereby breeding transgenic tetras that contain these particular transgenic integration events. Sperm from this line has been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Orange Pristella 1" (the deposit was designated accession number (PTA-127164)).

[0061] 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.

[0062] Example 3 - Purple Transgenic Pristella Transgenic fish are provided that exhibit a purple color. The particular transgenic event embodied in these fish is designated as the "Purple Pristella 1 transformation event." Sperm from these fish can be used to fertilize tetra eggs, thereby breeding transgenic tetras that contain these particular transgenic integration events. Sperm from this line has been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Purple Pristella 1" (the deposit was designated as Accession Number (PTA-127163)).

[0063] 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 purple color of the transgenic fish.

[0064] Example 4 - Red transgenic Pristella Transgenic fish are provided that exhibit a red color. The particular transgenic event embodied in these fish is designated as the "Red Pristella 1 transformation event." Sperm from these fish can be used to fertilize tetra eggs, thereby breeding transgenic tetras that contain these particular transgenic integration events. Sperm from this line has been deposited under the terms of the Budapest Treaty with the ATCC, Historic District, 10801 University Blvd, Manassas, VA 20110, as "Red Pristella 1" (the deposit was designated as Accession Number (PTA-127165)).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. A transgenic Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising a "Green Pristella 1 transformation event."

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

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

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

5. 1. A method for producing transgenic Pristella, comprising: (a) obtaining a Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising a "Green Pristella 1 transformation event"; (b) breeding the resulting Pristella with a second Pristella to provide a transgenic Pristella comprising the Green Pristella 1 transformation event.

6. 6. The method of claim 5, wherein the second Pristella is a non-transgenic Pristella.

7. 10. The progeny of the transgenic Pristella of claim 1, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said Pristella and progeny exhibit fluorescence and comprise said "Green Pristella 1 transformation event."

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

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

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

11. 1. A method for producing transgenic fish, comprising: (a) obtaining the transgenic fish of claim 1; (b) breeding the resulting fish with a second fish to provide a transgenic fish comprising the Green Pristella 1 transformation event.

12. The method of claim 11 , wherein the second fish is a non-transgenic fish.

13. A transgenic Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising an "Orange Pristella 1 transformation event."

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

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

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

17. 1. A method for producing transgenic Pristella, comprising: (a) obtaining a Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising an "Orange Pristella 1 transformation event"; (b) breeding the resulting Pristella with a second Pristella to provide a transgenic Pristella comprising the Orange Pristella 1 transformation event.

18. 18. The method of claim 17, wherein the second Pristella is a non-transgenic Pristella.

19. 14. Progeny of the transgenic Pristella of claim 13, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said Pristella and progeny exhibit fluorescence and comprise said "Orange Pristella 1 transformation event."

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

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

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

23. 1. A method for producing transgenic fish, comprising: (a) obtaining a transgenic fish according to claim 13; (b) breeding the resulting fish with a second fish to provide a transgenic fish comprising the Orange Pristella 1 transformation event.

24. 24. The method of claim 23, wherein the second fish is a non-transgenic fish.

25. A transgenic Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising a "Purple Pristella 1 transformation event."

26. 26. The transgenic Pristella of claim 25, further defined as a fertile transgenic Pristella.

27. 26. The transgenic Pristella of claim 25, wherein the fish is homozygous for the integrated expression cassette.

28. 26. The transgenic Pristella of claim 25, wherein the fish is heterozygous for the integrated expression cassette.

29. 1. A method for producing transgenic Pristella, comprising: (a) obtaining a Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising a "Purple Pristella 1 transformation event"; (b) breeding the resulting Pristella with a second Pristella to provide a transgenic Pristella comprising the Purple Pristella 1 transformation event.

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

31. 26. Progeny of the transgenic Pristella of claim 25, comprising a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said Pristella and progeny exhibit fluorescence and comprise said "Purple Pristella 1 transformation event."

32. 32. The progeny fish of claim 31, further defined as a fertile transgenic Pristella.

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. 1. A method for producing transgenic fish, comprising: (a) obtaining the transgenic fish of claim 25; (b) breeding the resulting fish with a second fish to provide a transgenic fish comprising the Purple Pristella 1 transformation event.

36. 36. The method of claim 35, wherein the second fish is a non-transgenic fish.

37. A transgenic Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising a "Red Pristella 1 transformation event."

38. 38. The transgenic Pristella of claim 37, further defined as a fertile transgenic Pristella.

39. 38. The transgenic Pristella of claim 37, wherein the fish is homozygous for the integrated expression cassette.

40. 38. The transgenic Pristella of claim 37, wherein the fish is heterozygous for the integrated expression cassette.

41. 1. A method for producing transgenic Pristella, comprising: (a) obtaining a Pristella comprising a chromosomally integrated expression cassette encoding a fluorescent protein, said Pristella comprising a "Red Pristella 1 transformation event"; (b) breeding the resulting Pristella with a second Pristella to provide a transgenic Pristella comprising the Red Pristella 1 transformation event.

42. 42. The method of claim 41, wherein the second Pristella is a non-transgenic Pristella.

43. 38. Progeny of the transgenic Pristella of claim 37, which comprises a chromosomally integrated expression cassette encoding a fluorescent protein, wherein said Pristella and progeny exhibit fluorescence and comprise said "Red Pristella 1 transformation event."

44. 44. The progeny fish of claim 43, further defined as a fertile transgenic Pristella.

45. 44. The progeny fish of claim 43, wherein the fish is homozygous for the integrated expression cassette.

46. 44. The progeny fish of claim 43, wherein the fish is heterozygous for the integrated expression cassette.

47. 1. A method for producing transgenic fish, comprising: (a) obtaining the transgenic fish of claim 37; (b) breeding the resulting fish with a second fish to provide a transgenic fish comprising the Red Pristella 1 transformation event.

48. 48. The method of claim 47, wherein the second fish is a non-transgenic fish.

49. 8. The transgenic Pristella of any one of claims 1, 5, or 7, wherein cryopreserved sperm containing the Green Pristella 1 transformation event has been deposited with the ATCC under accession number (PTA-127162).

50. 20. The transgenic Pristella of any one of claims 13, 17, or 19, wherein cryopreserved sperm containing the Orange Pristella 1 transformation event has been deposited with the ATCC under accession number (PTA-127164).

51. 32. The transgenic Pristella of any one of claims 25, 29, or 31, wherein cryopreserved sperm containing the Purple Pristella 1 transformation event has been deposited with the ATCC under accession number (PTA-127163).

52. 44. The transgenic Pristella of any one of claims 37, 41, and 43, wherein cryopreserved sperm containing the Red Pristella 1 transformation event has been deposited with the ATCC under accession number (PTA-127165).