TRANSGENIC RAINBOW SHARK

Abstract

The present invention relates to transgenic ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.

Claims

1.-80. (canceled)

81. A cell line for a transgenic rainbow shark comprising: a) an exogenous nucleic sequence encoding an orange fluorescent protein, wherein the cell line is deposited at the European Collection of Authenticated Cell Cultures (ECACC) as Accession Number 19010802; b) an exogenous nucleic sequence encoding a purple fluorescent protein, wherein the cell line is deposited at the ECACC as Accession Number 19010801; c) an exogenous nucleic sequence encoding a blue fluorescent protein, wherein the cell line is deposited at the ECACC as Accession Number 19010803; or d) an exogenous nucleic sequence encoding a green fluorescent protein, wherein the cell line is deposited at the ECACC as Accession Number 19010804, wherein the cell line upon fertilizing an egg of a rainbow shark produces a transgenic rainbow shark whose genome comprises a nucleic acid sequence encoding an orange, purple, blue, or green fluorescent protein.

82. The cell line of claim 81, wherein the transgenic rainbow shark is a fertile, transgenic rainbow shark.

83. The cell line of claim 81, wherein the transgenic rainbow shark is homozygous for the integrated expression cassette.

84. The cell line of claim 81, wherein the transgenic rainbow shark is heterozygous for the integrated expression cassette.

85. The method of claim 81, wherein the transgenic rainbow shark functionally expresses the orange, purple, blue, or green fluorescent protein.

86. A method of providing a transgenic rainbow shark to the ornamental fish market, comprising obtaining a transgenic rainbow shark obtained from a cell line in accordance with claim 81, and distributing the transgenic rainbow shark to the ornamental fish market.

87. The method of claim 86, wherein the transgenic rainbow shark are distributed by a grower to a commercial distributor.

88. The method of claim 86, wherein the transgenic rainbow shark are distributed by a grower or a commercial distributor to a retailer.

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

90. A method of producing a cell line comprising: (a) obtaining a first rainbow shark that comprises a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the rainbow shark comprises a transformation event selected from the group consisting of: the Orange rainbow shark 1 transformation event, sperm comprising the Orange rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010802); or the Purple rainbow shark 1 transformation event, sperm comprising the Purple rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010801); or the Blue rainbow 1 transformation event, sperm comprising the Blue rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010803); or the Green rainbow shark 1 transformation event, sperm comprising the Green rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010804), (b) breeding the first rainbow shark with a second rainbow shark to provide a third rainbow shark, wherein the third rainbow shark comprises the same transformation event of the first rainbow shark (c) obtaining sperm from the third rainbow shark, wherein the sperm genome comprises a nucleic acid sequence encoding an orange, purple, blue, or green fluorescent protein.

91. The method of claim 89, wherein the second rainbow shark is a non-transgenic rainbow shark.

92. A progeny transgenic rainbow shark obtained from the cell line of claim 81 that comprises a chromosomally integrated expression cassette encoding a fluorescent protein, wherein the rainbow shark and progeny therefrom exhibit fluorescence and comprise a transformation event selected from the group consisting of: the Orange rainbow shark 1 transformation event, sperm comprising the Orange rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010802); or the Purple rainbow shark 1 transformation event, sperm comprising the Purple rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010801); or the Blue rainbow 1 transformation event, sperm comprising the Blue rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010803); or the Green rainbow shark 1 transformation event, sperm comprising the Green rainbow shark 1 transformation event being deposited at the ECACC (Accession Number 19010804).

93. The progeny fish of claim 91, wherein the progeny is a fertile, transgenic rainbow shark.

94. The progeny fish of claim 91, wherein the progeny is homozygous for the integrated expression cassette.

95. The progeny fish of claim 91, wherein the progeny is heterozygous for the integrated expression cassette.

96. A method of providing a transgenic fish to the ornamental fish market, comprising obtaining a progeny in accordance with claim 91, and distributing the progeny to the ornamental fish market.

97. The method of claim 95, wherein the transgenic fish are distributed by a grower to a commercial distributor.

98. The method of claim 96, wherein the transgenic fish are distributed by a grower or a commercial distributor to a retailer.

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

100. A method of producing a transgenic fish comprising: (a) obtaining a first transgenic fish in accordance with claim 91; and (b) breeding the first transgenic fish with a second fish to provide a third fish, wherein the third fish comprises the same transformation event of the first transgenic fish.

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

Description

DETAILED DESCRIPTION

Transgenic Fish

[0031] In some aspects, the present disclosure regards transgenic fish. Methods of making transgenic fish are described in, for example, U.S. Pat. Nos. 7,135,613; 7,700,825; 7,834,239, each of which is incorporated by reference in its entirety. For example, a transgenic orange rainbow shark may be generated using an expression cassette encoding yellow fluorescent protein (YFP), such as TurboYFP an enhanced variant of the yellow fluorescent protein PhiYFP from jellyfish Phialidium sp. (Shagin et al., 2004). In other examples, a transgenic purple rainbow shark may be generated using an expression cassette encoding purple fluorescent protein (PFP). In other examples, a transgenic blue rainbow shark may be generated using an expression cassette encoding blue fluorescent protein (BFP), such as TagBFP, or TagBFP in combination with Non-fluorescent blue chromoprotein. In other examples, a transgenic green rainbow shark may be generated using an expression cassette encoding green fluorescent protein (GFP), such as zsGreen1.

[0032] It is preferred that fish belonging to species and varieties of fish of commercial value, particularly commercial value within the ornamental fish industry, be used. Such fish include but are not limited to catfish, zebrafish and other danios, medaka, carp, tilapia, goldfish, tetras, barbs, sharks (family cyprinidae, such as rainbow shark), angelfish, loach, koi, glassfish, discus, eel, goby, gourami, guppy, Xiphophorus, hatchet fish, Molly fish, or pangasius. A particular fish for use in the context of the present disclosure is a rainbow shark, Epalzeorhynchos frenatum. Rainbow shark are increasingly popular ornamental animals and would be of added commercial value in various colors. Rainbow shark embryos are easily accessible and nearly transparent. Rainbow shark skin color is determined by pigment cells in the skin, which contain pigment granules called melanosomes. The number, size, and density of the melanosomes per pigment cell influence the color of the fish skin.

In Vitro Fertilization

[0033] In commercial aquaculture, rainbow sharks, including orange, purple, blue and green rainbow sharks, are not spawned naturally, but are spawned in vitro using the same, long-standing, industry-standard process that has been used for the reproduction of their non-fluorescent counterparts over the last several decades. At the same time, rainbow sharks are an ideal candidate for hormone induction as they are large enough to be easily handled and fecund, with females producing a substantial number of eggs per spawning. Generally speaking, rainbow sharks are seasonal breeders and can be most easily spawned from approximately May to October. It takes roughly one year for rainbow sharks to reach sexual maturity. One year-old females can release up to one thousand eggs per spawn, while older females can release up to 10,000 eggs per spawn if well-conditioned for breeding. Females can be spawned one or two times per season. Males can be spawned as frequently as two or three times per month. The most commonly used hormone for the induction of ovulation, which has been approved for use with ornamental fish by FDA, is called Ovaprim. It contains a salmonid gonadotropin-releasing hormone analog and a dopamine antagonist. Ovaprim produces consistent results, is easy to dose, and it is widely commercially available. Industry-standard breeding information for this species (for both the fluorescent and non-fluorescent phenotype) follows below, however, exact details may vary slightly from one producer to another.

[0034] Shark brood stock may be kept in indoor tanks, preferably with a volume of at least 300 gallons, but are typically conditioned outdoors in earthen ponds. Once conditioned for breeding, rainbow sharks are moved from outdoor, earthen ponds into large indoor holding vats. For breeding, conditioned brood stock are removed from the holding vat and placed in water containing a sedative, such as Tricaine, which is FDA approved for this type of use. Fish are considered to be adequately sedated when they roll over. At this time, the fish in question is removed from the water containing the sedative, weighed, and injected with Ovaprim. Males may also receive an Ovaprim injection to induce spermiation. Females will generally begin ovulation about seven hours after injection; once ovulation begins, eggs will flow freely when the fish are gently squeezed. To prepare for in vitro fertilization, the eggs should then be stripped into a dry bowl. A few drops of milt from male rainbow sharks should be similarly stripped into the same bowl. Stirring eggs and sperm together with 0.5 mL (approximately 10 drops) of water will begin the fertilization process. After 20 seconds, another 2 mL of water should be added. This process will cause the eggs to be fertilized within approximately 30 seconds. Once fertilized, eggs can be placed in McDonald-type egg hatching jars. The eggs will swell and become buoyant over the first 30 minutes, so the flow on the hatching jars should be adjusted to be as low as possible initially to avoid loss of the eggs. The flow should be checked frequently and adjusted as needed. Fry should be fed newly hatched Artemia nauplii on day two post-hatch, and they should continue to be fed Artemia for one week. From that point, they can begin the transition to a prepared diet, overlapping with live feed for 5 days. Two weeks post-hatch, the fry can be moved to vats and/or ponds for continued growth through maturity.

Fertilization from Frozen Sperm

[0035] Fish sperm freezing methods are well-known in the art; see, e.g., Walker and Streisinger (1983) and Draper and Moens (2007), both of which are incorporated herein by reference in their entireties. To obtain the transgenic fish disclosed herein, frozen rainbow shark sperm may be used to fertilize eggs.

[0036] In at least one method, conditioned females are removed from the holding vat and placed in water containing a sedative, such as Tricaine, which is FDA approved for this type of use. Fish are considered to be adequately sedated when they roll over. Once sedated, the subject fish is removed from the water containing the sedative, weighed, and injected with Ovaprim. Injected females will generally begin ovulation about seven hours after injection; once ovulation begins, eggs will flow freely when the fish are gently squeezed. To prepare for in vitro fertilization, the eggs should then be stripped into a dry bowl. Eggs from several females may be pooled; the eggs can be kept unfertilized for several minutes. Frozen sperm is thawed at 33? C. in a water bath for 18-20 seconds. Once the sperm is thawed 70 ?L room temperature Hanks solution is added to the vial and mixed. The sperm is then immediately added to the eggs and gently mixed. Stirring eggs and sperm together with 0.5 mL (approximately 10 drops) of water will begin the fertilization process. After 20 seconds, another 2 mL of water should be added. This process will cause the eggs to be fertilized within approximately 30 seconds. Once fertilized, eggs can be placed in McDonald-type egg hatching jars. The eggs will swell and become buoyant over the first 30 minutes, so the flow on the hatching jars should be adjusted to be as low as possible initially to avoid loss of the eggs. The flow should be checked frequently and adjusted as needed. Fry should be fed newly hatched Artemia nauplii on day two post-hatch, and they should continue to be fed Artemia for one week. After the first week post hatch, fry can begin the transition to a prepared diet, overlapping with live feed for 5 days. Two weeks post-hatch, the fry can be moved to vats and/or ponds for continued growth through maturity. Parichy and Johnson, 2001, which is incorporated by reference in its entirety, provides additional examples regarding in vitro fertilization.

[0037] The present disclosure further encompasses progeny of a transgenic fish containing the Orange rainbow shark 1 transformation event, as well as such transgenic fish derived from a transgenic fish egg, sperm cell, embryo, or other cell containing a genomically integrated transgenic construct. Progeny, as the term is used herein, can result from breeding two transgenic fish of the invention, or from breeding a first transgenic fish of the invention to a second fish that is not a transgenic fish of the invention. In the latter case, the second fish can, for example, be a wild-type fish, a specialized strain of fish, a mutant fish, or another transgenic fish. The second fish may be of the same species, or may be of a different species or genus. The hybrid progeny of these matings have the benefits of the transgene for fluorescence combined with the benefits derived from these other lineages.

[0038] The simplest way to identify fish containing the Orange rainbow shark 1 transformation event is by visual inspection, as the fish in question would be orange colored and immediately distinguishable from non-transgenic fish.

[0039] The present disclosure further encompasses progeny of a transgenic fish containing the Purple rainbow shark 1 transformation event, as well as such transgenic fish derived from a transgenic fish egg, sperm cell, embryo, or other cell containing a genomically integrated transgenic construct. Progeny, as the term is used herein, can result from breeding two transgenic fish of the invention, or from breeding a first transgenic fish of the invention to a second fish that is not a transgenic fish of the invention. In the latter case, the second fish can, for example, be a wild-type fish, a specialized strain of fish, a mutant fish, or another transgenic fish. The second fish may be of the same species, or may be of a different species or genus. The hybrid progeny of these matings have the benefits of the transgene for fluorescence combined with the benefits derived from these other lineages.

[0040] The simplest way to identify fish containing the Purple rainbow shark 1 transformation event is by visual inspection, as the fish in question would be purple colored and immediately distinguishable from non-transgenic fish.

[0041] The present disclosure further encompasses progeny of a transgenic fish containing the Blue rainbow shark 1 transformation event, as well as such transgenic fish derived from a transgenic fish egg, sperm cell, embryo, or other cell containing a genomically integrated transgenic construct. Progeny, as the term is used herein, can result from breeding two transgenic fish of the invention, or from breeding a first transgenic fish of the invention to a second fish that is not a transgenic fish of the invention. In the latter case, the second fish can, for example, be a wild-type fish, a specialized strain of fish, a mutant fish, or another transgenic fish. The second fish may be of the same species, or may be of a different species or genus. The hybrid progeny of these matings have the benefits of the transgene for fluorescence combined with the benefits derived from these other lineages.

[0042] The simplest way to identify fish containing the Blue rainbow shark 1 transformation event is by visual inspection, as the fish in question would be blue colored and immediately distinguishable from non-transgenic fish.

[0043] The present disclosure further encompasses progeny of a transgenic fish containing the Green rainbow shark 1 transformation event, as well as such transgenic fish derived from a transgenic fish egg, sperm cell, embryo, or other cell containing a genomically integrated transgenic construct. Progeny, as the term is used herein, can result from breeding two transgenic fish of the invention, or from breeding a first transgenic fish of the invention to a second fish that is not a transgenic fish of the invention. In the latter case, the second fish can, for example, be a wild-type fish, a specialized strain of fish, a mutant fish, or another transgenic fish. The second fish may be of the same species, or may be of a different species or genus. The hybrid progeny of these matings have the benefits of the transgene for fluorescence combined with the benefits derived from these other lineages.

[0044] The simplest way to identify fish containing the Green rainbow shark 1 transformation event is by visual inspection, as the fish in question would be green colored and immediately distinguishable from non-transgenic fish.

Examples

[0045] Certain embodiments of the invention are further described with reference to the following examples. These examples are intended to be merely illustrative of the invention and are not intended to limit or restrict the scope of the present invention in any way and should not be construed as providing conditions, parameters, reagents, or starting materials that must be utilized exclusively in order to practice the art of the present invention.

Example 1-Orange Transgenic Rainbow Shark

[0046] Transgenic fish exhibiting an orange color are provided. The specific transgenic events embodied in these fish are designated the Orange rainbow shark 1 transformation event. Sperm from these fish may be used to fertilize rainbow shark eggs and thereby breed transgenic rainbow shark that comprise these specific transgenic integration events. Sperm from this line was deposited at the European Collection of Cell Cultures (ECACC (Accession Number 19010802)), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, under the provisions of the Budapest Treaty as Orange rainbow shark 1.

Example 2-Purple Transgenic Rainbow Shark

[0047] Transgenic fish exhibiting a purple color are provided. The specific transgenic events embodied in these fish are designated the Purple rainbow shark 1 transformation event. Sperm from these fish may be used to fertilize rainbow shark eggs and thereby breed transgenic rainbow shark that comprise these specific transgenic integration events. Sperm from this line was deposited at the European Collection of Cell Cultures (ECACC (Accession Number 19010801)), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, under the provisions of the Budapest Treaty as Purple rainbow shark 1.

Example 3-Blue Transgenic Rainbow Shark

[0048] Transgenic fish exhibiting a blue color are provided. The specific transgenic events embodied in these fish are designated the Blue rainbow shark 1 transformation event. Sperm from these fish may be used to fertilize rainbow shark eggs and thereby breed transgenic rainbow shark that comprise these specific transgenic integration events. Sperm from this line was deposited at the European Collection of Cell Cultures (ECACC (Accession Number 19010803)), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, under the provisions of the Budapest Treaty as Blue rainbow shark 1.

Example 4-Green Transgenic Rainbow Shark

[0049] Transgenic fish exhibiting a green color are provided. The specific transgenic events embodied in these fish are designated the Green rainbow shark 1 transformation event. Sperm from these fish may be used to fertilize rainbow shark eggs and thereby breed transgenic rainbow shark that comprise these specific transgenic integration events. Sperm from this line was deposited at the European Collection of Cell Cultures (ECACC (Accession Number 19010804)), Public Health England, CRYOSTORES, Bld. 17, Porton Down, Salisbury, SP4 OJG, United Kingdom, under the provisions of the Budapest Treaty as Green rainbow shark 1.

[0050] The fluorescent transgenic fish have use as ornamental fish in the market. Stably expressing transgenic lines can be developed by breeding a transgenic individual with a wild-type fish, mutant fish, or another transgenic fish. The desired transgenic fish can be distinguished from non-transgenic fish by observing the fish in white light, sunlight, ultraviolet light, blue light, or any other useful lighting condition that allows visualization of the orange, purple, green or blue color of the transgenic fish.

[0051] The fluorescent transgenic fish should also be valuable in the market for scientific research tools because they can be used for embryonic studies such as tracing cell lineage and cell migration. Additionally, these fish can be used to mark cells in genetic mosaic experiments and in fish cancer models.

[0052] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the 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.