WO2014145196A2 - Transgenic animals with customizable traits - Google Patents

Transgenic animals with customizable traits Download PDF

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Publication number
WO2014145196A2
WO2014145196A2 PCT/US2014/029918 US2014029918W WO2014145196A2 WO 2014145196 A2 WO2014145196 A2 WO 2014145196A2 US 2014029918 W US2014029918 W US 2014029918W WO 2014145196 A2 WO2014145196 A2 WO 2014145196A2
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WO
WIPO (PCT)
Prior art keywords
promoter
nucleic acid
animal
keratin
protein
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PCT/US2014/029918
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English (en)
French (fr)
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WO2014145196A3 (en
Inventor
James West
Original Assignee
Mice With Horns, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from US13/837,405 external-priority patent/US20130212723A1/en
Priority to US14/777,108 priority Critical patent/US20160044901A1/en
Priority to CA2906936A priority patent/CA2906936A1/en
Priority to BR112015023414A priority patent/BR112015023414A2/pt
Priority to CN201480024934.7A priority patent/CN105357960B/zh
Priority to AU2014233317A priority patent/AU2014233317B2/en
Application filed by Mice With Horns, Llc filed Critical Mice With Horns, Llc
Priority to EP14763711.0A priority patent/EP2967017A4/en
Priority to JP2016503281A priority patent/JP2016512697A/ja
Priority to MX2015013253A priority patent/MX2015013253A/es
Publication of WO2014145196A2 publication Critical patent/WO2014145196A2/en
Publication of WO2014145196A3 publication Critical patent/WO2014145196A3/en
Priority to HK16102648.4A priority patent/HK1214732A1/zh
Priority to AU2017213487A priority patent/AU2017213487B2/en
Priority to AU2019204084A priority patent/AU2019204084A1/en

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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027New or modified breeds of vertebrates
    • A01K67/0275Genetically modified vertebrates, e.g. transgenic
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/101Bovine
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/02Animal zootechnically ameliorated

Definitions

  • This invention relates to genetic modification of animals in order to effect controlled changes in specified traits such as, for example, changes in skin and/or fur pigmentation.
  • melanin The only pigment synthesized in most animal species, and the only pigment at all in mammalian species, is melanin.
  • melanin There are two classes of melanin: pheomelanin, which produces a blond or red color, and eumelanin, which produces a dark brown or black color. Both classes of melanin are synthesized from tyrosine, but their synthetic pathways diverge after production of dopaquinone.
  • MCIR melanocortin receptor
  • the melanocortin receptor can be activated by any of the melanocyte-stimulating hormones (MSH), most commonly by a-MSH, but also by ⁇ -defensin 103. Conversely, activation of MCIR can be inhibited through expression of the agouti signaling protein (ASP).
  • ASP agouti signaling protein
  • the ⁇ -defensin 103 signal is dominant over the ASP signal and the ASP signal is dominant over the MSH signal.
  • ASP agouti signaling protein
  • Plants make multiple additional classes of pigments, including chlorophyll, carotenoids, anthrocyanins, and betalains. Of these pigments, carotenoids can be most easily transferred to animals because production of carotenoid dyes in plants relies on a precursor that can also be found in animals.
  • Geranylgeranyl pyrophosphate is an intermediate in the HMG-CoA reductase pathway, and is used as a precursor for synthesizing steroids and sterols. With the addition of 4-5 plant proteins, geranylgeranyl pyrophosphate can instead be used as a precursor for synthesizing carotene yellow and orange, or torulene red.
  • the transfer of plant pigments to animals has occurred in nature: the aphid A. pisum has several genes somehow transferred from fungi. Carotenoids have also been produced in genetically-engineered yeast, which belong to the animal kingdom.
  • the subject invention provides materials and methods for creating customizable traits in animals.
  • the subject invention provides materials and methods for creating customizable patterns in the skin and/or fur of animals.
  • the customizable trait can involve the length and/or texture of animal skin or fur.
  • the subject invention can be used to effect controlled changes in the texture, structural strength, and/or length of animal nail, claw, and/or horn.
  • the methods of the subject invention comprise indtroducing into the cells of an animal a genetic construct comprising a keratin- 14 specific promoter, red fluorescent protein in a loxp cassette, dominant black (AG23) beta defensin 103 in a pigment cassette, and an SV40 (with intron) polyadenylation sequence.
  • a composition comprising Cre recombinase (or HTNCre) is then applied to the skin of an animal having the genetic construct, the fur of the animal will turn black where the composition was applied. In this way, the fur is permanently genetically modified to turn color in a desired shape.
  • the subject invention provides a method of creating customizable permanent patterns in the skin and/or fur of animals.
  • the subject invention provides methods for producing multicolor patterns in the skin and/or fur of animal species.
  • the subject invention provides a method of creating customizable patterns in the skin and/or fur of animal species such that the animal would continue to grow fur to sustain those colors throughout its lifetime.
  • the invention also provides methods of creating customizable predefined patterns of stripes that are heritable within that animal.
  • the present invention provides transdermal application of one or more activating factors to drive recombination and permanent transgene expression in the transgenic animals of the present invention.
  • the activating factors useful according to the present invention include, but are not limited to, recombinase proteins, small molecules (such as, doxycycline, cumate, ecdysone, etc) capable of inducing the expression of recombinase, viruses that capable of inducing the expression of recombinase, and nucleic acid (such as DNA) constructs that drive the expression of recombinase.
  • the activating factor is applied to the surface of the animal skin, either alone or in a carrier solution (e.g., liposomes, solvents, mixtures containing DMSO, etc.). In one embodiment, the activating factor is applied intradermally (such as with the use of a tattoo needle) or subdermally.
  • a carrier solution e.g., liposomes, solvents, mixtures containing DMSO, etc.
  • the activating factor is applied intradermally (such as with the use of a tattoo needle) or subdermally.
  • the transgenic animal comprises one or more exogenous nucleic acid molecules including, but not limited to, pigmentation-related genes, coat/hair quality genes (such as genes for controlling the length and/or curliness of animal hair), genes related to nail/claw or horn quality (such as a nucleic acid molecule encoding cross-linking keratin), and genes for synthesis and/or expression of plant pigments in animal cells.
  • exogenous nucleic acid molecules including, but not limited to, pigmentation-related genes, coat/hair quality genes (such as genes for controlling the length and/or curliness of animal hair), genes related to nail/claw or horn quality (such as a nucleic acid molecule encoding cross-linking keratin), and genes for synthesis and/or expression of plant pigments in animal cells.
  • promoters useful according to the present invention include, but are not limited to, skin-specific promoters (e.g., keratin specific promoter), melanocyte specific promoters (e.g., MCR promoter), constitutive promoters (e.g., beta-globin promoter, CMV promoter), and promoters responsive to circulating factors such as NF-kB, interferon gamma, estrogen, and glucocorticoids.
  • skin-specific promoters e.g., keratin specific promoter
  • melanocyte specific promoters e.g., MCR promoter
  • constitutive promoters e.g., beta-globin promoter, CMV promoter
  • promoters responsive to circulating factors such as NF-kB, interferon gamma, estrogen, and glucocorticoids.
  • the present invention provides the use of multiple types of recombinase targets to allow specific activation of different genes selectively through application of different recombinases.
  • multiple recombinase targets are used to allow multiple colors to be created after birth of the animal.
  • Cre recombinase activates the production of dog fur with black color
  • Flp recombinase activates the production of dog fur with red color
  • the present invention provides the use of native promoters to drive coat pigmentation without the need for an external activating factor.
  • the native promoter relates to defining somite boundaries in animal development.
  • the native heterologous promoter is used to create coat patterns in a different species, such as, for example, using the Tabby or Ticked promoters found in cats to drive coat coloration in dogs.
  • the present invention provides genetically modified animals with coat patterns that are permanently customizable after birth.
  • the present invention provides genetically modified animals with coat colors not normally found in mammals.
  • the present invention provides genetically modified cattle, sheep or other animals that have permanent identification marks (such as, a number or a bar code) growing in their coat.
  • the present invention provides genetically modified cattle, sheep or other animals that have "invisible” marks in their coat that can change color in response to changes in health or physiological conditions.
  • the transgenic cattle or sheep or other animals can express one symbol on the coat or fur, wherein that symbol can change color if the animal has chronic activation of NF-kB, and another symbol that can change color if the animal has chronic activation of interferon-gamma.
  • the present invention provides genetically modified animals born with coat colors and patterns not normally found in their native species.
  • the present invention provides a transgenic bovine animal of the black Angus breed with white or near-white coat or fur.
  • Figure 1 is a schematic depiction of an embodiment of a genetic construct for effecting color change in animals.
  • Figure 2 is a schematic depiction of an embodiment of a genetic construct for creating customizable patterns and color in animal skin or fur.
  • Figure 3 is a schematic depiction of an embodiment of genetic constructs for creating customizable patterns and color in animal skin or fur.
  • FIG 4 is a schematic depiction of one embodiment of a genetic construct for creating customizable patterns and color in the skin or fur of mice.
  • the construct comprises a Keratinl4 promoter, which is expressed in all skin fibroblasts, to drive the dominant black form of signaling molecule P-Defl03.
  • the expression of P-Defl03 is blocked by a LoxP excisable nucleic acid encoding ring finger protein (RFP), which is used as a marker.
  • RFP LoxP excisable nucleic acid encoding ring finger protein
  • the nucleic acid molecule encoding RFP is not required for the construct, and can be replaced by STOPs.
  • Figure 5 shows that beta-Defl03 expression activated by transdermal application of recombinase creates a genetically permanent alteration in the coat or fur of an adult mouse.
  • B Fine control of marking alteration can be achieved: a mouse with a single narrow black line is created by injection of recombinase. The change in skin/fur pattern has persisted through multiple cycles of coat regrowth, indicating successful alteration of resident stem cells.
  • Figure 6 shows that coat color of the transgenic mice can be titrated through the amount of recombinase applied to the mouse skin. Increasing the amounts of recombinase applied to the mice increases the level of transgene expression and eumelanin activation.
  • FIG 7 is a schematic depiction of one embodiment of a genetic construct for knock-in cattle with spermatozoa-specific expression of enhanced green fluorescent protein (EGFP), and recombinase-mediated melanocyte-specific expression of a dominant negative Rab7.
  • EGFP enhanced green fluorescent protein
  • Figure 8 shows a depiction of a Black Angus heifer genetically engineered to express a customizable identification pattern in the skin.
  • SEQ ID NO:l is the amino acid sequence of a bifunctional enzyme CarRP-like isoform 1 [Acyrthosiphon pisum] (GenBank Accession No. XP 001943170).
  • SEQ ID NO: 2 is the amino acid sequence of a bifunctional enzyme CarRP-like [Acyrthosiphon pisum] (GenBank Accession No. XP 001950787).
  • SEQ ID NO: 3 is the amino acid sequence of a lycopene cyclase / phytoene synthase- like [Acyrthosiphon pisum] (GenBank Accession No. XP 001950868).
  • SEQ ID NO:4 is the amino acid sequence of a phytoene dehydrogenase-like [Acyrthosiphon pisum] (GenBank Accession No. XP 001943225).
  • SEQ ID NO: 5 is the amino acid sequence of a phytoene dehydrogenase-like [Acyrthosiphon pisum] (GenBank Accession No. XP 001950764).
  • SEQ ID NO:6 is the amino acid sequence of a phytoene dehydrogenase-like
  • SEQ ID NO: 7 is the amino acid sequence of a phytoene dehydrogenase-like [Acyrthosiphon pisum] (Genbank Accession No. XP 001943938).
  • SEQ ID NO: 8 is the amino acid sequence of a melanocortin 1 receptor (GenBank Accession No . EDL 11741 ) .
  • SEQ ID NO: 9 is the amino acid sequence of an alpha melanocyte stimulating hormone (MSH).
  • SEQ ID NO: 10 is the amino acid sequence of a beta melanocyte stimulating hormone
  • SEQ ID NO: 11 is the amino acid sequence of a beta melanocyte stimulating hormone
  • SEQ ID NO: 12 is the amino acid sequence of a gamma melanocyte stimulating hormone (MSH).
  • SEQ ID NO: 13 is the amino acid sequence of a ⁇ -defensin protein (GenBank Accession No. AAT67592).
  • SEQ ID NO: 14 is the amino acid sequence of an agouti signaling protein precursor (GenBank Accession No. NP_056585).
  • SEQ ID NO: 15 is the amino acid sequence of a tyrosinase (TYR) (GenBank Accession No. BAA00341).
  • SEQ ID NO: 16 is the amino acid sequence of a melanocyte-specific transporter protein (GenBank Accession No. Q62052).
  • SEQ ID NO: 17 is the amino acid sequence of a rab protein geranylgeranyltransferase component A2 (GenBank Accession No. NP 067325).
  • SEQ ID NO:18 is the amino acid sequence of a ras-related protein Rab-7a (GenBank Accession No. NP_033031).
  • SEQ ID NO: 19 is the amino acid sequence of a probable E3 ubiquitin-protein ligase (HERC2) (GenBank Accession No. NP 084390).
  • SEQ ID NO: 20 is the amino acid sequence of a Pmell7 protein [Gallus gallus] (GenBank Accession No. AAT58250).
  • SEQ ID NO:21 is the amino acid sequence of a Pmell7 protein [Gallus gallus] (GenBank Accession No. AAT58246).
  • SEQ ID NO: 22 is the amino acid sequence of a Pmell7 protein [Gallus gallus] (GenBank Accession No. AAT58249).
  • the subject invention provides materials and methods for creating customizable traits in animals.
  • the subject invention provides materials and methods for creating customizable patterns in the skin and/or fur of animals.
  • the customizable trait can involve the length and/or texture of animal skin or fur.
  • the subject invention can be used to effect controlled changes in the texture, structural strength, and/or length of animal hair, nail, claw and/or horn.
  • the methods of the subject invention comprise introducing into the cells of an animal a genetic construct comprising a keratin- 14 specific promoter, red fluorescent protein in a loxp cassette, dominant black (AG23) beta defensin 103 in a pigment cassette, and an SV40 (with intron) polyadenylation sequence.
  • a composition comprising Cre recombinase (or HTNCre) is then applied to the skin of an animal having the genetic construct, the fur of the animal turns black where the composition is applied. In this way, the fur is permanently genetically modified to turn color in a desired shape.
  • the subject invention provides a method of creating customizable permanent patterns in the skin and/or fur of animals.
  • the subject invention provides methods for producing multicolor patterns in the skin and/or fur of animal species.
  • the subject invention provides a method of creating customizable patterns in the skin and/or fur of animal species such that the animal would continue to grow fur to sustain those colors throughout its lifetime.
  • the invention also provides methods of creating customizable predefined patterns of stripes that are heritable within that animal.
  • the transgenic animal has heritable soft claws or soft hair or fur.
  • the present invention provides cells, tissues, or parts (such as skin, hair, fur) of the transgenic animal, and uses thereof.
  • the transgenic animal has customizable color and/or patterns in the skin and/or fur, and the skin or fur can be subsequently removed from the transgenic animal for production of hides, fur, leather, etc, useful for production of clothing, rugs, shoes, horse tack, horse harness, upholstery, and other leather goods.
  • the present invention provides a transgenic animal with customizable traits, wherein the transgenic animal (such as in its genome) comprises:
  • nucleic acid molecule encoding a protein of interest, wherein the nucleic acid molecule is operably linked to a promoter and is under the control of an inducible gene expression system that requires the presence of an inducing agent to activate gene expression;
  • the exogenous nucleic acid molecule that encodes a protein is selected from pigment proteins; proteins involved in the synthesis and/or transport of pigments; luminescent (such as fluorescent) proteins; proteins involving the length and/or texture of animal skin or fur; and proteins involved in the texture, structural strength, and/or length of animal nail, claw, and/or horn.
  • the present invention provides a transgenic animal with customizable fur or skin pigmentation, wherein the genome of the transgenic animal comprises:
  • an exogenous nucleic acid molecule encoding a pigment protein of interest or a protein involved in the synthesis and/or transport of a pigment of interest, wherein the exogenous nucleic acid molecule is operably linked to a promoter and is under the control of an inducible gene expression system that is a site-specific recombination system,
  • site-specific recombination system inhibits the expression of the first nucleic acid molecule in the absence of site-specific recombination.
  • the expression of the exogenous nucleic acid molecule can be induced after the application of, for example, a recombinase to the transgenic animal.
  • the exogenous nucleic acid molecule, the promoter, and/or the site-specific recombination system are contained in a pigmentation construct.
  • the genome of the transgenic animal comprises an exogenous nucleic acid molecule whose expression is under the control of a Cre/LoxP recombination system, wherein the Cre/LoxP recombination system prevents the expression of the exogenous nucleic acid molecule.
  • the Cre/LoxP recombination system comprises a lox-stop-lox (LSL) sequence.
  • the pigmentation construct is transferred into cells, such as fertilized ova.
  • the pigmentation construct can be transferred into fertilized ova using any conventional means, including, but not limited to, lentivirii, pronuclear injections, and intracytoplasmic sperm injection (ICSI).
  • lentivirii lentivirii
  • pronuclear injections lentivirii
  • ICSI intracytoplasmic sperm injection
  • one or more pigmentation constructs are introduced into the genome of the transgenic animal.
  • the pigmentation construct can comprise more than one exogenous nucleic acid molecule, each nucleic acid molecule encoding a protein of interest.
  • the genome of the transgenic animal comprises more than one inducible gene expression systems to control the expression of the nucleic acid molecules of interest.
  • the present invention provides a transgenic animal with customizable traits (such as fur or skin pigmentation), wherein the genome of the transgenic animal comprises:
  • a first exogenous nucleic acid molecule encoding a protein of interest (such as a pigmentation protein) operably linked to a first promoter and under the control of a loxP site, wherein the loxP site prevents the expression of the first exogenous nucleic acid molecule in the absence of Cre recombinase protein;
  • a second nucleic acid molecule encoding a Cre recombinase protein, operably linked to a second promoter.
  • the present invention provides a transgenic animal with customizable traits (such as fur or skin pigmentation), wherein the genome of the transgenic animal comprises:
  • a first exogenous nucleic acid molecule encoding a protein of interest (such as pigmentation protein) operably linked to a first promoter and under the control of a loxP site, wherein the loxP site prevents the expression of the first nucleic acid molecule in the absence of Cre recombinase protein;
  • a protein of interest such as pigmentation protein
  • a second nucleic acid encoding a reverse tRA (rtTA), operably linked to a second promoter;
  • a third nucleic acid molecule encoding a Cre recombinase protein, operably linked to a third promoter under the control of a TetO operator.
  • Figures 2 and 3 show an embodiment of the expression constructs, wherein the expression of the exogenous nucleic acid molecule of interest (such as pigment proteins and proteins involved in the synthesis of biological pigments) is under the control of the Cre- LoxP recombination system and a tetracycline (Tet)-controlled transcription activation system.
  • the exogenous nucleic acid molecule of interest such as pigment proteins and proteins involved in the synthesis of biological pigments
  • doxycycline (or ecdysone, etc) mixed with DMSO carrier is applied to a transgenic animal with a gold fur color, whereby the color of the transgenic animal turns red; subsequently, Cre or HTNCre mixed with DMSO is applied to the transgenic animal to produce a black color.
  • the genome of the transgenic animal comprises an exogenous nucleic acid molecule the expression of which is under the control of a tetracycline (Tet)-controlled transcriptional activation system.
  • Tet tetracycline
  • the present invention provides a transgenic animal with customizable traits (such as fur or skin pigmentation), wherein the genome of the transgenic animal comprises: a first exogenous nucleic acid molecule encoding a protein of interest (such as a pigmentation protein) operably linked to a first promoter and under the control of an inducible gene expression system (e.g., a tetracycline (Tet)-controlled transcriptional activation system), wherein the inducible gene expression system, in its inactivated state (absent of induction), prevents the expression of the first nucleic acid molecule.
  • an inducible gene expression system e.g., a tetracycline (Tet)-controlled transcriptional activation system
  • the present invention provides a transgenic animal with customizable traits, wherein the transgenic animal (such as in its genome) comprises: a dominantly acting, exogenous nucleic acid molecule encoding a protein of interest, wherein the nucleic acid molecule is operably linked to a promoter.
  • the present invention provides a transgenic animal having a coat or fur color that is different from a wild-type animal of the same species, wherein the transgenic animal (such as in its genome) comprises: a dominantly acting, exogenous nucleic acid molecule encoding a protein selected from pigment proteins; proteins involved in the synthesis and/or transport of pigments; and luminescent (such as fluorescent) proteins.
  • the present invention provides a transgenic animal with coat or fur having a color of interest, wherein the transgenic animal comprises:
  • a dominantly acting, exogenous nucleic acid molecule operably linked to a promoter, wherein the exogenous nucleic acid encodes a protein selected from proteins involved in melanosome assembly, proteins involved in the synthesis of melanin, proteins involved in the transport of melanin, proteins involved in melanocyte development and/or migration, and proteins involved in the synthesis and/or transport of biological pigments; and/or an exogenous inhibitory RNA coding sequence of interest, operably linked to a promoter, wherein the exogenous inhibitory RNA coding sequence of interest interferes with the expression of a dominantly acting wild-type nucleic acid molecule of the animal, wherein the wild-type nucleic acid molecule encodes a protein selected from proteins involved in melanosome assembly, proteins involved in the synthesis of melanin, proteins involved in the transport of melanin, proteins involved in melanocyte development and/or migration, and proteins involved in the synthesis and/or transport of biological pigments.
  • the exogenous nucleic acid molecule is not present in the cells of the wild-type animal of the same species. In one embodiment, while the exogenous nucleic acid molecule is present in the cells of the wild-type animal of the same species, it is present at a different location in the wild-type genome. In one embodiment, while the exogenous nucleic acid molecule is present in the cells of the wild-type animal of the same species, it is expressed in different cell types in the wild-type animal. In another embodiment, while the exogenous nucleic acid molecule is present in the cells of the wild-type animal of the same species, the transgenic animal has additional copies of the exogenous nucleic acid, when compared to the wild-type animal of the same species.
  • the transgenic animal can be of any species, including, but not limited to, mammalian species including, but not limited to, domesticated and laboratory animals such as dogs, cats, mice, rats, guinea pigs, and hamsters; livestock such as horses, cattle, pigs, sheep, goats, ducks, geese, and chickens; primates such as apes, chimpanzees, orangutans, humans, and monkeys; fish; amphibians such as frogs and salamanders; reptiles such as snakes and lizards; and other animals such as fox, weasels, rabbits, mink, beavers, ermines, otters, sable, seals, coyotes, chinchillas, deer, muskrats, and possum.
  • the animal is not a human.
  • the present invention provides a transgenic bovine animal with white or near-white coat or fur, wherein the transgenic animal (such as in its genome) comprises:
  • a dominantly acting, exogenous nucleic acid molecule operably linked to a promoter, wherein the dominantly acting, exogenous nucleic acid encodes a protein selected from proteins that inhibit melanosome assembly, proteins that inhibit the synthesis of melanin, proteins that inhibit the transport of melanin, and proteins that inhibit melanocyte development and/or migration; and/or
  • an exogenous inhibitory RNA coding sequence of interest operably linked to a promoter, wherein the exogenous inhibitory RNA coding sequence encodes an inhibitory RNA that interferes with the expression of a dominantly acting wild-type nucleic acid molecule of the animal, wherein the wild-type nucleic acid molecule encodes a protein selected from proteins involved in melanosome assembly, proteins involved in the synthesis of melanin, proteins involved in the transport of melanin, and proteins involved in melanocyte development and/or migration.
  • transgenic bovine animals of the present invention can include, but are not limited to, domesticated cattle, bison, and buffalos (e.g., water buffalo, African buffalo).
  • the dominantly acting, exogenous nucleic acid molecule is a dominantly acting white allele.
  • allele refers to its ordinary meaning that is an alternative form of a gene, usually arising from mutations, that is located at a specific position on a specific chromosome.
  • Dominant white is a group of genetically related coat or color phenotypes that are present in various breeds of wild-type animals, such as for example, chicken, horses, mice, dogs, and cats.
  • the dominant white allele is a nucleic acid molecule encoding a Pmell7.
  • Pmell7 also called GP100 and Silv
  • Pmell7 proteins useful according to the present invention can be from, for example, chicken, horses, mice, dogs, and cats.
  • the dominant white allele encodes a Pmell7 protein of white chicken.
  • the dominant white allele is a nucleic acid molecule encoding
  • the exogenous inhibitory RNA interferes with the expression of melanocortin receptor (MC1R), melanocyte stimulating hormones (MSH) (e.g., a-MSH, ⁇ -MSH, ⁇ -MSH), ⁇ -defensin 103, agouti signaling protein (ASP), tyrosinase (TYR), melanocyte-specific transporter protein, Ras-related protein Rab-7, rab protein geranylgeranyltransferase component A2, and probable E3 ubiquitin-protein ligase (HERC2).
  • M1R melanocortin receptor
  • MSH melanocyte stimulating hormones
  • ASP agouti signaling protein
  • TMR tyrosinase
  • melanocyte-specific transporter protein Ras-related protein Rab-7
  • rab protein geranylgeranyltransferase component A2 rab protein geranylgeranyltransferase component A2
  • HERC2 probable E3
  • the transgenic bovine animal further comprises an inducible gene expression system.
  • the expression of the exogenous nucleic acid molecule or the expression of the exogenous inhibitory RNA coding sequence is under the control of the inducible system.
  • the inducible gene expression system is a site-specific recombination system (e.g., the Lox/P system).
  • the transgenic bovine animal has white or near-white coat color. In certain embodiments, the transgenic bovine animal has non-black colors, such as, gray, pink, brown, and yellow.
  • pigment protein refers to a protein comprising a pigment.
  • pigment refers to a material that does not emit light but changes the color of reflected or transmitted light as the result of wavelength-selective absorption; this physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which a material emits light.
  • Pigment proteins include, but are not limited to, chromoproteins such as cytochromes and fiavoproteins.
  • luminescent protein refers to a protein that emits light.
  • Luminescent proteins useful according to the present invention include, but are not limited to, fluorescent proteins including, but not limited to, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and red fluorescent protein; and phosphorescent proteins.
  • Fluorescent proteins are members of a class of proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of three amino acids within their own polypeptide sequence.
  • a variety of luminescent proteins, including fluorescent proteins are publicly known.
  • Fluorescent proteins useful according to the present invention include, but are not limited to, the fluorescent proteins disclosed in U.S. Patent No. 7,160,698, U.S. Application Publication Nos.
  • Proteins involved in the synthesis and/or transport of biological pigments include, but are not limited to, the wild-type or mutant forms of melanocortin receptor (MC1R), melanocyte stimulating hormones (MSH) (e.g., a-MSH, ⁇ -MSH, ⁇ -MSH), ⁇ -defensin 103, agouti signaling protein (ASP), tyrosinase (TYR), melanocyte-specific transporter protein, Ras-related protein Rab-7, rab protein geranylgeranyltransferase component A2, probable E3 ubiquitin-protein ligase (HERC2), Pmell7, and melanophilin (MLPH).
  • M1R melanocortin receptor
  • MSH melanocyte stimulating hormones
  • ASP agouti signaling protein
  • TMR tyrosinase
  • melanocyte-specific transporter protein Ras-related protein Rab-7
  • the genome of the transgenic animal comprises an exogenous nucleic acid molecule encoding a protein involved in the synthesis of a biological pigment.
  • Nucleic acid molecules encoding proteins involved in the synthesis and/or transport of biological pigments can be derived from genes including, but not limited to, the dominant MC1R E92K and the agouti gene.
  • the genome of the transgenic animal comprises a nucleic acid molecule encoding a protein involved in the synthesis and/or transport of biological pigments including, but not limited to, melanins (e.g., pheomelanin, eumelanin); urochrome; chlorophyll; bilirubin; biliverdin; phycobilin; phycoerythrobilin; stercobilin; urobilin; hemocyanin; hemoglobin; myoglobin; luciferins; carotenoids, including hematochromes, carotenes (e.g., alpha and beta carotene, lycopene, rhodopsin), xanthophylls (e.g., canthaxanthin
  • Carotenoid pigments in yellow, red, or orange can be synthesized in animals that express phytoenesynthases, desaturases, and cyclases, as described in Moran (2010) and Verdoes (2003).
  • the carotenoid dyes are synthesized using geranylgeranyl pyrophosphate as a substrate.
  • Proteins involved in the synthesis and/or transport of biological pigments include, but are not limited to, bifunctional enzyme CarRP-like isoform 1 [Acyrthosiphon pisum] (such as, GenBank Accession No. XP 001943170 (SEQ ID NO: l)), bifunctional enzyme CarRP-like [Acyrthosiphon pisum] (such as, GenBank Accession No. XP 001950787 (SEQ ID NO:2)), lycopene cyclase / phytoene synthase-like [Acyrthosiphon pisum] (such as GenBank Accession No.
  • XP 001950868 (SEQ ID NO:3)), phytoene dehydrogenase-like [Acyrthosiphon pisum] (such as, GenBank Accession No. XP 001943225 (SEQ ID NO:4)), phytoene dehydrogenase-like [Acyrthosiphon pisum] (such as, GenBank Accession No. XP 001950764 (SEQ ID NO:5)), phytoene dehydrogenase-like [Acyrthosiphon pisum] (such as, GenBank Accession No.
  • XP001946689 SEQ ID NO:6
  • phytoene dehydrogenase- like such as, GenBank Accession No. XP 001943938 (SEQ ID NO:7)
  • Proteins involved in the synthesis and/or transport of biological pigments can be of any animal origin (such as mouse, porcine, human, horse, dog, cat, mouse, chicken) including, but not limited to, melanocortin 1 receptor (such as, GenBank Accession No. EDL1 1741 (SEQ ID NO: 8)), alpha melanocyte stimulating hormones (MSH) (such as, SEQ ID NO:9), beta melanocyte stimulating hormones (MSH) (such as, SEQ ID NO: 10, SEQ ID NO: 1 1), gamma melanocyte stimulating hormones (MSH) (such as, SEQ ID NO: 12), ⁇ -defensin (such as, GenBank Accession No.
  • melanocortin 1 receptor such as, GenBank Accession No. EDL1 1741 (SEQ ID NO: 8)
  • alpha melanocyte stimulating hormones (MSH) such as, SEQ ID NO:9
  • beta melanocyte stimulating hormones such as, SEQ ID NO: 10
  • AAT67592 (SEQ ID NO: 13)), agouti signaling protein precursor (such as, GenBank Accession No. NP 056585 (SEQ ID NO: 14)), tyrosinase (TYR) (such as, GenBank Accession No. BAA00341 (SEQ ID NO: 15)), melanocyte-specific transporter protein (such as, GenBank Accession No. Q62052 (SEQ ID NO: 16)), rab proteins geranylgeranyltransferase component A2 (such as, GenBank Accession No. NP_067325 (SEQ ID NO: 17)), ras-related protein Rab-7a (such as, GenBank Accession No.
  • NP 033031 (SEQ ID NO: 18)
  • probable E3 ubiquitin-protein ligase (HERC2)
  • HERC2 E3 ubiquitin-protein ligase
  • MLPH melanophilin
  • proteins involved in the synthesis and/or transport of biological pigments can be proteins having at least 80% identity, or having any percent identity higher than 80% (such as at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), to any of SEQ ID NOs: 1-22.
  • Pmell7 proteins sequences are publicly available, such as via the GenBank database.
  • Pmell7 proteins useful according to the present invention include, but are not limited to, Gallus gallus Pmell7 having amino acid sequences, such as SEQ ID NOs: 20-22.
  • Proteins involved in the texture, structural strength, and/or length of hair, nail, claw and/or horn Proteins involved in the texture, structural strength, and/or length of hair, nail, claw and/or horn
  • the transgenic animal expresses a nucleic acid molecule encoding a protein that alters hair quality (such as straight or curly hair) or length.
  • conditional over-expression of WNT3 or DVL2 in the outer root sheath induces shorter hair in animals.
  • the transgenic animal expresses a nucleic acid molecule encoding a protein involves that the texture, structural strength, and/or length of animal hair, nail, claw and/or horn.
  • Proteins involved in controlling the texture, structural strength, and/or length of animal hair, nail, claw and/or horn include keratin proteins, including, but not limited to, keratin 1, keratin 2, keratin 2A, keratin HB6, keratin 3, keratin 4, keratin 5, keratin 6, keratin 7, keratin 8, keratin 9, keratin 10, keratin 11, keratin 12, keratin 13, keratin 14, keratin 15, keratin 16, keratin 17, keratin 18, keratin 19, keratin 20, keratin 23, keratin 24, keratin 25, keratin 26, keratin 27, keratin 28, keratin 31, keratin 32, keratin 33, keratin 34, keratin 35, keratin 36, keratin 37, keratin 38, keratin 39, keratin 40, keratin 71, kerat
  • the amino acid sequences of a variety of pigment proteins; proteins involved in the synthesis and/or transport of pigments; proteins involving the length and/or texture of animal skin or fur; and proteins involved in the texture, structural strength, and/or length of animal nail, claw, and/or horn, are publically available, such as via the GenBank database.
  • the present invention encompasses the use of such proteins.
  • Polynucleotides and polypeptides within the scope of the subject invention can also be defined in terms of identity with those sequences that are specifically exemplified herein.
  • sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%.
  • the identity of a sequence can be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
  • the nucleic acid molecule is operably linked to a constitutive, inducible, or tissue-specific promoter.
  • constitutive promoter refers to its ordinary meaning that is an unregulated promoter that allows for continual transcription of its associated gene.
  • Constitutive promoters useful according to the present invention include, but are not limited to, cytomegalovirus (CMV) promoter, CMV-chicken beta actin promoter, ubiquitin promoter,
  • Promoters useful according to the present invention include, but are not limited to, universal promoters (e.g., Rosa26); tissue-specific promoters, such as keratinocyte specific promoters (e.g., Keratin 14); melanocyte specific promoters (e.g., promoter of the melanocortin 1 receptor (MCR1) gene); and dermal papilla-specific promoters. Promoters useful according to the present invention include melanocyte specific promoters and matrix- cell specific promoters.
  • Keratinocyte specific promoters include, but are not limited to, promoters of keratin 1, keratin 2, keratin 2 A, keratin HB6, keratin 3, keratin 4, keratin 5, keratin 6, keratin 7, keratin 8, keratin 9, keratin 10, keratin 11, keratin 12, keratin 13, keratin 14, keratin 15, keratin 16, keratin 17, keratin 18, keratin 19, keratin 20, keratin 23, keratin 24, keratin 25, keratin 26, keratin 27, keratin 28, keratin 31, keratin 32, keratin 33, keratin 34, keratin 35, keratin 36, keratin 37, keratin 38, keratin 39, keratin 40, keratin 71, keratin 72, keratin 73, keratin 74, keratin
  • promoters useful according to the present invention include, but are not limited to, promoters inducing gene expression in the presence of an endogenous biological factor of interest, such as NF-KB, interferon-gamma, estrogen, and or glucocorticoids.
  • an endogenous biological factor of interest such as NF-KB, interferon-gamma, estrogen, and or glucocorticoids.
  • Promoters useful according to the present invention include, but are not limited to, promoters inducing gene expression in the presence of an infectious agent of interest, such as a virus, bacteria, and/or protozoa.
  • a dermal papilla-specific promoter is used for creating customizable pigmentation, color, or pattern in cells derived from somites; useful promoters include, but are not limited to, a Ripply2 promoter, a Tabby promoter, and a Ticked promoter. The choice of promoters can be determined by performing multiple species comparisons and/or using the extent of well-conserved promoter elements.
  • the promoter element further comprises a nucleic acid molecule encoding a reporter protein, which is expressed in response to the administration of drugs, and/or a metabolic state or circulating levels of biomarkers in the transgenic animal.
  • the promoter induces expression of a protein of interest (such as a pigment protein and/or a protein involved in the synthesis of a biological pigment) in response to the presence of a physiological state of interest in the transgenic animal, such as for example, cardiac stress, increased levels of circulating cytokines, and/or increased steroid presence or activity.
  • a protein of interest such as a pigment protein and/or a protein involved in the synthesis of a biological pigment
  • the inducible gene expression systems useful according the present invention include, but are not limited to, site-specific recombination systems including, but not limited to, a Cre- LoxP recombination system, a FLP-FRT recombination system; a tetracycline (Tet)- controlled transcription activation system; an ecdysone inducible system; a heat shock on/off system; a lacO/IPTG system; a cumate repressor protein CymR system; a nitroreductase system; coumermycin/novobiocin-regulated system; a RheoSwitch Ligand RSL1 system; a chimeric bipartite nuclear receptor expression system; a GAL4 system; sterol or steroid or synthetic steroid inducing/repressing system; and any combination thereof.
  • site-specific recombination systems including, but not limited to, a Cre- LoxP recombination system, a FLP-F
  • the inducible system useful according to the present invention is a Cre-LoxP recombination system.
  • the genome of the transgenic animal can comprise an exogenous nucleic acid molecule whose expression is under the control of a Cre/LoxP recombination system, wherein the Cre/LoxP recombination system prevents the expression of the exogenous nucleic acid molecule.
  • the Cre/LoxP recombination system comprises a lox-stop-lox (LSL) sequence.
  • the Cre-LoxP recombination system is a site-specific recombination technology useful for performing site-specific deletions, insertions, translocations, and inversions in the DNA of cells or transgenic animals.
  • the Cre recombinase protein (encoded by the locus originally named as "causes recombination") consists of four subunits and two domains: a larger carboxyl (C -terminal) domain and a smaller amino (N-terminal) domain.
  • the loxP locus of X-over PI
  • the results of Cre-recombinase-mediated recombination depend on the location and orientation of the loxP sites, which can be located cis or trans. In case of cis-localization, the orientation of the loxP sites can be the same or opposite. In case of trans-localization, the DNA strands involved can be linear or circular.
  • the results of Cre recombinase-mediated recombination can be excision (when the loxP sites are in the same orientation) or inversion (when the loxP sites are in the opposite orientation) of an intervening sequence in case of cis loxP sites, or insertion of one DNA into another or translocation between two molecules (chromosomes) in case of trans loxP sites.
  • the Cre-LoxP recombination system is known in the art, see, for example, Andras Nagy, Cre recombinase: the universal reagent for genome tailoring, Genesis 26:99-109 (2000).
  • the Lox-Stop-Lox (LSL) cassette prevents expression of the transgene in the absence of Cre-mediated recombination. In the presence of Cre recombinase, the LoxP sites recombine, and the stop cassette is deleted.
  • the Lox-Stop-Lox (LSL) cassette is known in the art. See, Allen Institute for Brain Science, Mouse Brain Connectivity Altas, Technical White Paper: Transgenic Characterization Overview (2012).
  • the loxP site further comprises a reporter gene encoding gene ⁇ e.g., lacz, GFP) and/or a nucleic acid molecule encoding a second pigmentation protein ⁇ e.g., if the first pigmentation contains a dominantly active MC1R, another agouti gene could be flanked by the loxP site).
  • a reporter gene encoding gene e.g., lacz, GFP
  • a nucleic acid molecule encoding a second pigmentation protein ⁇ e.g., if the first pigmentation contains a dominantly active MC1R, another agouti gene could be flanked by the loxP site.
  • Tetracycline (Tet)-controlled transcriptional activation is a method of inducible expression where transcription is reversibly controlled by the presence or absence of the antibiotic tetracycline or one of its derivatives ⁇ e.g., doxycycline).
  • Gene expression is activated as a result of binding of the Tet-off or Tet-on protein to tetracycline response elements (TREs) located within an inducible promoter. Both the Tet-on and Tet-off proteins activate gene expression.
  • the Tet-Off protein activates gene expression in the absence of a tetracycline derivative - doxycycline (Dox), whereas the Tet-on protein activates gene expression in the presence of Dox.
  • the tetracycline transactivator (tTA) protein which is created by fusing the TetR (tetracycline repressor) protein (obtainable from Escherichia coli bacteria) with the VP 16 protein (obtainable from the Herpes Simplex Virus), binds on DNA at a TetO operator. Once bound the TetO operator activates the promoter coupled to the TetO operator, thereby activating the transcription of the nearby gene. Tetracycline derivatives bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of target genes.
  • TetR tetracycline repressor
  • VP 16 protein obtainable from the Herpes Simplex Virus
  • the Tet-On system when the tTA protein is bound by doxycycline, the doxycycline-bound tTA is capable of binding the TetO operator.
  • the introduction of doxycyline to the system initiates the transcription of the genetic product.
  • the Tet-on system is sometimes preferred for the faster responsiveness.
  • rtTA reverse tTA
  • Dox Dox
  • Tet-on advanced transactivator also known as rtTA2 s -M2
  • rtTA2 s -M2 is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-on.
  • its expression is considered to be more stable in eukaryotic cells due to being human codon optimized and utilizing three minimal transcriptional activation domains.
  • Tet-on 3G (also known as rtTA-VIO) is similar to Tet-on Advanced, and is human codon optimized and composed of three minimal VP 16 activation domains.
  • the Tet-on 3G is sensitive to 100-fold less Dox than the original Tet-on.
  • a tetracycline-controlled reverse transactivator comprises a tetR (e.g., from Escherichia coli TnlO); a mammalian transcription factor VP 16 transactivating domain serving as an effector; and a tissue-specific promoter controlling the rtTA effector transcription.
  • the rtTA binds to a (TeTO) 7 operator (a seven tandemly repeated TetO sequence) placed upstream of a CMV promoter that drives expression of a transgene.
  • TeTO seven tandemly repeated TetO sequence
  • the present invention also provides expression constructs, vectors, as well as host cells useful for producing transgenic animals.
  • expression construct refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence.
  • Expression constructs of the invention also generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed.
  • regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.
  • the present invention provides expression constructs for customizing color and/or pattern of animals, including pigmentation constructs and patterning constructs.
  • Figure 1 shows an embodiment of a pigmentation construct comprising a promoter, a loxP cassette, a nucleic acid molecule encoding a pigment protein of interest or a protein involved in the synthesis and/or transport of a pigment of interest, and polyadenylation sequence.
  • the expression of the pigment protein of interest or a protein involved in the synthesis and/or transport of a pigment of interest is activated by application of Cre recombinase.
  • the present invention provides a pattern construct.
  • Figure 2 shows an embodiment of a patterning construct.
  • the promoter of the patterning construct is derived from a gene specific to somite boundary specification.
  • the promoter is selected from a Ripply! promoter, a Tabby promoter, and a Ticked promoter.
  • the transgenic animal comprises a pigmentation construct and a patterning construct.
  • the transgenic animal has customizable constitutive vertical stripes on the dorsal dermis.
  • the genome of the transgenic animal comprises:
  • pigmentation construct as shown in Fig. 1, wherein the promoter is a melanocyte - specific promoter and the nucleic acid molecule encodes a protein involved in the synthesis of a red pigment (such as ASIP or MC1R);
  • Fig. 3 a set of constructs as shown in Fig. 3, wherein the promoter is a melanocyte-specific promoter.
  • An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a peptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.
  • operably linked refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner.
  • operably linked components are in contiguous relation.
  • Sequence(s) operably-linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence.
  • a coding sequence is operably-linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
  • a “coding sequence” or “coding region” is a polynucleotide sequence that is transcribed into mRNA and/or translated into a polypeptide.
  • a coding sequence may encode a polypeptide of interest.
  • the boundaries of the coding sequence are determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3 '-terminus.
  • promoter refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule.
  • a promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors.
  • a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.
  • one or more enhancer sequences may be included such as, but not limited to, cytomegalovirus (CMV) early enhancer element and an SV40 enhancer element. Additional included sequences are an intron sequence such as the beta globin intron or a generic intron, a transcription termination sequence, and an mRNA polyadenylation (pA) sequence such as, but not limited to, SV40-pA, beta-globin-pA, the human growth hormone (hGH) pA and SCF-pA.
  • the expression construct comprises polyadenylation s equences, such as polyadenylation s equences derived from bovine growth hormone (BGH) and SV40.
  • BGH bovine growth hormone
  • polyA or "p(A)” or “pA” refers to nucleic acid sequences that signal for transcription termination and mRNA polyadenylation.
  • the polyA sequence is characterized by the hexanucleotide motif AAUAAA.
  • Commonly used polyadenylation signals are the SV40 pA, the human growth hormone (hGH) pA, the beta-actin pA, and beta-globin pA.
  • the sequences can range in length from 32 to 450 bp. Multiple pA signals may be used.
  • the genetic construct comprises a nucleic acid molecule encoding a selection marker, such as neomycin resistance biomarker protein, which can be excised through PIGGYBACTM transposons.
  • a selection marker such as neomycin resistance biomarker protein
  • the construct is flanked by short homology arms.
  • vector is used to refer to any molecule (e.g. , nucleic acid, plasmid, or virus) used to transfer coding information (e.g. , a polynucleotide of the invention) to a host cell.
  • expression vector and “transcription vector” are used interchangeably to refer to a vector that is suitable for use in a host cell (e.g., a subject's cell) and contains nucleic acid sequences that direct and/or control the expression of exogenous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
  • Vectors useful according to the present invention include plasmids, viruses, BACs, YACs, and the like. Particular viral vectors illustratively include those derived from adenovirus, adeno-associated virus and lentivirus.
  • isolated molecule refers to molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid construct in which two or more nucleic acids are linked and which are not found linked in nature.
  • nucleic acid refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide.
  • nucleotide sequence is used to refer to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.
  • expressed refers to transcription of a nucleic acid sequence to produce a corresponding mRNA and/or translation of the mRNA to produce the corresponding protein.
  • Expression constructs can be generated recombinantly or synthetically or by DNA synthesis using well-known methodology.
  • regulatory element refers to a nucleotide sequence which controls some aspect of the expression of an operably linked nucleic acid sequence.
  • exemplary regulatory elements illustratively include an enhancer, an internal ribosome entry site (IRES), an intron, an origin of replication, a polyadenylation signal (pA), a promoter, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid sequence.
  • pA polyadenylation signal
  • pA polyadenylation signal
  • promoter e.g., a transcription termination sequence
  • upstream regulatory domain which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid sequence.
  • the construct of the present invention comprises an internal ribosome entry site (IRES).
  • the expression construct comprises kozak consensus sequences.
  • reporter gene is included in the transgene construct.
  • reporter gene refers to a gene that is easily detectable when expressed, for example, via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, and the like.
  • Exemplary reporter genes include but are not limited to green fluorescent protein.
  • the vector may optionally contain flanking nucleic sequences that direct site-specific homologous recombination.
  • flanking DNA sequences to permit homologous recombination into a desired genetic locus is known in the art. At present it is preferred that up to several kilobases or more of flanking DNA corresponding to the chromosomal insertion site be present in the vector on both sides of the encoding sequence (or any other sequence of this invention to be inserted into a chromosomal location by homologous recombination) to assure precise replacement of chromosomal sequences with the exogenous DNA. See e.g. Deng et al, 1993, Mol. Cell.
  • Transformed host cells are cells which have been transformed or transfected with vectors containing nucleic acid constructs of the invention and may or may not transcribe or translate the operatively associated nucleic acid of interest.
  • RNA Interference Cassette for Customization of Animal Traits are cells which have been transformed or transfected with vectors containing nucleic acid constructs of the invention and may or may not transcribe or translate the operatively associated nucleic acid of interest.
  • the present invention provides a transgenic animal with customizable traits, wherein the genome of the transgenic animal comprises:
  • an exogenous inhibitory RNA coding sequence of interest operably linked to a promoter and under the control of an inducible gene expression system that requires the presence of an inducing agent to activate gene expression;
  • the exogenous inhibitory RNA coding sequence of interest interferes with the expression of a nucleic acid sequence encoding pigment proteins; proteins involved in the synthesis and/or transport of pigments; proteins involving the length and/or texture of animal skin or fur; luminescent (such as fluorescent) proteins; and proteins involved in the texture, structural strength, and/or length of animal nail, claw, or horn texture.
  • an exogenous inhibitory RNA coding sequence encodes an siRNA that interferes with the expression of cross-linking actin) in the nails, thereby producing genetically-engineered animals (such as cats) with nails that are soft instead of sharp.
  • the RNAi construct comprises an siRNA that interferes with the expression of a nucleic acid molecule encoding cross-linking keratin), operably linked to a promoter specific to the cross-linking keratin, and is under the control of a reporter gene flanked by loxP sites.
  • Keratin proteins involved in the texture, structural strength, and/or length of animal hair, nail, claw and/or horn include, but are not limited to, keratin 1 , keratin 2, keratin 2A, keratin HB6, keratin 3, keratin 4, keratin 5, keratin 6, keratin 7, keratin 8, keratin 9, keratin 10, keratin 1 1 , keratin 12, keratin 13, keratin 14, keratin 15, keratin 16, keratin 17, keratin 18, keratin 19, keratin 20, keratin 23, keratin 24, keratin 25, keratin 26, keratin 27, keratin 28, keratin 31 , keratin 32, keratin 33, keratin 34, keratin 35, keratin 36, keratin 37, keratin 38, keratin 39, keratin 40, keratin 71 ,
  • the present invention provides a microRNA cassette comprising the siRNA coding sequence and a 3 ' UTR sequence.
  • RNA interference refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of endogenous target genes, such as PKC-i.
  • small interfering RNA refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.
  • RNAi target-specific RNA interference
  • siRNA having a "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the siRNA has a sequence sufficient to trigger the destruction of the target mRNA (e.g., PKC- ⁇ mRNA) by the RNAi machinery or process.
  • target mRNA e.g., PKC- ⁇ mRNA
  • mRNA or messenger RNA or “transcript” is single- stranded RNA that specifies the amino acid sequence of one or more polypeptides. This information is translated during protein synthesis when ribosomes bind to the mRNA.
  • nucleotide refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety.
  • exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates.
  • polynucleotide and nucleic acid molecule are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms.
  • nucleic acid or “nucleic acid sequence” encompass an oligonucleotide, nucleotide, polynucleotide, or a fragment of any of these, DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent a sense or antisense strand, peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin.
  • PNA peptide nucleic acid
  • the nucleic acid is RNA
  • the deoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C, and U, respectively.
  • RNA or “RNA molecule” or “ribonucleic acid molecule” refers generally to a polymer of ribonucleotides.
  • DNA or “DNA molecule” or deoxyribonucleic acid molecule” refers generally to a polymer of deoxyribonucleotides.
  • DNA and RNA molecules can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA molecules can be post-transcriptionally modified. DNA and RNA molecules can also be chemically synthesized.
  • RNA molecules can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • RNA or "RNA molecule” or “ribonucleic acid molecule” can also refer to a polymer comprising primarily (i.e., greater than 80% or, preferably greater than 90%) ribonucleotides but optionally including at least one non-ribonucleotide molecule, for example, at least one deoxyribonucleotide and/or at least one nucleotide analog.
  • nucleotide analog also referred to herein as an “altered nucleotide” or “modified nucleotide,” refers to a non-standard nucleotide, including non- naturally occurring ribonucleotides or deoxyribonucleotides. Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function.
  • RNA analog refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA.
  • the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages.
  • Exemplary RNA analogues include sugar- and/or backbone -modified ribonucleotides and/or deoxyribonucleotides.
  • Such alterations or modifications can further include addition of non- nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA).
  • RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA interference or otherwise reduce target gene expression.
  • transgenic animals Any of various methods can be used to introduce a transgene into a non-human animal to produce a transgenic animal. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection, viral infection and transformation of embryonic stem cells and iPS cells. Methods for generating transgenic animals that can be used include, but are not limited to, those described in J. P. Sundberg and T. Ichiki, Eds., Genetically Engineered Mice Handbook, CRC Press; 2006; M. H. Hofker and I. van Deursen, Eds., Transgenic Mouse Methods and Protocols, Humana Press, 2002; A. L.
  • the genetically engineered animals with site-specific knock- ins can be created using spermatogonia! stem cells (SSCs), piggyBacTM mobile DNA technology using transposable elements, Xanthamonas transcription activator-like (TAL) Nucleases (XTNs) [aka TAL-effector nucleases (TALENs)], and a combination thereof.
  • SSCs spermatogonia! stem cells
  • piggyBacTM mobile DNA technology using transposable elements Xanthamonas transcription activator-like (TAL) Nucleases
  • XTNs Xanthamonas transcription activator-like Nucleases
  • TALENs TAL-effector nucleases
  • the present invention provides a method of customizing animal traits using the transgenic animal of the invention.
  • the method comprises:
  • nucleic acid molecule encoding a protein of interest, wherein the nucleic acid molecule is operably linked to a promoter and is under the control of an inducible gene expression system that requires the presence of an inducing agent to activate gene expression;
  • the exogenous nucleic acid molecule encodes a protein of interest, wherein the protein of interest is selected from pigment proteins; proteins involved in the synthesis and/or transport of pigments; luminescent (such as fluorescent) proteins; proteins involving the length and/or texture of animal skin or fur; and proteins involved in the texture, structural strength, and/or length of animal nail, claw, and/or horn.
  • the protein of interest is selected from pigment proteins; proteins involved in the synthesis and/or transport of pigments; luminescent (such as fluorescent) proteins; proteins involving the length and/or texture of animal skin or fur; and proteins involved in the texture, structural strength, and/or length of animal nail, claw, and/or horn.
  • the inducible gene expression systems useful according the present invention include, but are not limited to, site-specific recombination systems including, but not limited to, a Cre-LoxP recombination system, a FLP-FRT recombination system; a tetracycline (Tet)-controlled transcription activation system; an ecdysone inducible system; a heat shock on/off system; a lacO/IPTG system; a cumate repressor protein CymR system; a nitroreductase system; coumermycin/novobiocin-regulated system; a RheoSwitch Ligand RSL1 system; a chimeric bipartite nuclear receptor expression system; a GAL4 system; sterol or steroid or synthetic steroid inducing/repressing system; and any combinations thereof.
  • site-specific recombination systems including, but not limited to, a Cre-LoxP recombination system, a FLP
  • the inducing agents for gene expression useful according the present invention include, but are not limited to, ere recombinase, HTCre; FLP recombinase; tetracycline or its derivatives such as doxycycline; ecdysone; cumate; nitroreductase steroids; and any combinations thereof.
  • the transgenic animal comprises an exogenous nucleic acid molecule that is under the control of a site-specific recombination system, and the expression of the exogenous nucleic acid molecule is induced after the administration (such as via topical administration) of a recombinase protein, or the administration (such as via injection) of a nucleic molecule encoding a recombinase protein, to the transgenic animal.
  • the genome of the transgenic animal comprises an exogenous nucleic acid molecule whose expression is under the control of a Cre/LoxP recombination system, wherein the Cre/LoxP recombination system prevents the expression of the exogenous nucleic acid molecule, wherein the administration (such as via topical administration) of Cre recombinase and/or HTCre, or the administration (such as via injection) of a nucleic molecule encoding Cre recombinase and/or HTCre, to the transgenic animal, induces the expression of the exogenous nucleic acid molecule, thereby customizing the animal trait(s) of interest.
  • the method of customizing animal traits comprises: a) providing a transgenic animal whose genome comprises:
  • a first exogenous nucleic acid molecule encoding a protein of interest (such as pigmentation protein) operably linked to a first promoter and under the control of a loxP site, wherein the loxP site prevents the expression of the first nucleic acid molecule in the absence of Cre recombinase protein;
  • a protein of interest such as pigmentation protein
  • a second nucleic acid encoding a reverse tTA (rtTA), operably linked to a second promoter;
  • a third nucleic acid molecule encoding a Cre recombinase protein, operably linked to a third promoter under the control of a TetO operator;
  • the inducing agent for administration to the transgenic animal can be in a form that can be combined with a carrier.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the inducing agent and compositions can be administered to the transgenic animal by standard routes, including oral, inhalation, or parenteral administration including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection, infusion, and electroporation, as well as co-administration as a component of any medical device or object to be inserted (temporarily or permanently) into a transgenic animal.
  • parenteral administration including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epi
  • This Example provides embodiments of genetic constructs for customizing skin and fur pigmentation in animals.
  • C3H/HeJ murine strain with a brown (“Agouti” coloration) skin color are genetically engineered to express the following three constructs: 1) a construct comprising a keratin-14 specific promoter, a loxp cassette comprising a nucleic acid encoding a red fluorescent protein, a pigment cassette comprising a nucleic acid encoding a dominant black ( ⁇ 023) beta defensin 103 protein, and an SV40 (with intron) polyadenylation sequence; 2) a construct comprising a nucleic acid molecule encoding a reverse tetracycline transactivator (rtTA), operably linked to a keratin-14 specific promoter that initiates the transcription of the nucleic acid encoding rtTA, and an SV40 polyadenylation sequence; and 3) a construct comprising an agouti signaling protein (ASP), operably linked to a tetracycline-sensitive promoter (TetO)7 that initiates the transcription of
  • mice fur turns golden (this is the first demonstration of genetic modification of hair color after birth).
  • a carrier base e.g., protein carriers, such as lipid bilayers
  • Cre or HTNCre induces genetically permanent black coloration of fur in the area where Cre and/or HTNCre is applied.
  • mice born with brown fur can be modified to have golden fur with arbitrarily shaped black markings. For example, a black name or black logo can be created on mice fur with a gold background.
  • the animal could only express a pigmentation construct, and need not express a patterning construct (as shown in Figure 2).
  • the customization of pigmentations and patterns is activated directly by application of Cre and/or HTNCre.
  • heritable patterns can be created by genetically modifying the animals to express a patterning construct.
  • Figure 2 shows one embodiment of a patterning construct.
  • the promoter can be the Ripply2 promoter, or from any gene specific to somite boundary specification. Alternate promoters could be the Tabby or Ticked promoters.
  • the genetically-modified animal whose genome comprises a pigmentation construct and a patterning construct, has constitutive vertical stripes on the dorsal dermis.
  • Figure 3 illustrates certain embodiments of genetic constructs for creating complex or multicolored patterns in animal skin or fur.
  • complex patterns can be created with the use of a construct comprising an inducible system, such as promoters with mechanisms of inducibility.
  • Cre is activated by the presence of doxycycline only in tissues specific for the rtTA promoter.
  • tissue-specific promoter maintains somite border in animals; for example, the transgene can only be activated during the developmental period.
  • Inducible system can also be used to create multiple colors.
  • Inducible systems useful according to the present invention include, but are not limited to, tetracycline, ecdysone, and tamoxifen inducible systems; FLP-FRT recombination system; and Cre-LOX recombination system.
  • EXAMPLE 2 MICE WITH CUSTOMIZED FUR COLOR AND PATTERN
  • This Example shows the creation of genetically-modified mice whose fur color can be permanently altered through the transdermal application of HTNCre - a recombinase that can easily cross cell membranes.
  • Figure 4 shows a genetic construct for creating customized patterns and color in mouse skin or fur.
  • the construct comprises a Keratin 14 promoter, which is expressed in all skin fibroblasts, to drive the dominant black form of signaling molecule beta-Defl03; the expression of beta-Defl03 is blocked by a Loxp excisable nucleic acid encoding ring finger protein (RFP) (the RFP is used as a marker).
  • RFP Loxp excisable nucleic acid encoding ring finger protein
  • Agouti mice are genetically-engineered to express a transgene encoding the "dominant black” signaling molecule ⁇ 03, and the transgene expression is activated by the application of recombinase.
  • Figure 5A and B are photographs that show two genetically-engineered mice in which the expression of the dominant black pigment protein is activated by dermal or intradermal application of HTNCre in a carrier solution. Before the present invention, recombinase has never been applied in live animals.
  • FIG. 6 shows that the application of recombinase to genetically-engineered mice can result in dose dependent change in fur color.
  • the tip of Agouti mouse hairs are normally characterized by cells comprising yellow pheomelanin (A).
  • A yellow pheomelanin
  • B black eumelanin
  • C pure eumelanin
  • D melanin that the compartmentalized structure breaks down
  • transgenic cattle having customized skin color and patterns that can be used as a code (e.g., bar code) for cattle identification.
  • the transgenic cattle can be created using the method described in Example 2.
  • transdermal or intradermal application of recombinase to transgenic mice whose genomic comprises a Cre- LoxP recombination system induce customizable changes in coat color after birth.
  • Figure 7 shows a construct design for creating customized pattern or color identification in cattle.
  • the construct comprises a nucleic acid molecule encoding a dominant negative Rab7, operably linked to a MCIR promoter and under the control of the loxP- STOP-loxP sequence.
  • the expression of the Rab7 can be induced by the application of Cre recombinase.
  • the use of TAL nucleases allows site-specific knock-ins with short homology arms.
  • the acrosin promoter/EGFP arm on the construct allows flow sorting for genetically modified sperm, thereby ensuring 100% genetically modified offspring in the Fl generation.
  • MCIR melanocortin receptor
  • Figure 8 shows a depiction of a Black Angus heifer genetically engineered to express a customizable identification pattern in the skin.
  • transgenes are only expressed in the animal skin, which can be removed during food processing; therefore, the creation of cattle identification in the animal skin should not raise regulatory issues with governmental agencies such as USDA.
  • site-specific knock-in animals can be created using conventional technologies, including, but not limited to, spermatogonial stem cells (SSCs), piggyBacTM mobile DNA technology using transposable elements, Xanthamonas transcription activator- like (TAL) Nucleases (XTNs) [aka TAL-effector nucleases (TALENs)], and a combination thereof.
  • SSCs spermatogonial stem cells
  • piggyBacTM mobile DNA technology using transposable elements Xanthamonas transcription activator- like (TAL) Nucleases
  • TALENs TAL-effector nucleases
  • Black Angus coat color is post-natally modified with a heterozygous knock-in using a melanocyte-specific dominant negative Rab7, which is required for intracellular transport of the critical melanogenesis gene Tyrpl .
  • EXAMPLE 4 - CUSTOMIZED SKIN COLOR AND PATTERN FOR CATTLE DISEASE DETECTION Diseases are a concern for nearly every beef and dairy producer, and many common diseases can dramatically impact production without having overt clinical signs. Sudden death is often the first and only sign of clostridial diseases. Subclinical mastitis is a common and expensive problem in dairy production. Even rumors of bovine spongioform encephalopathy (BSE), which rarely has overt clinical signs, can cause severe economic damage to beef industry. In addition to infectious diseases, metabolic diseases in cattle can also cause economic damages: 1/3 of beef cattle have subclinical copper deficiency due to the presence of chelating agents in their diet. Changes in fur or skin color, in accordance with the present invention (e.g., using recombinase-activated coat-color specific markers) can be used to identify the presence of diseases in cattle.
  • the construct for cattle disease detection is identical to the construct as shown in Figure 7, except that the MC1R promoter is replaced with an alternate recombinase system (e.g., Flp-Frt recombination system) and/or disease-specific promoters, such as promoters responsive to NFkB, Ifny, or copper-deficiency targets.
  • an alternate recombinase system e.g., Flp-Frt recombination system
  • disease-specific promoters such as promoters responsive to NFkB, Ifny, or copper-deficiency targets.
  • cattle are painted with the recombinase to create the desired symbol.
  • the painted area would change color once the cattle develop inflammatory or metabolic stresses (the expression of pigment protein regulated by disease- specific promoter).
  • the cattle disease detection application can be used in combination with the cattle identification application through the use of different recombinases and recombinase targets.
  • Pugs have an intact melanocortin receptor with no endogenous expression of the "dominant black" signaling molecule PDefl03.
  • Dogs with customized fur color and pattern can be created using the methods described in Examples 3 and 4 for cattle, or through the process shown in Example 2 for mice.
  • genetically-engineered pugs whose genome comprises the construct as shown in Figure 7 have fur with customizable color and pattern including tiger stripes, logos, writing, and hearts.
  • Heat stress is a major cause of morbidity and mortality among cattle and has adverse economic impact on the cattle industry. Dark coat color substantially worsens the animal's ability to tolerate heat stress, with black-coated cattle showing more than five times the increase in core body temperature of white cattle.
  • Black Angus is the most popular breed of beef cattle in the United States. Partly due to its black coat, the Black Angus breed tolerates heat stress poorly and is not suited to live in hot climates. Problems of poor heat tolerance in Black Angus has been a long-standing problem in the cattle industry.
  • This Example provides a method of creating Angus cattle having a "white” coat via the introduction of a heterologous, non-native dominant white allele into the cattle.
  • a dominant white allele of Pmell7 (also called GP100 and Silv) in chicken is transferred to cattle.
  • Pmell7 relates to melanin assembly in melanosomes. While Charolais cattle with a recessive mutation in Pmell7 have white coat color, cattle with a dominant white allele are preferred. Dominantly acting Pmell7 alleles are found in mice, dogs, and horses. Transfer of these dominant white alleles to cattle can be accomplished via a knock-in of a dominant mutation to the recessive Pmell7 allele, or through introduction of a non-native dominant white Pmell7 at a locus different from the native Pmell7 allele.
  • Genes useful for creating animals (such as Angus cattle) with a white coat include, but are limited to, dominant alleles involved in melanosome assembly (such as Pmell7, MLPH, and the dominant negative Rab7 allele); and alleles encoding proteins that inhibit the transport of melanin to melanosomes.
  • dominant alleles involved in melanosome assembly such as Pmell7, MLPH, and the dominant negative Rab7 allele
  • alleles encoding proteins that inhibit the transport of melanin to melanosomes include, but are limited to, dominant alleles involved in melanosome assembly (such as Pmell7, MLPH, and the dominant negative Rab7 allele); and alleles encoding proteins that inhibit the transport of melanin to melanosomes.
  • transgenic animals with white coat color can be created by introducing dominant mutations in genes encoding proteins involved in melanocyte migration from the neural crest during development, thereby obtaining white animals (such as cattle) that lack melanocytes.
  • transgenic animals such as cattle with white coat color
  • siRNA constructs such as miRNA constructs
  • the present invention also provides transgenic animals (such as cattle) with white coat color, wherein genetic black markings can be created by dermal or transdermal application of recombinase proteins.
  • genes non-native to cattle can be used to create a "white" allele dominant over the constitutive ly active MCIR found in Black Angus, resulting in white or silver-haired offspring in an outbred cross between the engineered bull and a normal Black Angus cow.

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