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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L15/00—Egg products; Preparation or treatment thereof
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1036—Retroviridae, e.g. leukemia viruses
  • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
  • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/30—Bird
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/01—Animal expressing industrially exogenous proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/90—Vectors containing a transposable element
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
  • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
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  • C12N2830/00—Vector systems having a special element relevant for transcription
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
  • C12N2830/90—Vector systems having a special element relevant for transcription from vertebrates avian
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates generally to administration of a transposon-based vector to the reproductive tract in an animal.
• the reproductive tract includes an ovary, ova within an ovary, and an oviduct.
• Such administration results in incorporation of a gene of interest contained in the vector in the ovary, the oviduct or an ovum of the animal.
• the present invention further includes production of a protein encoded by the gene in an egg produced by the animal.
• Transgenic animals are desirable for a variety of reasons, including their potential as biological factories to produce desired molecules for pharmaceutical, diagnostic and industrial uses. This potential is attractive to the industry due to the inadequate capacity in facilities used for recombinant production of desired molecules and the increasing demand by the pharmaceutical industry for use of these facilities.
• transgenic animals Numerous attempts to produce transgenic animals have met several problems, including low rates of gene incorporation and unstable gene incorporation. Accordingly, improved gene technologies are needed for the development of transgenic animals for the production of desired molecules. Improved gene delivery technologies are also needed for the treatment of disease in animals and humans. Many diseases and conditions can be treated with gene-delivery technologies, which provide a gene of interest to a patient suffering from the disease or the condition. An example of such disease is Type 1 diabetes. Type 1 diabetes is an autoimmune disease that ultimately results in destruction of the insulin producing ⁇ -cells in the pancreas. Although patients with Type 1 diabetes may be treated adequately with insulin injections or insulin pumps, these therapies are only partially effective.
• Insulin replacement such as via insulin injection or pump administration, cannot fully reverse the defect in the vascular endothelium found in the hyperglycemic state (Pieper et al., 1996. Diabetes Res. Clin. Pract. Suppl. SI 57- S162).
• hyper- and hypoglycemia occurs frequently despite intensive home blood glucose monitoring.
• careful dietary constraints are needed to maintain an adequate ratio of calories consumed. This often causes major psychosocial stress for many diabetic patients.
• Development of gene therapies providing delivery of the insulin gene into the pancreas of diabetic patients could overcome many of these problems and result in improved life expectancy and quality of life.
• virus-based delivery vectors such as adeno and adeno-associated viruses, retroviruses, and other viruses, which have been attenuated to no longer replicate.
• viral vectors There are multiple problems associated with the use of viral vectors. Firstly, they are not tissue-specific. In fact, a gene therapy trial using adenovirus was recently halted because the vector was present in the patient's sperm (Gene trial to proceed despite fears that therapy could change child's genetic makeup. The New York Times, December 23, 2001). Secondly, viral vectors are likely to be transiently incorporated, which necessitates re-treating a patient at specified time intervals. (Kay, M.A., et al. 2001. Nature Medicine 7:33-40). Thirdly, there is a concern that a viral- based vector could revert to its virulent form and cause disease. Fourthly, viral-based vectors require a dividing cell for stable integration.
• inducible promoters are regulated by substances either produced or recognized by the transcription control elements within the cell in which the gene is incorporated.
• control of gene expression is desired in transgenic animals or humans so that incorporated genes are selectively activated at desired times and/or under the influence of specific substances. Accordingly, what is needed is a means to selectively activate genes introduced into the genome of cells of a transgenic animal or human.
• Transgenic animals include all egg-laying animals and milk-producing animals. Transgenic animals further include but are not limited to avians, fish, amphibians, reptiles, insects, mammals and humans.
• the animal is a milk-producing animal, including but not limited to bovine, porcine, ovine and equine animals.
• the animal is an avian animal.
• the animal is a mammal. Animals are made transgenic through administration of a composition comprising a transposon-based vector designed for incorporation of a gene of interest for production of a desired protein, together with an acceptable carrier.
• compositions of the present invention are introduced into the reproductive system of an animal.
• the compositions of the present invention are administered to a reproductive organ including, but not limited to, an oviduct, an ovary, or into the duct system of the mammary gland.
• the compositions of the present invention are may be administered to a reproductive organ of an animal through the cloaca.
• the compositions of the present invention may be directly administered to a reproductive organ or can be administered to an artery leading to the reproductive organ.
• the compositions of the present invention are introduced into the the reproductive system of an avian animal.
• the compositions of the present invention are introduced into the the intramammary duct system of a mammal.
• a transfection reagent is optionally added to the composition before administration.
• the transposon-based vectors of the present invention include a fransposase, operably-liriked to a first promoter, and a coding sequence for a protein or peptide of interest operably-linked to a second promoter, wherein the coding sequence for the protein or peptide of interest and its operably-linked promoter are flanked by fransposase insertion sequences recognized by the transposase.
• the transposon-based vector also includes the following characteristics: a) one or more modified Kozak sequences at the 3' end of the first promoter to enhance expression of the transposase; b) modifications of the codons for the first several N-terminal amino acids of the fransposase, wherein the nucleotide at the third base position of each codon is changed to an A or a T without changing the corresponding amino acid; c) addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) addition of an effective polyA sequence operably-linked to the transposase to further enhance expression of the fransposase gene.
• the effective polyA sequence is an avian optimized polyA sequence.
• the present invention also provides for tissue-specific incorporation and/or expression of a gene of interest.
• Tissue-specific incorporation of a gene of interest may be achieved by placing the transposase gene under the control of a tissue-specific promoter, whereas tissue-specific expression of a gene of interest may be achieved by placing the gene of interest under the control of a tissue-specific promoter.
• the gene of interest is transcribed under the influence of an ovalbumin, or other oviduct specific, promoter. Linking the gene of interest to an oviduct specific promoter in an egg-laying animal results in synthesis of a desired molecule and deposition of the desired molecule in a developing egg.
• the present invention advantageously produces a high number of fransgenic animals having a gene of interest stably incorporated.
• these transgenic animals successfully pass the desired gene to their progeny.
• the present invention can be used to obtain transgenic animals having the gene of interest incorporated into the germline through transfection of the ovary or the present invention can be used to obtain transgenic animals having the gene of interest incorporated into the oviduct in a tissue-specific manner.
• Both types of fransgenic animals of the present invention produce large amounts of a desired molecule encoded by the transgene.
• Transgenic egg-laying animals, particularly avians produce large amounts of a desired protein that is deposited in the egg for rapid harvest and purification.
• any desired gene may be incorporated into the novel transposon-based vectors of the present invention in order to synthesize a desired molecule in the transgenic animals.
• Proteins, peptides and nucleic acids are preferred desired molecules to be produced by the transgenic animals of the present invention.
• Particularly preferred proteins are antibody proteins and other immunopharmecuetical proteins.
• This invention provides a composition useful for the production of transgenic hens capable of producing substantially high amounts of a desired protein or peptide. Entire flocks of transgenic birds may be developed very quickly in order to produce industrial amounts of desired molecules.
• the present invention solves the problems inherent in the inadequate capacity of fermentation facilities used for bacterial production of molecules and provides a more efficient and economical way to produce desired molecules. Accordingly, the present invention provides a means to produce large amounts of therapeutic, diagnostic and reagent molecules.
• Transgenic chickens are excellent in terms of convenience and efficiency of manufacturing molecules such as proteins and peptides. Starting with a single transgenic rooster, thousands of transgenic offspring can be produced within a year. (In principle, up to forty million offspring could be produced in just three generations). Each fransgenic female is expected to lay at least 250 eggs/year, each potentially containing hundreds of milligrams of the selected protein. Flocks of chickens numbering in the hundreds of thousands are readily handled through established commercial systems. The technologies for obtaining eggs and fractionating them are also well known and widely accepted. Thus, for each therapeutic, diagnostic, or other protein of interest, large amounts of a substantially pure material can be produced at relatively low incremental cost.
• a wide range of recombinant peptides and proteins can be produced in transgenic egg-laying animals. Enzymes, hormones, antibodies, growth factors, semm proteins, commodity proteins, biological response modifiers, peptides and designed proteins may all be made through practice of the present invention. For example, rough estimates suggest that it is possible to produce in bulk growth hormone, insulin, or Factor VIII, and deposit them in egg whites, for an incremental cost in the order of one dollar per gram. At such prices it is feasible to consider administering such medical agents by inhalation or even orally, instead of through injection. Even if bioavailability rates through these avenues were low, the cost of a much higher effective-dose would not be prohibitive.
• the egg-laying fransgenic animal is an avian.
• the method of the present invention may be used in avians including Ratites, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, Cuculiformes, Columbiformes, Galliformes, Anseriformes, and Herodiones.
• the egg-laying fransgenic animal is a poultry bird. More preferably, the bird is a chicken, turkey, duck, goose or quail. Another preferred bird is a ratite, such as, an emu, an ostrich, a rhea, or a cassowary.
• Other preferred birds are partridge, pheasant, kiwi, parrot, parakeet, macaw, falcon, eagle, hawk, pigeon, cockatoo, song birds, jay bird, blackbird, finch, warbler, canary, toucan, mynah, or sparrow.
• Yet another object of the present invention is to provide a method to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector that are capable of producing a desired molecule, such as a protein, peptide or nucleic acid.
• Another object of the present invention is to provide a method to produce fransgenic animals through intraoviduct or intraovarian adminisfration of a transposon-based vector, wherein such administration results in modulation of endogenous gene expression.
• Still another object of the present invention is to provide a method to produce fransgenic avians through intraoviduct or intraovarian administration of a transposon- based vector that are capable of producing proteins or peptides and depositing these proteins or peptides in the egg.
• Another object of the present invention is to provide transgenic avians that contain a stably incorporated fransgene.
• Still another object of the present invention is to provide eggs containing desired proteins or peptides encoded by a fransgene incorporated into the transgenic avian that produces the egg.
• Another object of the present invention is to provide transgenic milk- producing animals that are capable of producing proteins or peptides and depositing these proteins or peptides in their milk.
• Yet another object of the present invention is to provide milk containing desired molecules encoded by a transgene incorporated into the transgenic milk- producing animals that produce the milk.
• Still another object of the present invention is to provide milk containing desired proteins or peptides encoded by a transgene incorporated into the transgenic milk-producing animals that produce the milk.
• An advantage of the present invention is that transgenic animals are produced with higher efficiencies than observed in the prior art.
• Another advantage of the present invention is that these transgenic animals possess high copy numbers of the transgene.
• Another advantage of the present invention is that the fransgenic animals produce large amounts of desired molecules encoded by the transgene.
• Still another advantage of the present invention is that desired molecules are produced by the transgenic animals much more efficiently and economically than prior art methods, thereby providing a means for large scale production of desired molecules, particularly proteins and peptides.
• Yet another advantage of the present invention is that the desired proteins and peptides are produced rapidly after making animals transgenic through introduction of the vectors of the present invention.
• Figure 1 depicts schematically a transposon-based vector containing a fransposase operably linked to a first promoter and a gene of interest operably-linked to a second promoter, wherein the gene of interest and its operably-linked promoter are flanked by insertion sequences (IS) recognized by the transposase.
• IS insertion sequences
• Pro designates a promoter.
• the size of the actual nucleotide sequence is not necessarily proportionate to the box representing that sequence.
• FIG. 2 depicts schematically a transposon-based vector for targeting deposition of a polypeptide in an egg white wherein Ov pro is the ovalbumin promoter, Ov protein is the ovalbumin protein and PolyA is a polyadenylation sequence.
• the TAG sequence includes a spacer sequence, the gp41 hairpin loop from
• HIV I HIV I and a protease cleavage site.
• Figure 3 depicts schematically a transposon-based vector for targeting deposition of a polypeptide in an egg white wherein Ovo pro is the ovomucoid promoter and Ovo SS is the ovomucoid signal sequence.
• the TAG sequence includes a spacer, the gp41 hairpin loop from HIV I and a protease cleavage site.
• Figure 4 depicts schematically a transposon based-vector for expression of an RNAi molecule.
• Tetj pro indicates a tetracycline inducible promoter whereas "pro” indicates the pro portion of a prepro sequence as described herein.
• Ovgen indicates approximately 60 base pairs of an ovalbumin gene
• Ovotrans indicates approximately 60 base pairs of an ovotransferrin gene
• Ovomucin indicates approximately 60 base pairs of an ovomucin gene.
• Figure 5 is a picture of an SDS-PAGE gel wherein a pooled fraction of an isolated proinsulin fusion protein was run in lanes 4 and 6. Lanes 1 and 10 of the gel contain molecular weight standards, lanes 2 and 8 contain non-frangenic chicken egg white, and lanes 3, 5, 7 and 9 are blank.
• the present invention provides a new, effective and efficient method of producing transgenic animals, particularly egg-laying animals and milk-producing animals, through administration of a composition comprising a transposon-based vector designed for incorporation of a gene of interest and production of a desired molecule.
• the transposon-based vectors are administered to a reproductive organ including, but not limited to, an oviduct, an ovary, or into the duct system of the mammary gland.
• the vectors may be directly administered to a reproductive organ or can be administered to an artery leading to the reproductive organ or to a lymph system proximate to the cells to be genetically altered.
• the vectors may be administered to a reproductive organ of an animal through the cloaca.
• One method of direct administration is by injection, and in one embodiment, the lumen of the magnum of the oviduct is injected with a transposon-based vector.
• Another method of direct administration is by injection, and in one embodiment, the lumen of the infundibulum of the oviduct is injected with a transposon-based vector.
• a preferred intrarterial administration is an administration into an artery that supplies the oviduct or the ovary.
• adminisfration of the transposon-based vector to an oviduct or an artery that leads to the oviduct results in incorporation of the vector into the epithelial and/or secretory cells of the oviduct.
• administration of the transposon-based vector to an ovary or an artery that leads to the ovary or a lymphatic system proximal to the ovary results in incorporation of the vector into an oocyte or a germinal disk inside the ovary.
• antibody is used interchangeably with the term “immunoglobulin” and is defined herein as a protein synthesized by an animal or a cell of the immune system in response to the presence of a foreign substance commonly referred to as an "antigen” or an “immunogen".
• antibody includes fragments of antibodies. Antibodies are characterized by specific affinity to a site on the antigen, wherein the site is referred to an "antigenic determinant” or an "epitope".
• Antigens can be naturally occurring or artificially engineered. Artificially engineered antigens include, but are not limited to, small molecules, such as small peptides, attached to haptens such as macromolecules, for example proteins, nucleic acids, or polysaccharides. Artificially designed or engineered variants of naturally occurring antibodies and artificially designed or engineered antibodies not occurring in nature are all included in the current definition. Such variants include conservatively substituted amino acids and other forms of substitution as described in the section concerning proteins and polypeptides.
• the term "egg-laying animal” includes all amniotes such as birds, turtles, lizards and monofeles. Monofeles are egg-laying mammals and include the platypus and echidna.
• the term "bird” or “fowl,” as used herein, is defined as a member of the Aves class of animals which are characterized as warmblooded, egg-laying vertebrates primarily adapted for flying.
• Avians include, without limitation, Ratites, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, Cuculiformes, Columbiformes, Galliformes, Anseriformes, and Herodiones.
• Ratite as used herein, is defined as a group of flightless, mostly large, running birds comprising several orders and including the emus, ostriches, kiwis, and cassowaries.
• Psittaciformes includes parrots and refers to a monofamilial order of birds that exhibit zygodactylism and have a strong hooked bill.
• a "parrot” is defined as any member of the avian family Psittacidae (the single family of the Psittaciformes), distinguished by the short, stout, strongly hooked beak.
• Avians include all poultry birds, especially chickens, geese, turkeys, ducks and quail.
• chickens used for table egg production such as egg-type chickens, chickens reared for public meat consumption, or broilers, and chickens reared for both egg and meat production ("dual-purpose” chickens).
• the term “chicken” also denotes chickens produced by primary breeder companies, or chickens that are the parents, grandparents, great-grandparents, etc. of those chickens reared for public table egg, meat, or table egg and meat consumption.
• egg is defined herein as including a large female sex cell enclosed in a porous, calcarous or leathery shell, produced by birds and reptiles.
• ovum is defined as a female gamete, and is also known as an egg. Therefore, egg production in all animals other than birds and reptiles, as used herein, is defined as the production and discharge of an ovum from an ovary, or "ovulation”. Accordingly, it is to be understood that the term “egg” as used herein is defined as a large female sex cell enclosed in a porous, calcarous or leathery shell, when a bird or reptile produces it, or it is an ovum when it is produced by all other animals.
• milk-producing animal refers herein to mammals including, but not limited to, bovine, ovine, porcine, equine, and primate animals. Milk-producing animals include but are not limited to cows, llamas, camels, goats, reindeer, zebu, water buffalo, yak, horses, pigs, rabbits, non-human primates, and humans.
• transgenic animal refers to an animal having at least a portion of the transposon-based vector DNA incorporated into its DNA. While a transgenic animal includes an animal wherein the transposon-based vector DNA is incorporated into the germline DNA, a transgenic animal also includes an animal having DNA in one or more cells that contain a portion of the transposon-based vector DNA for any period of time.
• a portion of the transposon-based vector comprises a gene of interest. More preferably, the gene of interest is incorporated into the animal's DNA for a period of at least five days, more preferably the reproductive life of the animal, and most preferably the life of the animal.
• the animal is an avian.
• vector is used interchangeably with the terms “construct”, “DNA construct” and “genetic construct” to denote synthetic nucleotide sequences used for manipulation of genetic material, including but not limited to cloning, subcloning, sequencing, or introduction of exogenous genetic material into cells, tissues or organisms, such as birds. It is understood by one skilled in the art that vectors may contain synthetic DNA sequences, naturally occurring DNA sequences, or both.
• the vectors of the present invention are transposon-based vectors as described herein.
• operably-linked is defined herein to mean that the two sequences are associated in a manner that allows the regulatory sequence to affect expression of the other nucleotide sequence. It is not required that the operably-linked sequences be directly adjacent to one another with no intervening sequence(s).
• regulatory sequence is defined herein as including promoters, enhancers and other expression confrol elements such as polyadenylation sequences, matrix attachment sites, insulator regions for expression of multiple genes on a single construct, ribosome entry/attachment sites, introns that are able to enhance expression, and silencers.
• DNA construct is an important factor in successfully producing fransgenic animals.
• the DNA (or RNA) constructs previously used often do not integrate into the host DNA, or integrate only at low frequencies. Other factors may have also played a part, such as poor entry of the vector into target cells.
• the present invention provides transposon-based vectors that can be administered to an animal that overcome the prior art problems relating to low transgene integration frequencies.
• pTnMCS SEQ ID NO:2
• pTnMod SEQ ID NO:3
• the transposon-based vectors of the present invention produce integration frequencies an order of magnitude greater than has been achieved with previous vectors. More specifically, intratesticular injections performed with a prior art transposon-based vector (described in U.S. Patent No. 5,719,055) resulted in 41% sperm positive roosters whereas intratesticular injections performed with the novel transposon-based vectors of the present invention resulted in 77% sperm positive roosters.
• Actual frequencies of integration were estimated by either or both comparative strength of the PCR signal from the sperm and histological evaluation of the testes and sperm by quantitative PCR.
• the transposon-based vectors of the present invention include a fransposase gene operably-linked to a first promoter, and a coding sequence for a desired protein or peptide operably-linked to a second promoter, wherein the coding sequence for the desired protein or peptide and its operably-linked promoter are flanked by transposase insertion sequences recognized by the transposase.
• the transposon-based vector also includes one or more of the following characteristics: a) one or more modified Kozak sequences comprising ACCATG (SEQ ID NO:l) at the 3' end of the first promoter to enhance expression of the transposase; b) modifications of the codons for the first several N-terminal amino acids of the transposase, wherein the third base of each codon was changed to an A or a T without changing the corresponding amino acid; c) addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) addition of an effective polyA sequence operably-linked to the fransposase to further enhance expression of the transposase gene.
• ACCATG SEQ ID NO:l
• the transposon-based vector may additionally or alternatively include one or more of the following Kozak sequences at the 3' end of any promoter, including the promoter operably- linked to the transposase: ACCATGG (SEQ ID NO:4), AAGATGT (SEQ ID NO:5), ACGATGA (SEQ ID NO:6), AAGATGG (SEQ ID NO:7), GACATGA (SEQ ID NO:8), ACCATGA (SEQ ID NO:9), and ACCATGA (SEQ ID NO:10), ACCATGT (SEQ ID NO:52).
• Figure 1 shows a schematic representation of several components of the transposon-based vector.
• the present invention further includes vectors containing more than one gene of interest, wherein a second or subsequent gene of interest is operably-linked to the second promoter or to a different promoter.
• the transposon-based vectors shown in the Figures are representative of the present invention and that the order of the vector elements may be different than that shown in the Figures, that the elements may be present in various orientations, and that the vectors may contain additional elements not shown in the Figures.
• the transposase found in the transposase-based vector is an altered target site (ATS) transposase and the insertion sequences are those recognized by the ATS fransposase.
• ATS target site
• the transposase located in the transposase-based vectors is not limited to a modified ATS transposase and can be derived from any transposase.
• Transposases known in the prior art include those found in AC7, Tn5SEQl, Tn916, Tn951, Tnl721, Tn 2410, Tnl681, Tnl, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, TnlO, Tn30, TnlOl, Tn903, Tn501, TnlOOO ( ⁇ ), Tnl681, Tn2901, AC transposons, Mp transposons, Spm transposons, En transposons, Dotted transposons, Mu transposons, Ds transposons, dSpm transposons and I transposons.
• these transposases and their regulatory sequences are modified for improved functioning as follows: a) the addition one or more modified Kozak sequences comprising ACCATG (SEQ ID NO:l) at the 3' end of the promoter operably-linked to the transposase; b) a change of the codons for the first several amino acids of the transposase, wherein the third base of each codon was changed to an A or a T without changing the corresponding amino acid; c) the addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) the addition of an effective polyA sequence operably-linked to the transposase to further enhance expression of the fransposase gene.
• ACCATG SEQ ID NO:l
• the modifications of the first several N-terminal codons of the fransposase gene increase transcription of the transposase gene, in part, by increasing strand dissociation. It is preferable that between approximately 1 and 20, more preferably 3 and 15, and most preferably between 4 and 12 of the first N-terminal codons of the transposase are modified such that the third base of each codon is changed to an A or a T without changing the encoded amino acid. In one embodiment, the first ten N- terminal codons of the transposase gene are modified in this manner. It is also preferred that the transposase contain mutations that make it less specific for preferred insertion sites and thus increases the rate of fransgene insertion as discussed in U.S. Patent No. 5,719,055.
• the transposon-based vectors are optimized for expression in a particular host by changing the methylation patterns of the vector DNA. For example, prokaryotic methylation may be reduced by using a methylation deficient organism for production of the transposon-based vector.
• the transposon- based vectors may also be methylated to resemble eukaryotic DNA for expression in a eukaryotic host.
• Transposases and insertion sequences from other analogous eukaryotic transposon-based vectors that can also be modified and used are, for example, the Drosophila P element derived vectors disclosed in U.S. Patent No. 6,291,243; the Drosophila mariner element described in Sherman et al. (1998); or the sleeping beauty transposon. See also Hackett et al. (1999); D. JLampe et al., 1999. Proc. Natl. Acad. Sci. USA, 96:11428-11433; S. Fischer et al., 2001. Proc. Natl. Acad. Sci. USA, 98:6759-6764; L. Zagoraiou et al., 2001. Proc.
• transposase-based vector will contain insertion sequences recognized by the particular transposase also found in the transposase-based vector.
• the insertion sequences have been shortened to about 70 base pairs in length as compared to those found in wild-type transposons that typically contain insertion sequences of well over 100 base pairs.
• the present invention also encompasses the use of a "rolling replication" type fransposon-based vector.
• Use of a rolling replication type transposon allows multiple copies of the transposon/transgene to be made from a single transgene construct and the copies inserted. This type of fransposon-based system thereby provides for insertion of multiple copies of a fransgene into a single genome.
• a rolling replication type transposon-based vector may be preferred when the promoter operably-linked to gene of interest is endogenous to the host cell and present in a high copy number or highly expressed.
• Tnl, Tn2, Tn3, Tn4, Tn5, Tn9, Tn21, Tn501, Tn551, Tn951, Tnl721, Tn2410 and Tn2603 are examples of a rolling replication type transposon, although Tn5 could be both a rolling replication and a cut and insert type transposon. Stop Codons and PolyA Sequences
• the transposon-based vector contains two stop codons operably-linked to the transposase and/or to the gene of interest.
• one stop codon of UAA or UGA is operably linked to the transposase and/or to the gene of interest.
• an "effective polyA sequence" refers to either a synthetic or non-synthetic sequence that contains multiple and sequential nucleotides containing an adenine base (an A polynucleotide string) and that increases expression of the gene to which it is operably-linked.
• a polyA sequence may be operably-linked to any gene in the transposon-based vector including, but not limited to, a transposase gene and a gene of interest.
• a preferred polyA sequence is optimized for use in the host animal or human.
• the polyA sequence is optimized for use in an avian species and more specifically, a chicken.
• An avian optimized polyA sequence generally contains a minimum of 40 base pairs, preferably between approximately 40 and several hundred base pairs, and more preferably approximately 75 base pairs that precede the A polynucleotide string and thereby separate the stop codon from the A polynucleotide string.
• the polyA sequence comprises a conalbumin polyA sequence as provided in SEQ ID NO:l 1 and as taken from GenBank accession # Y00407, base pairs 10651-11058.
• the polyA sequence comprises a synthetic polynucleotide sequence shown in SEQ ID NO: 12.
• the polyA sequence comprises an avian optimized polyA sequence provided in SEQ ID NO: 13.
• a chicken optimized polyA sequence may also have a reduced amount of CT repeats as compared to a synthetic polyA sequence.
• the present invention includes methods of or increasing incorporation of a gene of interest wherein the gene of interest resides in a transposon-based vector containing a transposase gene and wherein the transposase gene is operably linked to an avian optimized polyA sequence.
• the present invention also includes methods of increasing expression of a gene of interest in an avian that includes administering a gene of interest to the avian, wherein the gene of interest is operably-linked to an avian optimized polyA sequence.
• An avian optimized polyA nucleotide string is defined herein as a polynucleotide containing an A polynucleotide string and a minimum of 40 base pairs, preferably between approximately 40 and several hundred base pairs, and more preferably approximately 60 base pairs that precede the A polynucleotide string.
• the present invention further provides transposon-based vectors containing a gene of interest or transposase gene operably linked to an avian optimized polyA sequence.
• the first promoter operably-linked to the transposase gene and the second promoter operably-linked to the gene of interest can be a constitutive promoter or an inducible promoter.
• Constitutive promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter, herpes simplex virus 1 (HSV1) immediate early promoter, SV40 promoter, lysozyme promoter, early and late CMV promoters, early and late HSV promoters, ?-actin promoter, tubulin promoter, Rous-Sarcoma virus (RSV) promoter, and heat-shock protein (HSP) promoter.
• CMV immediate early cytomegalovirus
• HSV40 promoter herpes simplex virus 1 immediate early promoter
• lysozyme promoter early and late CMV promoters
• early and late HSV promoters early and late HSV promoters
• ?-actin promoter tubulin promoter
• Inducible promoters include tissue-specific promoters, developmentally-regulated promoters and chemically inducible promoters.
• tissue-specific promoters include the glucose 6 phosphate (G6P) promoter, vitellogenin promoter, ovalbumin promoter, ovomucoid promoter, conalbumin promoter, ovofransferrin promoter, prolactin promoter, kidney uromodulin promoter, and placental lactogen promoter.
• the vitellogenin promoter includes a polynucleotide sequence of SEQ ID NO: 14.
• the G6P promoter sequence may be deduced from a rat G6P gene untranslated upstream region provided in GenBank accession number U57552.1.
• Examples of developmentally-regulated promoters include the homeobox promoters and several hormone induced promoters.
• Examples of chemically inducible promoters include reproductive hormone induced promoters and antibiotic inducible promoters such as the tetracycline inducible promoter and the zinc-inducible metallothionine promoter.
• inducible promoter systems include the Lac operator repressor system inducible by IPTG (isopropyl beta-D-thiogalactoside) (Cronin, A. et al. 2001. Genes and Development, v. 15), ecdysone-based inducible systems (Hoppe, U. C. et al. 2000. Mol. Ther. 1:159-164); estrogen-based inducible systems (Braselmann, S. et al. 1993. Proc. Natl. Acad. Sci.
• progesterone-based inducible systems using a chimeric regulator, GLVP, which is a hybrid protein consisting of the GAL4 binding domain and the herpes simplex virus transcriptional activation domain, VP16, and a truncated form of the human progesterone receptor that retains tlie ability to bind ligand and can be turned on by RU486 (Wang, et al. 1994. Proc. Natl. Acad. Sci.
• GLVP chimeric regulator
• CID-based inducible systems using chemical inducers of dimerization (CIDs) to regulate gene expression, such as a system wherein rapamycin induces dimerization of the cellular proteins FKBP12 and FRAP (Belshaw, P. J. et al. 1996. J. Chem. Biol. 3:731-738; Fan, L. et al. 1999. Hum. Gene Ther. 10:2273-2285; Shariat, S.F. et al. 2001. Cancer Res. 61:2562-2571; Spencer, D.M. 1996. Curr. Biol. 6:839- 847).
• Chemical substances that activate the chemically inducible promoters can be administered to the animal containing the fransgene of interest via any method known to those of skill in the art.
• cell or tissue-specific and constitutive promoters include but are not limited to smooth-muscle SM22 promoter, including chimeric SM22alpha/telokin promoters (Hoggatt A.M. et al., 2002. Circ Res. 91(12): 1151-9); ubiquitin C promoter (Biochim Biophys Acta, 2003. Jan. 3;1625(l):52-63); Hsf2 promoter; murine COMP (cartilage oligomeric matrix protein) promoter; early B cell- specific mb-1 promoter (Sigvardsson M., et al., 2002. Mol. Cell Biol.
• PSA prostate specific antigen
• promoter of the human FAT/CD36 gene (Kuriki C, et al., 2002. Biol. Pharm. Bull. 25(11): 1476-8); VL30 promoter (Staplin W.R. et al., 2002. Blood October 24, 2002); and, IL-10 promoter (Brenner S., et al., 2002. J. Biol. Chem. December 18, 2002).
• avian promoters include, but are not limited to, promoters controlling expression of egg white proteins, such as ovalbumin, ovotransferrin (conalbumin), ovomucoid, lysozyme, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, ovostatin (ovomacroglobin), cystatin, avidin, thiamine-binding protein, glutamyl aminopeptidase minor glycoprotein 1, minor glycoprotein 2; and promoters controlling expression of egg-yolk proteins, such as vitellogenin, very low- density lipoproteins, low density lipoprotein, cobalamin-binding protein, riboflavin- binding protein, biotin-binding protein (Awade, 1996.
• egg white proteins such as ovalbumin, ovotransferrin (conalbumin), ovomucoid, lysozyme, ovomucin
• vitellogenin promoter is that it is active during the egg-laying stage of an animal's life-cycle, which allows for the production of the protein of interest to be temporally connected to the import of the protein of interest into the egg yolk when the protein of interest is equipped with an appropriate targeting sequence.
• the avian promoter is an oviduct-specific promoter.
• oviduct-specific promoter includes, but is not limited to, ovalbumin; ovotransferrin (conalbumin); ovomucoid; 01, 02, 03, 04 or 05 avidin; ovomucin; g2 ovoglobulin; g3 ovoglobulin; ovoflavoprotein; and ovostatin (ovomacroglobin) promoters.
• liver-specific promoters may be operably-linked to the gene of interest to achieve liver- specific expression of the transgene.
• Liver-specific promoters of the present invention include, but are not limited to, the following promoters, vitellogenin promoter, G6P promoter, cholesterol-7-alpha-hydroxylase (CYP7A) promoter, phenylalanine hydroxylase (PAH) promoter, protein C gene promoter, insulin-like growth factor I (IGF-I) promoter, bilirabin UDP-glucuronosylfransferase promoter, aldolase B promoter, furin promoter, metallothioneine promoter, albumin promoter, and insulin promoter.
• promoters that can be used to target expression of a protein of interest into the milk of a milk-producing animal including, but not limited to, ⁇ lactoglobin promoter, whey acidic protein promoter, lactalbumin promoter and casein promoter.
• immune system-specific promoters When germline transformation occurs via intraovarian administration, immune system-specific promoters may be operably-linked to the gene of interest to achieve immune system-specific expression of the transgene. Accordingly, promoters associated with cells of the immune system may also be used. Acute phase promoters such as interleukin (IL)-1 and IL-2 may be employed. Promoters for heavy and light chain Ig may also be employed. The promoters of the T cell receptor components CD4 and CD8, B cell promoters and the promoters of CR2 (complement receptor type 2) may also be employed. Immune system promoters are preferably used when the desired protein is an antibody protein.
• modified promoters/enhancers wherein elements of a single promoter are duplicated, modified, or otherwise changed.
• steroid hormone-binding domains of the ovalbumin promoter are moved from about -6.5 kb to within approximately the first 1000 base pairs of the gene of interest. Modifying an existing promoter with promoter/enhancer elements not found naturally in the promoter, as well as building an entirely synthetic promoter, or drawing promoter/enhancer elements from various genes together on a non-natural backbone, are all encompassed by the current invention.
• the promoters contained within the fransposon-based vectors of the present invention may be entire promoter sequences or fragments of promoter sequences.
• the promoter operably linked to a gene of interest is an approximately 900 base pair fragment of a chicken ovalbumin promoter (SEQ ID NO: 15).
• the constitutive and inducible promoters contained within the transposon-based vectors may also be modified by the addition of one or more modified Kozak sequences of ACCATG (SEQ ID NO:l).
• the present invention includes fransposon-based vectors containing one or more enhancers.
• enhancers may or may not be operably- linked to their native promoter and may be located at any distance from their operably-linked promoter.
• a promoter operably-linked to an enhancer and a promoter modified to eliminate repressive regulatory effects are referred to herein as an "enhanced promoter.”
• the enhancers contained within the transposon-based vectors are preferably enhancers found in birds, and more preferably, an ovalbumin enhancer, but are not limited to these types of enhancers.
• an approximately 675 base pair enhancer element of an ovalbumin promoter is cloned upstream of an ovalbumin promoter with 300 base pairs of spacer DNA separating the enhancer and promoter.
• the enhancer used as a part of the present invention comprises base pairs 1-675 of a chicken ovalbumin enhancer from GenBank accession #S82527.1. The polynucleotide sequence of this enhancer is provided in SEQ ID NO: 16.
• cap sites and fragments of cap sites are also included in some of the transposon-based vectors of the present invention.
• approximately 50 base pairs of a 5' untranslated region wherem the capsite resides are added on the 3' end of an enhanced promoter or promoter.
• An exemplary 5' untranslated region is provided in SEQ ID NO: 17.
• a putative cap-site residing in this 5' untranslated region preferably comprises the polynucleotide sequence provided in SEQ ID NO: 18.
• the first promoter operably-linked to the transposase gene is a constitutive promoter and the second promoter operably- linked to the gene of interest is a tissue-specific promoter.
• the first constitutive promoter allows for constitutive activation of the fransposase gene and incorporation of the gene of interest into virtually all cell types, including the germline of the recipient animal. Although the gene of interest is incorporated into the germline generally, the gene of interest may only be expressed in a tissue-specific manner.
• a fransposon-based vector having a constitutive promoter operably-linked to the transposase gene can be administered by any route, and in one embodiment, the vector is administered to an ovary, to an artery leading to the ovary or to a lymphatic system or fluid proximal to the ovary.
• cell- or tissue-specific expression as described herein does not require a complete absence of expression in cells or tissues other than the preferred cell or tissue. Instead, "cell-specific” or “tissue-specific” expression refers to a majority of the expression of a particular gene of interest in the preferred cell or tissue, respectively.
• the first promoter operably-linked to the transposase gene can be a tissue-specific promoter.
• tissue-specific promoter For example, transfection of a fransposon-based vector containing a fransposase gene operably-linked to an oviduct specific promoter such as the ovalbumin promoter provides for activation of the transposase gene and incorporation of the gene of interest in the cells of the oviduct but not into the germline and other cells generally.
• the second promoter operably-linked to the gene of interest can be a constitutive promoter or an inducible promoter. In a preferred embodiment, both the first promoter and the second promoter are an ovalbumin promoter.
• the fransposon-based vector is administered directly to the tissue of interest, to an artery leading to the tissue of interest or to fluids surrounding the tissue of interest.
• the tissue of interest is the oviduct and administration is achieved by direct injection into the oviduct or an artery leading to the oviduct.
• adminisfration is achieved by direct injection into the lumen of the magnum or the infundibulum of the oviduct. Indirect administration to the oviduct may occur through the cloaca.
• cell specific promoters may be used to enhance transcription in selected tissues.
• promoters that are found in cells of the fallopian tube such as ovalbumin, conalbumin, ovomucoid and/or lysozyme, are used in the vectors to ensure transcription of the gene of interest in the epithelial cells and tubular gland cells of the fallopian tube, leading to synthesis of the desired protein encoded by the gene and deposition into the egg white.
• promoters specific for the epithelial cells of the alveoli of the mammary gland such as prolactin, insulin, beta lactoglobin, whey acidic protein, lactalbumin, casein, and/or placental lactogen, are used in the design of vectors used for transfection of these cells for the production of desired proteins for deposition into the milk.
• the G6P promoter may be employed to drive transcription of the gene of interest for protein production. Proteins made in the liver of birds may be delivered to the egg yolk.
• the promoter and other regulatory sequences operably-linked to the transposase gene may be those derived from the host.
• host specific regulatory sequences can be tissue specific as described above or can be of a constitutive nature.
• an avian actin promoter and its associated polyA sequence can be operably- linked to a transposase in a transposase-based vector for transfection into an avian.
• Examples of other host specific promoters that could be operably-linked to the transposase include the myosin and DNA or RNA polymerase promoters.
• Directing Sequences In some embodiments of the present invention, the gene of interest is operably-linked to a directing sequence or a sequence that provides proper conformation to the desired protein encoded by the gene of interest.
• the term "directing sequence" refers to both signal sequences and targeting sequences.
• An egg directing sequence includes, but is not limited to, an ovomucoid signal sequence, an ovalbumin signal sequence, a cecropin pre pro signal sequence, and a vitellogenin targeting sequence.
• the term "signal sequence” refers to an amino acid sequence, or the polynucleotide sequence that encodes the amino acid sequence, that directs the protein to which it is linked to the endoplasmic reticulum in a eukaryote, and more preferably the translocational pores in the endoplasmic reticulum, or the plasma membrane in a prokaryote, or mitochondria, such as for the purpose of gene therapy for mitochondrial diseases.
• Signal and targeting sequences can be used to direct a desired protein into, for example, the milk, when the fransposon-based vectors are administered to a milk-producing animal.
• Signal sequences can also be used to direct a desired protein into, for example, a secretory pathway for incorporation into the egg yolk or the egg white, when the fransposon-based vectors are administered to a bird or other egg-laying animal.
• a transposon-based vector is provided in Figure 3 wherein the gene of interest is operably linked to the ovomucoid signal sequence.
• the present invention also includes a gene of interest operably-linked to a second gene containing a signal sequence.
• Figure 2 An example of such an embodiment is shown in Figure 2 wherein the gene of interest is operably-linked to the ovalbumin gene that contains an ovalbumin signal sequence.
• the signal sequence is an ovalbumin signal sequence including a sequence shown in SEQ ID NO: 19.
• the signal sequence is a modified ovalbumin signal sequence including a sequence shown in SEQ ID NO:20 or SEQ ID NO:21.
• targeting sequence refers to an amino acid sequence, or the polynucleotide sequence encoding the amino acid sequence, which amino acid sequence is recognized by a receptor located on the exterior of a cell. Binding of the receptor to the targeting sequence results in uptake of the protein or peptide operably-linked to the targeting sequence by the cell.
• a targeting sequence is a vitellogenin targeting sequence that is recognized by a vitellogenin receptor (or the low density lipoprotein receptor) on the exterior of an oocyte.
• the vitellogenin targeting sequence includes the polynucleotide sequence of SEQ ID NO: 22.
• the vitellogenin targeting sequence includes all or part of the vitellogenin gene.
• VLDL and Apo E are also capable of binding the vitellogenin receptor. Since the ApoE protein is not endogenously expressed in birds, its presence may be used advantageously to identify birds carrying the transposon- based vectors of the present invention.
• a gene of interest selected for stable incorporation is designed to encode any desired protein or peptide or to regulate any cellular response.
• the desired proteins or peptides are deposited in an egg or in milk.
• the present invention encompasses fransposon-based vectors containing multiple genes of interest.
• the multiple genes of interest may each be operably-linked to a separate promoter and other regulatory sequence(s) or may all be operably-linked to the same promoter and other regulatory sequences(s).
• multiple gene of interest are linked to a single promoter and other regulatory sequence(s) and each gene of interest is separated by a cleavage site or a pro portion of a signal sequence.
• a gene of interest may contain modifications of the codons for the first several N-terminal amino acids of the gene of interest, wherein the third base of each codon is changed to an A or a T without changing the corresponding amino acid.
• Protein and peptide hormones are a preferred class of proteins in the present invention.
• Such protein and peptide hormones are synthesized throughout the endocrine system and include, but are not limited to, hypothalamic hormones and hypophysiofropic hormones, anterior, intermediate and posterior pituitary hormones, pancreatic islet hormones, hormones made in the gastrointestinal system, renal hormones, thymic hormones, parathyroid hormones, adrenal cortical and medullary hormones.
• hormones that can be produced using the present invention include, but are not limited to, chorionic gonadotropin, corticotropin, erythropoietin, glucagons, IGF-1, oxytocin, platelet-derived growth factor, calcitonin, follicle- stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, insulin, gonadotropin-releasing hormone and its analogs, vasopressin, octreotide, somatostatin, prolactin, adrenocorticofropic hormone, antidiuretic hormone, thyrotropin-releasing hormone (TRH), growth hormone-releasing hormone (GHRH), dopamine, melatonin, thyroxm (T ), parathyroid hormone (PTH), glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, androgens such as testosterone, adrenaline (epinephrine), noradrenaline (nor
• the gene of interest is a proinsulin gene and the desired molecule is insulin.
• Proinsulin consists of three parts: a C- peptide and two strands of amino acids (the alpha and beta chains) that later become linked together to form the insulin molecule.
• Figures 2 and 3 are schematics of transposon-based vector constructs containing a proinsulin gene operably-linked to an ovalbumin promoter and ovalbumin protein or an ovomucoid promoter and ovomucoid signal sequence, respectively.
• proinsulin is expressed in the oviduct tubular gland cells and then deposited in the egg white.
• SEQ ID NO: 23 One example of a proinsulin polynucleotide sequence is shown in SEQ ID NO: 23, wherem the C-peptide cleavage site spans from Arg at position 31 to Arg at position 65.
• Semm proteins including lipoproteins such as high density lipoprotein (HDL), HDL-Milano and low density lipoprotein, albumin, clotting cascade factors, factor VIII, factor IX, fibrinogen, and globulins are also included in the group of desired proteins of the present invention.
• Immunoglobulins are one class of desired globulin molecules and include but are not limited to IgG, IgM, IgA, IgD, IgE, IgY, lambda chains, kappa chains and fragments thereof; Fc fragments, and Fab fragments. Desired antibodies include, but are not limited to, naturally occurring antibodies, human antibodies, humanized antibodies, and hybrid antibodies.
• transposon-based vectors of the present invention may be incorporated into the transposon-based vectors of the present invention. Desired antibodies also include antibodies with the ability to bind specific ligands, for example, antibodies against proteins associated with cancer-related molecules, such as anti-her 2, or anti- CA125. Accordingly, the present invention encompasses a fransposon-based vector containing one or more genes encoding a heavy immunoglobulin (Ig) chain and a light Ig chain. Further, more than one gene encoding for more than one antibody may be administered in one or more transposon-based vectors of the present invention. In this manner, an egg may contain more than one type of antibody in the egg white, the egg yolk or both. In one embodiment, a transposon-based vector contains a heavy Ig chain and a light Ig chain, both operably linked to a promoter.
• Ig immunoglobulin
• a transposon-based vector contains a heavy Ig chain and a light Ig chain, both operably linked to a
• Antibodies used as therapeutic reagents include but are not limited to antibodies for use in cancer immunotherapy agamst specific antigens, or for providing passive immunity to an animal or a human against an infectious disease or a toxic agent.
• Antibodies used as diagnostic reagents include, but are not limited to antibodies that may be labeled and detected with a detector, for example antibodies with a fluorescent label attached that may be detected following exposure to specific wavelengths.
• Such labeled antibodies may be primary antibodies directed to a specific antigen, for example, rhodamine-labeled rabbit anti-growth hormone, or may be labeled secondary antibodies, such as fluorescein-labeled goat-anti chicken IgG.
• Such labeled antibodies are known to one of ordinary skill in the art.
• Labels useful for attachment to antibodies are also known to one of ordinary skill in the art. Some of these labels are described in the "Handbook of Fluorescent Probes and Research Products", ninth edition, Richard P. Haugland (ed) Molecular Probes, Inc. Eugene, OR), which is incorporated herein in its entirety.
• Antibodies produced with using the present invention may be used as laboratory reagents for numerous applications including radioimmunoassay, western blots, dot blots, ELISA, immunoaffinity columns and other procedures requiring antibodies as known to one of ordinary skill in the art.
• Such antibodies include primary antibodies, secondary antibodies and tertiary antibodies, which may be labeled or unlabeled.
• Antibodies that may be made with the practice of the present invention include, but are not limited to primary antibodies, secondary antibodies, designer antibodies, anti-protein antibodies, anti-peptide antibodies, anti-DNA antibodies, anti- RNA antibodies, anti-hormone antibodies, anti-hypophysiotropic peptides, antibodies against non-natural antigens, anti-anterior pituitary hormone antibodies, anti-posterior pituitary hormone antibodies, anti-venom antibodies, anti-tumor marker antibodies, antibodies directed against epitopes associated with infectious disease, including, antiviral, anti-bacterial, anti-protozoal, anti-fungal, anti-parasitic, anti-receptor, anti-lipid, anti-phospholipid, anti-growth factor, anti-cytokine, anti-monokine, anti-idiotype, and anti-accessory (presentation) protein antibodies. Antibodies made with the present invention, as well as light chains or heavy chains, may also be used to inhibit enzyme activity.
• Antibodies that may be produced using the present invention include, but are not limited to, antibodies made against the following proteins: Bovine ⁇ -Globulin, Semm; Bovine IgG, Plasma; Chicken ⁇ -Globulin, Serum; Human ⁇ -Globulin, Serum; Human IgA, Plasma; Human IgAi, Myeloma; Human IgA 2 , Myeloma; Human IgA 2 , Plasma; Human IgD, Plasma; Human IgE, Myeloma; Human IgG, Plasma; Human IgG, Fab Fragment, Plasma; Human IgG, F(ab') 2 Fragment, Plasma; Human IgG, Fc Fragment, Plasma; Human IgGi, Myeloma; Human IgG 2 , Myeloma; Human IgG 3 , Myeloma; Human IgG 4 , Myeloma; Human IgM, Myeloma; Human Ig
• the transposon-based vector comprises the coding sequence of light and heavy chains of a murine monoclonal antibody that shows specificity for human seminoprotein (GenBank Accession numbers AY129006 and AY129304 for the light and heavy chains, respectively).
• a further non-limiting list of antibodies that recognize other antibodies is as follows: Anti-Chicken IgG, heavy (H) & light (L) Chain Specific (Sheep); Anti-Goat ⁇ -Globulin (Donkey); Anti-Goat IgG, Fc Fragment Specific (Rabbit); Anti-Guinea Pig ⁇ -Globulin (Goat); Anti-Human Ig, Light Chain, Type K Specific; Anti-Human Ig, Light Chain, Type ⁇ Specific; Anti-Human IgA, o.-Chain Specific (Goat); Anti- Human IgA, Fab Fragment Specific; Anti-Human IgA, Fc Fragment Specific; Anti- Human IgA, Secretory; Anti-Human IgE, e-Chain Specific (Goat); Anti-Human IgE, Fc Fragment Specific; Anti-Human IgG, Fc Fragment Specific (Goat); Anti-Human IgG, ⁇ -Chain Specific (Goat); Anti
• Antibodies made by the transgenic animals of the present invention include antibodies that may be used as therapeutic reagents, for example in cancer immunotherapy against specific antigens, as diagnostic reagents and as laboratory reagents for numerous applications including immunoneutralization, radioimmunoassay, western blots, dot blots, ELISA, immunoprecipitation and immunoaffinity columns.
• antibodies include, but are not limited to, antibodies which bind the following ligands: adrenomedulin, a ylin, calcitonin, amyloid, calcitonin gene-related peptide, cholecystokinin, gastrin, gastric inhibitory peptide, gastrin releasing peptide, interleukin, interferon, cortistatin, somatostatin, endothelin, sarafotoxin, glucagon, glucagon-like peptide, insulin, atrial natriuretic peptide, BNP, CNP, neurokinin, substance P, leptin, neuropeptide Y, melanin concentrating hormone, melanocyte stimulating hormone, orphanin, endorphin, dynorphin, enkephalin, enkephalin, leumorphin, peptide F, PACAP, PACAP-related peptide, parathyroid hormone, urocortin, cortico
• abciximab (ReoPro), abciximab anti- platelet aggregation monoclonal antibody, anti-CDlla (hull24), anti-CD18 antibody, anti-CD20 antibody, anti-cytomegalovirus (CMV) antibody, anti-digoxin antibody, anti-hepatitis B antibody, anti-HER-2 antibody, anti-idiotype antibody to GD3 glycolipid, anti-IgE antibody, anti-IL-2R antibody, antimetastatic cancer antibody (mAb 17-1A), anti-rabies antibody, anti-respiratory syncytial virus (RSV) antibody, anti-Rh antibody, anti-TCR, anti-TNF antibody, anti-VEGF antibody and fab fragment thereof, rattlesnake venom antibody, black widow spider venom antibody, coral snake venom antibody, antibody against very late antigen-4 (VLA-4), C225 humanized antibody to EGF receptor, chimeric (human & mouse
• the antibodies prepared using the methods of the present invention may also be designed to possess specific labels that may be detected through means known to one of ordinary skill in the art.
• the antibodies may also be designed to possess specific sequences useful for purification through means known to one of ordinary skill in the art.
• Specialty antibodies designed for binding specific antigens may also be made in transgenic animals using the transposon-based vectors of the present invention.
• Production of a monoclonal antibody using the transposon-based vectors of the present invention can be accomplished in a variety of ways. In one embodiment, two vectors may be constructed: one that encodes the light chain, and a second vector that encodes the heavy chain of the monoclonal antibody. These vectors may then be incorporated into the genome of the target animal by methods disclosed herein.
• sequences encoding light and heavy chains of a monoclonal antibody may be included on a single DNA construct.
• the coding sequence of light and heavy chains of a murine monoclonal antibody that show specificity for human seminoprotein can be expressed using fransposon-based constructs of the present invention (GenBank Accession numbers AY129006 and AY129304 for the light and heavy chains, respectively).
• proteins and peptides synthesized by the immune system including those synthesized by the thymus, lymph nodes, spleen, and the gastrointestinal associated lymph tissues (GALT) system.
• the immune system proteins and peptides proteins that can be made in fransgenic animals using the transposon-based vectors of the present invention include, but are not limited to, alpha-interferon, beta-interferon, gamma-interferon, alpha-interferon A, alpha-interferon 1, G-CSF, GM-CSF, interlukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF- ⁇ , and TNF- ⁇ .
• cytokines included in the present invention include cardiotrophin, sfromal cell derived factor, macrophage derived chemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophage inflammatory proteins 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 4 and 5.
• Lytic peptides such as pi 46 are also included in the desired molecules of the present invention.
• the pl46 peptide comprises an amino acid sequence of SEQ ID NO: 24.
• the present invention also encompasses a transposon- based vector comprising a pl46 nucleic acid comprising a polynucleotide sequence of SEQ ID NO:25.
• Enzymes are another class of proteins that may be made through the use of the fransposon-based vectors of the present invention.
• Such enzymes include but are not limited to adenosine deaminase, alpha-galactosidase, cellulase, collagenase, dnasel, hyaluronidase, lactase, L-asparaginase, pancreatin, papain, sfreptokinase B, subtilisin, superoxide dismutase, thrombin, trypsin, urokinase, fibrinolysin, glucocerebrosidase and plasminogen activator.
• additional amino acids and a protease cleavage site are added to the carboxy end of the enzyme of interest in order to prevent expression of a functional enzyme. Subsequent digestion of the enzyme with a protease results in activation of the enzyme.
• Extracellular matrix proteins are one class of desired proteins that may be made through the use of the present invention. Examples include but are not limited to collagen, fibrin, elastin, laminin, and fibronectin and subtypes thereof. Intracellular proteins and structural proteins are other classes of desired proteins in the present invention.
• Growth factors are another desired class of proteins that may be made through the use of the present invention and include, but are not limited to, transforming growth factor- ⁇ ("TGF- ⁇ ”), transforming growth factor- ⁇ (TGF- ⁇ ), platelet-derived growth factors (PDGF), fibroblast growth factors (FGF), including FGF acidic isoforms 1 and 2, FGF basic form 2 and FGF 4, 8, 9 and 10, nerve growth factors (NGF) including NGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brain derived neurotrophic factor, cartilage derived factor, growth factors for stimulation of the production of red blood cells, growth factors for stimulation of the production of white blood cells, bone growth factors (BGF), basic fibroblast growth factor, vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), insulin like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF),
• Another desired class of proteins that may be made may be made through the use of the present invention include, but are not limited to, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, ENBREL, angiostatin, endostatin, thrombospondin, osteogenic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, and osteocalcin.
• LIF leukemia inhibitory factor
• ENBREL ENBREL
• angiostatin endostatin
• thrombospondin thrombospondin
• osteogenic protein-1 bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, and osteocalcin.
• Yet another desired class of proteins are blood proteins or clotting cascade protein including albumin, Prekallikrein, High molecular weight kininogen (HMWK) (contact activation cofactor; Fitzgerald, Flaujeac Williams factor), Factor I (Fibrinogen), Factor II (prothrombin), Factor III (Tissue Factor), Factor TV (calcium), Factor V (proaccelerin, labile factor, accelerator (Ac-) globulin), Factor VI (Va) (accelerin), Factor VII (proconvertin), serum prothrombin conversion accelerator (SPCA), cothromboplastin), Factor VIII (antihemophiliac factor A, antihemophilic globulin (AHG)), Factor IX (Christmas Factor, antihemophilic factor B,plasma thromboplastin component (PTC)), Factor X (Stuart-Prower Factor), Factor XI (Plasma thromboplastin antecedent (PTA)), Factor XI
• a non-limiting list of the peptides and proteins that may be made may be made through the use of the present invention is provided in product catalogs of companies such as Phoenix Pharmaceuticals, Inc. (www.phoenixpeptide.com; 530 Harbor Boulevard, Belmont, CA), Peninsula Labs (San Carlos CA), SIGMA, (St.Louis, MO www.sigma-aldrich.com), Cappel ICN (Irvine, California, www.icnbiomed.com), and Calbiochem (La Jolla, California, www.calbiochem.com).
• the polynucleotide sequences encoding these proteins and peptides of interest may be obtained from the scientific literature, from patents, and from databases such as GenBank. Alternatively, one of ordinary skill in the art may design the polynucleotide sequence to be incorporated into the genome by choosing the codons that encode for each amino acid in the desired protein or peptide.
• fransgenic animals of the present invention include bacitracin, polymixin b, vancomycin, cyclosporine, anti- RSV antibody, alpha- 1 antitrypsin (AAT), anti-cytomegalovirus antibody, anti- hepatitis antibody, anti-inhibitor coagulant complex, anti-rabies antibody, anti-Rh(D) antibody, adenosine deaminase, anti-digoxin antibody, antivenin crotalidae (rattlesnake venom antibody), antivenin lafrodectus (black widow spider venom antibody), antivenin micrurus (coral snake venom antibody), aproti in, corticofropin (ACTH), diphtheria antitoxin, lymphocyte immune globulin (anti-thymocyte antibody), protamine, thyrotropin, capreomycin, ⁇ -galactosidase, gramicidin, strepto
• IR502 IR502
• IR501 Bl 1050/1272 mAb against very late antigen-4 (VLA-4)
• C225 humanized mAb to EGF receptor anti-idiotype antibody to GD3 glycolipid, antibacterial peptide against H. pylori, MDX-447 bispecific humanized mAb to EGF receptor, anti-cytomegalovirus (CMV), Medi-491 B19 parvo virus vaccine, humanized recombinant mAb (IgG Ik) against respiratory syncytial vims (RSV), urinary tract infection vaccine (against "pili” on Escherechia coli strains), proteins of lyme disease vaccine against B.
• VLA-4 very late antigen-4
• burgdorferi protein DbpA
• proteins of Medi-501 human papilloma virus- 11 vaccine HPV
• Streptococcus pneumoniae vaccine Medi-507 mAb (humanized form of BTI-322) against CD2 receptor on T-cells
• MDX-33 mAb to Fc ⁇ Rl receptor MDX-RA immunotoxin (ricin A linked) mAb
• MDX-210 bi- specific mAb against HER-2 MDX-447 bi-specific mAb against EGF receptor
• MDX-22 MDX-220 bi-specific mAb against TAG-72 on tumors
• colony-stimulating factor (CSF) molgramostim
• humanized mAb to the IL-2 R c-chain basiciliximab
• mAb to IgE IGE 025A
• myelin basic protein-altered peptide MSP771A
• humanized mAb agamst the epidermal growth receptor-2 humanized mAb against the c
• the peptides and proteins made using the present invention may be labeled using labels and techniques known to one of ordinary skill in the art. Some of these labels are described in the "Handbook of Fluorescent Probes and Research Products", ninth edition, Richard P. Haugland (ed) Molecular Probes, Inc. Eugene, OR), which is incorporated herein in its entirety. Some of these labels may be genetically engineered into the polynucleotide sequence for the expression of the selected protein or peptide. The peptides and proteins may also have label-incorporation "handles" incorporated to allow labeling of an otherwise difficult or impossible to label protein. It is to be understood that the various classes of desired peptides and proteins, as well as specific peptides and proteins described in this section may be modified as described below by inserting selected codons for desired amino acid substitutions into the gene incorporated into the transgenic animal.
• the present invention may also be used to produce desired molecules other than proteins and peptides including, but not limited to, lipoproteins such as high density lipoprotein (HDL), HDL-Milano, and low density lipoprotein, lipids, carbohydrates, siRNA and ribozymes.
• lipoproteins such as high density lipoprotein (HDL), HDL-Milano, and low density lipoprotein, lipids, carbohydrates, siRNA and ribozymes.
• a gene of interest encodes a nucleic acid molecule or a protein that directs production of the desired molecule.
• the present invention further encompasses the use of inhibitory molecules to inhibit endogenous (i.e., non-vector) protein production.
• inhibitory molecules include antisense nucleic acids, siRNA and inhibitory proteins.
• the endogenous protein whose expression is inhibited is an egg white protein including, but not limited to ovalbumin, ovofransferrin, and ovomucin.
• a transposon-based vector containing an ovalbumin DNA sequence, that upon transcription forms a double stranded RNA molecule, is transfected into an animal such as a bird and the bird's production of endogenous ovalbumin protein is reduced by the interference RNA mechanism (RNAi).
• RNAi interference RNA mechanism
• a fransposon-based vector encodes an inhibitory RNA molecule that inhibits the expression of more than one egg white protein.
• One exemplary construct is provided in Figure 4 wherein "Ovgen” indicates approximately 60 base pairs of an ovalbumin gene, “Ovotrans” indicates approximately 60 base pairs of an ovofransferrin gene and “Ovomucin” indicates approximately 60 base pairs of an ovomucin gene.
• ovalbumin, ovofransferrin and ovomucin can be from any avian species, and in some embodiments, are from a chicken or quail.
• the term "pro” indicates the pro portion of a prepro sequence.
• One exemplary prepro sequence is that of cecropin and comprising base pairs 563-733 of the Cecropin cap site and Prepro provided in Genbank accession number X07404. Additional cecropin prepro and pro sequences are provided in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO: 50, and SEQ ID NO:51. Additionally, inducible knockouts or knockdowns of the endogenous protein may be created to achieve a reduction or inhibition of endogenous protein production. Endogenous egg white production can be inhibited in an avian at any time, but is preferably inhibited preceding, or immediately preceding, the harvest of eggs. Modified Desired Proteins and Peptides
• Proteins are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid.
• the terminal amino acid at one end of the chain i.e., the amino terminal
• the terminal amino acid at the other end of the chain i.e., the carboxy terminal
• amino terminus refers to the free alpha-amino group on the amino acid at the amino terminal of the protein, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the protein.
• carboxy terminus refers to the free carboxyl group on the amino acid at the carboxy terminus of a protein, or to the carboxyl group of an amino acid at any other location within the protein.
• amino acids making up a protein are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the protein.
• “residue” is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a protein by an amide bond.
• the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics).
• an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
• a conservative substitution is a substitution in which the substituting amino acid (naturally occurring or modified) is structurally related to the amino acid being substituted, i.e., has about the same size and electronic properties as the amino acid being substituted. Thus, the substituting amino acid would have the same or a similar functional group in the side chain as the original amino acid.
• a “conservative substitution” also refers to utilizing a substituting amino acid which is identical to the amino acid being substituted except that a functional group in the side chain is protected with a suitable protecting group. Suitable protecting groups are described in Green and Wuts, "Protecting
• Preferred protecting groups are those which facilitate transport of the peptide through membranes, for example, by reducing the hydrophilicity and increasing the lipophilicity of the peptide, and which can be cleaved, either by hydrolysis or enzymatically (Ditter et al., 1968. J. Pharm. Sci. 57:783; Ditter et al., 1968. J. Pharm. Sci. 57:828; Ditter et al., 1969. J. Pharm. Sci. 58:557; King et al., 1987.
• Suitable hydroxyl protecting groups include ester, carbonate and carbamate protecting groups.
• Suitable amine protecting groups include acyl groups and alkoxy or aryloxy carbonyl groups, as described above for N-terminal protecting groups.
• Suitable carboxylic acid protecting groups include aliphatic, benzyl and aryl esters, as described below for C-terminal protecting groups.
• the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residues in a peptide of the present invention is protected, preferably as a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.
• Group I includes leucine, isoleucine, valine, methionine and modified amino acids having the following side chains: ethyl, n-propyl n-butyl.
• Group I includes leucine, isoleucine, valine and methionine.
• Group II includes glycine, alanine, valine and a modified amino acid having an ethyl side chain.
• Group II includes glycine and alanine.
• Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl glycme, and modified amino residues having substituted benzyl or phenyl side chains.
• Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, — NH 2 , methoxy, ethoxy and — CN.
• Group III includes phenylalanine, tyrosine and tryptophan.
• Group IV includes glutamic acid, aspartic acid, a substitated or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n-propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, — CO — NH — alkylated glutamine or asparagines (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain — (CH 2 ) 3 — COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof.
• glutamic acid e.g., methyl, ethyl,
• Group IV includes glutamic acid, aspartic acid, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate, glutamine and asparagine.
• Group V includes histidine, lysine, omithine, arginine, N-nifroarginine, ⁇ - cycloarginine, ⁇ -hydroxyarginine, N-amidinocitruline and 2-amino-4- guanidinobutanoic acid, homologs of lysine, homologs of arginine and homologs of ornithine.
• Group V includes histidine, lysine, arginine and omithine.
• a homolog of an amino acid includes from 1 to about
• Group VI includes serine, threonine, cysteine and modified amino acids having Cl-
• Group VI includes serine, cysteine or threonine.
• suitable substitutions for amino acid residues include “severe” substitutions.
• a "severe substitution” is a substitation in which the substituting amino acid (naturally occurring or modified) has significantly different size and/or electronic properties compared with the amino acid being substitated.
• the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
• severe substitutions of this type include the substitution of phenylalanine or cyclohexylmethyl glycine for alanme, isoleucine for glycine, a D amino acid for the corresponding L amino acid, or — NH — CH[( — CH 2 ) 5 — COOH] — CO — for aspartic acid.
• a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group.
• Examples of severe substitutions of this type include adding of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine, or deleting the amine group in the side chain of lysine or omithine.
• the side chain of the substituting amino acid can have significantly different steric and electronic properties that the functional group of the amino acid being substituted. Examples of such modifications include tryptophan for glycine, lysine for aspartic acid and — (CH 2 ) 4 COOH for the side chain of serine. These examples are not meant to be limiting.
• the individual amino acids may be substitated according in the following manner:
• AAi is serine, glycine, alanine, cysteine or threonine
• AA 2 is alanine, threonine, glycine, cysteine or serine
• AA 3 is valine, arginine, leucine, isoleucine, methionine, omithine, lysine, N- nitroarginine, ⁇ -cycloarginine, ⁇ -hydroxyarginine, N-amidmocitruline or 2-amino-4- guanidinobutanoic acid;
• AA-i is proline, leucine, valine, isoleucine or methionine;
• AA 5 is tryptophan, alanine, phenylalanine, tyrosine or glycine;
• AA 6 is serine, glycme, alanine, cysteine or threonine;
• AA 7 is proline, leucine, valine, isoleucine or methionine;
• AA 8 is alanine, threonine, glycine, cysteine or serine;
• AA 9 is alanine, threonine, glycme, cysteine or serine; AAio is leucine, isoleucine, methionine or valine;
• AA ⁇ is serine, glycme, alanine, cysteine or threonine
• AAj 2 is leucine, isoleucine, methionine or valine;
• AAj 3 is leucine, isoleucine, methionine or valine;
• AAj 4 is glutamine, glutamic acid, aspartic acid, asparagine, or a substitated or unsubstitated aliphatic or aryl ester of glutamic acid or aspartic acid;
• AAi 5 is arginine, N-nitroarginine, ⁇ -cycloarginine, ⁇ -hydroxy-arginine, N- amidinocitruline or 2-amino-4-guanidino-butanoic acid
• AA ⁇ 6 is proline, leucine, valine, isoleucine or methionine;
• AA ⁇ 7 is serine, glycine, alanine, cysteine or threonine;
• AA 18 is glutamic acid, aspartic acid, asparagine, glutamine or a substitated or unsubstitated aliphatic or aryl ester of glutamic acid or aspartic acid;
• AA ⁇ 9 is aspartic acid, asparagine, glutamic acid, glutamine, leucine, valine, isoleucine, methionine or a substituted or unsubstitated aliphatic or aryl ester of glutamic acid or aspartic acid;
• AA 20 is valine, arginine, leucine, isoleucine, methionine, omithine, lysine, N- nitroarginine, ⁇ -cycloarginine, ⁇ -hydroxyarginine, N-amidinocitruline or 2-amino-4- guanidinobutanoic acid;
• AA 2 ⁇ is alanine, threonine, glycine, cysteine or serine;
• AA 22 is alanine, threonine, glycine, cysteine or serine; AA 23 is histidine, serine, threonine, cysteine, lysine or omithine;
• AA 24 is threonine, aspartic acid, serine, glutamic acid or a substitated or unsubstitated aliphatic or aryl ester of glutamic acid or aspartic acid;
• AA 25 is asparagine, aspartic acid,, glutamic acid, glutamine, leucine, valine, isoleucine, methionine or a substitated or unsubstitated aliphatic or aryl ester of glutamic acid or aspartic acid; and
• AA 26 is cysteine, histidine, serine, threonine, lysine or omithine. It is to be understood that these amino acid substitutions may be made for longer or shorter peptides than the 26 mer in the preceding example above, and for proteins.
• codons for the first several N- tertninal amino acids of the fransposase are modified such that the third base of each codon is changed to an A or a T without changing the corresponding amino acid. It is preferable that between approximately 1 and 20, more preferably 3 and 15, and most preferably between 4 and 12 of the first N-terminal codons of the gene of interest are modified such that the third base of each codon is changed to an A or a T without changing the corresponding amino acid. In one embodiment, the first ten N-terminal codons of the gene of interest are modified in this manner.
• proteins, protein fragments or peptides may be separated by a spacer molecule such as, for example, a peptide, consisting of one or more amino acids.
• a spacer molecule such as, for example, a peptide, consisting of one or more amino acids.
• the spacer will have no specific biological activity other than to join the desired proteins, protein fragments or peptides together, or to preserve some minimum distance or other spatial relationship between them.
• the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
• the spacer may also be contained within a nucleotide sequence with a purification handle or be flanked by cleavage sites, such as proteolytic cleavage sites.
• polypeptide spacers may have from about 5 to about 40 amino acid residues.
• the spacers in a polypeptide are independently chosen, but are preferably all the same.
• the spacers should allow for flexibility of movement in space and are therefore typically rich in small amino acids, for example, glycine, serine, proline or alanine.
• peptide spacers contain at least 60%, more preferably at least 80% glycine or alanine.
• peptide spacers generally have little or no biological and antigenic activity.
• Preferred spacers are (Gly-Pro-Gly-Gly) x (SEQ ID NO:26) and (Gly 4 -Ser) y , wherein x is an integer from about 3 to about 9 and y is an integer from about 1 to about 8.
• suitable spacers include (Gly-Pro-Gly-Gly) 3
• Nucleotide sequences encoding for the production of residues which may be useful in purification of the expressed recombinant protein may also be built into the vector. Such sequences are known in the art and include the glutathione binding domain from glutathione S-fransferase, polylysine, hexa-histidine or other cationic amino acids, thioredoxin, hemagglutinin antigen and maltose binding protein.
• nucleotide sequences may be inserted into the gene of interest to be incorporated so that the protein or peptide can also include from one to about six amino acids that create signals for proteolytic cleavage.
• specific nucleotide sequences encoding for amino acids recognized by enzymes may be inco ⁇ orated into the gene to facilitate cleavage of the large protein or peptide sequence into desired peptides or proteins or both.
• nucleotides encoding a proteolytic cleavage site can be introduced into the gene of interest so that a signal sequence can be cleaved from a protein or peptide encoded by the gene of interest.
• Nucleotide sequences encoding other amino acid sequences which display pH sensitivity or chemical sensitivity may also be added to the vector to facilitate separation of the signal sequence from the peptide or protein of interest.
• Proteolytic cleavage sites include cleavage sites recognized by exopeptidases such as carboxypeptidase A, carboxypeptidase B, aminopeptidase I, and dipeptidylaminopeptidase; endopeptidases such as trypsin, V8-protease, enterokinase, factor Xa, coUagenase, endoproteinase, subtilisin, and thombin; and proteases such as Protease 3C IgA protease (Igase) Rhinovirus 3C(preScission)protease . Chemical cleavage sites are also included in the defintion of cleavage site as used herein.
• Chemical cleavage sites include, but are not limited to, site cleaved by cyanogen bromide, hydroxylamine, formic acid, and acetic acid.
• a TAG sequence is linked to the gene of interest.
• the TAG sequence serves three pu ⁇ oses: 1) it allows free rotation of the peptide or protein to be isolated so there is no interference from the native protein or signal sequence, i.e. vitellogenin, 2) it provides a "purification handle" to isolate the protein using column purification, and 3) it includes a cleavage site to remove the desired protein from the signal and purification sequences.
• a TAG sequence includes a spacer sequence, a purification handle and a cleavage site.
• the spacer sequences in the TAG proteins contain one or more repeats shown in SEQ ID NO:30.
• a preferred spacer sequence comprises the sequence provided in SEQ ID NO:31.
• One example of a purification handle is the gp41 hai ⁇ in loop from HIV I.
• Exemplary gp41 polynucleotide and polypeptide sequences are provided in SEQ ID NO:32 and SEQ ID NO:33, respectively.
• any antigenic region may be used as a purification handle, including any antigenic region of gp41.
• Preferred purification handles are those that elicit highly specific antibodies.
• the cleavage site can be any protein cleavage site known to one of ordinary skill in the art and includes an enterokinase cleavage site comprising the Asp Asp Asp Asp Lys sequence (SEQ ID NO: 34) and a furin cleavage site.
• enterokinase cleavage site comprising the Asp Asp Asp Asp Lys sequence (SEQ ID NO: 34) and a furin cleavage site.
• Constructs containing a TAG sequence are shown in Figures 2 and 3.
• the TAG sequence comprises a polynucleotide sequence of SEQ ID NO:35.
• the present invention also includes methods of administering the transposon-based vectors to an animal, methods of producing a transgenic animal wherein a gene of interest is inco ⁇ orated into the germline of the animal and methods of producing a transgenic animal wherein a gene of interest is inco ⁇ orated into cells other than the germline cells (somatic cells) of the animal.
• the transposon-based vectors of the present invention are administered to a reproductive organ of an animal via any method known to those of skill in the art.
• Preferred reproductive organs include an ovary, an oviduct, a mammary gland, and a fallopian tube.
• a transposon-based vector is directly administered to the reproductive organ.
• Direct administration encompasses injection into the organ, and in a preferred embodiment, a transposon-based vector is injected into the lumen of the oviduct, and more preferably, the lumen of the magnum or the infundibulum of the oviduct.
• the transposon-based vectors may additionally or alternatively be placed in an artery supplying the reproductive organ. Administering the vectors to the artery supplying the ovary results in transfection of follicles and oocytes in the ovary to create a germline transgenic animal. Alternatively, supplying the vectors through an artery leading to the oviduct would preferably transfect the tabular gland and epithelial cells.
• transfected cells could manufacture a desired protein or peptide for deposition in the egg white.
• a transposon-based vector is administered into the lumen of the magnum or the infundibulum of the oviduct and to an artery supplying the oviduct.
• Indirect administration to the oviduct epithelium may occur through the cloaca.
• Direct administration into the mammary gland comprises introduction into the duct system of the mammary gland.
• transposon-based vectors may occur in arteries supplying the ovary and or through direct intrathecal administration into the ovary through injection.
• the transposon-based vectors may be administered in a single administration, multiple administrations, continuously, or intermittently.
• the fransposon-based vectors may be administered by injection, via a catheter, an osmotic mini-pump or any other method.
• the transposon-based vector is administered to an animal in multiple administrations, each administration containing the vector and a different transfecting reagent.
• the transposon-based vectors may be administered to the animal at any point during the lifetime of the animal, however, it is preferable that the vectors are administered prior to the animal reaching sexual maturity.
• the fransposon-based vectors are preferably administered to a chicken between approximately 14 and 16 weeks of age and to a quail between approximately 5 and 10 weeks of age, more preferably 5 and 8 weeks of age, and most preferably between 5 and 6 weeks of age, when standard poultry rearing practices are used.
• the vectors may be administered at earlier ages when exogenous hormones are used to induce early sexual maturation in the bird.
• the transposon-based vector is administered to an animal following an increase in proliferation of the oviduct epithelial cells and/or the tabular gland cells.
• the fransposon-based vector is administered following an increase in proliferation of the oviduct epithelial cells and before the avian begins to produce egg white constituents.
• the animal is an egg-laying animal, and more preferably, an avian.
• between approximately 1 and 150 ⁇ g, 1 and 100 ⁇ g, 1 and 50 ⁇ g, preferably between 1 and 20 ⁇ g, and more preferably between 5 and 10 ⁇ g of transposon-based vector DNA is administered to the oviduct of a bird.
• Optimal ranges depend upon the type of bird and the bird's stage of sexual maturity.
• Intraoviduct administration of the transposon-based vectors of the present invention result in inco ⁇ oration of the gene of interest into the cells of the oviduct as evidenced by a PCR positive signal in the oviduct tissue.
• the fransposon-based vector is administered to an artery that supplies the oviduct.
• the transposon-based vector is administered in conjunction with an acceptable carrier and/or transfection reagent.
• Acceptable carriers include, but are not limited to, water, saline, Hanks Balanced Salt Solution (HBSS), Tris-EDTA (TE) and lyotropic liquid crystals.
• Transfection reagents commonly known to one of ordinary skill in the art that may be employed include, but are not limited to, the following: cationic lipid fransfection reagents, cationic lipid mixtures, polyamine reagents, liposomes and combinations thereof; SUPERFECT®, Cytofectene, BioPORTER®, GenePORTER®, NeuroPORTER®, and perfectin from Gene Therapy Systems; lipofectamine, cellfectin, DMRIE-C oligofectamine, TROJENE® and PLUS reagent from InVitrogen; Xtreme gene, fugene, DOSPER and DOTAP from Roche; Lipotaxi and Genejammer from Sfrategene; and Escort from SIGMA.
• the fransfection reagent is SUPERFECT®.
• the ratio of DNA to transfection reagent may vary based upon the method of administration.
• the transposon-based vector is administered to the oviduct and the ratio of DNA to fransfection reagent can be from 1 : 1.5 to 1 : 15, preferably 1 :2 to 1:5, all expressed as wt vol.
• Transfection may also be accomplished using other means known to one of ordinary skill in the art, including without limitation electroporation, gene guns, injection of naked DNA, and use of dimethyl sulfoxide (DMSO).
• DMSO dimethyl sulfoxide
• the form of the transposon-based vector may be important.
• Plasmids harvested from bacteria are generally closed circular supercoiled molecules, and this is the preferred state of a vector for gene delivery because of the ease of preparation.
• transposase expression and insertion may be more efficient in a relaxed, closed circular configuration or in a linear configuration.
• a purified transposase protein may be co-injected with a transposon-based vector containing the gene of interest for more immediate insertion. This could be accomplished by using a fransfection reagent complexed with both the purified transposase protein and the fransposon-based vector.
• DNA is extracted from the animal to confirm integration of the gene of interest.
• Advantages provided by the present invention include the high rates of integration, or inco ⁇ oration, and transcription of the gene of interest when administered to a bird via an intraoviduct or intraovarian route (including infraarterial administrations to arteries leading to the oviduct or ovary).
• Example 6 below describes isolation of a proinsulin/ENT TAG protein from a transgenic hen following ammonium sulfate precipitation and ion exchange chromatography.
• Figure 5 demonstrates successful adminisfration of a transposon-based vector to a hen, successful integration of the gene of interest, successful production of a protein encoded by the gene of interest, and successful deposition of the protein in egg white produced by the transgenic hen.
• PRINS primed in sita hybridization technique
• the transposon-based vectors of the present invention may be administered to a bird for production of desired proteins or peptides in the egg white.
• These transposon-based vectors preferably contain one or more of an ovalbumin promoter, an ovomucoid promoter, an ovalbumin signal sequence and an ovomucoid signal sequence.
• Oviduct-specific ovalbumin promoters are described in B. O'Malley et al., 1987. EMBO J., vol. 6, pp. 2305-12; A. Qiu et al., 1994. Proc. Nat. Acad. Sci. (USA), vol. 91, pp. 4451-4455; D. Monroe et al., 2000. Biochim.
• the present invention is particularly advantageous for production of recombinant peptides and proteins of low solubility in the egg yolk.
• proteins include, but are not limited to, membrane-associated or membrane-bound proteins, lipophilic compounds; attachment factors, receptors, and components of second messenger transduction machinery.
• Low solubility peptides and proteins are particularly challenging to produce using conventional recombinant protein production techniques (cell and tissue cultures) because they aggregate in water- based, hydrophilic environments. Such aggregation necessitates denataration and refolding of the recombinantly-produced proteins, which may deleteriously affect their structure and function.
• the present invention provides an advantageous resolution of the problem of protein and peptide solubility during production of large amounts of recombinant proteins.
• deposition of a desired protein into the egg yolk is accomplished in offspring by attaching a sequence encoding a protein capable of binding to the yolk vitellogenin receptor to a gene of interest that encodes a desired protein.
• This transposon-based vector can be used for the receptor-mediated uptake of the desired protein by the oocytes.
• the sequence ensuring the binding to the vitellogenin receptor is a targeting sequence of a vitellogenin protein.
• the invention encompasses various vitellogenin proteins and their targeting sequences.
• a chicken vitellogenin protein targeting sequence is used, however, due to the high degree of conservation among vitellogenin protein sequences and known cross- species reactivity of vitellogenin targeting sequences with their egg-yolk receptors, other vitellogenin targeting sequences can be substitated.
• a construct for use in the transposon-based vectors of the present invention and for deposition of an insulin protein in an egg yolk is a fransposon-based vector containing a vitellogenin promoter, a vitellogenin targeting sequence, a TAG sequence, a pro- insulin sequence and a synthetic polyA sequence.
• the present invention includes, but is not limited to, vitellogenin targeting sequences residing in the N-terminal domain of vitellogenin, particularly in lipovitellin I.
• vitellogenin targeting sequence contains the polynucleotide sequence of SEQ ID NO:22.
• the transposon-based vector contains a transposase gene operably-linked to a constitative promoter and a gene of interest operably-linked to a liver-specific promoter and a vitellogenin targeting sequence.
• an animal breeding stock that is homozygous for the transgene is preferred.
• Such homozygous individuals are obtained and identified through, for example, standard animal breeding procedures or PCR protocols.
• peptides, polypeptides and proteins can be purified according to standard procedures known to one of ordinary skill in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel elecfrophoresis, high performance liquid chromatography, immunoprecipitation and the like. Substantially pure compositions of about 50 to 99% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.
• the animal in which the desired protein is produced is an egg-laying animal.
• the animal is an avian and a desired peptide, polypeptide or protein is isolated from an egg white.
• Egg white containing the exogenous protein or peptide is separated from the yolk and other egg constituents on an industrial scale by any of a variety of methods known in the egg industry. See, e.g., W. Stadelman et al. (Eds.), Egg Science & Technology, Haworth Press, Binghamton, NY (1995).
• Isolation of the exogenous peptide or protein from the other egg white constituents is accomplished by any of a number of polypeptide isolation and purification methods well known to one of ordinary skill in the art. These techniques include, for example, chromatographic methods such as gel permeation, ion exchange, affinity separation, metal chelation, HPLC, and the like, either alone or in combination. Another means that may be used for isolation or purification, either in lieu of or in addition to chromatographic separation methods, includes electrophoresis. Successful isolation and purification is confirmed by standard analytic techniques, including HPLC, mass specfroscopy, and spectrophotometry. These separation methods are often facilitated if the first step in the separation is the removal of the endogenous ovalbumin fraction of egg white, as doing so will reduce the total protein content to be further purified by about 50%.
• transposon- based vectors may include one or more additional epitopes or domains.
• epitopes or domains include DNA sequences encoding enzymatic or chemical cleavage sites including, but not limited to, an enterokinase cleavage site; the glutathione binding domain from glutathione S-fransferase; polylysine; hexa-histidine or other cationic amino acids; thioredoxin; hemagglutinin antigen; maltose binding protein; a fragment of gp41 from HIV; and other purification epitopes or domains commonly known to one of skill in the art.
• purification of desired proteins from egg white utilizes the antigenicity of the ovalbumin carrier protein and particular attributes of a TAG linker sequence that spans ovalbumin and the desired protein.
• the TAG sequence is particularly useful in this process because it contains 1) a highly antigenic epitope, a fragment of gp41 from HIV, allowing for stringent affinity purification, and, 2) a recognition site for the protease enterokinase immediately juxtaposed to the desired protein.
• the TAG sequence comprises approximately 50 amino acids.
• a representative TAG sequence is provided below.
• the underlined sequences were taken from the hai ⁇ in loop domain of HIV gp-41 (SEQ ID NO:33). Sequences in italics represent the cleavage site for enterokinase (SEQ ID NO: 34).
• the spacer sequence upstream of the loop domain was made from repeats of (Pro Ala Asp Asp Ala) (SEQ ID NO:31) to provide free rotation and promote surface availability of the hai ⁇ in loop from the ovalbumin carrier protein. Isolation and purification of a desired protein is performed as follows:
• Cleavage of the transgene product can be accomplished in at least one of two ways: a. The fransgenic ovalbumin-TAG-desired protein is left attached to the gp41 affinity resin (beads) from step 4 and the protease enterokinase is added. This liberates the transgene target protein from the gp41 affinity resin while the ovalbumin-TAG sequence is retained. Separation by centrifugation (in a batch process) or flow through (in a column purification), leaves the desired protein together with enterokinase in solution. Enterokinase is recovered and reused. b.
• enterokinase is immobilized on resin (beads) by the addition of poly-lysine moieties to a non-catalytic area of the protease.
• the transgenic ovalbumin-TAG-desired protein eluted from the affinity column of step 4 is then applied to the protease resin.
• Protease action cleaves the ovalbumin-TAG sequence from the desired protein and leaves both entities in solution.
• the immobilized enterokinase resin is recharged and reused.
• the choice of these alternatives is made depending upon the size and chemical composition of the transgene target protein. 6.
• a final separation of either of these two (5a or 5b) protein mixtures is made using size exclusion, or enterokinase affinity chromatography. This step allows for desalting, buffer exchange and/or polishing, as needed.
• One non-limiting example of a method for isolating a desired peptide, polypeptide or protein from an egg white is as follows. It is to be understood that this method may be employed to isolate any desired peptide, polypeptide or protein from the eggs of transgenic animals of the present invention. This present example involved transgenes that used a portion of or the entire ovalbumin protein, or specific ovalbumin epitopes, as a carrier, linked to the protein of interest via the specified
• TAG sequence or another affinity/cleavage sequence.
• the TAG sequence contains the hai ⁇ in loop epitope from HIV I followed by an enterokinase cleavage site.
• the viscosity of the egg white was lowered by subjecting the egg white to low shear forces of 3140 cps (Tung et al., 1969). The resulting pourable solution was then filtered to remove chalazae. An ammonium sulfate precipitation was then used to enrich the fraction of transgenic protein (see, for example, Practical Protein Chemistry A Handbook A. Darbre (Ed.), John Wiley & Sons Ltd., 1986). Other methods of crude fractionation known in the art are also used as needed. The supernatant of this separation was then fractionated using size-exclusion chromatography, further enriching the transgenic fusion protein fraction and eliminating the ammonium sulfate from the material.
• the fusion protein was isolated by anti-ovalbumin affinity chromatography (batch or column) using methods known to one skilled in the art. This step may capture native ovalbumin in addition to an ovalbumin-transgene fusion protein. After elution from the anti-ovalbumin affinity resin, the transgenic protein was specifically isolated using anti-gp41 affinity chromatography (batch or column) using methods known to one skilled in the art.
• Cleavage of the fransgene product from the carrier and the TAG sequences was accomplished in one of at least two ways: 1) The transgenic ovalbumin-TAG-transgene target protein was left attached to the gp41 affinity resin and the protease enterokinase was added. Cleavage of the transgene by enterokinase liberated the fransgene target protein from the gp41 affinity resin while the ovalbumin-TAG sequence was retained. Separation by centrifugation (in a batch process) or flow through (in a column purification), kept the transgene target protein together with enterokinase in solution. Enterokinase was recovered and reused.
• enterokinase was immobilized on resin (beads) by the addition of poly-lysine moieties to a non-catalytic area of the protease.
• the fransgenic ovalbumin-TAG-transgene target protein was eluted from the gp41 affinity chromatography resin and then applied to the protease resin. Protease action cleaved the ovalbumin-TAG sequence from the transgene target protein and left both entities in solution.
• the immobilized enterokinase resin was recharged and reused. The choice between these alternatives is made on a case-by case basis, depending upon the size and chemical composition of the transgene target protein.
• a final separation of either of these two (process 1 or 2) protein mixtures was made using size exclusion chromatography, or enterokinase affinity chromatography. This step also allows for desalting, concentrating, buffer exchange and/or polishing, as needed. It is believed that a typical chicken egg produced by a transgenic animal of the present invention will contain at least 0.001 mg, from about 0.001 to 1.0 mg, or from about 0.001 to 100.0 mg of exogenous protein, peptide or polypeptide, in addition to the normal constituents of egg white (or possibly replacing a small fraction of the latter). In some embodiments, a chicken egg will contain between 50 and 75 mg of exogenous protein.
• the desired proteins, fragments thereof and peptides may possess a conformation substantially different than the native conformations of the proteins, fragments thereof and peptides. In this case, it is often necessary to denature and reduce protein and then to cause the protein to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art.
• Production of Protein or Peptide in Milk In addition to methods of producing eggs containing transgenic proteins or peptides, the present invention encompasses methods for the production of milk containing fransgenic proteins or peptides.
• the transposon-based vector contains a fransposase operably- linked to a constitative promoter and a gene of interest operably-linked to mammary specific promoter. Genes of interest can include, but are not limited to antiviral and antibacterial proteins and immunoglobulins.
• a transposon- based vector is administered to the ovary of an animal and germline transformation is obtained. In these embodiments, offspring of the transfected animal express a gene of interest in the mammary gland under the control of a mammary gland-specific promoter.
• Quail or chicken were selected for administration of the fransposon-based vectors of the present invention. Feathers were removed from the area where surgery was performed and the area was cleansed and sterilized by rinsing it with ethanol (alcohol) and 0.5% chlorhexidine. Using the scalpel, a dorsolateral incision was made through the skin over the ovary approximately 2 cm in length. Using blunt scissors, a second incision was made through the muscle between the last two ribs to expose the oviduct beneath. A small animal refractor was used to spread the last two ribs, exposing the oviduct beneath. The oviduct was further exposed using retractors to pull the intestines to one side.
• a delivery solution containing a transposon-based vector and SUPERFECT® was prepared fresh immediately before surgery. Specific ratios of vector and SUPERFECT® that were used in each experiment are provided in the Examples below.
• the delivery solution was warmed to room temperatare prior to injection into the bird. Approximately 250-500 ⁇ l of the delivery solution was injected into the lumen of the magnum of the oviduct using a 1 cc syringe with a 27 gauge needle attached. The wound was closed and antibiotic cream liberally applied to the area surrounding the wound.
• a vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells, given below as SEQ ID NO:3.
• This vector employed a cytomegalovirus (CMV) promoter.
• CMV cytomegalovirus
• a modified Kozak sequence (ACCATG) (SEQ ID NO:l) was added to the promoter.
• the nucleotide in the wobble position in nucleotide triplet codons encoding the first 10 amino acids of fransposase was changed to an adenine (A) or thymine (T), which did not alter the amino acid encoded by this codon.
• Two stop codons were added and a synthetic polyA was used to provide a strong termination sequence.
• This vector uses a promoter designed to be active soon after entering the cell (without any induction) to increase the likelihood of stable integration. The additional stop codons and synthetic polyA insures proper termination without read through to potential genes downstream.
• the first step in constructing this vector was to modify the fransposase to have the desired changes. Modifications to the transposase were accomplished with the primers High Efficiency forward primer (Hef) Altered fransposase (ATS)-Hef 5' ATCTCGAGACCATGTGTGAACTTGATATTTTACATGATTCTCTTTACC 3 ' (SEQ ID NO: 36) and Altered transposase- High efficiency reverse primer (Her) 5' GATTGATCATTATCATAATTTCCCCAAAGCGTAACC 3' (SEQ ID NO:37, a reverse complement primer).
• Hef High Efficiency forward primer
• ATS Altered fransposase
• ATS Altered fransposase
• ATS ATCTCGAGACCATGTGTGAACTTGATATTTTACATGATTCTCTTTACC 3 '
• Altered transposase- High efficiency reverse primer Her 5' GATTGATCATTATCATAATTTCCCCAAAGCGTAACC 3' (SEQ ID NO:37
• the sequence CTCGAG (SEQ ID NO:38) is the recognition site for the restriction enzyme Xho I, which permits directional cloning of the amplified gene.
• the sequence ACCATG (SEQ ID NO:l) contains the Kozak sequence and start codon for the fransposase and the underlined bases represent changes in the wobble position to an A or T of codons for the first 10 amino acids (without changing the amino acid coded by the codon).
• Primer ATS-Her (SEQ ID NO:37) contains an additional stop codon TAA in addition to native stop codon TGA and adds a Bel I restriction site, TGATCA (SEQ ID NO:39), to allow directional cloning.
• pTnLac plasmid
• tn defines transposon
• lac defines the beta fragment of the lactose gene, which contains a multiple cloning site
• pTnLac plasmid
• tn defines transposon
• lac defines the beta fragment of the lactose gene, which contains a multiple cloning site
• Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet fransilluminator.
• a band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, CA).
• Purified DNA was digested with restriction enzymes Xho I (5') and Bel I (3') (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research). Plasmid gWhiz (Gene Therapy Systems, San Diego, CA) was digested with restriction enzymes Sal I and BamH I (New England Biolabs), which are compatible with Xho I and Bel I, but destroy the restriction sites.
• Digested gWhiz was separated on an agarose gel, the desired band excised and purified as described above. Cutting the vector in this manner facilitated directional cloning of the modified fransposase (mATS) between the CMV promoter and synthetic polyA.
• mATS modified fransposase
• a Sfratagene T4 Ligase Kit (Stratagene, Inc. La Jolla, CA) was used and the ligation set up according to the manufacturer's protocol.
• Ligated product was transformed into E. coli Top 10 competent cells (Invifrogen Life Technologies, Carlsbad, CA) using chemical transformation according to Invifrogen' s protocol.
• Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT# 15544- 042) medium for 1 hour at 37° C before being spread to LB (Luria-Bertani media (broth or agar)) plates supplemented with 100 ⁇ g/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37° C and resulting colonies picked to LB/amp broth for overnight growth at 37° C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al., 1989), elecfrophoresed on a 1% agarose gel, and visualized on a UN. fransilluminator after ethidium bromide staining.
• Colonies producing a plasmid of the expected size were cultured in at least 250 ml of LB/amp broth and plasmid D ⁇ A harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, CA).
• Qiagen Maxi-Prep Kit column purification
• Column purified D ⁇ A was used as template for sequencing to verify the changes made in the transposase were the desired changes and no further changes or mutations occurred due to PCR amplification.
• Perkin-Elmer's Big Dye Sequencing Kit was used. All samples were sent to the Gene Probes and Expression Laboratory (LSU School of Veterinary Medicine) for sequencing on a Perkin-Elmer Model 377 Automated Sequencer.
• primers CMVf- ⁇ goM IV (5' TTGCCGGCATCAGATTGGCTAT (SEQ ID ⁇ O:40); underlined bases denote a NgoM IV recognition site) and Syn-polyA- BstE II (5' AGAGGTCACCGGGTCAATTCTTCAGCACCTGGTA (SEQ ID NO:41); underlined bases denote a BstE II recognition site) were used to PCR amplify the entire CMV promoter, mATS, and synthetic polyA for cloning upstream of the transposon in pTnLac.
• PCR was conducted with FailSafeTM as described above, purified using the Zymo Clean and Concentrator kit, the ends digested with NgoM IV and BstE II (New England Biolabs), purified with the Zymo kit again and cloned upstream of the fransposon in pTnLac as described below.
• Plasmid pTnLac was digested with NgoM IV and BstE II to remove the ptac promoter and fransposase and the fragments separated on an agarose gel.
• the band corresponding to the vector and transposon was excised, purified from the agarose, and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs) to prevent self-annealing.
• the enzyme was removed from the vector using a Zymo DNA Clean and Concentrator-5.
• the purified vector and CMVp/mATS/polyA were ligated together using a Stratagene T4 Ligase Kit and transformed into E. coli as described above.
• Base pairs 1-130 are a remainder of Fl(-) on from pBluescriptll sk(-) (Stratagene), corresponding to base pairs 1-130 of pBluescriptll sk(-).
• Base pairs 131 - 132 are a residue from ligation of restriction enzyme sites used in constructing the vector.
• Base pairs 133 -1777 are the CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems), corresponding to bp 229-1873 of pGWiz.
• the CMV promoter was modified by the addition of an ACC sequence upstream of ATG.
• Base pairs 1778-1779 are a residue from ligation of restriction enzyme sites used in constructing the vector.
• Base pairs 1780 - 2987 are the coding sequence for the fransposase, modified from TnlO (GenBank accession JO 1829) by optimizing codons for stability of the fransposase mRNA and for the expression of protein. More specifically, in each of the codons for the first ten amino acids of the transposase, G or C was changed to A or T when such a substitation would not alter the amino acid that was encoded.
• Base pairs 2988-2993 are two engineered stop codons.
• Base pair 2994 is a residue from ligation of restriction enzyme sites used in constructing the vector.
• Base pairs 2995 - 3410 are a synthetic polyA sequence taken from the pGWiz vector (Gene Therapy Systems), corresponding to bp 1922-2337 of 10 pGWiz.
• Base pairs 3415 - 3718 are non-coding DNA that is residual from vector pNK2859.
• Base pairs 3719 - 3761 are non-coding ⁇ DNA that is residual from pNK2859.
• Base pairs 3762 - 3831 are the 70 bp of the left insertion sequence recognized by the fransposon TnlO.
• Base pairs 3832-3837 are a residue from ligation of restriction enzyme sites used in constructing the vector.
• Base pairs 3838 - 4527 are the multiple cloning site from pBluescriptll sk(20), corresponding to bp 924-235 of pBluescriptll sk(-). This multiple cloning site may be used to insert any coding sequence of interest into the vector.
• Base pairs 4528-4532 are a residue from ligation of restriction enzyme sites used in constructing the vector.
• Base pairs 4533 - 4602 are the 70 bp of the right insertion sequence recognized by the transposon TnlO.
• Base pairs 4603 - 4644 are non-coding ⁇ DNA that is residual from pNK2859.
• Base pairs 4645 - 5488 are non-coding DNA that is residual from pNK2859.
• Base pairs 5489 - 7689 are from the pBluescriptll sk(-) base vector - (Stratagene, Inc.), corresponding to bp 761-2961 of pBluescriptll sk(-).
• Completing pTnMod is a pBlueScript backbone that contains a colE I origin of replication and an antibiotic resistance marker (ampicillin).
• Plasmid DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37°C overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, CA) according to the manufactarer's protocol. Plasmid DNA was resuspended in 500 ⁇ L of PCR-grade water and stored at -20°C until used.
• EXAMPLE 3 Transposon-Based Vector pTnMCS Another transposon-based vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells. This vector was termed pTnMCS and its constitaents are provided below.
• the sequence of the pTnMCS vector is provided in SEQ ID NO:2.
• the pTnMCS vector contains an avian optimized polyA sequence operably-linked to the transposase gene.
• the avian optimized polyA sequence contains approximately 40 nucleotides that precede the A nucleotide string.
• EXAMPLE 4 Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/Prolns/PA) ⁇ Chicken A vector was designed to insert a humsan proinsulin coding sequence under the confrol of a chicken ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird given below as SEQ ID NO:42.
• Base pairs 1 - 130 are a remainder of Fl(-) ori of pBluescriptll sk(-) (Stratagene) corresponding to base pairs 1 - 130 of pBluescriptll sk(-) .
• Base pairs 133 - 1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
• Base pairs 1780 - 2987 are a transposase, modified from TnlO (GenBank accession number J01829).
• Base pairs 2988-2993 are two engineered stop codons.
• Base pairs 2995 - 3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922- 2337 of pGWiz.
• Base pairs 3415 - 3718 are non coding DNA that is residual from vector pNK2859.
• Base pairs 3719 - 3761 are ⁇ DNA that is residual from pNK2859.
• Base pairs 3762 - 3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon TnlO.
• Base pairs 3838 - 4044 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 924-718 of pBluescriptll sk(-) .
• Base pairs 4050 - 4951 are a chicken ovalbumin promoter (including SDRE) that corresponds to base pairs 431-1332 of the chicken ovalbumin promoter in GenBank Accession Number J00895 M24999.
• Base pairs 4958 - 6115 are a chicken ovalbumin signal sequence and ovalbumin gene that correspond to base pairs 66-1223 of GenBank Accession Number V00383.1. (The STOP codon being omitted).
• Base pairs 6122 - 6271 are a TAG sequence containing a gp41 hai ⁇ in loop from HIV I, an enterokinase cleavage site and a spacer (synthetic).
• Base pairs 6272 - 6531 are a proinsulin gene.
• Base pairs 6539 - 6891 are a synthetic polyadenylation sequence from pGWiz
• Base pairs 6897 - 7329 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 667-235 of pBluescriptll sk(-).
• Base pairs 7335- 7404 are the 70 base pairs of the right insertion sequence (IS 10) recognized by the transposon Tnl 0.
• Base pairs 7405 - 7446 are ⁇ DNA that is residual from pNK2859.
• Base pairs 7447 - 8311 are non coding DNA that is residual from pNK2859.
• Base pairs 8312 - 10512 are pBlueScript sk(-) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961of pBluescriptll sk(-).
• a vector was designed to insert a proinsulin coding sequence under the control of a quail ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird given below as SEQ ID NO:43.
• EXAMPLE 6 Transfection of Japanese Quail using a Transposon-based Vector containing a Proinsulin Gene via Oviduct Injections
• Two experiments were conducted in Japanese quail using transpson-based vectors containing either Oval promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A (SEQ ID NO:42) or CMV promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A (SEQ ID NO:43).
• the Oval promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A containing constract was injected into the lumen of the oviduct of sexually mature quail; three hens received 5 ⁇ g at a 1:3 SUPERFECT® ratio and three received 10 ⁇ g at a 1:3 SUPERFECT® ratio.
• at least one bird that received above-mentioned construct was producing human proinsulin in egg white (other birds remain to be tested).
• each quail egg contains approximately 1.4 ⁇ g/ml of the proinsulin protein. It is also estimated that each transgenic chicken egg contains 50-75 mg of protein encoded by the gene of interest.
• the transposon-based vector containing CMV promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A was injected into the lumen of the oviduct of sexually immature Japanese quail. A total of 9 birds were injected.
• This blocking solution was subsequently decanted and replaced with a solution of the primary antibody (Goat Anti-GP41 TAG) diluted in fresh PBS/BSA/Tween 20. After a two hour period of incubation with the primary antibody, each plate was washed with a solution of PBS and 0.05% Tween 20 in an automated plate washer to remove unbound antibody. Next, the secondary antibody, Rabbit anti-Goat Alkaline Phosphatase-conjugated, was diluted in PBS/BSA/Tween 20 and allowed to incubate 1 hour. The plates were then subjected to a second wash with PBS/Tween 20.
• the primary antibody Goat Anti-GP41 TAG
• Antigen was detected using a solution of ?-Nitrophenyl Phosphate in Diethanolamine Substrate Buffer for Alkaline Phosphatase and measuring the absorbance at 30 minutes and 1 hour. Additionally, a proinsulin fusion protein produced using a construct described above was isolated from egg white using ammonium sulfate precipitation and ion exchange chromotgraphy. A pooled fraction of the isolated fusion protein was run on an SDS-PAGE gel shown in Figure 5, lanes 4 and 6. Lanes 1 and 10 of the gel contain molecular weight standards, lanes 2 and 8 contain non-frangenic chicken egg white, whereas lanes 3, 5, 7 and 9 are blank.
• EXAMPLE 7 Isolation of Human Proinsulin Using Anti-TAG Column Chromotography
• a HiTrap NHS-activated 1 mL column (Amersham) was charged with a 30 amino acid peptide that contained the gp-41 epitope containing gp-41 's native disulfide bond that stabilizes the formation of the gp-41 hai ⁇ in loop.
• the 30 amino acid gp41 peptide is provided as SEQ ID NO:32.
• Approximately 10 mg of the peptide was dissolved in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3 and the ligand was circulated on the column for 2 hours at room temperatare at 0.5 mL/minute.
• Antibodies to gp-41 were raised in goats by inoculation with the gp-41 peptide described above. More specifically, goats were inoculated, given a booster injection of the gp-41 peptide and blood samples were obtained by veinupunctare. Serum was harvested by centrifugation. Approximately 30 mL of goat serum was filtered to 0.45 uM and passed over a TAG column at a rate of 0.5 mL/min. The column was washed with 75 mM Tris, pH 8.0 until absorbance at 280 nm reached a baseline.
• the pooled fractions from the Anti-TAG affinity column were characterized by SDS-PAGE and western blot analysis.
• SDS-PAGE of the pooled fractions revealed a 60 kDal molecular weight band not present in control egg white fluid, consistent with the predicted molecular weight of the transgenic protein. Although some contaminating bands were observed, the 60 kDal species was greatly enriched compared to the other proteins.
• An aliquot of the pooled fractions was cleaved overnight at room temperatare with the protease, enterokinase.
• SDS-PAGE analysis of the cleavage product revealed a band not present in the uncut material that co- migrated with a commercial human proinsulin positive control.
• Eggs from hens positively identified as producing RPI are pooled for RPI purification.
• the initial purification step involved diluting the pool 1:1 with 100 mM Tris-HCl, pH 8 for a final concentration of 50 mM Tris-HCl.
• the pH of this solution was then adjusted to 6 and ovomucin was allowed to precipitate at 4°C for a minimum of 3hrs (preferably overnight) with constant stirring.
• the precipitated ovomucin was then pelleted and removed by centrifugation at 2400 x g. After collection of the RPI containing supernatant, the pH of this solution was readjusted to 8.
• the ovomucin precipitated egg white solution was subjected to successive filtration steps with the pore size of the filtration membrane decreasing at each step. This procedure involved time and dilution of the egg white solution to reach 0.45 um filtration.
• Amersham's hollow-fiber ultrafilfration apparatus was used to produced a column-ready solution filtered down to ⁇ 0.2 um with an undiluted starting solution. This approach minimized the time and the solution dilution needed to prepare the egg white solution for column loading.
• initial purification schemes involved developing a one- step column purification procedure for the RPI.
• Goats immunized with the synthetic Ent peptide were employed to produce anti-Ent Tag antiserum which was used in the egg screening ELISAs followed by antibody purification.
• the purified goat Anti-Ent Tag antibodies were covalently bound to the matrix of HiTrap NHS-activated HP columns (Amersham) and subsequently used to specifically bind and purify the RPI.
• the present purification scheme employed a combination of classical techniques such as ammonium sulfate precipitation, ion exchange, and gel filtration chromatography.
• the egg white solution was subjected to protein precipitation using a 40% ammonium sulfate fractionation.
• the precipitated protein was subsequently collected via centrifugation and resuspended in 50 mM Tris-HCl, pH 8.
• the resuspended protein solution was dialyzed to remove residual NH 4 ) 2 SO 4 or subjected to gel filtration to remove the (NH 4 ) 2 SO 4 and partially isolate the RPI from the remaining egg white protein.
• the RPI was further isolated via anion exchange chromatography using a 0 to 0.5M NaCl gradient in 50 mM Tris-HCl, pH 8. Two possible elution profiles were observed.
• Cleavage of the RPI Enterokinase recognition site was accomplished using purified enterokinase from Sigma. Enterokinase, 0.004 Unit/ ⁇ l per reaction, was applied to the pooled and, if necessary, concentrated protein solution. The digestion reaction was incubated at room temperatare (up to 30°C in a rolling hybridization oven) for a minimum of 16 h and in some cases up to 48 hrs of incubation. The digestion efficiency was followed using 16.5% Tris-Tricine SDS-PAGE peptide gels. All gel staining utilized Simply Blue Coomassie Staining Solutions. Free Proinsulin was observed on gels after digestion.
• fransfection rates of oviduct cells in a flock of chicken or quail hens are enhanced by synchronizing the development of the oviduct and ovary within the flock.
• timing of injections is optimized to transfect the greatest number of cells. Accordingly, oviduct development is synchronized as described below to ensure that a large and uniform proportion of oviduct secretory cells are transfected with the gene of interest.
• the lumens of the oviducts of freated hens are injected with the transposon-based vector. Hens are subjected to additional esfrogen stimulation after an optimized time during which the transposon-based vector is taken up into oviduct secretory cells. Re-stimulation by estrogen activates transposon expression, causing the integration of the gene of interest into the host genome. Esfrogen stimulation is then withdrawn and hens continue normal sexual development. If a developmentally regulated promoter such as the ovalbumin promoter is used, expression of the transposon-based vector initiates in the oviduct at the time of sexual maturation. Intra-ovarian artery injection during this window allows for high and uniform transfection efficiencies of ovarian follicles to produce germ-line fransfections and possibly oviduct expression.
• EXAMPLE 10 Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/Prolns/PA) — Quail
• a vector is designed for inserting a proinsulin gene under the control of a quail ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird given below as SEQ ID NO:44.
• Base pairs 1 -130 are a remainder of Fl(-) ori of pBluescriptll sk(-) (Stratagene) corresponding to base pairs 1-130 of pBluescriptll sk(-).
• Base pairs 133 - 1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
• Base pairs 1780 - 2987 are a transposase, modified from TnlO (GenBank accession number JO 1829).
• Base pairs 2988-2993 are an engineered stop codon.
• Base pairs 2995 - 3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922- 2337 of pGWiz.
• Base pairs 3415 - 3718 are non coding DNA that is residual from vector pNK2859.
• Base pairs 3719 - 3761 are ⁇ DNA that is residual from ⁇ NK2859.
• Base pairs 3762 - 3831 are the 70 base pairs of the left insertion sequence (IS 10) recognized by the fransposon TnlO.
• Base pairs 3838 - 4044 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 924-718 of pBluescriptll sk(-).
• Base pairs 4050 - 4938 are the Japanese quail ovalbumin promoter (including
• the Japanese quail ovalbumin promoter was isolated by its high degree of homology to the chicken ovalbumin promoter (GenBank accession number J00895 M24999, base pairs 431-1332). Some deletions were noted in the quail sequence, as compared to the chicken sequence.
• Base pairs 4945 - 6092 are a quail ovalbumin signal sequence and ovalbumin gene that corresponds to base pairs 54 - 1201 of GenBank accession number X53964.1. (The STOP codon being omitted).
• Base pairs 6093 - 6246 are a TAG sequence containing a gp41 hai ⁇ in loop from HIV I an enterokinase cleavage site and a spacer (synthetic).
• Base pairs 6247 - 6507 are a proinsulin gene.
• Base pairs 6514 - 6866 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920 - 2272of pGWiz.
• Base pairs 6867 - 7303 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 667-235 of pBluescriptll sk(-).
• Base pairs 7304- 7379 are the 70 base pairs of the right insertion sequence (IS 10) recognized by the transposon TnlO.
• Base pairs 7380 - 7421 are ⁇ DNA that is residual from ⁇ NK2859.
• Base pairs 7422 - 8286 are non coding DNA that is residual from pNK2859.
• Base pairs 8287 - 10487 are pBlueScript sk(-) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptll sk(-).
• EXAMPLE 11 Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/pl46/PA) - Chicken
• a vector was designed for inserting a pi 46 gene under the confrol of a chicken ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird.
• the vector sequence is provided below as SEQ ID NO:45.
• Base pairs 1 - 130 are a remainder of Fl(-) ori of pBluescriptll sk(-) (Stratagene) corresponding to base pairs 1-130 of pBluescriptll sk(-).
• Base pairs 133 - 1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
• Base pairs 1780 - 2987 are a fransposase, modified from TnlO (GenBank accession number JO 1829).
• Base pairs 2988-2993 are an engineered stop codon.
• Base pairs 2995 - 3410 are a synthetic polyA from pGWiz (Gene Therapy
• Base pairs 3415 - 3718 are non coding DNA that is residual from vector pNK2859.
• Base pairs 3719 - 3761 are ⁇ DNA that is residual from pNK2859.
• Base pairs 3762 - 3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the fransposon TnlO.
• Base pairs 3838 - 4044 are a multiple cloning site from pBlueScript ⁇ sk(-) corresponding to base pairs 924-718 of pBluescriptll sk(-).
• Base pairs 4050 - 4951 are a chicken ovalbumin promoter (including SDRE, steroid-dependent response element) that corresponds to base pairs 431-1332 of the chicken ovalbumin promoter in GenBank Accession Number J00895 M24999.
• Base pairs 4958 - 6115 are a chicken ovalbumin signal sequence and Ovalbumin gene that correspond to base pairs 66-1223 of GenBank Accession Number V00383.1 (The STOP codon being omitted).
• Base pairs 6122 - 6271 are a TAG sequence containing a gp41 hai ⁇ in loop from HIV I, an enterokinase cleavage site and a spacer (synthetic).
• Base pairs 6272 - 6316 are a pl46 sequence (synthetic) with 2 added stop codons.
• Base pairs 6324 - 6676 are a synthetic polyadenylation sequence from pGWiz
• Base pairs 6682 - 7114 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 667-235 of pBluescriptll sk(-).
• Base pairs 7120- 7189 are the 70 base pairs of the right insertion sequence (IS10) recognized by the fransposon TnlO.
• Base pairs 7190 - 7231 are ⁇ DNA that is residual from pNK2859.
• Base pairs 7232 - 8096 are non coding DNA that is residual from pNK2859.
• Base pairs 8097 - 10297 are pBlueScript sk(-) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptll sk(-).
• Base pairs 133 - 1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
• Base pairs 1780 - 2987 are a fransposase, modified from TnlO (GenBank accession number JO 1829).
• Base pairs 2988-2993 are an engineered stop codon.
• Base pairs 2995 - 3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz.
• Base pairs 3415 - 3718 are non coding DNA that is residual from vector pNK2859.
• Base pairs 3719 - 3761 are ⁇ DNA that is residual from pNK2859.
• Base pairs 3762 - 3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon TnlO.
• Base pairs 3838 - 4044 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 924-718 of pBluescriptll sk(-).
• Base pairs 4050 - 4938 are the Japanese quail ovalbumin promoter (including SDRE, steroid-dependent response element).
• the Japanese quail ovalbumin promoter was isolated by its high degree of homology to the chicken ovalbumin promoter (GenBank accession number J00895 M24999, base pairs 431-1332).
• Bp 4945 - 6092 are a quail ovalbumin signal sequence and ovalbumin gene that corresponds to base pairs 54 - 1201 of GenBank accession number X53964.1. (The STOP codon being omitted).
• Base pairs 6097 - 6246 are a TAG sequence containing a gp41 hai ⁇ in loop from HIV I, an enterokinase cleavage site and a spacer (synthetic).
• Base pairs 6247 - 6291 are a pi 46 sequence (synthetic) with 2 added stop codons.
• Base pairs 6299 - 6651 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920 - 2272of pGWiz.
• Base pairs 6657 - 7089 are a multiple cloning site from pBlueScriptll sk(-) corresponding to base pairs 667-235 of pBluescriptll sk(-).
• Base pairs 7095- 7164 are the 70 base pairs of the right insertion sequence (IS 10) recognized by the fransposon TnlO.
• Base pairs 7165 - 7206 are ⁇ DNA that is residual from pNK2859.
• Base pairs 7207 - 8071 are non coding DNA that is residual from pNK2859.
• Base pairs 8072 - 10272 are pBlueScript sk(-) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptll sk(-).
• the following example provides a description of various fransposon-based vectors of the present invention and several constructs that have been made for insertion into the transposon-based vectors of the present invention. These examples are not meant to be limiting in any way.
• the constructs for insertion into a fransposon-based vector are provided in a cloning vector pTnMCS or pTnMod, both described above.
• pTnMCS (Ouail OVep+OVg'+ENT+proins+svn polvA ⁇ ) Bp 1 - 3670 from cloning vector pTnMCS, bp 1 - 3670 Bp 3676 - 4333 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upsfream chicken ovalbumin enhancer, GenBank accession # S82527.1, bp 1-675.
• Bp 4340 - 5705 Quail Ovalbumin promoter 1366 bp sequence, amplified in-house from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitutions and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.) Bp 5712 - 6910 Quail Ovalbumin gene, EMBL accession # X53964, bp 1-1199.
• GenBank accession #S82527.1, bp 1-675 GenBank accession #S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)
• Quail Ovalbumin promoter 1366 bp sequence, amplified from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitutions and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)
• Bp 4051 - 5695 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1864
• Quail Ovalbumin enhancer 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upsfream chicken ovalbumin enhancer, GenBank accession # S82527.1, bp 1-675. (There are multiple base pair substitations and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)
• Quail Ovalbumin promoter 1366 bp sequence, amplified in-house from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter,
• GenBank accession # J00895-M24999 bp 1-1336 accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitations and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)
• Quail Ovalbumin enhancer 658 bp sequence, amplified in- housefrom quail genomic DNA, roughly equivalent to the far-upsfream chicken ovalbumin enhancer, GenBank accession #S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)
• Quail Ovalbumin promoter 1366 bp sequence, amplified in-house from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitations and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)
• pTnMOD CMV-prepro-ent-hGH-CPA Bp 1-4045 from vector PTnMOD, bp 1 - 4045
• Bp 4051-5694 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1873
• BP 133-1777 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems) bp 229-1873.
• BP 3344-3386 Lambda DNA from pNK2859.
• BP 7345-7752 ConPolyA from Chicken conalbumin polyA from GenBank # Y00407 bp 10651-11058. (5'SacII, 3'XhoI) BP 7753-8195 multiple cloning site from pBluescriptll sk(-) bp 677-235.

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