US20040172667A1 - Administration of transposon-based vectors to reproductive organs - Google Patents

Administration of transposon-based vectors to reproductive organs Download PDF

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US20040172667A1
US20040172667A1 US10/746,149 US74614903A US2004172667A1 US 20040172667 A1 US20040172667 A1 US 20040172667A1 US 74614903 A US74614903 A US 74614903A US 2004172667 A1 US2004172667 A1 US 2004172667A1
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method
promoter
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transposon
sequence
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Richard Cooper
William Fioretti
Gary Cadd
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Louisiana State University and Agricultural and Mechanical College
TransGenRx Inc
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Louisiana State University and Agricultural and Mechanical College
TransGenRx Inc
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Priority to US44137703P priority
Priority to US44140503P priority
Priority to US44138103P priority
Priority to US44144703P priority
Priority to US44139203P priority
Priority to US10/609,019 priority patent/US7527966B2/en
Priority to US10/746,149 priority patent/US20040172667A1/en
Application filed by Louisiana State University and Agricultural and Mechanical College, TransGenRx Inc filed Critical Louisiana State University and Agricultural and Mechanical College
Assigned to BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COLLEGE, THE reassignment BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COLLEGE, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COOPER, RICHARD K.
Assigned to TRANSGENRX, INC. reassignment TRANSGENRX, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CADD, GARY C., FIORETTI, WILLIAM C.
Publication of US20040172667A1 publication Critical patent/US20040172667A1/en
Assigned to TRANSGENRX, INC. reassignment TRANSGENRX, INC. TO CORRECT NAME OF ASSIGNOR GARY C. CADD TO GARY G. CADD ON NOTICE OF ASSIGNMENT RECORDATION DOCUMENT ON REEL 015010, FRAME 0117; RECORDED ON 02/26/2004 Assignors: CADD, GARY G., FIORETTI, WILLIAM C.
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/62Insulins
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • A01K67/0275Genetically modified vertebrates, e.g. transgenic
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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    • C07K16/26Immunoglobulins [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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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/105Murine
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/30Bird
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/01Animal expressing industrially exogenous proteins
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23Immunoglobulins specific features characterized by taxonomic origin from birds
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    • C12N2800/00Nucleic acids vectors
    • C12N2800/90Vectors containing a transposable element
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    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/001Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003Vector 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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    • C12N2830/00Vector systems having a special element relevant for transcription
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    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/80Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/90Vector systems having a special element relevant for transcription from vertebrates avian
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    • C12N2840/00Vectors comprising a special translation-regulating system
    • C12N2840/20Vectors comprising a special translation-regulating system translation of more than one cistron

Abstract

Methods and compositions for the administration of transposon-based vectors to the reproductive organs of animals and the creation of transgenic animals. Preferred methods involve administration of the transposon-based vectors to the lumen of the oviduct of an avian, expression of a vector derived transgene in the avian, and deposition of the resultant polypeptide in an egg. This invention allows for large amounts of protein to be deposited in the egg.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • The present application is a continuation-in-part of U.S. patent application No. 10/609,019 filed on Jun. 26, 2003, and claims the priority benefit of U.S. Provisional Patent Application No. 60/441,392 filed Jan. 21, 2003; U.S. Provisional Patent Application No. 60/441,377 filed Jan. 21, 2003; U.S. Provisional Patent Application No. 60/441,502 filed Jan. 21, 2003; U.S. Provisional Patent Application No. 60/441,405 filed Jan. 21, 2003; U.S. Provisional Patent Application No. 60/441,447 filed Jan. 21, 2003; U.S. Provisional Patent Application No. 60/441,381 filed Jan. 21, 2003; and U.S. Provisional Patent Application No. 60/392,415 filed Jun. 26, 2002.[0001]
  • [0002] The U.S. Government has certain rights in this invention. The development of this invention was partially funded by the United States Government under a HATCH grant from the United States Department of Agriculture, partially funded by the United States Government with Formula 1433 funds from the United States Department of Agriculture and partially funded by the United States Government under contract DAAD 19-02016 awarded by the Army.
  • FIELD OF THE INVENTION
  • 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. In some embodiments, the present invention further includes production of a protein encoded by the gene in an egg produced by the animal. [0003]
  • BACKGROUND OF THE INVENTION
  • 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. 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. [0004]
  • 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 β-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. S157-S162). In addition, hyper- and hypoglycemia occurs frequently despite intensive home blood glucose monitoring. Finally, 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. [0005]
  • Several of the prior art gene delivery technologies employed viruses that are associated with potentially undesirable side effects and safety concerns. The majority of current gene-delivery technologies useful for gene therapy rely on virus-based delivery vectors, such as adeno and adeno-associated viruses, retroviruses, and other viruses, which have been attenuated to no longer replicate. (Kay, M. A., et al. 2001. Nature Medicine 7:33-40). [0006]
  • 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, Dec. 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. Fifthly, viral-based vectors indiscriminately integrate into various cells, which can result in undesirable germline integration. Sixthly, the required high titers needed to achieve the desired effect have resulted in the death of one patient and they are believed to be responsible for induction of cancer in a separate study. (Science, News of the Week, Oct. 4, 2002). [0007]
  • Accordingly, what is needed is a new method to produce transgenic animals and humans with stably incorporated genes, in which the vector containing those genes does not cause disease or other unwanted side effects. There is also a need for DNA constructs that would be stably incorporated into the tissues and cells of animals and humans, including cells in the resting state that are not replicating. There is a further recognized need in the art for DNA constructs capable of delivering genes to specific tissues and cells of animals and humans. [0008]
  • When incorporating a gene of interest into an animal for the production of a desired protein or when incorporating a gene of interest in an animal or human for the treatment of a disease, it is often desirable to selectively activate incorporated genes using inducible promoters. These inducible promoters are regulated by substances either produced or recognized by the transcription control elements within the cell in which the gene is incorporated. In many instances, 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. This can be taken a step further to cause incorporation to be tissue-specific, which prevents widespread gene incorporation throughout a patient's body (animal or human). This decreases the amount of DNA needed for a treatment, decreases the chance of incorporation in gametes, and targets gene delivery, incorporation, and expression to the desired tissue where the gene is needed to function. What is also needed is a rapid expression method for rapidly producing a protein or peptide of interest in eggs and milk of transgenic animals. [0009]
  • SUMMARY OF THE INVENTION
  • The present invention addresses the problems described above by providing new, effective and efficient compositions for producing transgenic animals and for treating disease in animals or humans. 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. In another preferred embodiment, the animal is a milk-producing animal, including but not limited to bovine, porcine, ovine and equine animals. In a preferred embodiment, the animal is an avian animal. In another preferred embodiment, 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. The 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. In a preferred embodiment, the compositions of the present invention are introduced into the the reproductive system of an avian animal. In another preferred embodiment, 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. [0010]
  • The transposon-based vectors of the present invention include a transposase, operably-linked 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 transposase 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 transposase, 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 transposase gene. In some embodiments, the effective polyA sequence is an avian optimized polyA sequence. [0011]
  • 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. In some embodiments, 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. [0012]
  • The present invention advantageously produces a high number of transgenic animals having a gene of interest stably incorporated. In some embodiments wherein the transposon-based vector is administered to the ovary, these transgenic animals successfully pass the desired gene to their progeny. Accordingly, 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 transgenic 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. [0013]
  • 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. [0014]
  • 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. [0015]
  • 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 transgenic 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. [0016]
  • A wide range of recombinant peptides and proteins can be produced in transgenic egg-laying animals. Enzymes, hormones, antibodies, growth factors, serum 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. [0017]
  • In one embodiment, the egg-laying transgenic 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. Preferably, the egg-laying transgenic 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. [0018]
  • Accordingly, it is an object of the present invention to provide novel transposon-based vectors. [0019]
  • It is another object of the present invention to provide novel transposon-based vectors that encode for the production of desired proteins or peptides in cells. [0020]
  • It is an object of the present invention to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector. [0021]
  • Another object of the present invention is to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector, wherein the transgenic animals produce desired proteins or peptides. [0022]
  • It is further an object of the present invention to provide a method to produce transgenic animals through intraovarian administration of a transposon-based vector that are capable of producing transgenic progeny. [0023]
  • 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. [0024]
  • 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, wherein such administration results in modulation of endogenous gene expression. [0025]
  • It is yet another object of the present invention to provide a method to produce transgenic avians through intraoviduct or intraovarian administration of a transposon-based vector that are capable of producing proteins, peptides or nucleic acids. [0026]
  • It is another object of the present invention to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector encoding an antibody or a fragment thereof. [0027]
  • Still another object of the present invention is to provide a method to produce transgenic 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. [0028]
  • Another object of the present invention is to provide transgenic avians that contain a stably incorporated transgene. [0029]
  • Still another object of the present invention is to provide eggs containing desired proteins or peptides encoded by a transgene incorporated into the transgenic avian that produces the egg. [0030]
  • It is further an object of the present invention to provide a method to produce transgenic milk-producing animals through administration of a transposon-based vector that are capable of producing proteins, peptides or nucleic acids. [0031]
  • Still another object of the present invention is to provide a method to produce transgenic milk-producing animals through administration of a transposon-based vector that are capable of producing proteins or peptides and depositing these proteins or peptides in their milk. [0032]
  • Another object of the present invention is to provide transgenic milk-producing animals that contain a stably incorporated transgene. [0033]
  • 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. [0034]
  • 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. [0035]
  • 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. [0036]
  • An advantage of the present invention is that transgenic animals are produced with higher efficiencies than observed in the prior art. [0037]
  • Another advantage of the present invention is that these transgenic animals possess high copy numbers of the transgene. [0038]
  • Another advantage of the present invention is that the transgenic animals produce large amounts of desired molecules encoded by the transgene. [0039]
  • 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. [0040]
  • 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. [0041]
  • These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and claims. [0042]
  • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 depicts schematically a transposon-based vector containing a transposase 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. “Pro” designates a promoter. In this and subsequent figures, the size of the actual nucleotide sequence is not necessarily proportionate to the box representing that sequence. [0043]
  • 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 and a protease cleavage site. [0044]
  • FIG. 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. [0045]
  • FIG. 4 depicts schematically a transposon based-vector for expression of an RNAi molecule. “Tet[0046] i 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 and “Ovomucin” indicates approximately 60 base pairs of an ovomucin gene.
  • FIG. 5 is a picture of an SDS-PAGE gel wherein a pooled fraction of an isolated proinsulin fusion protein was run in lanes [0047] 4 and 6. Lanes 1 and 10 of the gel contain molecular weight standards, lanes 2 and 8 contain non-trangenic chicken egg white, and lanes 3, 5, 7 and 9 are blank.
  • DETAILED DESCRIPTION OF THE INVETION
  • 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. In some embodiments, administration 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. In other embodiments, 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. [0048]
  • Definitions
  • It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized. [0049]
  • The term “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”. The term 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. [0050]
  • As used herein, the term “egg-laying animal” includes all amniotes such as birds, turtles, lizards and monotremes. Monotremes 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 warm-blooded, 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. The term “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. The term “Psittaciformes”, as used herein, 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. The term “chicken” as used herein denotes 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. [0051]
  • The term “egg” is defined herein as including a large female sex cell enclosed in a porous, calcarous or leathery shell, produced by birds and reptiles. The term “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. [0052]
  • The term “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. [0053]
  • The term “gene” is defined herein to include a coding region for a protein, peptide or polypeptide. [0054]
  • The term “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. In a preferred embodiment, 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. In a further preferred embodiment, the animal is an avian. [0055]
  • The term “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. [0056]
  • When referring to two nucleotide sequences, one being a regulatory sequence, the term “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). [0057]
  • The term “regulatory sequence” is defined herein as including promoters, enhancers and other expression control 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. [0058]
  • Transposon-Based Vectors
  • While not wanting to be bound by the following statement, it is believed that the nature of the DNA construct is an important factor in successfully producing transgenic animals. The “standard” types of plasmid and viral vectors that have previously been almost universally used for transgenic work in all species, especially avians, have low efficiencies and may constitute a major reason for the low rates of transformation previously observed. 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. Two preferred transposon-based vectors of the present invention in which a tranposase, gene of interest and other polynucleotide sequences may be introduced are termed pTnMCS (SEQ ID NO:2) and pTnMod (SEQ ID NO:3). [0059]
  • 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. Pat. 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. [0060]
  • The transposon-based vectors of the present invention include a transposase 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:1) 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 transposase to further enhance expression of the transposase gene. 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). [0061]
  • FIG. 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. It is also to be understood that 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. [0062]
  • Transposases and Insertion Sequences
  • In a further embodiment of the present invention, 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 transposase. However, 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, Tn5SEQ1, Tn916, Tn951, Tn1721, Tn 2410, Tn1681, Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn903, Tn501, Tn1000 (γδ), Tn1681, Tn2901, AC transposons, Mp transposons, Spm transposons, En transposons, Dotted transposons, Mu transposons, Ds transposons, dSpm transposons and I transposons. According to the present invention, 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:1) 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 transposase gene. [0063]
  • Although not wanting to be bound by the following statement, it is believed that the modifications of the first several N-terminal codons of the transposase 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 transgene insertion as discussed in U.S. Pat. No. 5,719,055. [0064]
  • In some embodiments, 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. [0065]
  • 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. Pat. 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. Lampe 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. Natl. Acad. Sci. USA, 98:11474-11478; and D. Berg et al. (Eds.), Mobile DNA, Amer. Soc. Microbiol. (Washington, D.C., 1989). However, it should be noted that bacterial transposon-based elements are preferred, as there is less likelihood that a eukaryotic transposase in the recipient species will recognize prokaryotic insertion sequences bracketing the transgene. [0066]
  • Many transposases recognize different insertion sequences, and therefore, it is to be understood that a transposase-based vector will contain insertion sequences recognized by the particular transposase also found in the transposase-based vector. In a preferred embodiment of the invention, 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. [0067]
  • While the examples provided below incorporate a “cut and insert” Tn10 based vector that is destroyed following the insertion event, the present invention also encompasses the use of a “rolling replication” type transposon-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 transposon-based system thereby provides for insertion of multiple copies of a transgene 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. However, use of a rolling replication system may require tight control to limit the insertion events to non-lethal levels. Tn1, Tn2, Tn3, Tn4, Tn5, Tn9, Tn21, Tn501, Tn551, Tn951, Tn1721, 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. [0068]
  • Stop Codons and PolyA Sequences
  • In one embodiment, the transposon-based vector contains two stop codons operably-linked to the transposase and/or to the gene of interest. In an alternate embodiment, one stop codon of UAA or UGA is operably linked to the transposase and/or to the gene of interest. [0069]
  • As used herein 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. In one embodiment, 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. In one embodiment of the present invention, the polyA sequence comprises a conalbumin polyA sequence as provided in SEQ ID NO:11 and as taken from GenBank accession #Y00407, base pairs 10651-11058. In another embodiment, the polyA sequence comprises a synthetic polynucleotide sequence shown in SEQ ID NO:12. In yet another embodiment, 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. [0070]
  • It is a surprising discovery of the present invention that such an avian optimized poly A sequence increases expression of a polynucleotide to which it is operably-linked in an avian as compared to a non-avian optimized polyA sequence. Accordingly, 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. [0071]
  • Promoters and Enhancers
  • 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, β-actin promoter, tubulin promoter, Rous-Sarcoma virus (RSV) promoter, and heat-shock protein (HSP) promoter. Inducible promoters include tissue-specific promoters, developmentally-regulated promoters and chemically inducible promoters. Examples of tissue-specific promoters include the glucose 6 phosphate (G6P) promoter, vitellogenin promoter, ovalbumin promoter, ovomucoid promoter, conalbumin promoter, ovotransferrin promoter, prolactin promoter, kidney uromodulin promoter, and placental lactogen promoter. In one embodiment, 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. [0072]
  • Other 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. 90:1657-1661); 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 the ability to bind ligand and can be turned on by RU486 (Wang, et al. 1994. Proc. Natl. Acad. Sci. 91:8180-8184); 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 transgene of interest via any method known to those of skill in the art. [0073]
  • Other examples of 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. 22(24):8539-51); prostate specific antigen (PSA) promoter (Yoshimura I. et al., 2002, J. Urol. 168(6):2659-64); exorh promoter and pineal expression-promoting element (Asaoka Y., et al., 2002. Proc. Natl. Acad. Sci. 99(24):15456-61); neural and liver ceramidase gene promoters (Okino N. et al., 2002. Biochem. Biophys. Res. Commun. 299(1):160-6); PSP94 gene promoter/enhancer (Gabril M. Y. et al., 2002. Gene Ther. 9(23): 1589-99); 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 Oct. 24, 2002); and, IL-10 promoter (Brenner S., et al., 2002. J. Biol. Chem. Dec. 18, 2002). [0074]
  • Examples of 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. Z. Lebensm. Unters. Forsch. 202:1-14). An advantage of using the 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. In some embodiments, the avian promoter is an oviduct-specific promoter. As used herein, the term “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. [0075]
  • When germline transformation occurs via intraovarian administration, 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, bilirubin UDP-glucuronosyltransferase promoter, aldolase B promoter, furin promoter, metallothioneine promoter, albumin promoter, and insulin promoter. [0076]
  • Also included in the present invention are 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, β lactoglobin promoter, whey acidic protein promoter, lactalbumin promoter and casein promoter. [0077]
  • 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. [0078]
  • Also included in this invention are modified promoters/enhancers wherein elements of a single promoter are duplicated, modified, or otherwise changed. In one embodiment, 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. [0079]
  • Accordingly, it is to be understood that the promoters contained within the transposon-based vectors of the present invention may be entire promoter sequences or fragments of promoter sequences. For example, in one embodiment, 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:1). [0080]
  • As indicated above, the present invention includes transposon-based vectors containing one or more enhancers. These 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. In one embodiment, 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. In one embodiment, 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. [0081]
  • Also included in some of the transposon-based vectors of the present invention are cap sites and fragments of cap sites. In one embodiment, approximately 50 base pairs of a 5′ untranslated region wherein 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. [0082]
  • In one embodiment of the present invention, 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. In the second embodiment, use of the first constitutive promoter allows for constitutive activation of the transposase 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 transposon-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. [0083]
  • It should be noted that 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. [0084]
  • When incorporation of the gene of interest into the germline is not preferred, the first promoter operably-linked to the transposase gene can be a tissue-specific promoter. For example, transfection of a transposon-based vector containing a transposase 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. In this embodiment, 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. In embodiments wherein tissue-specific expression or incorporation is desired, it is preferred that the transposon-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. In a preferred embodiment, the tissue of interest is the oviduct and administration is achieved by direct injection into the oviduct or an artery leading to the oviduct. In a further preferred embodiment, administration 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. [0085]
  • Accordingly, cell specific promoters may be used to enhance transcription in selected tissues. In birds, for example, 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. In mammals, 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. In liver cells, 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. [0086]
  • In order to achieve higher or more efficient expression of the transposase gene, the promoter and other regulatory sequences operably-linked to the transposase gene may be those derived from the host. These host specific regulatory sequences can be tissue specific as described above or can be of a constitutive nature. For example, 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. [0087]
  • Directing Sequences [0088]
  • 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. As used herein, 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 transposon-based vectors are administered to a milk-producing animal. [0089]
  • 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 transposon-based vectors are administered to a bird or other egg-laying animal. One example of such a transposon-based vector is provided in FIG. 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. An example of such an embodiment is shown in FIG. 2 wherein the gene of interest is operably-linked to the ovalbumin gene that contains an ovalbumin signal sequence. Other signal sequences that can be included in the transposon-based vectors include, but are not limited to the ovotransferrin and lysozyme signal sequences. In one embodiment, the signal sequence is an ovalbumin signal sequence including a sequence shown in SEQ ID NO:19. In another embodiment, the signal sequence is a modified ovalbumin signal sequence including a sequence shown in SEQ ID NO:20 or SEQ ID NO:21. [0090]
  • As also used herein, the term “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. One example of 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. In one embodiment, the vitellogenin targeting sequence includes the polynucleotide sequence of SEQ ID NO:22. In another embodiment, the vitellogenin targeting sequence includes all or part of the vitellogenin gene. Other targeting sequences include VLDL and Apo E, which 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. [0091]
  • Genes of Interest Encoding Desired Proteins
  • A gene of interest selected for stable incorporation is designed to encode any desired protein or peptide or to regulate any cellular response. In some embodiments, the desired proteins or peptides are deposited in an egg or in milk. It is to be understood that the present invention encompasses transposon-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). In one embodiment, 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. [0092]
  • 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 hypophysiotropic 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. Specifically, 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, adrenocorticotropic hormone, antidiuretic hormone, thyrotropin-releasing hormone (TRH), growth hormone-releasing hormone (GHRH), dopamine, melatonin, thyroxin (T[0093] 4), parathyroid hormone (PTH), glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, androgens such as testosterone, adrenaline (epinephrine), noradrenaline (norepinephrine), estrogens such as estradiol, progesterone, glucagons, calcitrol, calciferol, atrial-natriuretic peptide, gastrin, secretin, cholecystokinin (CCK), neuropeptide Y, ghrelin, PYY3-36, angiotensinogen, thrombopoietin, and leptin. By using appropriate polynucleotide sequences, species-specific hormones may be made by transgenic animals.
  • In one embodiment of the present invention, 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. FIGS. 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. In these embodiments, proinsulin is expressed in the oviduct tubular gland cells and then deposited in the egg white. One example of a proinsulin polynucleotide sequence is shown in SEQ ID NO:23, wherein the C-peptide cleavage site spans from Arg at position 31 to Arg at position 65. [0094]
  • Serum 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. Genes encoding modified versions of naturally occurring antibodies or fragments thereof and genes encoding artificially designed antibodies or fragments thereof 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 transposon-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. [0095]
  • Antibodies used as therapeutic reagents include but are not limited to antibodies for use in cancer immunotherapy against 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, Oreg.), which is incorporated herein in its entirety. [0096]
  • 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. [0097]
  • 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, anti-viral, 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. [0098]
  • Antibodies that may be produced using the present invention include, but are not limited to, antibodies made against the following proteins: Bovine γ-Globulin, Serum; Bovine IgG, Plasma; Chicken γ-Globulin, Serum; Human γ-Globulin, Serum; Human IgA, Plasma; Human IgA[0099] 1, Myeloma; Human IgA2, Myeloma; Human IgA2, 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 IgG1, Myeloma; Human IgG2, Myeloma; Human IgG3, Myeloma; Human IgG4, Myeloma; Human IgM, Myeloma; Human IgM, Plasma; Human Immunoglobulin, Light Chain K, Urine; Human Immunoglobulin, Light Chains κ and λ, Plasma; Mouse γ-Globulin, Serum; Mouse IgG, Serum; Mouse IgM, Myeloma; Rabbit γ-Globulin, Serum; Rabbit IgG, Plasma; and Rat γ-Globulin, Serum. In one embodiment, 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 γ-Globulin (Donkey); Anti-Goat IgG, Fc Fragment Specific (Rabbit); Anti-Guinea Pig γ-Globulin (Goat); Anti-Human Ig, Light Chain, Type κ Specific; Anti-Human Ig, Light Chain, Type λ Specific; Anti-Human IgA, α-Chain Specific (Goat); Anti-Human IgA, Fab Fragment Specific; Anti-Human IgA, Fc Fragment Specific; Anti-Human IgA, Secretory; Anti-Human IgE, ε-Chain Specific (Goat); Anti-Human IgE, Fc Fragment Specific; Anti-Human IgG, Fc Fragment Specific (Goat); Anti-Human IgG, γ-Chain Specific (Goat); Anti-Human IgG, Fc Fragment Specific; Anti-Human IgG, Fd Fragment Specific; Anti-Human IgG, H & L Chain Specific (Goat); Anti-Human IgG[0100] 1, Fc Fragment Specific; Anti-Human IgG2, Fc Fragment Specific; Anti-Human IgG2, Fd Fragment Specific; Anti-Human IgG3, Hinge Specific; Anti-Human IgG4, Fc Fragment Specific; Anti-Human IgM, Fc Fragment Specific; Anti-Human IgM, μ-Chain Specific; Anti-Mouse IgE, ε-Chain Specific; Anti-Mouse γ-Globulin (Goat); Anti-Mouse IgG, γ-Chain Specific (Goat); Anti-Mouse IgG, γ-Chain Specific (Goat) F(ab′)2 Fragment; Anti-Mouse IgG, H & L Chain Specific (Goat); Anti-Mouse IgM, μ-Chain Specific (Goat); Anti-Mouse IgM, H & L Chain Specific (Goat); Anti-Rabbit γ-Globulin (Goat); Anti-Rabbit IgG, Fc Fragment Specific (Goat); Anti-Rabbit IgG, H & L Chain Specific (Goat); Anti-Rat γ-Globulin (Goat); Anti-Rat IgG, H & L Chain Specific; Anti-Rhesus Monkey γ-Globulin (Goat); and, Anti-Sheep IgG, H & L Chain Specific.
  • Another non-limiting list of the antibodies that may be produced using the present invention is provided in product catalogs of companies such as Phoenix Pharmaceuticals, Inc. (www.phoenixpeptide.com; 530 Harbor Boulevard, Belmont, Calif.), Peninsula Labs (San Carlos Calif.), SIGMA (St.Louis, Mo. www.sigma-aldrich.com), Cappel ICN (Irvine, Calif., www.icnbiomed.com), and Calbiochem (La Jolla, Calif., www.calbiochem.com), which are all incorporated herein by reference in their entirety. The polynucleotide sequences encoding these antibodies 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 antibody. 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. Some of these antibodies include, but are not limited to, antibodies which bind the following ligands: adrenomedulin, amylin, 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, corticotrophin releasing hormone, PHM, PHI, vasoactive intestinal polypeptide, secretin, ACTH, angiotensin, angiostatin, bombesin, endostatin, bradykinin, FMRF amide, galanin, gonadotropin releasing hormone (GnRH) associated peptide, GnRH, growth hormone releasing hormone, inhibin, granulocyte-macrophage colony stimulating factor (GM-CSF), motilin, neurotensin, oxytocin, vasopressin, osteocalcin, pancreastatin, pancreatic polypeptide, peptide YY, proopiomelanocortin, transforming growth factor, vascular endothelial growth factor, vesicular monoamine transporter, vesicular acetylcholine transporter, ghrelin, NPW, NPB, C3d, prokinetican, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, prolactin, growth hormone, beta-lipotropin, melatonin, kallikriens, kinins, prostaglandins, erythropoietin, p146 (SEQ ID NO:24 amino acid sequence, SEQ ID NO:25, nucleotide sequence), estrogen, testosterone, corticosteroids, mineralocorticoids, thyroid hormone, thymic hormones, connective tissue proteins, nuclear proteins, actin, avidin, activin, agrin, albumin, and prohormones, propeptides, splice variants, fragments and analogs thereof. [0101]
  • The following is yet another non-limiting list of antibodies that can be produced by the methods of present invention: abciximab (ReoPro), abciximab anti-platelet aggregation monoclonal antibody, anti-CD11a (hu1124), anti-CD 18 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) antibody against TNFo, antibody directed against GPIIb/IIIa receptor on human platelets, gamma globulin, anti-hepatitis B immunoglobulin, human anti-D immunoglobulin, human antibodies against S aureus, human tetanus immunoglobulin, humanized antibody against the epidermal growth receptor-2, humanized antibody against the a subunit of the interleukin-2 receptor, humanized antibody CTLA4IG, humanized antibody to the IL-2 R α-chain, humanized anti-CD40-ligand monoclonal antibody (5c8), humanized mAb against the epidermal growth receptor-2, humanized mAb to rous sarcoma virus, humanized recombinant antibody (IgGlk) against respiratory syncytial virus (RSV), lymphocyte immunoglobulin (anti-thymocyte antibody), lymphocyte immunoglobulin, mAb against factor VII, MDX-210 bi-specific antibody against HER-2, MDX-22, MDX-220 bi-specific antibody against TAG-72 on tumors, MDX-33 antibody to FcγR1 receptor, MDX-447 bi-specific antibody against EGF receptor, MDX-447 bispecific humanized antibody to EGF receptor, MDX-RA immunotoxin (ricin A linked) antibody, Medi-507 antibody (humanized form of BTI-322) against CD2 receptor on T-cells, monoclonal antibody LDP-02, muromonab-CD3(OKT3) antibody, OKT3 (“muromomab-CD3”) antibody, PRO 542 antibody, ReoPro (“abciximab”) antibody, and TNF-IgG fusion protein. [0102]
  • 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. [0103]
  • 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. In an alternative embodiment, the sequences encoding light and heavy chains of a monoclonal antibody may be included on a single DNA construct. For example, the coding sequence of light and heavy chains of a murine monoclonal antibody that show specificity for human seminoprotein can be expressed using transposon-based constructs of the present invention (GenBank Accession numbers AY129006 and AY129304 for the light and heavy chains, respectively). [0104]
  • Further included in the present invention are 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 transgenic 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, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-α, and TNF-β. Other cytokines included in the present invention include cardiotrophin, stromal 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. [0105]
  • Lytic peptides such as p146 are also included in the desired molecules of the present invention. In one embodiment, the p146 peptide comprises an amino acid sequence of SEQ ID NO:24. The present invention also encompasses a transposon-based vector comprising a p146 nucleic acid comprising a polynucleotide sequence of SEQ ID NO:25. [0106]
  • Enzymes are another class of proteins that may be made through the use of the transposon-based vectors of the present invention. Such enzymes include but are not limited to adenosine deaminase, alpha-galactosidase, cellulase, collagenase, dnaseI, hyaluronidase, lactase, L-asparaginase, pancreatin, papain, streptokinase B, subtilisin, superoxide dismutase, thrombin, trypsin, urokinase, fibrinolysin, glucocerebrosidase and plasminogen activator. In some embodiments wherein the enzyme could have deleterious effects, 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. [0107]
  • 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. [0108]
  • 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-α (“TGF-α”), transforming growth factor-β (TGF-β), 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), transforming growth factors (TGF), including TGFs alpha, beta, beta1, beta2, beta3, skeletal growth factor, bone matrix derived growth factors, bone derived growth factors, erythropoietin (EPO) and mixtures thereof. [0109]
  • 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. [0110]
  • 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 IV (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 XII (Hageman Factor), Factor XIII (rotransglutaminase, fibrin stabilizing factor (FSF), fibrinoligase), von Willebrand factor, Protein C, Protein S, Thrombomodulin, Antithrombin III. [0111]
  • 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, Calif.), Peninsula Labs (San Carlos Calif.), SIGMA, (St.Louis, Mo. www.sigma-aldrich.com), Cappel ICN (Irvine, Calif., www.icnbiomed.com), and Calbiochem (La Jolla, Calif., 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. [0112]
  • Some of these desired proteins or peptides that may be made through the use of the present invention include but are not limited to the following: adrenomedulin, amylin, 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, leumorphin, peptide F, PACAP, PACAP-related peptide, parathyroid hormone, urocortin, corticotrophin releasing hormone, PHM, PHI, vasoactive intestinal polypeptide, secretin, ACTH, angiotensin, angiostatin, bombesin, endostatin, bradykinin, FMRF amide, galanin, gonadotropin releasing hormone (GnRH) associated peptide, GnRH, growth hormone releasing hormone, inhibin, granulocyte-macrophage colony stimulating factor (GM-CSF), motilin, neurotensin, oxytocin, vasopressin, osteocalcin, pancreastatin, pancreatic polypeptide, peptide YY, proopiomelanocortin, transforming growth factor, vascular endothelial growth factor, vesicular monoamine transporter, vesicular acetylcholine transporter, ghrelin, NPW, NPB, C3d, prokinetican, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, prolactin, growth hormone, beta-lipotropin, melatonin, kallikriens, kinins, prostaglandins, erythropoietin, p146 (SEQ ID NO:24, amino acid sequence, SEQ ID NO:25, nucleotide sequence), thymic hormones, connective tissue proteins, nuclear proteins, actin, avidin, activin, agrin, albumin, apolipoproteins, apolipoprotein A, apolipoprotein B, and prohormones, propeptides, splice variants, fragments and analogs thereof. [0113]
  • Other desired proteins that may be made by the transgenic 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 latrodectus (black widow spider venom antibody), antivenin micrurus (coral snake venom antibody), aprotinin, corticotropin (ACTH), diphtheria antitoxin, lymphocyte immune globulin (anti-thymocyte antibody), protamine, thyrotropin, capreomycin, α-galactosidase, gramicidin, streptokinase, tetanus toxoid, tyrothricin, IGF-1, proteins of varicella vaccine, anti-TNF antibody, anti-IL-2r antibody, anti-HER-2 antibody, OKT3 (“muromonab-CD3”) antibody, TNF-IgG fusion protein, ReoPro (“abciximab”) antibody, ACTH fragment 1-24, desmopressin, gonadotropin-releasing hormone, histrelin, leuprolide, lypressin, nafarelin, peptide that binds GPIIb/GPIIIa on platelets (integrilin), goserelin, capreomycin, colistin, anti-respiratory syncytial virus, lymphocyte immune globulin (Thymoglovin, Atgam), panorex, alpha-antitrypsin, botulinin, lung surfactant protein, tumor necrosis receptor-IgG fusion protein (enbrel), gonadorelin, proteins of influenza vaccine, proteins of rotavirus vaccine, proteins of haemophilus b conjugate vaccine, proteins of poliovirus vaccine, proteins of pneumococcal conjugate vaccine, proteins of meningococcal C vaccine, proteins of influenza vaccine, megakaryocyte growth and development factor (MGDF), neuroimmunophilin ligand-A (NIL-A), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), leptin (native), leptin B, leptin C, IL-1RA (interleukin-1RA), R-568, novel erythropoiesis-stimulating protein (NESP), humanized mAb to rous sarcoma virus (MEDI-493), glutamyl-tryptophan dipeptide IM862, LFA-3TIP immunosuppressive, humanized anti-CD40-ligand monoclonal antibody (5c8), gelsonin enzyme, tissue factor pathway inhibitor (TFPI), proteins of meningitis B vaccine, antimetastatic cancer antibody (mAb 17-1A), chimeric (human & mouse) mAb against TNFα, mAb against factor VII, relaxin, capreomycin, glycopeptide (LY333328), recombinant human activated protein C (rhAPC), humanized mAb against the epidermal growth receptor-2, altepase, anti-CD20 antigen, C2B8 antibody, insulin-like growth factor-1, atrial natriuretic peptide (anaritide), tenectaplase, anti-CD11a antibody (hu 1124), anti-CD18 antibody, mAb LDP-02, anti-VEGF antibody, fab fragment of anti-VEGF Ab, AP02 ligand (tumor necrosis factor-related apoptosis-inducing ligand), rTGF-β (transforming growth factor-β), alpha-antitrypsin, ananain (a pineapple enzyme), humanized mAb CTLA4IG, PRO 542 (mAb), D2E7 (mAb), calf intestine alkaline phosphatase, α-L-iduronidase, α-L-galactosidase (humanglutamic acid decarboxylase, acid sphingomyelinase, bone morphogenetic protein-2 (rhBMP-2), proteins of HIV vaccine, T cell receptor (TCR) peptide vaccine, TCR peptides, V beta 3 and V beta 13.1. (IR502), (IR501), BI 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 [0114] H. pylori, MDX-447 bispecific humanized mAb to EGF receptor, anti-cytomegalovirus (CMV), Medi-491 B 19 parvovirus vaccine, humanized recombinant mAb (IgG1k) against respiratory syncytial virus (RSV), urinary tract infection vaccine (against “pili” on Escherechia coli strains), proteins of lyme disease vaccine against B. 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 FcγR1 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 α-chain (basiliximab), mAb to IgE (IGE 025A), myelin basic protein-altered peptide (MSP771A), humanized mAb against the epidermal growth receptor-2, humanized mAb against the α subunit of the interleukin-2 receptor, low molecular weight heparin, anti-hemophillic factor, and bactericidal/permeability-increasing protein (r-BPI).
  • 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, Oreg.), 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. [0115]
  • 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. [0116]
  • 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. In these embodiments, a gene of interest encodes a nucleic acid molecule or a protein that directs production of the desired molecule. [0117]
  • The present invention further encompasses the use of inhibitory molecules to inhibit endogenous (i.e., non-vector) protein production. These inhibitory molecules include antisense nucleic acids, siRNA and inhibitory proteins. In a preferred embodiment, the endogenous protein whose expression is inhibited is an egg white protein including, but not limited to ovalbumin, ovotransferrin, and ovomucin. In one embodiment, 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). In other embodiments, a transposon-based vector encodes an inhibitory RNA molecule that inhibits the expression of more than one egg white protein. One exemplary construct is provided in FIG. 4 wherein “Ovgen” indicates approximately 60 base pairs of an ovalbumin gene, “Ovotrans” indicates approximately 60 base pairs of an ovotransferrin gene and “Ovomucin” indicates approximately 60 base pairs of an ovomucin gene. These ovalbumin, ovotransferrin 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. [0118]
  • Modified Desired Proteins and Peptides
  • “Proteins”, “peptides,” “polypeptides” and “oligopeptides” 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) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term “amino terminus” (abbreviated N-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. Similarly, the term “carboxy terminus” (abbreviated C-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. [0119]
  • Typically, the 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. Thus, when one amino acid is said to “follow” another, that amino acid is positioned closer to the carboxy terminal of the protein than the preceding amino acid. [0120]
  • The term “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. As such, 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). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art. [0121]
  • Furthermore, one of skill will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than about 5%, more typically less than about 1%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: [0122]
  • 1) Alanine (A), Serine (S), Threonine (T); [0123]
  • 2) Aspartic acid (D), Glutamic acid (E); [0124]
  • 3) Asparagine (N), Glutamine (Q); [0125]
  • 4) Arginine (R), Lysine (K); [0126]
  • 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and [0127]
  • 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). [0128]
  • 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. [0129]
  • Suitable protecting groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. 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. Biochemistry 26:2294; Lindberg et al., 1989. Drug Metabolism and Disposition 17:311; Tunek et al., 1988. Biochem. Pharm. 37:3867; Anderson et al., 1985 Arch. Biochem. Biophys. 239:538; and Singhal et al., 1987. FASEB J. 1:220). 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. In one embodiment, 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. [0130]
  • Provided below are groups of naturally occurring and modified amino acids in which each amino acid in a group has similar electronic and steric properties. Thus, a conservative substitution can be made by substituting an amino acid with another amino acid from the same group. It is to be understood that these groups are non-limiting, i.e. that there are additional modified amino acids which could be included in each group. [0131]
  • Group I includes leucine, isoleucine, valine, methionine and modified amino acids having the following side chains: ethyl, n-propyl n-butyl. Preferably, Group I includes leucine, isoleucine, valine and methionine. [0132]
  • Group II includes glycine, alanine, valine and a modified amino acid having an ethyl side chain. Preferably, Group II includes glycine and alanine. [0133]
  • Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl glycine, 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[0134] 2, methoxy, ethoxy and —CN. Preferably, Group III includes phenylalanine, tyrosine and tryptophan.
  • Group IV includes glutamic acid, aspartic acid, a substituted 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[0135] 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. Preferably, 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, ornithine, arginine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitruline and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and homologs of ornithine. Preferably, Group V includes histidine, lysine, arginine and ornithine. A homolog of an amino acid includes from 1 to about 3 additional or subtracted methylene units in the side chain. [0136]
  • Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with —OH or —SH, for example, —CH[0137] 2CH2OH, —CH2CH2CH2OH or —CH2CH2OHCH3. Preferably, Group VI includes serine, cysteine or threonine.
  • In another aspect, suitable substitutions for amino acid residues include “severe” substitutions. A “severe substitution” is a substitution in which the substituting amino acid (naturally occurring or modified) has significantly different size and/or electronic properties compared with the amino acid being substituted. Thus, 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. Examples of severe substitutions of this type include the substitution of phenylalanine or cyclohexylmethyl glycine for alanine, isoleucine for glycine, a D amino acid for the corresponding L amino acid, or —NH—CH[(—CH[0138] 2)5—COOH]—CO— for aspartic acid. Alternatively, 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 ornithine. In yet another alternative, 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 —(CH2)4COOH for the side chain of serine. These examples are not meant to be limiting.
  • In another embodiment, for example in the synthesis of a peptide 26 amino acids in length, the individual amino acids may be substituted according in the following manner: [0139]
  • AA[0140] 1 is serine, glycine, alanine, cysteine or threonine;
  • AA[0141] 2 is alanine, threonine, glycine, cysteine or serine;
  • AA[0142] 3 is valine, arginine, leucine, isoleucine, methionine, omithine, lysine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitruline or 2-amino-4-guanidinobutanoic acid;
  • AA[0143] 4 is proline, leucine, valine, isoleucine or methionine;
  • AA[0144] 5 is tryptophan, alanine, phenylalanine, tyrosine or glycine;
  • AA[0145] 6 is serine, glycine, alanine, cysteine or threonine;
  • AA[0146] 7 is proline, leucine, valine, isoleucine or methionine;
  • AA[0147] 8 is alanine, threonine, glycine, cysteine or serine;
  • AA[0148] 9 is alanine, threonine, glycine, cysteine or serine;
  • AA[0149] 10 is leucine, isoleucine, methionine or valine;
  • AA[0150] 11 is serine, glycine, alanine, cysteine or threonine;
  • AA[0151] 12 is leucine, isoleucine, methionine or valine;
  • AA[0152] 13 is leucine, isoleucine, methionine or valine;
  • AA[0153] 14 is glutamine, glutamic acid, aspartic acid, asparagine, or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
  • AA[0154] 15 is arginine, N-nitroarginine, β-cycloarginine, γ-hydroxy-arginine, N-amidinocitruline or 2-amino4-guanidino-butanoic acid
  • AA[0155] 16 is proline, leucine, valine, isoleucine or methionine;
  • AA[0156] 17 is serine, glycine, alanine, cysteine or threonine;
  • AA[0157] 18 is glutamic acid, aspartic acid, asparagine, glutamine or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
  • AA[0158] 19 is aspartic acid, asparagine, glutamic acid, glutamine, leucine, valine, isoleucine, methionine or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
  • AA[0159] 20 is valine, arginine, leucine, isoleucine, methionine, ornithine, lysine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitruline or 2-amino-4-guanidinobutanoic acid;
  • AA[0160] 21 is alanine, threonine, glycine, cysteine or serine;
  • AA[0161] 22 is alanine, threonine, glycine, cysteine or serine;
  • AA[0162] 23 is histidine, serine, threonine, cysteine, lysine or ornithine;
  • AA[0163] 24 is threonine, aspartic acid, serine, glutamic acid or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
  • AA[0164] 25 is asparagine, aspartic acid, glutamic acid, glutamine, leucine, valine, isoleucine, methionine or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid; and
  • AA[0165] 26 is cysteine, histidine, serine, threonine, lysine or ornithine.
  • 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. [0166]
  • In one embodiment of the present invention, codons for the first several N-terminal amino acids of the transposase 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. [0167]
  • When several desired proteins, protein fragments or peptides are encoded in the gene of interest to be incorporated into the genome, one of skill in the art will appreciate that the 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. Generally, 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. However, 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. [0168]
  • Such 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. Preferably, peptide spacers contain at least 60%, more preferably at least 80% glycine or alanine. In addition, peptide spacers generally have little or no biological and antigenic activity. Preferred spacers are (Gly-Pro-Gly-Gly)[0169] x (SEQ ID NO:26) and (Gly4-Ser)y, wherein x is an integer from about 3 to about 9 and y is an integer from about 1 to about 8. Specific examples of suitable spacers include
    (Gly-Pro-Gly-Gly)3
    SEQ ID NO:27 Gly Pro Gly Gly Gly Pro Gly Gly Gly Pro Gly Gly
    (Gly4-Ser)3
    SEQ ID NO:28 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
    or (Gly4-Ser)4
    SEQ ID NO:29 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
    Gly Gly Gly Gly Ser.
  • 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-transferase, polylysine, hexa-histidine or other cationic amino acids, thioredoxin, hemagglutinin antigen and maltose binding protein. [0170]
  • Additionally, 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. In this manner, if a gene is designed to make one or more peptides or proteins of interest in the transgenic animal, specific nucleotide sequences encoding for amino acids recognized by enzymes may be incorporated into the gene to facilitate cleavage of the large protein or peptide sequence into desired peptides or proteins or both. For example, 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. [0171]
  • 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, collagenase, 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. [0172]
  • In one embodiment of the present invention, a TAG sequence is linked to the gene of interest. The TAG sequence serves three purposes: 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. Accordingly, as used herein, 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 hairpin loop from HIV I. Exemplary gp41 polynucleotide and polypeptide sequences are provided in SEQ ID NO:32 and SEQ ID NO:33, respectively. However, it should be understood that 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. Additionally, 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. Constructs containing a TAG sequence are shown in FIGS. 2 and 3. In one embodiment of the present invention, the TAG sequence comprises a polynucleotide sequence of SEQ ID NO:35. [0173]
  • Methods of Administering Transposon-Based Vectors
  • In addition to the transposon-based vectors described above, 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 incorporated into the germline of the animal and methods of producing a transgenic animal wherein a gene of interest is incorporated 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. [0174]
  • In some embodiments, 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 infindibulum 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 tubular gland and epithelial cells. Such transfected cells could manufacture a desired protein or peptide for deposition in the egg white. In one embodiment, 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. [0175]
  • Administration of transposon-based vectors may occur in arteries supplying the ovary and or through direct intrathecal administration into the ovary through injection. [0176]
  • The transposon-based vectors may be administered in a single administration, multiple administrations, continuously, or intermittently. The transposon-based vectors may be administered by injection, via a catheter, an osmotic mini-pump or any other method. In some embodiments, the transposon-based vector is administered to an animal in multiple administrations, each administration containing the vector and a different transfecting reagent. [0177]
  • 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 transposon-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. In some embodiments, the transposon-based vector is administered to an animal following an increase in proliferation of the oviduct epithelial cells and/or the tubular gland cells. Such an increase in proliferation normally follows an influx of reproductive hormones in the area of the oviduct. When the animal is an avian, the transposon-based vector is administered following an increase in proliferation of the oviduct epithelial cells and before the avian begins to produce egg white constituents. [0178]
  • In a preferred embodiment, the animal is an egg-laying animal, and more preferably, an avian. In one embodiment, between approximately 1 and 150 μg, 1 and 100 μg, 1 and 50 μg, preferably between 1 and 20 μg, and more preferably between 5 and 10 μg 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. In a chicken, it is preferred that between approximately 1 and 100 μg, or 5 and 50 μg are administered. In a quail, it is preferred that between approximately 5 and 10 μg are administered. Intraoviduct administration of the transposon-based vectors of the present invention result in incorporation of the gene of interest into the cells of the oviduct as evidenced by a PCR positive signal in the oviduct tissue. In other embodiments, the transposon-based vector is administered to an artery that supplies the oviduct. These methods of administration may also be combined with any methods for facilitating transfection, including without limitation, electroporation, gene guns, injection of naked DNA, and use of dimethyl sulfoxide (DMSO). [0179]
  • According to the present invention, 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 transfection 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 Strategene; and Escort from SIGMA. In one embodiment, the transfection reagent is SUPERFECT®. The ratio of DNA to transfection reagent may vary based upon the method of administration. In one embodiment, the transposon-based vector is administered to the oviduct and the ratio of DNA to transfection 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). [0180]
  • Depending upon the cell or tissue type targeted for transfection, 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. In some instances, transposase expression and insertion may be more efficient in a relaxed, closed circular configuration or in a linear configuration. In still other instances, 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 transfection reagent complexed with both the purified transposase protein and the transposon-based vector. [0181]
  • Testing for and Breeding Animals Carrying the Transgene
  • Following administration of a transposon-based vector to an animal, 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 incorporation, and transcription of the gene of interest when administered to a bird via an intraoviduct or intraovarian route (including intraarterial 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. FIG. 5 demonstrates successful administration 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. [0182]
  • Actual frequencies of integration may be estimated both by comparative strength of the PCR signal, and by histological evaluation of the tissues by quantitative PCR. Another method for estimating the rate of transgene insertion is the so-called primed in situ hybridization technique (PRINS). This method determines not only which cells carry a transgene of interest, but also into which chromosome the gene has inserted, and even what portion of the chromosome. Briefly, labeled primers are annealed to chromosome spreads (affixed to glass slides) through one round of PCR, and the slides are then developed through normal in situ hybridization procedures. This technique combines the best features of in situ PCR and fluorescence in situ hybridization (FISH) to provide distinct chromosome location and copy number of the gene in question. [0183]
  • Breeding experiments are also conducted to determine if germline transmission of the transgene has occurred. In a general bird breeding experiment performed according to the present invention, each male bird was exposed to 2-3 different adult female birds for 3-4 days each. This procedure was continued with different females for a total period of 6-12 weeks. Eggs ae collected daily for up to 14 days after the last exposure to the transgenic male, and each egg is incubated in a standard incubator. The resulting embryos are examined for transgene presence at day 3 or 4 using PCR. It is to be understood that the above procedure can be modified to suit animals other than birds and that selective breeding techniques may be performed to amplify gene copy numbers and protein output. [0184]
  • Production of Desired Proteins or Peptides in Egg White
  • In one embodiment, 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. Biophys. Acta, 1517 (1):27-32; H. Park et al., 2000. Biochem., 39:8537-8545; and T. Muramatsu et al., 1996. Poult. Avian Biol. Rev., 6:107-123. Examples of transposon-based vectors designed for production of a desired protein in an egg white are shown in FIGS. 2 and 3. [0185]
  • Production of Desired Proteins or Peptides in Egg Yolk
  • The present invention is particularly advantageous for production of recombinant peptides and proteins of low solubility in the egg yolk. Such 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 denaturation and re-folding of the recombinantly-produced proteins, which may deleteriously affect their structure and function. Moreover, even highly soluble recombinant peptides and proteins may precipitate and require denaturation and renaturation when produced in sufficiently high amounts in recombinant protein production systems. The present invention provides an advantageous resolution of the problem of protein and peptide solubility during production of large amounts of recombinant proteins. [0186]
  • In one embodiment of the present invention wherein germline transfection is obtained via intraovarian administration of the transposon-based vector, 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. In a preferred embodiment, 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. In a preferred embodiment, 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 substituted. One example of 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 transposon-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. In one embodiment, the vitellogenin targeting sequence contains the polynucleotide sequence of SEQ ID NO:22. In a preferred embodiment, the transposon-based vector contains a transposase gene operably-linked to a constitutive promoter and a gene of interest operably-linked to a liver-specific promoter and a vitellogenin targeting sequence. [0187]
  • Isolation and Purification of Desired Protein or Peptide
  • For large-scale production of protein, 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. [0188]
  • Once expressed, 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 electrophoresis, 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. [0189]
  • In one embodiment of the present invention, the animal in which the desired protein is produced is an egg-laying animal. In a preferred embodiment of the present invention, 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, N.Y. (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 spectroscopy, 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%. [0190]
  • To facilitate or enable purification of a desired protein or peptide, transposon-based vectors may include one or more additional epitopes or domains. Such 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-transferase; 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. [0191]
  • In one representative embodiment, 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. In a preferred embodiment, the TAG sequence comprises approximately 50 amino acids. A representative TAG sequence is provided below. [0192]
  • Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp [0193] Ala Thr Thr Cys Ile Leu Lys Gly Ser Cys Gly Trp, Ile Gly Leu Leu Asp Asp Asp Asp Lys (SEQ ID NO:35)
  • The underlined sequences were taken from the hairpin 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 hairpin loop from the ovalbumin carrier protein. [0194]
  • Isolation and purification of a desired protein is performed as follows: [0195]
  • 1. Enrichment of the egg white protein fraction containing ovalbumin and the transgenic ovalbumin-TAG-desired protein. [0196]
  • 2. Size exclusion chromatography to isolate only those proteins within a narrow range of molecular weights (a further enrichment of step 1). [0197]
  • 3. Ovalbumin affinity chromatography. Highly specific antibodies to ovalbumin will eliminate virtually all extraneous egg white proteins except ovalbumin and the transgenic ovalbumin-TAG-desired protein. [0198]
  • 4. gp41 affinity chromatography using anti-gp41 antibodies. Stringent application of this step will result in virtually pure transgenic ovalbumin-TAG-desired protein. [0199]
  • 5. Cleavage of the transgene product can be accomplished in at least one of two ways: [0200]
  • a. The transgenic 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. [0201]
  • b. Alternatively, 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. [0202]
  • c. The choice of these alternatives is made depending upon the size and chemical composition of the transgene target protein. [0203]
  • 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. [0204]
  • Cleavage of the transgene product (ovalbumin-TAG-desired protein) by enterokinase, then, results in two products: ovalbumin-TAG and the desired protein. More specific methods for isolation using the TAG label is provided in the Examples. Some desired proteins may require additions or modifications of the above-described approach as known to one of ordinary skill in the art. The method is scaleable from the laboratory bench to pilot and production facility largely because the techniques applied are well documented in each of these settings. [0205]
  • In another representative embodiment, egg whites containing a protein of interest were pooled and separated, in any order, from the yolks and other egg constituents by methods known to one skilled in the art. A variety of such methods is described in manuals known in the art, such as [0206] Egg Science & Technology, W. Stadelman, et al. (Eds.), Haworth Press, Binghamton, N.Y. (1995).
  • 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 hairpin loop epitope from HIV I followed by an enterokinase cleavage site. [0207]
  • First, 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, [0208] 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 transgene product from the carrier and the TAG sequences was accomplished in one of at least two ways: [0209]
  • 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 transgene 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. [0210]
  • 2) Alternatively, enterokinase was immobilized on resin (beads) by the addition of poly-lysine moieties to a non-catalytic area of the protease. The transgenic 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. [0211]
  • 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. [0212]
  • 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. [0213]
  • One of skill in the art will recognize that after biological expression or purification, 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. [0214]
  • 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 transgenic proteins or peptides. These methods include the administration of a transposon-based vector described above to a mammal through the duct system. In one embodiment, the transposon-based vector contains a transposase operably-linked to a constitutive 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. In other embodiments, a transposon-based vector is administered to the ovary of an animal and gerrnline 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. [0215]
  • The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that resort may be had to various embodiments, modifications and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention. [0216]
  • EXAMPLE 1 IntraOviduct Administration of Transposon-Based Vectors
  • Quail or chicken were selected for administration of the transposon-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 retractor 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. [0217]
  • 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 temperature prior to injection into the bird. Approximately 250-500 μ1 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. [0218]
  • EXAMPLE 2 Preparation of Transposon-Based Vector pTnMod
  • A vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells, given below as SEQ ID NO:3. The vector of SEQ ID NO:3, termed pTnMod, was constructed and its sequence verified. [0219]
  • This vector employed a cytomegalovirus (CMV) promoter. A modified Kozak sequence (ACCATG) (SEQ ID NO:1) was added to the promoter. The nucleotide in the wobble position in nucleotide triplet codons encoding the first 10 amino acids of transposase 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. [0220]
  • The first step in constructing this vector was to modify the transposase to have the desired changes. Modifications to the transposase were accomplished with the primers High Efficiency forward primer (Hef) Altered transposase (ATS)-Hef 5′ ATCTCGAGACCATGTG[0221] TGAACTTGATATTTTACATGATTCTCTTTACC 3′ (SEQ ID NO:36) and Altered transposase-High efficiency reverse primer (Her) 5′ GATTGATCATTATCATAATTTCCCCAAAGCGTAACC 3′ (SEQ ID NO:37, a reverse complement primer). In the 5′ forward primer ATS-Hef, 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:1) contains the Kozak sequence and start codon for the transposase 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 Bcl I restriction site, TGATCA (SEQ ID NO:39), to allow directional cloning. These primers were used in a PCR reaction with pTnLac (p defines plasmid, tn defines transposon, and lac defines the beta fragment of the lactose gene, which contains a multiple cloning site) as the template for the transposase and a FailSafe™ PCR System (which includes enzyme, buffers, dNTP's, MgCl2 and PCR Enhancer; Epicentre Technologies, Madison, Wis.). Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. 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, Calif.). Purified DNA was digested with restriction enzymes Xho 1 (5′) and Bcl 1 (3′) (New England Biolabs, Beverly, Mass.) 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, Calif.) was digested with restriction enzymes Sal I and BamH I (New England Biolabs), which are compatible with Xho I and Bcl 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 transposase (mATS) between the CMV promoter and synthetic polyA. [0222]
  • To insert the mATS between the CMV promoter and synthetic polyA in gWhiz, a Stratagene T4 Ligase Kit (Stratagene, Inc. La Jolla, Calif.) was used and the ligation set up according to the manufacturer's protocol. Ligated product was transformed into [0223] E. coli Top10 competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) using chemical transformation according to Invitrogen'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 μg/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), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size (approximately 6.4 kbp) were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, Calif.). Column purified DNA 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. For sequencing, 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.
  • Once a clone was identified that contained the desired mATS in the correct orientation, primers CMVf-NgoM IV (5′ TT[0224] GCCGGCATCAGATTGGCTAT (SEQ ID NO: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. The PCR was conducted with FailSafe™ 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 transposon in pTnLac as described below.
  • Plasmid pTnLac was digested with NgoM IV and BstE II to remove the ptac promoter and transposase 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 [0225] E. coli as described above.
  • Colonies resulting from this transformation were screened (mini-preps) as describe above and clones that were the correct size were verified by DNA sequence analysis as described above. The vector was given the name pTnMod (SEQ ID NO:3) and includes the following components: [0226]
  • Base pairs 1-130 are a remainder of F1(−) on from pBluescriptII sk(−) (Stratagene), corresponding to base pairs 1-130 of pBluescriptII sk(−). [0227]
  • Base pairs 131-132 are a residue from ligation of restriction enzyme sites used in constructing the vector. [0228]
  • 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. [0229]
  • Base pairs 1778-1779 are a residue from ligation of restriction enzyme sites used in constructing the vector. [0230]
  • Base pairs 1780-2987 are the coding sequence for the transposase, modified from Tn10 (GenBank accession J01829) by optimizing codons for stability of the transposase 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 substitution would not alter the amino acid that was encoded. [0231]
  • Base pairs 2988-2993 are two engineered stop codons. [0232]
  • Base pair 2994 is a residue from ligation of restriction enzyme sites used in constructing the vector. [0233]
  • 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. [0234]
  • Base pairs 3415-3718 are non-coding DNA that is residual from vector pNK2859. [0235]
  • Base pairs 3719-3761 are non-coding λ DNA that is residual from pNK2859. [0236]
  • Base pairs 3762-3831 are the 70 bp of the left insertion sequence recognized by the transposon Tn10. [0237]
  • Base pairs 3832-3837 are a residue from ligation of restriction enzyme sites used in constructing the vector. [0238]
  • Base pairs 3838-4527 are the multiple cloning site from pBluescriptII 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. [0239]
  • Base pairs 4528-4532 are a residue from ligation of restriction enzyme sites used in constructing the vector. [0240]
  • Base pairs 4533-4602 are the 70 bp of the right insertion sequence recognized by the transposon Tn10. [0241]
  • Base pairs 4603-4644 are non-coding λ DNA that is residual from pNK2859. [0242]
  • Base pairs 4645-5488 are non-coding DNA that is residual from pNK2859. [0243]
  • Base pairs 5489-7689 are from the pBluescriptII sk(−) base vector—(Stratagene, Inc.), corresponding to bp 761-2961 of pBluescriptII sk(−). [0244]
  • Completing pTnMod is a pBlueScript backbone that contains a colE I origin of replication and an antibiotic resistance marker (ampicillin). [0245]
  • It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants. [0246]
  • All plasmid DNA was isolated by standard procedures. Briefly, [0247] 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, Calif.) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 μL of PCR-grade water and stored at −20° C. until used.
  • EXAMPLE 3 Transposon-Based VectorpTnMCS
  • 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 constituents 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. [0248]
  • Bp 1-130 Remainder of F1 (−) ori of pBluescriptII sk(−) (Stratagene) bp1-130 [0249]
  • Bp 133-1777 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems) bp 229-1873 [0250]
  • Bp 1783-2991 Transposase, from Tn10 (GenBank accession #J01829) bp 108-1316 [0251]
  • Bp 2992-3344 Non coding DNA from vector pNK2859 [0252]
  • Bp 3345-3387 Lambda DNA from pNK2859 [0253]
  • Bp 3388-3457 70 bp of IS10 left from Tn10 [0254]
  • Bp 3464-3670 Multiple cloning site from pBluescriptII sk(−), thru the XmaI site bp 924-718 [0255]
  • Bp 3671-3715 Multiple cloning site from pBluescriptII sk(−), from the XmaI site thru the XhoI site. These base pairs are usually lost when cloning into pTnMCS bp 717-673 [0256]
  • Bp 3716-4153 Multiple cloning site from pBluescriptII sk(−), from the XhoI site bp 672-235 [0257]
  • Bp 4159-4228 70 bp of IS10 right from Tn10 [0258]
  • Bp 4229-4270 Lambda DNA from pNK2859 [0259]
  • Bp 4271-5114 Non-coding DNA from pNK2859 [0260]
  • Bp 5115-7315 pBluescript sk(−) base vector (Stratagene, Inc.) bp 761-2961. [0261]
  • EXAMPLE 4 Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/ProIns/PA)—Chicken
  • A vector was designed to insert a humsan proinsulin coding sequence under the control 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. [0262]
  • Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptll sk(−). [0263]
  • Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz. [0264]
  • Base pairs 1780-2987 are a transposase, modified from Tn10 (GenBank accession number J01829). [0265]
  • Base pairs 2988-2993 are two engineered stop codons. [0266]
  • Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz. [0267]
  • Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859. [0268]
  • Base pairs 3719-3761 are λ DNA that is residual from pNK2859. [0269]
  • Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10. [0270]
  • Base pairs 3838-4044 are a multiple cloning site from pBluescriptII sk(−) corresponding to base pairs 924-718 of pBluescriptII sk(−). [0271]
  • 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. [0272]
  • 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). [0273]
  • Base pairs 6122-6271 are a TAG sequence containing a gp41 hairpin loop from HIV I, an enterokinase cleavage site and a spacer (synthetic). [0274]
  • Base pairs 6272-6531 are a proinsulin gene. [0275]
  • Base pairs 6539-6891 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz. [0276]
  • Base pairs 6897-7329 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptll sk(−). [0277]
  • Base pairs 7335-7404 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10. [0278]
  • Base pairs 7405-7446 are λ DNA that is residual from pNK2859. [0279]
  • Base pairs 7447-8311 are non coding DNA that is residual from pNK2859. [0280]
  • Base pairs 8312-10512 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptll sk(−). [0281]
  • It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants. [0282]
  • EXAMPLE 5 Transposon-Based Vector pTnMOD (CMV-CHOVg-ent-ProInsulin-synPA)
  • 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. [0283]
  • Bp 1-4045 from vector pTnMod, bp 1-4045 [0284]
  • Bp 4051-5695 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1864 [0285]
  • Bp 5702-6855 Chicken ovalbumin gene taken from GenBank accession #V00383, bp 66-1219 [0286]
  • Bp 6862-7011 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0287]
  • Bp 7012-7272 Human Proinsulin taken from GenBank accession #NM000207, bp 117-377 [0288]
  • Bp 7273-7317 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems) [0289]
  • Bp 7318-7670 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271 [0290]
  • Bp 7672-11271 from cloning vector pTnMCS, bp 3716-7315 [0291]
  • 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). [0292]
  • In the first experiment, the Oval promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A containing construct was injected into the lumen of the oviduct of sexually mature quail; three hens received 5 μg at a 1:3 SUPERFECT® ratio and three received 10 μg at a 1:3 SUPERFECT® ratio. As of the writing of the present application, at least one bird that received above-mentioned construct was producing human proinsulin in egg white (other birds remain to be tested). This experiment indicates that 1) the DNA has been stable for at least 3 months; 2) protein levels are comparable to those observed with a constitutive promoter such as the CMV promoter; and 3) sexually mature birds can be injected and results obtained without the need for cell culture. It is estimated that each quail egg contains approximately 1.4 μg/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. [0293]
  • In the second experiment, 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. Of the 8 survivors, 3 produced human proinsulin in the white of their eggs for over 6 weeks. An ELISA assay described in detail below was developed to detect GP41 in the fusion peptide (Oval gene/GP41 Enterokinase TAG/Proinsulin) since the GP41 peptide sequence is unique and not found as part of normal egg white protein. In all ELISA assays, the same birds produced positive results and all controls worked as expected. [0294]
  • ELISA Procedure: Individual egg white samples were diluted in sodium carbonate buffer, pH 9.6, and added to individual wells of 96 well microtiter ELISA plates at a total volume of 0.1 ml. These plates were then allowed to coat overnight at 4° C. Prior to ELISA development, the plates were allowed warm to room temperature. Upon decanting the coating solutions and blotting away any excess, non-specific binding of antibodies was blocked by adding a solution of phosphate buffered saline (PBS), 1% (w/v) BSA, and 0.05% (v/v) Tween 20 and allowing it to incubate with shaking for a minimum of 45 minutes. 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. Antigen was detected using a solution of p-Nitrophenyl Phosphate in Diethanolamine Substrate Buffer for Alkaline Phosphatase and measuring the absorbance at 30 minutes and 1 hour. [0295]
  • 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 FIG. 5, lanes [0296] 4 and 6. Lanes 1 and 10 of the gel contain molecular weight standards, lanes 2 and 8 contain non-trangenic chicken egg white, whereas lanes 3, 5, 7 and 9 are blank.
  • EXAMPLE7 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 hairpin 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 temperature at 0.5 mL/minute. Excess active groups were then deactivated using 6 column volumes of 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 and the column was washed alternately with 6 column volumes of acetate buffer (0.1 M acetate, 0.5 M NaCl, pH 4.0) and ethanolamine (above). The column was neutralized using 1×PBS. The column was then washed with buffers to be used in affinity purification: 75 mM Tris, pH 8.0 and elution buffer, 100 mM glycine-HCl, 0.5 M NaCl, pH 2.7. Finally, the column was equilibrated in 75 mM Tris buffer, pH 8.0. [0297]
  • 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 veinupuncture. 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. Three column volumes (3 mL) of elution buffer (100 mM glycine, 0.5 M NaCl, pH 2.7) was applied, followed by 75 mM Tris buffer, pH 8.0, all at a rate of 0.5 mL/min. One milliliter fractions were collected. Fractions were collected into 200 uL 1 M Tris, pH 9.0 to neutralize acidic factions as rapidly as possible. A large peak eluted from the column, coincident with the application the elution buffer. Fractions were pooled. Analysis by SDS-PAGE showed a high molecular weight species that separated into two fragments under reducing condition, in keeping with the heavy and light chain structure of IgG. [0298]
  • Pooled antibody fractions were used to charge two 1 mL HiTrap NHS-activated columns, attached in series. Coupling was carried out in the same manner as that used for charging the TAG column. [0299]
  • Isolation of Ovalbumin-TAG-Proinsulin from Egg White
  • Egg white from quail and chickens treated by intra-oviduct injection of the CMV-ovalbumin-TAG-proinsulin construct were pooled. Viscosity was lowered by subjecting the allantoid fluid to successively finer pore sizes using negative pressure filtration, finishing with a 0.22 μM pore size. Through the process, egg white was diluted approximately 1:16. The clarified sample was loaded on the Anti-TAG column and eluted in the same manner as described for the purification of the anti-TAG antibodies. A peak of absorbance at 280 nm, coincident with the application of the elution buffer, indicated that protein had been specifically eluted from the Anti-TAG column. Fractions containing the eluted peak were pooled for analysis. [0300]
  • 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 temperature 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. Western blot analysis showed specific binding to the 60 kDal species under non-reducing condition (which preserved the hairpin epitope of gp-41 by retaining the disulfide bond). Western analysis of the low molecular weight species that appeared upon cleavage with an anti-human proinsulin antibody, conclusively identified the cleaved fragment as human proinsulin. [0301]
  • EXAMPLE8 Purification Procedures for Insulin
  • I. ELISA data for egg characterization/identification [0302]
  • An ELISA was employed for the initial screening of eggs and, thereby, identification of hens producing positive eggs. With further modifications this procedure was used for the initial quantification of recombinant protein amounts. These procedures were aided by the successful purification of an initial stock of the recombinant proinsulin (RPI). This stock of protein is used in the development of a double antibody assay that increases the sensitivity and reduces the background in the assay. Subsequent identification of hens producing positive eggs obviate the need to screen each egg collected. Only periodic checks are needed to determine if production levels are consistent. [0303]
  • II. Egg White (EW) or Albumin Preparation [0304]
  • A. Clarification—Ovomucin precipitation [0305]
  • 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 3 hrs (preferably overnight) with constant stirring. The precipitated ovomucin was then pelleted and removed by centrifugation at 2400×g. After collection of the RPI containing supernatant, the pH of this solution was readjusted to 8. [0306]
  • B. Filtration [0307]
  • To prepare the egg white for loading onto the column and, thereby, minimize the potential for clogging the columns during loading, the egg white solution was filtered to at least 0.45 um. [0308]
  • Initially, 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. [0309]
  • Amersham's hollow-fiber ultrafiltration 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. [0310]
  • III. Purification [0311]
  • A. Affinity Chromatography [0312]
  • Using antibody with specificity to a synthetic peptide modeled after the enterokinase recognition site, initial purification schemes involved developing a one-step column purification procedure for the RPI. [0313]
  • 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. [0314]
  • An initial attempt was made to direct the first purification step against the ovalbumin portion of the recombinant protein using an antibody specific for the ovalbumin portion. The present purification scheme employed a combination of classical techniques such as ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. [0315]
  • After the initial ovomucin precipitation, 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[0316] 4)2SO4 or subjected to gel filtration to remove the (NH4)2SO4 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. One at approximately 25% of the 0.5 M NaCl gradient without (NH4)2SO4 precipitation. The second was observed at less than 16% gradient (approximately 7%) following 40% (NH4)2SO4 precipitation and a longer gradient. Fractions containing RPI were identified by SDS-PAGE analysis and pooled.
  • Three gel filtration columns, differing by column size and fractionation range, were employed in RPI purification and/or desalting: Superdex 75 10/300 GL, Hiload 26/60 Superdex 75, and Hiload 26/60 Superdex 200. Using these individual columns at different steps in the purification scheme increased the efficiency of the process. Fractions containing RPI were identified by SDS-PAGE analysis and pooled. [0317]
  • Cleavage of the RPI Enterokinase recognition site was accomplished using purified enterokinase from Sigma. Enterokinase, 0.004 Unit/μl per reaction, was applied to the pooled and, if necessary, concentrated protein solution. The digestion reaction was incubated at room temperature (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. [0318]
  • A subsequent gel filtration separation was employed to obtain purified Proinsulin, and to remove the remaining Ovalbumin portion of the RPI and residual native EW proteins. Select steps in the purification process were analyzed using the 2-dimensional Beckman Coulter ProteomeLab PF2D Protein Fractionation System. [0319]
  • EXAMPLE9 Optimization of Intra-oviduct and Intra-ovarian Arterial Injections
  • Overall transfection 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. When the development of the oviducts and ovaries are uniform across a group of hens and when the stage of oviduct and ovarian development can be determined or predicted, 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. [0320]
  • Hens are treated with estradiol to stimulate oviduct maturation as described in Oka and Schimke (T. Oka and RT Schimke, J. Cell Biol., 41, 816 (1969)), Palmiter, Christensen and Schimke (J Biol. Chem. 245(4):833-845, 1970). Specifically, repeated daily injections of 1 mg estradiol benzoate are performed sometime before the onset of sexual maturation, a period ranging from 1-14 weeks of age. After a stimulation period sufficient to maximize development of the oviduct, hormone treatment is withdrawn thereby causing regression in oviduct secretory cell size but not cell number. At an optimum time after hormone withdrawal, the lumens of the oviducts of treated hens are injected with the transposon-based vector. Hens are subjected to additional estrogen 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. Estrogen 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 transfections and possibly oviduct expression. [0321]
  • Other means are also used to synchronize the development, or regression, of the oviduct and ovary to allow high and uniform transfection efficiencies. Alterations of lighting and/or feed regimens, for example, cause hens to ‘molt’ during which time the oviduct and ovary regress. Molting is used to synchronize hens for transfection, and may be used in conjunction with other hormonal methods to control regression and/or development of the oviduct and ovary. [0322]
  • EXAMPLE10 Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/ProIns/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. [0323]
  • Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptII sk(−). [0324]
  • Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz. [0325]
  • Base pairs 1780-2987 are a transposase, modified from Tn10 (GenBank accession number J01829). [0326]
  • Base pairs 2988-2993 are an engineered stop codon. [0327]
  • Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz. [0328]
  • Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859. [0329]
  • Base pairs 3719-3761 are λ DNA that is residual from pNK2859. [0330]
  • Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10. [0331]
  • Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptII sk(−). [0332]
  • 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). Some deletions were noted in the quail sequence, as compared to the chicken sequence. [0333]
  • 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). [0334]
  • Base pairs 6093-6246 are a TAG sequence containing a gp41 hairpin loop from HIV I an enterokinase cleavage site and a spacer (synthetic). [0335]
  • Base pairs 6247-6507 are a proinsulin gene. [0336]
  • Base pairs 6514-6866 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz. [0337]
  • Base pairs 6867-7303 are a multiple cloning site from pBlueScriptll sk(−) corresponding to base pairs 667-235 of pBluescriptII sk(−). [0338]
  • Base pairs 7304-7379 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10. [0339]
  • Base pairs 7380-7421 are λ DNA that is residual from pNK2859. [0340]
  • Base pairs 7422-8286 are non coding DNA that is residual from pNK2859. [0341]
  • Base pairs 8287-10487 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptII sk(−). [0342]
  • It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants. [0343]
  • EXAMPLE11 Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/p146/PA)—Chicken
  • A vector was designed for inserting a p146 gene under the control 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. [0344]
  • Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptlI sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptll sk(−). [0345]
  • Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz. [0346]
  • Base pairs 1780-2987 are a transposase, modified from Tn10 (GenBank accession number J01829). [0347]
  • Base pairs 2988-2993 are an engineered stop codon. [0348]
  • Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz. [0349]
  • Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859. [0350]
  • Base pairs 3719-3761 are λ DNA that is residual from pNK2859. [0351]
  • Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10. [0352]
  • Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptll sk(−). [0353]
  • 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. [0354]
  • 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). [0355]
  • Base pairs 6122-6271 are a TAG sequence containing a gp41 hairpin loop from HIV I, an enterokinase cleavage site and a spacer (synthetic). [0356]
  • Base pairs 6272-6316 are a p146 sequence (synthetic) with 2 added stop codons. [0357]
  • Base pairs 6324-6676 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz. [0358]
  • Base pairs 6682-7114 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptll sk(−). [0359]
  • Base pairs 7120-7189 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10. [0360]
  • Base pairs 7190-7231 are λ DNA that is residual from pNK2859. [0361]
  • Base pairs 7232-8096 are non coding DNA that is residual from pNK2859. [0362]
  • Base pairs 8097-10297 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptll sk(−). [0363]
  • It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants. [0364]
  • EXAMPLE12 Preparation of Transposon-Based VectorpTnMod(Oval/ENT TAG/p146/PA)—Quail
  • A vector was designed for inserting a p146 gene under the control of a quail ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird. The vector sequence is given below as SEQ ID NO:46. [0365]
  • Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptll sk(−). [0366]
  • Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz. [0367]
  • Base pairs 1780-2987 are a transposase, modified from Tn10 (GenBank accession number J01829). [0368]
  • Base pairs 2988-2993 are an engineered stop codon. [0369]
  • Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz. [0370]
  • Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859. [0371]
  • Base pairs 3719-3761 are λ DNA that is residual from pNK2859. [0372]
  • Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10. [0373]
  • Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptll sk(−). [0374]
  • 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). [0375]
  • 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). [0376]
  • Base pairs 6097-6246 are a TAG sequence containing a gp41 hairpin loop from HIV I, an enterokinase cleavage site and a spacer (synthetic). [0377]
  • Base pairs 6247-6291 are a p146 sequence (synthetic) with 2 added stop codons. [0378]
  • Base pairs 6299-6651 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272of pGWiz. [0379]
  • Base pairs 6657-7089 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptll sk(−). [0380]
  • Base pairs 7095-7164 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10. [0381]
  • Base pairs 7165-7206 are λ DNA that is residual from pNK2859. [0382]
  • Base pairs 7207-8071 are non coding DNA that is residual from pNK2859. [0383]
  • Base pairs 8072-10272 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961of pBluescriptll sk(−). [0384]
  • It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants. [0385]
  • EXAMPLE13 Additional Transposon-Based Vectors for Administration to an Animal
  • The following example provides a description of various transposon-based vectors of the present invention and several constructs that have been made for insertion into the transposon-based vectors of the present invention, all for intraoviduct administration. These examples are not meant to be limiting in any way. The constructs for insertion into a transposon-based vector are provided in a cloning vector pTnMCS or pTnMod, both described above. [0386]
  • pTnMCS (CMV-CHOVg-ent-Prolnsulin-synPA) (SEQ ID NO:47) [0387]
  • Bp 1-3670 from vector PTnMCS, bp 1-3670 [0388]
  • Bp 3676-5320 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems), bp 230-1864 [0389]
  • Bp 5327-6480 Chicken ovalbumin gene taken from GenBank accession #V00383, bp 66-1219 [0390]
  • Bp 6487-6636 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0391]
  • Bp 6637-6897 Human Proinsulin taken from GenBank accession #NM000207, bp 117-377 [0392]
  • Bp 6898-6942 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems) [0393]
  • Bp 6943-7295 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271 [0394]
  • Bp 7296-10895 from cloning vector pTnMCS, bp 3716-7315 [0395]
  • pTnMCS (CMV-prepro-ent-ProInsulin-synPA) [0396]
  • Bp 1-3670 from vector PTnMCS, bp 1-3670 [0397]
  • Bp 3676-5320 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems), bp 230-1864 [0398]
  • Bp 5326-5496 Capsite/prepro taken fron GenBank accession #X07404, bp 563-733 [0399]
  • Bp 5504-5652 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0400]
  • Bp 5653-5913 Human Proinsulin taken from GenBank accession #NM000207, bp 117-377 [0401]
  • Bp 5914-5958 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems) [0402]
  • Bp 5959-6310 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271 [0403]
  • Bp 6313-9912 from cloning vector pTnMCS, bp 3716-7315 [0404]
  • pTnMCS(Chicken OVep+OVg′+ENT+proins+syn polyA) [0405]
  • Bp 1-3670 from vector pTnMCS, bp 1-3670 [0406]
  • Bp 3676-4350 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1 bp 1-675 [0407]
  • Bp 4357-5692 Chicken Ovalbumin promoter taken from GenBank accession #J00895M24999 bp 1-1336 [0408]
  • Bp 5699-6917 Chicken Ovalbumin gene from GenBank Accession #V00383.1 bp 2-1220. (This sequence includes the 5′UTR, containing putative cap site, bp 5699-5762.) [0409]
  • Bp 6924-7073 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0410]
  • Bp 7074-7334 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0411]
  • Bp 7335-7379 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0412]
  • Bp 7380-7731 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0413]
  • Bp 7733-11332 from vector pTnMCS, bp 3716-7315 [0414]
  • pTnMCS(Chicken OVep+prepro+ENT+proins+syn polyA) [0415]
  • Bp 1-3670 from cloning vector pTnMCS, bp 1-3670 [0416]
  • Bp 3676-4350 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1 bp 1-675 [0417]
  • Bp 4357-5692 Chicken Ovalbumin promoter taken from GenBank accession #J00895-M24999bp 1-1336 [0418]
  • Bp 5699-5869 Cecropin cap site and prepro, Genbank accession #X07404 bp 563-733 [0419]
  • Bp 5876-6025 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0420]
  • Bp 6026-6286 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0421]
  • Bp 6287-6331 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0422]
  • Bp 6332-6683 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0423]
  • Bp 6685-10284 from cloning vector pTnMCS, bp 3716-7315 [0424]
  • pTnMCS(Quail OVep+OVg′+ENT+proins+syn polyA) [0425]
  • Bp 1-3670 from cloning vector pTnMCS, bp 1-3670 [0426]
  • Bp 3676-4333 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upstream chicken ovalbumin enhancer, GenBank accession #S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative tochicken, so the number of bases does not correspond exactly.) [0427]
  • 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.) [0428]
  • Bp 5712-6910 Quail Ovalbumin gene, EMBL accession #X53964, bp 1-1199. (This sequence includes the 5′UTR, containing putative cap site bp 5712-5764.) [0429]
  • Bp 6917-7066 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0430]
  • Bp 7067-7327 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0431]
  • Bp 7328-7372 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0432]
  • Bp 7373-7724 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0433]
  • Bp 7726-11325 from cloning vector pTnMCS, bp 3716-7315 [0434]
  • pTnMCS(Ouail OVep+prepro+ENT+proins+syn polyA) [0435]
  • Bp 1-3670 from cloning vector pTnMCS, bp 1-3670 [0436]
  • Bp 3676-4333 Quail Ovalbumin enhancer: 658 bp sequence, amplified from quail genomic DNA, roughly equivalent to the far-upstream 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.) [0437]
  • Bp 4340-5705 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.) [0438]
  • Bp 5712-5882 Cecropin cap site and prepro, Genbank accession #X07404 bp 563-733 [0439]
  • Bp 5889-6038 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0440]
  • Bp 6039-6299 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0441]
  • Bp 6300-6344 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0442]
  • Bp 6345-6696 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0443]
  • Bp 6698-10297 from cloning vector pTnMCS, bp 3716-7315. [0444]
  • pTnMOD (CMV-prepro-ent-proins-synPA) [0445]
  • Bp 1-4045 from vector PTnMCS, bp 1-4045 [0446]
  • Bp 4051-5695 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1864 [0447]
  • Bp 5701-5871 Capsite/prepro taken from GenBank accession #X07404, bp 563-733 [0448]
  • Bp 5879-6027 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0449]
  • Bp 6028-6288 Human Proinsulin taken from GenBank accession #NM000207, bp 117-377 [0450]
  • Bp 6289-6333 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems) [0451]
  • Bp 6334-6685 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271 [0452]
  • Bp 6687-10286 from cloning vector pTnMCS, bp 3716-7315 [0453]
  • pTnMOD(Chicken OVep+OVg′+ENT+proins+syn polyA) [0454]
  • Bp 1-4045 from cloning vector pTnMod, bp 1-4045 [0455]
  • Bp 4051-4725 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1 bp 1-675 [0456]
  • Bp 4732-6067 Chicken Ovalbumin promoter taken from GenBank accession #J00895-M24999 bp 1-1336 [0457]
  • Bp 6074-7292 Chicken Ovalbumin gene from GenBank Accession #V00383.1 bp 2-1220. (This sequence includes the 5′UTR, containing putative cap site bp 6074-6137.) [0458]
  • Bp 7299-7448 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0459]
  • Bp 7449-7709 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0460]
  • Bp 7710-7754 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0461]
  • Bp 7755-8106 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0462]
  • Bp 8108-11707 from cloning vector pTnMod, bp 3716-7315 [0463]
  • pTnMOD(Chicken OVep+prepro+ENT+proins+syn polyA) [0464]
  • Bp 1-4045 from cloning vector pTnMCS, bp 1-4045 [0465]
  • Bp 4051-4725 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1 bp 1-675 [0466]
  • Bp 4732-6067 Chicken Ovalbumin promoter taken from GenBank accession #J00895-M24999 bp 1-1336 [0467]
  • Bp 6074-6244 Cecropin cap site and prepro, Genbank accession #X07404 bp 563-733 [0468]
  • Bp 6251-6400 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0469]
  • Bp 6401-6661 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0470]
  • Bp 6662-6706 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0471]
  • Bp 6707-7058 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0472]
  • Bp 7060-10659 from cloning vector pTnMCS, bp 3716-7315 [0473]
  • pTnMOD(Quail OVep+OVg′+ENT+proins+syn polyA) [0474]
  • Bp 1-4045 from cloning vector pTnMCS, bp 1-4045 [0475]
  • Bp 4051-4708 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upstream 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.) [0476]
  • Bp 4715-6080 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.) [0477]
  • Bp 6087-7285 Quail Ovalbumin gene, EMBL accession #X53964, bp 1-1199. (This sequence includes the 5′UTR, containing putative cap site bp 6087-6139.) [0478]
  • Bp 7292-7441 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0479]
  • Bp 7442-7702 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0480]
  • Bp 7703-7747 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0481]
  • Bp 7748-8099 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0482]
  • Bp 8101-11700 from cloning vector pTnMCS, bp 3716-7315 [0483]
  • pTnMOD(Quail OVep+prepro+ENT+proins+svn polyA) [0484]
  • Bp 1-4045 from cloning vector pTnMCS, bp 1-4045 [0485]
  • Bp 4051-4708 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-housefrom quail genomic DNA, roughly equivalent to the far-upstream 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.) [0486]
  • Bp 4715-6080 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.) [0487]
  • Bp 6087-6257 Cecropin cap site and Prepro, Genbank accession #X07404 bp 563-733 [0488]
  • Bp 6264-6413 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0489]
  • Bp 6414-6674 Human proinsulin GenBank Accession #NM000207 bp 117-377 [0490]
  • Bp 6675-6719 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems) [0491]
  • Bp 6720-7071 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271 [0492]
  • Bp 7073-10672 from cloning vector pTnMCS, bp 3716-7315 [0493]
  • pTnMOD (CMV-prepro-ent-hGH-CPA) [0494]
  • Bp 1-4045 from vector PTnMOD, bp 1-4045 [0495]
  • Bp 4051-5694 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1873 [0496]
  • Bp 5701-5871 Capsite/Prepro taken fron GenBank accession #X07404, bp 563-733 [0497]
  • Bp 5878-6012 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0498]
  • Bp 6013-6666 Human growth hormone taken from GenBank accession #V00519, bp 1-654 [0499]
  • Bp 6673-7080 Conalbumin polyA taken from GenBank accession #Y00407, bp 10651-11058 [0500]
  • Bp 7082-10681 from cloning vector pTnMOD, bp 4091-7690 [0501]
  • pTnMCS (CHOVep-prepro-ent-hGH-CPA) [0502]
  • Bp 1-3670 from vector PTnMCS, bp 1-3670 [0503]
  • Bp 3676-4350 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1, bp 1-675 [0504]
  • Bp 4357-5692 Chicken Ovalbumin promoter taken from GenBank accession #J00899-M24999, bp 1-1336 [0505]
  • Bp 5699-5869 Capsite/Prepro taken fron GenBank accession #X07404, bp 563-733 [0506]
  • Bp 5876-6010 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0507]
  • Bp 6011-6664 Human growth hormone taken from GenBank accession #V00519, bp 1-654 [0508]
  • Bp 6671-7078 Conalbumin polyA taken from GenBank accession #Y00407, bp 10651-11058 [0509]
  • Bp 7080-10679 from cloning vector pTnMCS, bp 3716-7315 [0510]
  • pTnMCS (CMV-prepro-ent-hGH-CPA) [0511]
  • Bp 1-3670 from vector PTnMCS, bp 1-3670 [0512]
  • Bp 3676-5319 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1873 [0513]
  • Bp 5326-5496 Capsite/Prepro taken fron GenBank accession #X07404, bp 563-733 [0514]
  • Bp 5503-5637 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0515]
  • Bp 5638-6291 Human growth hormone taken from GenBank accession #V00519, bp 1-654 [0516]
  • Bp 6298-6705 Conalbumin polyA taken from GenBank accession #Y00407, bp 10651-11058 [0517]
  • Bp 6707-10306 from cloning vector pTnMCS, bp 3716-7315 [0518]
  • pTnMOD (CHOVep-prepro-ent-hGH-CPA) [0519]
  • Bp 1-4045 from vector PTnMOD, bp 1-4045 [0520]
  • Bp 4051-4725 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1, bp 1-675 [0521]
  • Bp 4732-6067 Chicken Ovalbumin promoter taken from GenBank accession #J00899-M24999, bp 1-1336 [0522]
  • Bp 6074-6244 Capsite/Prepro taken fron GenBank accession #X07404, bp 563-733 [0523]
  • Bp 6251-6385 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site [0524]
  • Bp 6386-7039 Human growth hormone taken from GenBank accession #V00519, bp 1-654 [0525]
  • Bp 7046-7453 Conalbumin polyA taken from GenBank accession #Y00407, bp 10651-11058 [0526]
  • Bp 7455-11054 from cloning vector pTnMOD, bp 4091-7690 [0527]
  • PTnMod(CMV/Transposase/ChickOvep/prepro/ProteinA/ConpolyA) [0528]
  • BP 1-130 remainder of F1 (−) ori of pBluescriptII sk(−) (Stragagene) bp 1-130. [0529]
  • BP 133-1777 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems) bp 229-1873. [0530]
  • BP 1780-2987 Transposase, modified from Tn10 (GenBank #J01829). [0531]
  • BP 2988-2993 Engineered DOUBLE stop codon. [0532]
  • BP 2994-3343 non coding DNA from vector pNK2859. [0533]
  • BP 3344-3386 Lambda DNA from pNK2859. [0534]
  • BP 3387-3456 70bp of IS10 left from Tn10. [0535]
  • BP 3457-3674 multiple cloning site from pBluescriptII sk(−) bp 924-707. [0536]
  • BP 3675-5691 Chicken Ovalbumin enhancer plus promoter from a Topo Clone 10 maxi 040303 (5′ XmaI, 3′ BamHI) [0537]
  • BP 5698-5865 prepro with Cap site amplified from cecropin of pMON200 GenBank #X07404 (5′BamHI, 3′KpnI) [0538]
  • BP 5872-7338 Protein A gene from GenBank#J01786, mature peptide bp 292-1755 (5′Kpnl, 3′SacII) [0539]
  • BP 7345-7752 ConPolyA from Chicken conalbumin polyA from GenBank #Y00407 bp 10651-11058. (5′SacII, 3′XhoI) [0540]
  • BP 7753-8195 multiple cloning site from pBluescriptII sk(−) bp 677-235. [0541]
  • BP 8196-8265 70 bp of IS10 left from Tn10. [0542]
  • BP 8266-8307 Lamda DNA from pNK2859 [0543]
  • BP 8308-9151 noncoding DNA from pNK2859 [0544]
  • BP 9152-11352 pBluescriptIl sk(−) base vector (Stratagene, INC.) bp 761-2961 [0545]
  • All patents, publications and abstracts cited above are incorporated herein by reference in their entirety. It should be understood that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the present invention as defined in the following claims. [0546]
  • 1 52 1 680 DNA Gallus sp. 1 ccgggctgca gaaaaatgcc aggtggacta tgaactcaca tccaaaggag cttgacctga 60 tacctgattt tcttcaaact ggggaaacaa cacaatccca caaaacagct cagagagaaa 120 ccatcactga tggctacagc accaaggtat gcaatggcaa tccattcgac attcatctgt 180 gacctgagca aaatgattta tctctccatg aatggttgct tctttccctc atgaaaaggc 240 aatttccaca ctcacaatat gcaacaaaga caaacagaga acaattaatg tgctccttcc 300 taatgtcaaa attgtagtgg caaagaggag aacaaaatct caagttctga gtaggtttta 360 gtgattggat aagaggcttt gacctgtgag ctcacctgga cttcatatcc ttttggataa 420 aaagtgcttt tataactttc aggtctccga gtctttattc atgagactgt tggtttaggg 480 acagacccac aatgaaatgc ctggcatagg aaagggcagc agagccttag ctgacctttt 540 cttgggacaa gcattgtcaa acaatgtgtg acaaaactat ttgtactgct ttgcacagct 600 gtgctgggca gggcaatcca ttgccaccta tcccaggtaa ccttccaact gcaagaagat 660 tgttgcttac tctctctaga 680 2 7315 DNA Artificial Sequence Synthetic 2 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgcga actcgatatt 1800 ttacacgact ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggcaata cgatgcccat tgtacttgtt gactggtctg atattcgtga gcaaaaacga 2100 cttatggtat tgcgagcttc agtcgcacta cacggtcgtt ctgttactct ttatgagaaa 2160 gcgttcccgc tttcagagca atgttcaaag aaagctcatg accaatttct agccgacctt 2220 gcgagcattc taccgagtaa caccacaccg ctcattgtca gtgatgctgg ctttaaagtg 2280 ccatggtata aatccgttga gaagctgggt tggtactggt taagtcgagt aagaggaaaa 2340 gtacaatatg cagacctagg agcggaaaac tggaaaccta tcagcaactt acatgatatg 2400 tcatctagtc actcaaagac tttaggctat aagaggctga ctaaaagcaa tccaatctca 2460 tgccaaattc tattgtataa atctcgctct aaaggccgaa aaaatcagcg ctcgacacgg 2520 actcattgtc accacccgtc acctaaaatc tactcagcgt cggcaaagga gccatgggtt 2580 ctagcaacta acttacctgt tgaaattcga acacccaaac aacttgttaa tatctattcg 2640 aagcgaatgc agattgaaga aaccttccga gacttgaaaa gtcctgccta cggactaggc 2700 ctacgccata gccgaacgag cagctcagag cgttttgata tcatgctgct aatcgccctg 2760 atgcttcaac taacatgttg gcttgcgggc gttcatgctc agaaacaagg ttgggacaag 2820 cacttccagg ctaacacagt cagaaatcga aacgtactct caacagttcg cttaggcatg 2880 gaagttttgc ggcattctgg ctacacaata acaagggaag acttactcgt ggctgcaacc 2940 ctactagctc aaaatttatt cacacatggt tacgctttgg ggaaattatg aggggatcgc 3000 tctagagcga tccgggatct cgggaaaagc gttggtgacc aaaggtgcct tttatcatca 3060 ctttaaaaat aaaaaacaat tactcagtgc ctgttataag cagcaattaa ttatgattga 3120 tgcctacatc acaacaaaaa ctgatttaac aaatggttgg tctgccttag aaagtatatt 3180 tgaacattat cttgattata ttattgataa taataaaaac cttatcccta tccaagaagt 3240 gatgcctatc attggttgga atgaacttga aaaaaattag ccttgaatac attactggta 3300 aggtaaacgc cattgtcagc aaattgatcc aagagaacca acttaaagct ttcctgacgg 3360 aatgttaatt ctcgttgacc ctgagcactg atgaatcccc taatgatttt ggtaaaaatc 3420 attaagttaa ggtggataca catcttgtca tatgatcccg gtaatgtgag ttagctcact 3480 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg 3540 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 3600 aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg ctctagaact 3660 agtggatccc ccgggctgca ggaattcgat atcaagctta tcgataccgc tgacctcgag 3720 ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt 3780 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 3840 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 3900 acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt gttaaaattc 3960 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 4020 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 4080 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 4140 gatggcccac tactccggga tcatatgaca agatgtgtat ccaccttaac ttaatgattt 4200 ttaccaaaat cattagggga ttcatcagtg ctcagggtca acgagaatta acattccgtc 4260 aggaaagctt atgatgatga tgtgcttaaa aacttactca atggctggtt atgcatatcg 4320 caatacatgc gaaaaaccta aaagagcttg ccgataaaaa aggccaattt attgctattt 4380 accgcggctt tttattgagc ttgaaagata aataaaatag ataggtttta tttgaagcta 4440 aatcttcttt atcgtaaaaa atgccctctt gggttatcaa gagggtcatt atatttcgcg 4500 gaataacatc atttggtgac gaaataacta agcacttgtc tcctgtttac tcccctgagc 4560 ttgaggggtt aacatgaagg tcatcgatag caggataata atacagtaaa acgctaaacc 4620 aataatccaa atccagccat cccaaattgg tagtgaatga ttataaataa cagcaaacag 4680 taatgggcca ataacaccgg ttgcattggt aaggctcacc aataatccct gtaaagcacc 4740 ttgctgatga ctctttgttt ggatagacat cactccctgt aatgcaggta aagcgatccc 4800 accaccagcc aataaaatta aaacagggaa aactaaccaa ccttcagata taaacgctaa 4860 aaaggcaaat gcactactat ctgcaataaa tccgagcagt actgccgttt tttcgcccat 4920 ttagtggcta ttcttcctgc cacaaaggct tggaatactg agtgtaaaag accaagaccc 4980 gtaatgaaaa gccaaccatc atgctattca tcatcacgat ttctgtaata gcaccacacc 5040 gtgctggatt ggctatcaat gcgctgaaat aataatcaac aaatggcatc gttaaataag 5100 tgatgtatac cgatcagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 5160 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5220 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 5280 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5340 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5400 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5460 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5520 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5580 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5640 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5700 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 5760 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5820 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5880 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5940 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6000 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6060 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6120 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6180 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6240 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6300 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6360 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6420 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6480 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6540 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6600 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6660 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6720 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 6780 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 6840 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 6900 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 6960 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7020 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7080 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7140 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7200 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7260 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac 7315 3 7689 DNA Artificial Sequence Synthetic 3 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt 1800 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggaatac gatgcccatt gtacttgttg actggtctga tattcgtgag caaaaacgac 2100 ttatggtatt gcgagcttca gtcgcactac acggtcgttc tgttactctt tatgagaaag 2160 cgttcccgct ttcagagcaa tgttcaaaga aagctcatga ccaatttcta gccgaccttg 2220 cgagcattct accgagtaac accacaccgc tcattgtcag tgatgctggc tttaaagtgc 2280 catggtataa atccgttgag aagctgggtt ggtactggtt aagtcgagta agaggaaaag 2340 tacaatatgc agacctagga gcggaaaact ggaaacctat cagcaactta catgatatgt 2400 catctagtca ctcaaagact ttaggctata agaggctgac taaaagcaat ccaatctcat 2460 gccaaattct attgtataaa tctcgctcta aaggccgaaa aaatcagcgc tcgacacgga 2520 ctcattgtca ccacccgtca cctaaaatct actcagcgtc ggcaaaggag ccatgggttc 2580 tagcaactaa cttacctgtt gaaattcgaa cacccaaaca acttgttaat atctattcga 2640 agcgaatgca gattgaagaa accttccgag acttgaaaag tcctgcctac ggactaggcc 2700 tacgccatag ccgaacgagc agctcagagc gttttgatat catgctgcta atcgccctga 2760 tgcttcaact aacatgttgg cttgcgggcg ttcatgctca gaaacaaggt tgggacaagc 2820 acttccaggc taacacagtc agaaatcgaa acgtactctc aacagttcgc ttaggcatgg 2880 aagttttgcg gcattctggc tacacaataa caagggaaga cttactcgtg gctgcaaccc 2940 tactagctca aaatttattc acacatggtt acgctttggg gaaattatga taatgatcca 3000 gatcacttct ggctaataaa agatcagagc tctagagatc tgtgtgttgg ttttttgtgg 3060 atctgctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3120 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3180 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcagcaca gcaaggggga 3240 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacctctct 3300 ctctctctct ctctctctct ctctctctct ctctcggtac ctctctctct ctctctctct 3360 ctctctctct ctctctctct cggtaccagg tgctgaagaa ttgacccggt gaccaaaggt 3420 gccttttatc atcactttaa aaataaaaaa caattactca gtgcctgtta taagcagcaa 3480 ttaattatga ttgatgccta catcacaaca aaaactgatt taacaaatgg ttggtctgcc 3540 ttagaaagta tatttgaaca ttatcttgat tatattattg ataataataa aaaccttatc 3600 cctatccaag aagtgatgcc tatcattggt tggaatgaac ttgaaaaaaa ttagccttga 3660 atacattact ggtaaggtaa acgccattgt cagcaaattg atccaagaga accaacttaa 3720 agctttcctg acggaatgtt aattctcgtt gaccctgagc actgatgaat cccctaatga 3780 ttttggtaaa aatcattaag ttaaggtgga tacacatctt gtcatatgat cccggtaatg 3840 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 3900 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 3960 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 4020 gccgctctag aactagtgga tcccccgggc tgcaggaatt cgatatcaag cttatcgata 4080 ccgctgacct cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg 4140 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 4200 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 4260 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata 4320 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 4380 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 4440 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 4500 ccgtctatca gggcgatggc ccactactcc gggatcatat gacaagatgt gtatccacct 4560 taacttaatg atttttacca aaatcattag gggattcatc agtgctcagg gtcaacgaga 4620 attaacattc cgtcaggaaa gcttatgatg atgatgtgct taaaaactta ctcaatggct 4680 ggttatgcat atcgcaatac atgcgaaaaa cctaaaagag cttgccgata aaaaaggcca 4740 atttattgct atttaccgcg gctttttatt gagcttgaaa gataaataaa atagataggt 4800 tttatttgaa gctaaatctt ctttatcgta aaaaatgccc tcttgggtta tcaagagggt 4860 cattatattt cgcggaataa catcatttgg tgacgaaata actaagcact tgtctcctgt 4920 ttactcccct gagcttgagg ggttaacatg aaggtcatcg atagcaggat aataatacag 4980 taaaacgcta aaccaataat ccaaatccag ccatcccaaa ttggtagtga atgattataa 5040 ataacagcaa acagtaatgg gccaataaca ccggttgcat tggtaaggct caccaataat 5100 ccctgtaaag caccttgctg atgactcttt gtttggatag acatcactcc ctgtaatgca 5160 ggtaaagcga tcccaccacc agccaataaa attaaaacag ggaaaactaa ccaaccttca 5220 gatataaacg ctaaaaaggc aaatgcacta ctatctgcaa taaatccgag cagtactgcc 5280 gttttttcgc ccatttagtg gctattcttc ctgccacaaa ggcttggaat actgagtgta 5340 aaagaccaag acccgtaatg aaaagccaac catcatgcta ttcatcatca cgatttctgt 5400 aatagcacca caccgtgctg gattggctat caatgcgctg aaataataat caacaaatgg 5460 catcgttaaa taagtgatgt ataccgatca gcttttgttc cctttagtga gggttaattg 5520 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 5580 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 5640 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 5700 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 5760 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 5820 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 5880 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 5940 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 6000 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 6060 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 6120 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 6180 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 6240 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 6300 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 6360 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 6420 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 6480 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 6540 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 6600 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 6660 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 6720 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 6780 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 6840 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 6900 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 6960 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 7020 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 7080 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 7140 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 7200 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 7260 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 7320 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 7380 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 7440 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 7500 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 7560 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 7620 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 7680 aagtgccac 7689 4 7 DNA Artificial Sequence Synthetic 4 accatgg 7 5 7 DNA Artificial Sequence Synthetic 5 accatgt 7 6 7 DNA Artificial Sequence Synthetic 6 aagatgt 7 7 7 DNA Artificial Sequence Synthetic 7 acgatga 7 8 7 DNA Artificial Sequence Synthetic 8 aagatgg 7 9 7 DNA Artificial Sequence Synthetic 9 gacatga 7 10 7 DNA Artificial Sequence Synthetic 10 accatga 7 11 315 DNA Gallus sp. 11 tctgccattg ctgcttcctc tgcccttcct cgtcactctg aatgtggctt cttcgctact 60 gccacagcaa gaaataaaat ctcaacatct aaatgggttt cctgaggttt ttcaagagtc 120 gttaagcaca ttccttcccc agcacccctt gctgcaggcc agtgccaggc accaacttgg 180 ctactgctgc ccatgagaga aatccagttc aatattttcc aaagcaaaat ggattacata 240 tgccctagat cctgattaac aggcgtttgt attatctagt gctttcgctt cacccagatt 300 atcccattgc ctccc 315 12 361 DNA Artificial Sequence Synthetic 12 ggcgcctgga tccagatcac ttctggctaa taaaagatca gagctctaga gatctgtgtg 60 ttggtttttt gtggatctgc tgtgccttct agttgccagc catctgttgt ttgcccctcc 120 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 180 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 240 cacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 300 atgggtacct ctctctctct ctctctctct ctctctctct ctctctctcg gtacctctct 360 c 361 13 350 DNA Artificial Sequence Synthetic 13 ggggatcgct ctagagcgat ccgggatctc gggaaaagcg ttggtgacca aaggtgcctt 60 ttatcatcac tttaaaaata aaaaacaatt actcagtgcc tgttataagc agcaattaat 120 tatgattgat gcctacatca caacaaaaac tgatttaaca aatggttggt ctgccttaga 180 aagtatattt gaacattatc ttgattatat tattgataat aataaaaacc ttatccctat 240 ccaagaagtg atgcctatca ttggttggaa tgaacttgaa aaaaattagc cttgaataca 300 ttactggtaa ggtaaacgcc attgtcagca aattgatcca agagaaccaa 350 14 908 DNA Artificial Sequence Synthetic 14 tgaatgtgtt cttgtgttat caatataaat cacagttagt gatgaagttg gctgcaagcc 60 tgcatcagtt cagctacttg gctgcatttt gtatttggtt ctgtaggaaa tgcaaaaggt 120 tctaggctga cctgcacttc tatccctctt gccttactgc tgagaatctc tgcaggtttt 180 aattgttcac attttgctcc catttacttt ggaagataaa atatttacag aatgcttatg 240 aaacctttgt tcatttaaaa atattcctgg tcagcgtgac cggagctgaa agaacacatt 300 gatcccgtga tttcaataaa tacatatgtt ccatatattg tttctcagta gcctcttaaa 360 tcatgtgcgt tggtgcacat atgaatacat gaatagcaaa ggtttatctg gattacgctc 420 tggcctgcag gaatggccat aaaccaaagc tgagggaaga gggagagtat agtcaatgta 480 gattatactg attgctgatt gggttattat cagctagata acaacttggg tcaggtgcca 540 ggtcaacata acctgggcaa aaccagtctc atctgtggca ggaccatgta ccagcagcca 600 gccgtgaccc aatctaggaa agcaagtagc acatcaattt taaatttatt gtaaatgccg 660 tagtagaagt gttttactgt gatacattga aacttctggt caatcagaaa aaggtttttt 720 atcagagatg ccaaggtatt atttgatttt ctttattcgc cgtgaagaga atttatgatt 780 gcaaaaagag gagtgtttac ataaactgat aaaaaacttg aggaattcag cagaaaacag 840 ccacgtgttc ctgaacattc ttccataaaa gtctcaccat gcctggcaga gccctattca 900 ccttcgct 908 15 901 DNA Gallus 15 gaggtcagaa tggtttcttt actgtttgtc aattctatta tttcaataca gaacaatagc 60 ttctataact gaaatatatt tgctattgta tattatgatt gtccctcgaa ccatgaacac 120 tcctccagct gaatttcaca attcctctgt catctgccag gccattaagt tattcatgga 180 agatctttga ggaacactgc aagttcatat cataaacaca tttgaaattg agtattgttt 240 tgcattgtat ggagctatgt tttgctgtat cctcagaaaa aaagtttgtt ataaagcatt 300 cacacccata aaaagataga tttaaatatt ccagctatag gaaagaaagt gcgtctgctc 360 ttcactctag tctcagttgg ctccttcaca tgcatgcttc tttatttctc ctattttgtc 420 aagaaaataa taggtcacgt cttgttctca cttatgtcct gcctagcatg gctcagatgc 480 acgttgtaga tacaagaagg atcaaatgaa acagacttct ggtctgttac tacaaccata 540 gtaataagca cactaactaa taattgctaa ttatgttttc catctctaag gttcccacat 600 ttttctgttt tcttaaagat cccattatct ggttgtaact gaagctcaat ggaacatgag 660 caatatttcc cagtcttctc tcccatccaa cagtcctgat ggattagcag aacaggcaga 720 aaacacattg ttacccagaa ttaaaaacta atatttgctc tccattcaat ccaaaatgga 780 cctattgaaa ctaaaatcta acccaatccc attaaatgat ttctatggcg tcaaaggtca 840 aacttctgaa gggaacctgt gggtgggtca caattcaggc tatatattcc ccagggctca 900 g 901 16 680 DNA Gallus 16 ccgggctgca gaaaaatgcc aggtggacta tgaactcaca tccaaaggag cttgacctga 60 tacctgattt tcttcaaact ggggaaacaa cacaatccca caaaacagct cagagagaaa 120 ccatcactga tggctacagc accaaggtat gcaatggcaa tccattcgac attcatctgt 180 gacctgagca aaatgattta tctctccatg aatggttgct tctttccctc atgaaaaggc 240 aatttccaca ctcacaatat gcaacaaaga caaacagaga acaattaatg tgctccttcc 300 taatgtcaaa attgtagtgg caaagaggag aacaaaatct caagttctga gtaggtttta 360 gtgattggat aagaggcttt gacctgtgag ctcacctgga cttcatatcc ttttggataa 420 aaagtgcttt tataactttc aggtctccga gtctttattc atgagactgt tggtttaggg 480 acagacccac aatgaaatgc ctggcatagg aaagggcagc agagccttag ctgacctttt 540 cttgggacaa gcattgtcaa acaatgtgtg acaaaactat ttgtactgct ttgcacagct 600 gtgctgggca gggcaatcca ttgccaccta tcccaggtaa ccttccaact gcaagaagat 660 tgttgcttac tctctctaga 680 17 72 DNA Artificial Sequence Synthetic 17 gtggatcaac atacagctag aaagctgtat tgcctttagc actcaagctc aaaagacaac 60 tcagagttca cc 72 18 62 DNA Artificial Sequence Synthetic 18 acatacagct agaaagctgt attgccttta gcactcaagc tcaaaagaca actcagagtt 60 ca 62 19 1158 DNA Gallus 19 atgggctcca tcggcgcagc aagcatggaa ttttgttttg atgtattcaa ggagctcaaa 60 gtccaccatg ccaatgagaa catcttctac tgccccattg ccatcatgtc agctctagcc 120 atggtatacc tgggtgcaaa agacagcacc aggacacaga taaataaggt tgttcgcttt 180 gataaacttc caggattcgg agacagtatt gaagctcagt gtggcacatc tgtaaacgtt 240 cactcttcac ttagagacat cctcaaccaa atcaccaaac caaatgatgt ttattcgttc 300 agccttgcca gtagacttta tgctgaagag agatacccaa tcctgccaga atacttgcag 360 tgtgtgaagg aactgtatag aggaggcttg gaacctatca actttcaaac agctgcagat 420 caagccagag agctcatcaa ttcctgggta gaaagtcaga caaatggaat tatcagaaat 480 gtccttcagc caagctccgt ggattctcaa actgcaatgg ttctggttaa tgccattgtc 540 ttcaaaggac tgtgggagaa aacatttaag gatgaagaca cacaagcaat gcctttcaga 600 gtgactgagc aagaaagcaa acctgtgcag atgatgtacc agattggttt atttagagtg 660 gcatcaatgg cttctgagaa aatgaagatc ctggagcttc catttgccag tgggacaatg 720 agcatgttgg tgctgttgcc tgatgaagtc tcaggccttg agcagcttga gagtataatc 780 aactttgaaa aactgactga atggaccagt tctaatgtta tggaagagag gaagatcaaa 840 gtgtacttac ctcgcatgaa gatggaggaa aaatacaacc tcacatctgt cttaatggct 900 atgggcatta ctgacgtgtt tagctcttca gccaatctgt ctggcatctc ctcagcagag 960 agcctgaaga tatctcaagc tgtccatgca gcacatgcag aaatcaatga agcaggcaga 1020 gaggtggtag ggtcagcaga ggctggagtg gatgctgcaa gcgtctctga agaatttagg 1080 gctgaccatc cattcctctt ctgtatcaag cacatcgcaa ccaacgccgt tctcttcttt 1140 ggcagatgtg tttcccct 1158 20 53 DNA Gallus 20 atgggctcca tcggcgcagc aagcatggaa ttttgttttg atgtattcaa gga 53 21 103 DNA Gallus 21 atgggctcca tcggcgcagc aagcatggaa ttttgttttg atgtattcaa ggagctcaaa 60 gtccaccatg ccaatgagaa catcttctac tgccccattg cca 103 22 63 DNA Artificial Sequence Synthetic 22 atgaggggga tcatactggc attagtgctc acccttgtag gcagccagaa gtttgacatt 60 ggt 63 23 260 DNA Artificial Sequence Synthetic 23 tttgtgaacc aacacctgtg cggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact ctgcaactag 260 24 9 DNA Artificial Sequence Synthetic 24 kykkakkak 9 25 39 DNA Artificial Sequence Synthetic 25 aaatacaaaa aagcactgaa aaaactggca aaactgctg 39 26 4 PRT Artificial Sequence Synthetic 26 Gly Pro Gly Gly 1 27 12 PRT Artificial Sequence Synthetic 27 Gly Pro Gly Gly Gly Pro Gly Gly Gly Pro Gly Gly 1 5 10 28 15 PRT Artificial Sequence Synthetic 28 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 29 20 PRT Artificial Sequence Synthetic 29 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 30 5 PRT Artificial Sequence Synthetic 30 Pro Ala Asp Asp Ala 1 5 31 29 PRT Artificial Sequence Synthetic 31 Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro 1 5 10 15 Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp 20 25 32 16 PRT Artificial Sequence Synthetic 32 Ala Thr Thr Cys Ile Leu Lys Gly Ser Cys Gly Trp Ile Gly Leu Leu 1 5 10 15 33 30 PRT Artificial Sequence Synthetic 33 Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Thr Thr Cys Ile Leu Lys 1 5 10 15 Gly Ser Cys Gly Trp Ile Gly Leu Leu Asp Asp Asp Asp Lys 20 25 30 34 5 PRT Artificial Sequence Synthetic 34 Asp Asp Asp Asp Lys 1 5 35 50 PRT Artificial Sequence Synthetic 35 Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro 1 5 10 15 Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Thr Thr 20 25 30 Cys Ile Leu Lys Gly Ser Cys Gly Trp Ile Gly Leu Leu Asp Asp Asp 35 40 45 Asp Lys 50 36 48 DNA Artificial Sequence Synthetic 36 atctcgagac catgtgtgaa cttgatattt tacatgattc tctttacc 48 37 36 DNA Artificial Sequence Synthetic 37 gattgatcat tatcataatt tccccaaagc gtaacc 36 38 6 DNA Artificial Sequence Synthetic 38 ctcgag 6 39 6 DNA Artificial Sequence Synthetic 39 tgatca 6 40 22 DNA Artificial Sequence Synthetic 40 ttgccggcat cagattggct at 22 41 34 DNA Artificial Sequence Synthetic 41 agaggtcacc gggtcaattc ttcagcacct ggta 34 42 10512 DNA Artificial Sequence Synthetic 42 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt 1800 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggaatac gatgcccatt gtacttgttg actggtctga tattcgtgag caaaaacgac 2100 ttatggtatt gcgagcttca gtcgcactac acggtcgttc tgttactctt tatgagaaag 2160 cgttcccgct ttcagagcaa tgttcaaaga aagctcatga ccaatttcta gccgaccttg 2220 cgagcattct accgagtaac accacaccgc tcattgtcag tgatgctggc tttaaagtgc 2280 catggtataa atccgttgag aagctgggtt ggtactggtt aagtcgagta agaggaaaag 2340 tacaatatgc agacctagga gcggaaaact ggaaacctat cagcaactta catgatatgt 2400 catctagtca ctcaaagact ttaggctata agaggctgac taaaagcaat ccaatctcat 2460 gccaaattct attgtataaa tctcgctcta aaggccgaaa aaatcagcgc tcgacacgga 2520 ctcattgtca ccacccgtca cctaaaatct actcagcgtc ggcaaaggag ccatgggttc 2580 tagcaactaa cttacctgtt gaaattcgaa cacccaaaca acttgttaat atctattcga 2640 agcgaatgca gattgaagaa accttccgag acttgaaaag tcctgcctac ggactaggcc 2700 tacgccatag ccgaacgagc agctcagagc gttttgatat catgctgcta atcgccctga 2760 tgcttcaact aacatgttgg cttgcgggcg ttcatgctca gaaacaaggt tgggacaagc 2820 acttccaggc taacacagtc agaaatcgaa acgtactctc aacagttcgc ttaggcatgg 2880 aagttttgcg gcattctggc tacacaataa caagggaaga cttactcgtg gctgcaaccc 2940 tactagctca aaatttattc acacatggtt acgctttggg gaaattatga taatgatcca 3000 gatcacttct ggctaataaa agatcagagc tctagagatc tgtgtgttgg ttttttgtgg 3060 atctgctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3120 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3180 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcagcaca gcaaggggga 3240 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacctctct 3300 ctctctctct ctctctctct ctctctctct ctctcggtac ctctctctct ctctctctct 3360 ctctctctct ctctctctct cggtaccagg tgctgaagaa ttgacccggt gaccaaaggt 3420 gccttttatc atcactttaa aaataaaaaa caattactca gtgcctgtta taagcagcaa 3480 ttaattatga ttgatgccta catcacaaca aaaactgatt taacaaatgg ttggtctgcc 3540 ttagaaagta tatttgaaca ttatcttgat tatattattg ataataataa aaaccttatc 3600 cctatccaag aagtgatgcc tatcattggt tggaatgaac ttgaaaaaaa ttagccttga 3660 atacattact ggtaaggtaa acgccattgt cagcaaattg atccaagaga accaacttaa 3720 agctttcctg acggaatgtt aattctcgtt gaccctgagc actgatgaat cccctaatga 3780 ttttggtaaa aatcattaag ttaaggtgga tacacatctt gtcatatgat cccggtaatg 3840 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 3900 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 3960 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 4020 gccgctctag aactagtgga tcccccgggg aggtcagaat ggtttcttta ctgtttgtca 4080 attctattat ttcaatacag aacaatagct tctataactg aaatatattt gctattgtat 4140 attatgattg tccctcgaac catgaacact cctccagctg aatttcacaa ttcctctgtc 4200 atctgccagg ccattaagtt attcatggaa gatctttgag gaacactgca agttcatatc 4260 ataaacacat ttgaaattga gtattgtttt gcattgtatg gagctatgtt ttgctgtatc 4320 ctcagaaaaa aagtttgtta taaagcattc acacccataa aaagatagat ttaaatattc 4380 cagctatagg aaagaaagtg cgtctgctct tcactctagt ctcagttggc tccttcacat 4440 gcatgcttct ttatttctcc tattttgtca agaaaataat aggtcacgtc ttgttctcac 4500 ttatgtcctg cctagcatgg ctcagatgca cgttgtagat acaagaagga tcaaatgaaa 4560 cagacttctg gtctgttact acaaccatag taataagcac actaactaat aattgctaat 4620 tatgttttcc atctctaagg ttcccacatt tttctgtttt cttaaagatc ccattatctg 4680 gttgtaactg aagctcaatg gaacatgagc aatatttccc agtcttctct cccatccaac 4740 agtcctgatg gattagcaga acaggcagaa aacacattgt tacccagaat taaaaactaa 4800 tatttgctct ccattcaatc caaaatggac ctattgaaac taaaatctaa cccaatccca 4860 ttaaatgatt tctatggcgt caaaggtcaa acttctgaag ggaacctgtg ggtgggtcac 4920 aattcaggct atatattccc cagggctcag cggatccatg ggctccatcg gcgcagcaag 4980 catggaattt tgttttgatg tattcaagga gctcaaagtc caccatgcca atgagaacat 5040 cttctactgc cccattgcca tcatgtcagc tctagccatg gtatacctgg gtgcaaaaga 5100 cagcaccagg acacagataa ataaggttgt tcgctttgat aaacttccag gattcggaga 5160 cagtattgaa gctcagtgtg gcacatctgt aaacgttcac tcttcactta gagacatcct 5220 caaccaaatc accaaaccaa atgatgttta ttcgttcagc cttgccagta gactttatgc 5280 tgaagagaga tacccaatcc tgccagaata cttgcagtgt gtgaaggaac tgtatagagg 5340 aggcttggaa cctatcaact ttcaaacagc tgcagatcaa gccagagagc tcatcaattc 5400 ctgggtagaa agtcagacaa atggaattat cagaaatgtc cttcagccaa gctccgtgga 5460 ttctcaaact gcaatggttc tggttaatgc cattgtcttc aaaggactgt gggagaaaac 5520 atttaaggat gaagacacac aagcaatgcc tttcagagtg actgagcaag aaagcaaacc 5580 tgtgcagatg atgtaccaga ttggtttatt tagagtggca tcaatggctt ctgagaaaat 5640 gaagatcctg gagcttccat ttgccagtgg gacaatgagc atgttggtgc tgttgcctga 5700 tgaagtctca ggccttgagc agcttgagag tataatcaac tttgaaaaac tgactgaatg 5760 gaccagttct aatgttatgg aagagaggaa gatcaaagtg tacttacctc gcatgaagat 5820 ggaggaaaaa tacaacctca catctgtctt aatggctatg ggcattactg acgtgtttag 5880 ctcttcagcc aatctgtctg gcatctcctc agcagagagc ctgaagatat ctcaagctgt 5940 ccatgcagca catgcagaaa tcaatgaagc aggcagagag gtggtagggt cagcagaggc 6000 tggagtggat gctgcaagcg tctctgaaga atttagggct gaccatccat tcctcttctg 6060 tatcaagcac atcgcaacca acgccgttct cttctttggc agatgtgttt cccctccgcg 6120 gccagcagat gacgcaccag cagatgacgc accagcagat gacgcaccag cagatgacgc 6180 accagcagat gacgcaccag cagatgacgc aacaacatgt atcctgaaag gctcttgtgg 6240 ctggatcggc ctgctggatg acgatgacaa atttgtgaac caacacctgt gcggctcaca 6300 cctggtggaa gctctctacc tagtgtgcgg ggaacgaggc ttcttctaca cacccaagac 6360 ccgccgggag gcagaggacc tgcaggtggg gcaggtggag ctgggcgggg gccctggtgc 6420 aggcagcctg cagcccttgg ccctggaggg gtccctgcag aagcgtggca ttgtggaaca 6480 atgctgtacc agcatctgct ccctctacca gctggagaac tactgcaact agggcgcctg 6540 gatccagatc acttctggct aataaaagat cagagctcta gagatctgtg tgttggtttt 6600 ttgtggatct gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 6660 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 6720 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc agcacagcaa 6780 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac 6840 ctctctctct ctctctctct ctctctctct ctctctctct cggtacctct ctcgaggggg 6900 ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt 6960 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 7020 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 7080 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt 7140 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt 7200 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc 7260 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg 7320 gcccactact ccgggatcat atgacaagat gtgtatccac cttaacttaa tgatttttac 7380 caaaatcatt aggggattca tcagtgctca gggtcaacga gaattaacat tccgtcagga 7440 aagcttatga tgatgatgtg cttaaaaact tactcaatgg ctggttatgc atatcgcaat 7500 acatgcgaaa aacctaaaag agcttgccga taaaaaaggc caatttattg ctatttaccg 7560 cggcttttta ttgagcttga aagataaata aaatagatag gttttatttg aagctaaatc 7620 ttctttatcg taaaaaatgc cctcttgggt tatcaagagg gtcattatat ttcgcggaat 7680 aacatcattt ggtgacgaaa taactaagca cttgtctcct gtttactccc ctgagcttga 7740 ggggttaaca tgaaggtcat cgatagcagg ataataatac agtaaaacgc taaaccaata 7800 atccaaatcc agccatccca aattggtagt gaatgattat aaataacagc aaacagtaat 7860 gggccaataa caccggttgc attggtaagg ctcaccaata atccctgtaa agcaccttgc 7920 tgatgactct ttgtttggat agacatcact ccctgtaatg caggtaaagc gatcccacca 7980 ccagccaata aaattaaaac agggaaaact aaccaacctt cagatataaa cgctaaaaag 8040 gcaaatgcac tactatctgc aataaatccg agcagtactg ccgttttttc gccccattta 8100 gtggctattc ttcctgccac aaaggcttgg aatactgagt gtaaaagacc aagacccgct 8160 aatgaaaagc caaccatcat gctattccat ccaaaacgat tttcggtaaa tagcacccac 8220 accgttgcgg gaatttggcc tatcaattgc gctgaaaaat aaataatcaa caaaatggca 8280 tcgttttaaa taaagtgatg tataccgaat tcagcttttg ttccctttag tgagggttaa 8340 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8400 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8460 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8520 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 8580 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8640 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 8700 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8760 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8820 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 8880 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 8940 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 9000 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9060 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 9120 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 9180 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 9240 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9300 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9360 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9420 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 9480 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9540 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9600 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 9660 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9720 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9780 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9840 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 9900 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 9960 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 10020 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 10080 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 10140 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 10200 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10260 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 10320 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 10380 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 10440 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10500 gaaaagtgcc ac 10512 43 11255 DNA Artificial Sequence Synthetic 43 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt 1800 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggcaata cgatgcccat tgtacttgtt gactggtctg atattcgtga gcaaaaacga 2100 cttatggtat tgcgagcttc agtcgcacta cacggtcgtt ctgttactct ttatgagaaa 2160 gcgttcccgc tttcagagca atgttcaaag aaagctcatg accaatttct agccgacctt 2220 gcgagcattc taccgagtaa caccacaccg ctcattgtca gtgatgctgg ctttaaagtg 2280 ccatggtata aatccgttga gaagctgggt tggtactggt taagtcgagt aagaggaaaa 2340 gtacaatatg cagacctagg agcggaaaac tggaaaccta tcagcaactt acatgatatg 2400 tcatctagtc actcaaagac tttaggctat aagaggctga ctaaaagcaa tccaatctca 2460 tgccaaattc tattgtataa atctcgctct aaaggccgaa aaaatcagcg ctcgacacgg 2520 actcattgtc accacccgtc acctaaaatc tactcagcgt cggcaaagga gccatgggtt 2580 ctagcaacta acttacctgt tgaaattcga acacccaaac aacttgttaa tatctattcg 2640 aagcgaatgc agattgaaga aaccttccga gacttgaaaa gtcctgccta cggactaggc 2700 ctacgccata gccgaacgag cagctcagag cgttttgata tcatgctgct aatcgccctg 2760 atgcttcaac taacatgttg gcttgcgggc gttcatgctc agaaacaagg ttgggacaag 2820 cacttccagg ctaacacagt cagaaatcga aacgtactct caacagttcg cttaggcatg 2880 gaagttttgc ggcattctgg ctacacaata acaagggaag acttactcgt ggctgcaacc 2940 ctactagctc aaaatttatt cacacatggt tacgctttgg ggaaattatg ataatgatcc 3000 agatcacttc tggctaataa aagatcagag ctctagagat ctgtgtgttg gttttttgtg 3060 gatctgctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 3120 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 3180 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcagcac agcaaggggg 3240 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggtacctctc 3300 tctctctctc tctctctctc tctctctctc tctctcggta cctctctctc tctctctctc 3360 tctctctctc tctctctctc tcggtaccag gtgctgaaga attgacccgg tgaccaaagg 3420 tgccttttat catcacttta aaaataaaaa acaattactc agtgcctgtt ataagcagca 3480 attaattatg attgatgcct acatcacaac aaaaactgat ttaacaaatg gttggtctgc 3540 cttagaaagt atatttgaac attatcttga ttatattatt gataataata aaaaccttat 3600 ccctatccaa gaagtgatgc ctatcattgg ttggaatgaa cttgaaaaaa attagccttg 3660 aatacattac tggtaaggta aacgccattg tcagcaaatt gatccaagag aaccaactta 3720 aagctttcct gacggaatgt taattctcgt tgaccctgag cactgatgaa tcccctaatg 3780 attttggtaa aaatcattaa gttaaggtgg atacacatct tgtcatatga tcccggtaat 3840 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 3900 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 3960 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctcca ccgcggtggc 4020 ggccgctcta gaactagtgg atcccccggg catcagattg gctattggcc attgcatacg 4080 ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt 4140 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 4200 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 4260 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 4320 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 4380 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 4440 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 4500 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 4560 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 4620 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 4680 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 4740 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga 4800 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 4860 gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg 4920 tttttggctt ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta 4980 gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc 5040 cattactaat ccataacatg gctctttgcc acaactatct ctattggcta tatgccaata 5100 ctctgtcctt cagagactga cacggactct gtatttttac aggatggggt cccatttatt 5160 atttacaaat tcacatatac aacaacgccg tcccccgtgc ccgcagtttt tattaaacat 5220 agcgtgggat ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta 5280 gcggcggagc ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg 5340 gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg cccaccacca 5400 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag cgtggagatt 5460 gggctcgcac ggctgacgca gatggaagac ttaaggcagc ggcagaagaa gatgcaggca 5520 gctgagttgt tgtattctga taagagtcag aggtaactcc cgttgcggtg ctgttaacgg 5580 tggagggcag tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata 5640 gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcggatca 5700 atgggctcca tcggtgcagc aagcatggaa ttttgttttg atgtattcaa ggagctcaaa 5760 gtccaccatg ccaatgagaa catcttctac tgccccattg ccatcatgtc agctctagcc 5820 atggtatacc tgggtgcaaa agacagcacc aggacacaaa taaataaggt tgttcgcttt 5880 gataaacttc caggattcgg agacagtatt gaagctcagt gtggcacatc tgtaaacgtt 5940 cactcttcac ttagagacat cctcaaccaa atcaccaaac caaatgatgt ttattcgttc 6000 agccttgcca gtagacttta tgctgaagag agatacccaa tcctgccaga atacttgcag 6060 tgtgtgaagg aactgtatag aggaggcttg gaacctatca actttcaaac agctgcagat 6120 caagccagag agctcatcaa ttcctgggta gaaagtcaga caaatggaat tatcagaaat 6180 gtccttcagc caagctccgt ggattctcaa actgcaatgg ttctggttaa tgccattgtc 6240 ttcaaaggac tgtgggagaa agcatttaag gatgaagaca cacaagcaat gcctttcaga 6300 gtgactgagc aagaaagcaa acctgtgcag atgatgtacc agattggttt atttagagtg 6360 gcatcaatgg cttctgagaa aatgaagatc ctggagcttc catttgccag tgggacaatg 6420 agcatgttgg tgctgttgcc tgatgaagtc tcaggccttg agcagcttga gagtataatc 6480 aactttgaaa aactgactga atggaccagt tctaatgtta tggaagagag aagatcaaag 6540 tgtacttacc tcgcatgaag atggaggaaa aatacaacct cacatctgtc ttaatggcta 6600 tgggcattac tgacgtgttt agctcttcag ccaatctgtc tggcatctcc tcagcagaga 6660 gcctgaagat atctcaagct gtccatgcag cacatgcaga aatcaatgaa gcaggcagag 6720 aggtggtagg gtcagcagag gctggagtgg atgctgcaag cgtctctgaa gaatttaggg 6780 ctgaccatcc attcctcttc tgtatcaagc acatcgcaac caacgccgtt ctcttctttt 6840 ggcagatgtg tttcccgcgg ccagcagatg acgcaccagc agatgacgca ccagcagatg 6900 acgcaccagc agatgacgca ccagcagatg acgcaacaac atgtatcctg aaaggctctt 6960 gtggctggat cggcctgctg gatgacgatg acaaatttgt gaaccaacac ctgtgcggct 7020 cacacctggt ggaagctctc tacctagtgt gcggggaacg aggcttcttc tacacaccca 7080 agacccgccg ggaggcagag gacctgcagg tggggcaggt ggagctgggc gggggccctg 7140 gtgcaggcag cctgcagccc ttggccctgg aggggtccct gcagaagcgt ggcattgtgg 7200 aacaatgctg taccagcatc tgctccctct accagctgga gaactactgc aactagggcg 7260 cctaaagggc gaattatcgc ggccgctcta gaccaggcgc ctggatccag atcacttctg 7320 gctaataaaa gatcagagct ctagagatct gtgtgttggt tttttgtgga tctgctgtgc 7380 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 7440 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 7500 ggtgtcattc tattctgggg ggtggggtgg ggcagcacag caagggggag gattgggaag 7560 acaatagcag gcatgctggg gatgcggtgg gctctatggg tacctctctc tctctctctc 7620 tctctctcac tctctctctc tctcggtacc tctcctcgag ggggggcccg gtacccaatt 7680 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact 7740 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 7800 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 7860 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 7920 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata 7980 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt 8040 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tactccggga 8100 tcatatgaca agatgtgtat ccaccttaac ttaatgattt ttaccaaaat cattagggga 8160 ttcatcagtg ctcagggtca acgagaatta acattccgtc aggaaagctt atgatgatga 8220 tgtgcttaaa aacttactca atggctggtt atgcatatcg caatacatgc gaaaaaccta 8280 aaagagcttg ccgataaaaa aggccaattt attgctattt accgcggctt tttattgagc 8340 ttgaaagata aataaaatag ataggtttta tttgaagcta aatcttcttt atcgtaaaaa 8400 atgccctctt gggttatcaa gagggtcatt atatttcgcg gaataacatc atttggtgac 8460 gaaataacta agcacttgtc tcctgtttac tcccctgagc ttgaggggtt aacatgaagg 8520 tcatcgatag caggataata atacagtaaa acgctaaacc aataatccaa atccagccat 8580 cccaaattgg tagtgaatga ttataaataa cagcaaacag taatgggcca ataacaccgg 8640 ttgcattggt aaggctcacc aataatccct gtaaagcacc ttgctgatga ctctttgttt 8700 ggatagacat cactccctgt aatgcaggta aagcgatccc accaccagcc aataaaatta 8760 aaacagggaa aactaaccaa ccttcagata taaacgctaa aaaggcaaat gcactactat 8820 ctgcaataaa tccgagcagt actgccgttt tttcgcccat ttagtggcta ttcttcctgc 8880 cacaaaggct tggaatactg agtgtaaaag accaagaccc gtaatgaaaa gccaaccatc 8940 atgctattca tcatcacgat ttctgtaata gcaccacacc gtgctggatt ggctatcaat 9000 gcgctgaaat aataatcaac aaatggcatc gttaaataag tgatgtatac cgatcagctt 9060 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc 9120 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 9180 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 9240 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 9300 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 9360 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 9420 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 9480 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 9540 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 9600 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 9660 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 9720 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 9780 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 9840 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 9900 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 9960 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 10020 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 10080 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 10140 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 10200 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 10260 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 10320 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 10380 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 10440 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 10500 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 10560 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 10620 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 10680 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 10740 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 10800 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 10860 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 10920 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 10980 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 11040 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 11100 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 11160 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 11220 aggggttccg cgcacatttc cccgaaaagt gccac 11255 44 10487 DNA Artificial Sequence Synthetic 44 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt 1800 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggaatac gatgcccatt gtacttgttg actggtctga tattcgtgag caaaaacgac 2100 ttatggtatt gcgagcttca gtcgcactac acggtcgttc tgttactctt tatgagaaag 2160 cgttcccgct ttcagagcaa tgttcaaaga aagctcatga ccaatttcta gccgaccttg 2220 cgagcattct accgagtaac accacaccgc tcattgtcag tgatgctggc tttaaagtgc 2280 catggtataa atccgttgag aagctgggtt ggtactggtt aagtcgagta agaggaaaag 2340 tacaatatgc agacctagga gcggaaaact ggaaacctat cagcaactta catgatatgt 2400 catctagtca ctcaaagact ttaggctata agaggctgac taaaagcaat ccaatctcat 2460 gccaaattct attgtataaa tctcgctcta aaggccgaaa aaatcagcgc tcgacacgga 2520 ctcattgtca ccacccgtca cctaaaatct actcagcgtc ggcaaaggag ccatgggttc 2580 tagcaactaa cttacctgtt gaaattcgaa cacccaaaca acttgttaat atctattcga 2640 agcgaatgca gattgaagaa accttccgag acttgaaaag tcctgcctac ggactaggcc 2700 tacgccatag ccgaacgagc agctcagagc gttttgatat catgctgcta atcgccctga 2760 tgcttcaact aacatgttgg cttgcgggcg ttcatgctca gaaacaaggt tgggacaagc 2820 acttccaggc taacacagtc agaaatcgaa acgtactctc aacagttcgc ttaggcatgg 2880 aagttttgcg gcattctggc tacacaataa caagggaaga cttactcgtg gctgcaaccc 2940 tactagctca aaatttattc acacatggtt acgctttggg gaaattatga taatgatcca 3000 gatcacttct ggctaataaa agatcagagc tctagagatc tgtgtgttgg ttttttgtgg 3060 atctgctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3120 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3180 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcagcaca gcaaggggga 3240 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacctctct 3300 ctctctctct ctctctctct ctctctctct ctctcggtac ctctctctct ctctctctct 3360 ctctctctct ctctctctct cggtaccagg tgctgaagaa ttgacccggt gaccaaaggt 3420 gccttttatc atcactttaa aaataaaaaa caattactca gtgcctgtta taagcagcaa 3480 ttaattatga ttgatgccta catcacaaca aaaactgatt taacaaatgg ttggtctgcc 3540 ttagaaagta tatttgaaca ttatcttgat tatattattg ataataataa aaaccttatc 3600 cctatccaag aagtgatgcc tatcattggt tggaatgaac ttgaaaaaaa ttagccttga 3660 atacattact ggtaaggtaa acgccattgt cagcaaattg atccaagaga accaacttaa 3720 agctttcctg acggaatgtt aattctcgtt gaccctgagc actgatgaat cccctaatga 3780 ttttggtaaa aatcattaag ttaaggtgga tacacatctt gtcatatgat cccggtaatg 3840 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 3900 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 3960 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 4020 gccgctctag aactagtgga tcccccgggg aggtcagaat ggtttcttta ctgtttgtca 4080 attctattat ttcaatacag aacaaaagct tctataactg aaatatattt gctattgtat 4140 attatgattg tccctcgaac catgaacact cctccagctg aatttcacaa ttcctctgtc 4200 atctgccagg ctggaagatc atggaagatc tctgaggaac attgcaagtt cataccataa 4260 actcatttgg aattgagtat tattttgctt tgaatggagc tatgttttgc agttccctca 4320 gaagaaaagc ttgttataaa gcgtctacac ccatcaaaag atatatttaa atattccaac 4380 tacagaaaga ttttgtctgc tcttcactct gatctcagtt ggtttcttca cgtacatgct 4440 tctttatttg cctattttgt caagaaaata ataggtcaag tcctgttctc acttatctcc 4500 tgcctagcat ggcttagatg cacgttgtac attcaagaag gatcaaatga aacagacttc 4560 tggtctgtta caacaaccat agtaataaac agactaacta ataattgcta attatgtttt 4620 ccatctctaa ggttcccaca tttttctgtt ttaagatccc attatctggt tgtaactgaa 4680 gctcaatgga acatgaacag tatttctcag tcttttctcc agcaatcctg acggattaga 4740 agaactggca gaaaacactt tgttacccag aattaaaaac taatatttgc tctcccttca 4800 atccaaaatg gacctattga aactaaaatc tgacccaatc ccattaaatt atttctatgg 4860 cgtcaaaggt caaacttttg aagggaacct gtgggtgggt cccaattcag gctatatatt 4920 ccccagggct cagccagtgg atccatgggc tccatcggtg cagcaagcat ggaattttgt 4980 tttgatgtat tcaaggagct caaagtccac catgccaatg acaacatgct ctactccccc 5040 tttgccatct tgtcaactct ggccatggtc ttcctaggtg caaaagacag caccaggacc 5100 cagataaata aggttgttca ctttgataaa cttccaggat tcggagacag tattgaagct 5160 cagtgtggca catctgtaaa tgttcactct tcacttagag acatactcaa ccaaatcacc 5220 aaacaaaatg atgcttattc gttcagcctt gccagtagac tttatgctca agagacatac 5280 acagtcgtgc cggaatactt gcaatgtgtg aaggaactgt atagaggagg cttagaatcc 5340 gtcaactttc aaacagctgc agatcaagcc agaggcctca tcaatgcctg ggtagaaagt 5400 cagacaaacg gaattatcag aaacatcctt cagccaagct ccgtggattc tcaaactgca 5460 atggtcctgg ttaatgccat tgccttcaag ggactgtggg agaaagcatt taaggctgaa 5520 gacacgcaaa caataccttt cagagtgact gagcaagaaa gcaaacctgt gcagatgatg 5580 taccagattg gttcatttaa agtggcatca atggcttctg agaaaatgaa gatcctggag 5640 cttccatttg ccagtggaac aatgagcatg ttggtgctgt tgcctgatga tgtctcaggc 5700 cttgagcagc ttgagagtat aatcagcttt gaaaaactga ctgaatggac cagttctagt 5760 attatggaag agaggaaggt caaagtgtac ttacctcgca tgaagatgga ggagaaatac 5820 aacctcacat ctctcttaat ggctatggga attactgacc tgttcagctc ttcagccaat 5880 ctgtctggca tctcctcagt agggagcctg aagatatctc aagctgtcca tgcagcacat 5940 gcagaaatca atgaagcggg cagagatgtg gtaggctcag cagaggctgg agtggatgct 6000 actgaagaat ttagggctga ccatccattc ctcttctgtg tcaagcacat cgaaaccaac 6060 gccattctcc tctttggcag atgtgtttct ccgcggccag cagatgacgc accagcagat 6120 gacgcaccag cagatgacgc accagcagat gacgcaccag cagatgacgc accagcagat 6180 gacgcaacaa catgtatcct gaaaggctct tgtggctgga tcggcctgct ggatgacgat 6240 gacaaatttg tgaaccaaca cctgtgcggc tcacacctgg tggaagctct ctacctagtg 6300 tgcggggaac gaggcttctt ctacacaccc aagacccgcc gggaggcaga ggacctgcag 6360 gtggggcagg tggagctggg cgggggccct ggtgcaggca gcctgcagcc cttggccctg 6420 gaggggtccc tgcagaagcg tggcattgtg gaacaatgct gtaccagcat ctgctccctc 6480 taccagctgg agaactactg caactagggc gcctggatcc agatcacttc tggctaataa 6540 aagatcagag ctctagagat ctgtgtgttg gttttttgtg gatctgctgt gccttctagt 6600 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 6660 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 6720 tctattctgg ggggtggggt ggggcagcac agcaaggggg aggattggga agacaatagc 6780 aggcatgctg gggatgcggt gggctctatg ggtacctctc tctctctctc tctctctctc 6840 tctctctctc tctctcggta cctctctcga gggggggccc ggtacccaat tcgccctata 6900 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 6960 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 7020 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga 7080 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt 7140 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat 7200 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa 7260 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctactccggg atcatatgac 7320 aagatgtgta tccaccttaa cttaatgatt tttaccaaaa tcattagggg attcatcagt 7380 gctcagggtc aacgagaatt aacattccgt caggaaagct tatgatgatg atgtgcttaa 7440 aaacttactc aatggctggt tatgcatatc gcaatacatg cgaaaaacct aaaagagctt 7500 gccgataaaa aaggccaatt tattgctatt taccgcggct ttttattgag cttgaaagat 7560 aaataaaata gataggtttt atttgaagct aaatcttctt tatcgtaaaa aatgccctct 7620 tgggttatca agagggtcat tatatttcgc ggaataacat catttggtga cgaaataact 7680 aagcacttgt ctcctgttta ctcccctgag cttgaggggt taacatgaag gtcatcgata 7740 gcaggataat aatacagtaa aacgctaaac caataatcca aatccagcca tcccaaattg 7800 gtagtgaatg attataaata acagcaaaca gtaatgggcc aataacaccg gttgcattgg 7860 taaggctcac caataatccc tgtaaagcac cttgctgatg actctttgtt tggatagaca 7920 tcactccctg taatgcaggt aaagcgatcc caccaccagc caataaaatt aaaacaggga 7980 aaactaacca accttcagat ataaacgcta aaaaggcaaa tgcactacta tctgcaataa 8040 atccgagcag tactgccgtt ttttcgcccc atttagtggc tattcttcct gccacaaagg 8100 cttggaatac tgagtgtaaa agaccaagac ccgctaatga aaagccaacc atcatgctat 8160 tccatccaaa acgattttcg gtaaatagca cccacaccgt tgcgggaatt tggcctatca 8220 attgcgctga aaaataaata atcaacaaaa tggcatcgtt ttaaataaag tgatgtatac 8280 cgaattcagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc 8340 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 8400 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 8460 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 8520 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 8580 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 8640 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 8700 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 8760 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 8820 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 8880 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 8940 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 9000 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 9060 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 9120 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 9180 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 9240 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 9300 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 9360 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 9420 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 9480 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 9540 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 9600 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 9660 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 9720 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 9780 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 9840 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 9900 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 9960 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 10020 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 10080 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 10140 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 10200 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 10260 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 10320 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 10380 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 10440 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac 10487 45 10297 DNA Artificial Sequence Synthetic 45 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt 1800 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggaatac gatgcccatt gtacttgttg actggtctga tattcgtgag caaaaacgac 2100 ttatggtatt gcgagcttca gtcgcactac acggtcgttc tgttactctt tatgagaaag 2160 cgttcccgct ttcagagcaa tgttcaaaga aagctcatga ccaatttcta gccgaccttg 2220 cgagcattct accgagtaac accacaccgc tcattgtcag tgatgctggc tttaaagtgc 2280 catggtataa atccgttgag aagctgggtt ggtactggtt aagtcgagta agaggaaaag 2340 tacaatatgc agacctagga gcggaaaact ggaaacctat cagcaactta catgatatgt 2400 catctagtca ctcaaagact ttaggctata agaggctgac taaaagcaat ccaatctcat 2460 gccaaattct attgtataaa tctcgctcta aaggccgaaa aaatcagcgc tcgacacgga 2520 ctcattgtca ccacccgtca cctaaaatct actcagcgtc ggcaaaggag ccatgggttc 2580 tagcaactaa cttacctgtt gaaattcgaa cacccaaaca acttgttaat atctattcga 2640 agcgaatgca gattgaagaa accttccgag acttgaaaag tcctgcctac ggactaggcc 2700 tacgccatag ccgaacgagc agctcagagc gttttgatat catgctgcta atcgccctga 2760 tgcttcaact aacatgttgg cttgcgggcg ttcatgctca gaaacaaggt tgggacaagc 2820 acttccaggc taacacagtc agaaatcgaa acgtactctc aacagttcgc ttaggcatgg 2880 aagttttgcg gcattctggc tacacaataa caagggaaga cttactcgtg gctgcaaccc 2940 tactagctca aaatttattc acacatggtt acgctttggg gaaattatga taatgatcca 3000 gatcacttct ggctaataaa agatcagagc tctagagatc tgtgtgttgg ttttttgtgg 3060 atctgctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3120 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3180 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcagcaca gcaaggggga 3240 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacctctct 3300 ctctctctct ctctctctct ctctctctct ctctcggtac ctctctctct ctctctctct 3360 ctctctctct ctctctctct cggtaccagg tgctgaagaa ttgacccggt gaccaaaggt 3420 gccttttatc atcactttaa aaataaaaaa caattactca gtgcctgtta taagcagcaa 3480 ttaattatga ttgatgccta catcacaaca aaaactgatt taacaaatgg ttggtctgcc 3540 ttagaaagta tatttgaaca ttatcttgat tatattattg ataataataa aaaccttatc 3600 cctatccaag aagtgatgcc tatcattggt tggaatgaac ttgaaaaaaa ttagccttga 3660 atacattact ggtaaggtaa acgccattgt cagcaaattg atccaagaga accaacttaa 3720 agctttcctg acggaatgtt aattctcgtt gaccctgagc actgatgaat cccctaatga 3780 ttttggtaaa aatcattaag ttaaggtgga tacacatctt gtcatatgat cccggtaatg 3840 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 3900 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 3960 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 4020 gccgctctag aactagtgga tcccccgggg aggtcagaat ggtttcttta ctgtttgtca 4080 attctattat ttcaatacag aacaatagct tctataactg aaatatattt gctattgtat 4140 attatgattg tccctcgaac catgaacact cctccagctg aatttcacaa ttcctctgtc 4200 atctgccagg ccattaagtt attcatggaa gatctttgag gaacactgca agttcatatc 4260 ataaacacat ttgaaattga gtattgtttt gcattgtatg gagctatgtt ttgctgtatc 4320 ctcagaaaaa aagtttgtta taaagcattc acacccataa aaagatagat ttaaatattc 4380 cagctatagg aaagaaagtg cgtctgctct tcactctagt ctcagttggc tccttcacat 4440 gcatgcttct ttatttctcc tattttgtca agaaaataat aggtcacgtc ttgttctcac 4500 ttatgtcctg cctagcatgg ctcagatgca cgttgtagat acaagaagga tcaaatgaaa 4560 cagacttctg gtctgttact acaaccatag taataagcac actaactaat aattgctaat 4620 tatgttttcc atctctaagg ttcccacatt tttctgtttt cttaaagatc ccattatctg 4680 gttgtaactg aagctcaatg gaacatgagc aatatttccc agtcttctct cccatccaac 4740 agtcctgatg gattagcaga acaggcagaa aacacattgt tacccagaat taaaaactaa 4800 tatttgctct ccattcaatc caaaatggac ctattgaaac taaaatctaa cccaatccca 4860 ttaaatgatt tctatggcgt caaaggtcaa acttctgaag ggaacctgtg ggtgggtcac 4920 aattcaggct atatattccc cagggctcag cggatccatg ggctccatcg gcgcagcaag 4980 catggaattt tgttttgatg tattcaagga gctcaaagtc caccatgcca atgagaacat 5040 cttctactgc cccattgcca tcatgtcagc tctagccatg gtatacctgg gtgcaaaaga 5100 cagcaccagg acacagataa ataaggttgt tcgctttgat aaacttccag gattcggaga 5160 cagtattgaa gctcagtgtg gcacatctgt aaacgttcac tcttcactta gagacatcct 5220 caaccaaatc accaaaccaa atgatgttta ttcgttcagc cttgccagta gactttatgc 5280 tgaagagaga tacccaatcc tgccagaata cttgcagtgt gtgaaggaac tgtatagagg 5340 aggcttggaa cctatcaact ttcaaacagc tgcagatcaa gccagagagc tcatcaattc 5400 ctgggtagaa agtcagacaa atggaattat cagaaatgtc cttcagccaa gctccgtgga 5460 ttctcaaact gcaatggttc tggttaatgc cattgtcttc aaaggactgt gggagaaaac 5520 atttaaggat gaagacacac aagcaatgcc tttcagagtg actgagcaag aaagcaaacc 5580 tgtgcagatg atgtaccaga ttggtttatt tagagtggca tcaatggctt ctgagaaaat 5640 gaagatcctg gagcttccat ttgccagtgg gacaatgagc atgttggtgc tgttgcctga 5700 tgaagtctca ggccttgagc agcttgagag tataatcaac tttgaaaaac tgactgaatg 5760 gaccagttct aatgttatgg aagagaggaa gatcaaagtg tacttacctc gcatgaagat 5820 ggaggaaaaa tacaacctca catctgtctt aatggctatg ggcattactg acgtgtttag 5880 ctcttcagcc aatctgtctg gcatctcctc agcagagagc ctgaagatat ctcaagctgt 5940 ccatgcagca catgcagaaa tcaatgaagc aggcagagag gtggtagggt cagcagaggc 6000 tggagtggat gctgcaagcg tctctgaaga atttagggct gaccatccat tcctcttctg 6060 tatcaagcac atcgcaacca acgccgttct cttctttggc agatgtgttt cccctccgcg 6120 gccagcagat gacgcaccag cagatgacgc accagcagat gacgcaccag cagatgacgc 6180 accagcagat gacgcaccag cagatgacgc aacaacatgt atcctgaaag gctcttgtgg 6240 ctggatcggc ctgctggatg acgatgacaa aaaatacaaa aaagcactga aaaaactggc 6300 aaaactgctg taatgagggc gcctggatcc agatcacttc tggctaataa aagatcagag 6360 ctctagagat ctgtgtgttg gttttttgtg gatctgctgt gccttctagt tgccagccat 6420 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 6480 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 6540 ggggtggggt ggggcagcac agcaaggggg aggattggga agacaatagc aggcatgctg 6600 gggatgcggt gggctctatg ggtacctctc tctctctctc tctctctctc tctctctctc 6660 tctctcggta cctctctcga gggggggccc ggtacccaat tcgccctata gtgagtcgta 6720 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 6780 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 6840 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaagc 6900 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 6960 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 7020 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 7080 cgaaaaaccg tctatcaggg cgatggccca ctactccggg atcatatgac aagatgtgta 7140 tccaccttaa cttaatgatt tttaccaaaa tcattagggg attcatcagt gctcagggtc 7200 aacgagaatt aacattccgt caggaaagct tatgatgatg atgtgcttaa aaacttactc 7260 aatggctggt tatgcatatc gcaatacatg cgaaaaacct aaaagagctt gccgataaaa 7320 aaggccaatt tattgctatt taccgcggct ttttattgag cttgaaagat aaataaaata 7380 gataggtttt atttgaagct aaatcttctt tatcgtaaaa aatgccctct tgggttatca 7440 agagggtcat tatatttcgc ggaataacat catttggtga cgaaataact aagcacttgt 7500 ctcctgttta ctcccctgag cttgaggggt taacatgaag gtcatcgata gcaggataat 7560 aatacagtaa aacgctaaac caataatcca aatccagcca tcccaaattg gtagtgaatg 7620 attataaata acagcaaaca gtaatgggcc aataacaccg gttgcattgg taaggctcac 7680 caataatccc tgtaaagcac cttgctgatg actctttgtt tggatagaca tcactccctg 7740 taatgcaggt aaagcgatcc caccaccagc caataaaatt aaaacaggga aaactaacca 7800 accttcagat ataaacgcta aaaaggcaaa tgcactacta tctgcaataa atccgagcag 7860 tactgccgtt ttttcgcccc atttagtggc tattcttcct gccacaaagg cttggaatac 7920 tgagtgtaaa agaccaagac ccgctaatga aaagccaacc atcatgctat tccatccaaa 7980 acgattttcg gtaaatagca cccacaccgt tgcgggaatt tggcctatca attgcgctga 8040 aaaataaata atcaacaaaa tggcatcgtt ttaaataaag tgatgtatac cgaattcagc 8100 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt 8160 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 8220 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 8280 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 8340 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 8400 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 8460 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 8520 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 8580 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 8640 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 8700 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 8760 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 8820 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 8880 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 8940 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 9000 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 9060 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 9120 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 9180 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 9240 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 9300 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 9360 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 9420 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 9480 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 9540 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 9600 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 9660 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 9720 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 9780 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 9840 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 9900 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 9960 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 10020 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 10080 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 10140 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 10200 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 10260 ataggggttc cgcgcacatt tccccgaaaa gtgccac 10297 46 10272 DNA Artificial Sequence Synthetic 46 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt 1800 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggaatac gatgcccatt gtacttgttg actggtctga tattcgtgag caaaaacgac 2100 ttatggtatt gcgagcttca gtcgcactac acggtcgttc tgttactctt tatgagaaag 2160 cgttcccgct ttcagagcaa tgttcaaaga aagctcatga ccaatttcta gccgaccttg 2220 cgagcattct accgagtaac accacaccgc tcattgtcag tgatgctggc tttaaagtgc 2280 catggtataa atccgttgag aagctgggtt ggtactggtt aagtcgagta agaggaaaag 2340 tacaatatgc agacctagga gcggaaaact ggaaacctat cagcaactta catgatatgt 2400 catctagtca ctcaaagact ttaggctata agaggctgac taaaagcaat ccaatctcat 2460 gccaaattct attgtataaa tctcgctcta aaggccgaaa aaatcagcgc tcgacacgga 2520 ctcattgtca ccacccgtca cctaaaatct actcagcgtc ggcaaaggag ccatgggttc 2580 tagcaactaa cttacctgtt gaaattcgaa cacccaaaca acttgttaat atctattcga 2640 agcgaatgca gattgaagaa accttccgag acttgaaaag tcctgcctac ggactaggcc 2700 tacgccatag ccgaacgagc agctcagagc gttttgatat catgctgcta atcgccctga 2760 tgcttcaact aacatgttgg cttgcgggcg ttcatgctca gaaacaaggt tgggacaagc 2820 acttccaggc taacacagtc agaaatcgaa acgtactctc aacagttcgc ttaggcatgg 2880 aagttttgcg gcattctggc tacacaataa caagggaaga cttactcgtg gctgcaaccc 2940 tactagctca aaatttattc acacatggtt acgctttggg gaaattatga taatgatcca 3000 gatcacttct ggctaataaa agatcagagc tctagagatc tgtgtgttgg ttttttgtgg 3060 atctgctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3120 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3180 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcagcaca gcaaggggga 3240 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtacctctct 3300 ctctctctct ctctctctct ctctctctct ctctcggtac ctctctctct ctctctctct 3360 ctctctctct ctctctctct cggtaccagg tgctgaagaa ttgacccggt gaccaaaggt 3420 gccttttatc atcactttaa aaataaaaaa caattactca gtgcctgtta taagcagcaa 3480 ttaattatga ttgatgccta catcacaaca aaaactgatt taacaaatgg ttggtctgcc 3540 ttagaaagta tatttgaaca ttatcttgat tatattattg ataataataa aaaccttatc 3600 cctatccaag aagtgatgcc tatcattggt tggaatgaac ttgaaaaaaa ttagccttga 3660 atacattact ggtaaggtaa acgccattgt cagcaaattg atccaagaga accaacttaa 3720 agctttcctg acggaatgtt aattctcgtt gaccctgagc actgatgaat cccctaatga 3780 ttttggtaaa aatcattaag ttaaggtgga tacacatctt gtcatatgat cccggtaatg 3840 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 3900 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 3960 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 4020 gccgctctag aactagtgga tcccccgggg aggtcagaat ggtttcttta ctgtttgtca 4080 attctattat ttcaatacag aacaaaagct tctataactg aaatatattt gctattgtat 4140 attatgattg tccctcgaac catgaacact cctccagctg aatttcacaa ttcctctgtc 4200 atctgccagg ctggaagatc atggaagatc tctgaggaac attgcaagtt cataccataa 4260 actcatttgg aattgagtat tattttgctt tgaatggagc tatgttttgc agttccctca 4320 gaagaaaagc ttgttataaa gcgtctacac ccatcaaaag atatatttaa atattccaac 4380 tacagaaaga ttttgtctgc tcttcactct gatctcagtt ggtttcttca cgtacatgct 4440 tctttatttg cctattttgt caagaaaata ataggtcaag tcctgttctc acttatctcc 4500 tgcctagcat ggcttagatg cacgttgtac attcaagaag gatcaaatga aacagacttc 4560 tggtctgtta caacaaccat agtaataaac agactaacta ataattgcta attatgtttt 4620 ccatctctaa ggttcccaca tttttctgtt ttaagatccc attatctggt tgtaactgaa 4680 gctcaatgga acatgaacag tatttctcag tcttttctcc agcaatcctg acggattaga 4740 agaactggca gaaaacactt tgttacccag aattaaaaac taatatttgc tctcccttca 4800 atccaaaatg gacctattga aactaaaatc tgacccaatc ccattaaatt atttctatgg 4860 cgtcaaaggt caaacttttg aagggaacct gtgggtgggt cccaattcag gctatatatt 4920 ccccagggct cagccagtgg atccatgggc tccatcggtg cagcaagcat ggaattttgt 4980 tttgatgtat tcaaggagct caaagtccac catgccaatg acaacatgct ctactccccc 5040 tttgccatct tgtcaactct ggccatggtc ttcctaggtg caaaagacag caccaggacc 5100 cagataaata aggttgttca ctttgataaa cttccaggat tcggagacag tattgaagct 5160 cagtgtggca catctgtaaa tgttcactct tcacttagag acatactcaa ccaaatcacc 5220 aaacaaaatg atgcttattc gttcagcctt gccagtagac tttatgctca agagacatac 5280 acagtcgtgc cggaatactt gcaatgtgtg aaggaactgt atagaggagg cttagaatcc 5340 gtcaactttc aaacagctgc agatcaagcc agaggcctca tcaatgcctg ggtagaaagt 5400 cagacaaacg gaattatcag aaacatcctt cagccaagct ccgtggattc tcaaactgca 5460 atggtcctgg ttaatgccat tgccttcaag ggactgtggg agaaagcatt taaggctgaa 5520 gacacgcaaa caataccttt cagagtgact gagcaagaaa gcaaacctgt gcagatgatg 5580 taccagattg gttcatttaa agtggcatca atggcttctg agaaaatgaa gatcctggag 5640 cttccatttg ccagtggaac aatgagcatg ttggtgctgt tgcctgatga tgtctcaggc 5700 cttgagcagc ttgagagtat aatcagcttt gaaaaactga ctgaatggac cagttctagt 5760 attatggaag agaggaaggt caaagtgtac ttacctcgca tgaagatgga ggagaaatac 5820 aacctcacat ctctcttaat ggctatggga attactgacc tgttcagctc ttcagccaat 5880 ctgtctggca tctcctcagt agggagcctg aagatatctc aagctgtcca tgcagcacat 5940 gcagaaatca atgaagcggg cagagatgtg gtaggctcag cagaggctgg agtggatgct 6000 actgaagaat ttagggctga ccatccattc ctcttctgtg tcaagcacat cgaaaccaac 6060 gccattctcc tctttggcag atgtgtttct ccgcggccag cagatgacgc accagcagat 6120 gacgcaccag cagatgacgc accagcagat gacgcaccag cagatgacgc accagcagat 6180 gacgcaacaa catgtatcct gaaaggctct tgtggctgga tcggcctgct ggatgacgat 6240 gacaaaaaat acaaaaaagc actgaaaaaa ctggcaaaac tgctgtaatg agggcgcctg 6300 gatccagatc acttctggct aataaaagat cagagctcta gagatctgtg tgttggtttt 6360 ttgtggatct gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 6420 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 6480 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc agcacagcaa 6540 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac 6600 ctctctctct ctctctctct ctctctctct ctctctctct cggtacctct ctcgaggggg 6660 ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt 6720 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 6780 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 6840 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt 6900 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt 6960 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc 7020 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg 7080 gcccactact ccgggatcat atgacaagat gtgtatccac cttaacttaa tgatttttac 7140 caaaatcatt aggggattca tcagtgctca gggtcaacga gaattaacat tccgtcagga 7200 aagcttatga tgatgatgtg cttaaaaact tactcaatgg ctggttatgc atatcgcaat 7260 acatgcgaaa aacctaaaag agcttgccga taaaaaaggc caatttattg ctatttaccg 7320 cggcttttta ttgagcttga aagataaata aaatagatag gttttatttg aagctaaatc 7380 ttctttatcg taaaaaatgc cctcttgggt tatcaagagg gtcattatat ttcgcggaat 7440 aacatcattt ggtgacgaaa taactaagca cttgtctcct gtttactccc ctgagcttga 7500 ggggttaaca tgaaggtcat cgatagcagg ataataatac agtaaaacgc taaaccaata 7560 atccaaatcc agccatccca aattggtagt gaatgattat aaataacagc aaacagtaat 7620 gggccaataa caccggttgc attggtaagg ctcaccaata atccctgtaa agcaccttgc 7680 tgatgactct ttgtttggat agacatcact ccctgtaatg caggtaaagc gatcccacca 7740 ccagccaata aaattaaaac agggaaaact aaccaacctt cagatataaa cgctaaaaag 7800 gcaaatgcac tactatctgc aataaatccg agcagtactg ccgttttttc gccccattta 7860 gtggctattc ttcctgccac aaaggcttgg aatactgagt gtaaaagacc aagacccgct 7920 aatgaaaagc caaccatcat gctattccat ccaaaacgat tttcggtaaa tagcacccac 7980 accgttgcgg gaatttggcc tatcaattgc gctgaaaaat aaataatcaa caaaatggca 8040 tcgttttaaa taaagtgatg tataccgaat tcagcttttg ttccctttag tgagggttaa 8100 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8160 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8220 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8280 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 8340 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8400 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 8460 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8520 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8580 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 8640 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 8700 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 8760 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 8820 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 8880 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 8940 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 9000 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9060 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9120 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9180 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 9240 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9300 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9360 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 9420 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9480 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9540 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9600 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 9660 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 9720 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 9780 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 9840 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 9900 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 9960 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10020 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 10080 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 10140 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 10200 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10260 gaaaagtgcc ac 10272 47 10880 DNA Artificial Sequence Synthetic 47 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa 180 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac 240 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 300 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 360 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 420 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 480 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 540 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 600 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 660 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 720 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 780 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 840 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 900 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 960 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct 1020 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 1080 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac 1140 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1200 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata 1260 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg 1320 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca 1380 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta 1440 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag 1500 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac 1560 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc 1620 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc 1680 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga 1740 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgcga actcgatatt 1800 ttacacgact ctctttacca attctgcccc gaattacact taaaacgact caacagctta 1860 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt 1920 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt 1980 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt 2040 tcgggcaata cgatgcccat tgtacttgtt gactggtctg atattcgtga gcaaaaacga 2100 cttatggtat tgcgagcttc agtcgcacta cacggtcgtt ctgttactct ttatgagaaa 2160 gcgttcccgc tttcagagca atgttcaaag aaagctcatg accaatttct agccgacctt 2220 gcgagcattc taccgagtaa caccacaccg ctcattgtca gtgatgctgg ctttaaagtg 2280 ccatggtata aatccgttga gaagctgggt tggtactggt taagtcgagt aagaggaaaa 2340 gtacaatatg cagacctagg agcggaaaac tggaaaccta tcagcaactt acatgatatg 2400 tcatctagtc actcaaagac tttaggctat aagaggctga ctaaaagcaa tccaatctca 2460 tgccaaattc tattgtataa atctcgctct aaaggccgaa aaaatcagcg ctcgacacgg 2520 actcattgtc accacccgtc acctaaaatc tactcagcgt cggcaaagga gccatgggtt 2580 ctagcaacta acttacctgt tgaaattcga acacccaaac aacttgttaa tatctattcg 2640 aagcgaatgc agattgaaga aaccttccga gacttgaaaa gtcctgccta cggactaggc 2700 ctacgccata gccgaacgag cagctcagag cgttttgata tcatgctgct aatcgccctg 2760 atgcttcaac taacatgttg gcttgcgggc gttcatgctc agaaacaagg ttgggacaag 2820 cacttccagg ctaacacagt cagaaatcga aacgtactct caacagttcg cttaggcatg 2880 gaagttttgc ggcattctgg ctacacaata acaagggaag acttactcgt ggctgcaacc 2940 ctactagctc aaaatttatt cacacatggt tacgctttgg ggaaattatg aggggatcgc 3000 tctagagcga tccgggatct cgggaaaagc gttggtgacc aaaggtgcct tttatcatca 3060 ctttaaaaat aaaaaacaat tactcagtgc ctgttataag cagcaattaa ttatgattga 3120 tgcctacatc acaacaaaaa ctgatttaac aaatggttgg tctgccttag aaagtatatt 3180 tgaacattat cttgattata ttattgataa taataaaaac cttatcccta tccaagaagt 3240 gatgcctatc attggttgga atgaacttga aaaaaattag ccttgaatac attactggta 3300 aggtaaacgc cattgtcagc aaattgatcc aagagaacca acttaaagct ttcctgacgg 3360 aatgttaatt ctcgttgacc ctgagcactg atgaatcccc taatgatttt ggtaaaaatc 3420 attaagttaa ggtggataca catcttgtca tatgatcccg gtaatgtgag ttagctcact 3480 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg 3540 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 3600 aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg ctctagaact 3660 agtggatccc ccgggcatca gattggctat tggccattgc atacgttgta tccatatcat 3720 aatatgtaca tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg 3780 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 3840 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 3900 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 3960 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 4020 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 4080 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 4140 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 4200 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 4260 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 4320 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 4380 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc 4440 cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat 4500 agactctata ggcacacccc tttggctctt atgcatgcta tactgttttt ggcttggggc 4560 ctatacaccc ccgcttcctt atgctatagg tgatggtata gcttagccta taggtgtggg 4620 ttattgacca ttattgacca ctcccctatt ggtgacgata ctttccatta ctaatccata 4680 acatggctct ttgccacaac tatctctatt ggctatatgc caatactctg tccttcagag 4740 actgacacgg actctgtatt tttacaggat ggggtcccat ttattattta caaattcaca 4800 tatacaacaa cgccgtcccc cgtgcccgca gtttttatta aacatagcgt gggatctcca 4860 cgcgaatctc gggtacgtgt tccggacatg ggctcttctc cggtagcggc ggagcttcca 4920 catccgagcc ctggtcccat gcctccagcg gctcatggtc gctcggcagc tccttgctcc 4980 taacagtgga ggccagactt aggcacagca caatgcccac caccaccagt gtgccgcaca 5040 aggccgtggc ggtagggtat gtgtctgaaa atgagcgtgg agattgggct cgcacggctg 5100 acgcagatgg aagacttaag gcagcggcag aagaagatgc aggcagctga gttgttgtat 5160 tctgataaga gtcagaggta actcccgttg cggtgctgtt aacggtggag ggcagtgtag 5220 tctgagcagt actcgttgct gccgcgcgcg ccaccagaca taatagctga cagactaaca 5280 gactgttcct ttccatgggt cttttctgca gtcaccgtcg gatcaatggg ctccatcggt 5340 gcagcaagca tggaattttg ttttgatgta ttcaaggagc tcaaagtcca ccatgccaat 5400 gagaacatct tctactgccc cattgccatc atgtcagctc tagccatggt atacctgggt 5460 gcaaaagaca gcaccaggac acaaataaat aaggttgttc gctttgataa acttccagga 5520 ttcggagaca gtattgaagc tcagtgtggc acatctgtaa acgttcactc ttcacttaga 5580 gacatcctca accaaatcac caaaccaaat gatgtttatt cgttcagcct tgccagtaga 5640 ctttatgctg aagagagata cccaatcctg ccagaatact tgcagtgtgt gaaggaactg 5700 tatagaggag gcttggaacc tatcaacttt caaacagctg cagatcaagc cagagagctc 5760 atcaattcct gggtagaaag tcagacaaat ggaattatca gaaatgtcct tcagccaagc 5820 tccgtggatt ctcaaactgc aatggttctg gttaatgcca ttgtcttcaa aggactgtgg 5880 gagaaagcat ttaaggatga agacacacaa gcaatgcctt tcagagtgac tgagcaagaa 5940 agcaaacctg tgcagatgat gtaccagatt ggtttattta gagtggcatc aatggcttct 6000 gagaaaatga agatcctgga gcttccattt gccagtggga caatgagcat gttggtgctg 6060 ttgcctgatg aagtctcagg ccttgagcag cttgagagta taatcaactt tgaaaaactg 6120 actgaatgga ccagttctaa tgttatggaa gagagaagat caaagtgtac ttacctcgca 6180 tgaagatgga ggaaaaatac aacctcacat ctgtcttaat ggctatgggc attactgacg 6240 tgtttagctc ttcagccaat ctgtctggca tctcctcagc agagagcctg aagatatctc 6300 aagctgtcca tgcagcacat gcagaaatca atgaagcagg cagagaggtg gtagggtcag 6360 cagaggctgg agtggatgct gcaagcgtct ctgaagaatt tagggctgac catccattcc 6420 tcttctgtat caagcacatc gcaaccaacg ccgttctctt cttttggcag atgtgtttcc 6480 cgcggccagc agatgacgca ccagcagatg acgcaccagc agatgacgca ccagcagatg 6540 acgcaccagc agatgacgca acaacatgta tcctgaaagg ctcttgtggc tggatcggcc 6600 tgctggatga cgatgacaaa tttgtgaacc aacacctgtg cggctcacac ctggtggaag 6660 ctctctacct agtgtgcggg gaacgaggct tcttctacac acccaagacc cgccgggagg 6720 cagaggacct gcaggtgggg caggtggagc tgggcggggg ccctggtgca ggcagcctgc 6780 agcccttggc cctggagggg tccctgcaga agcgtggcat tgtggaacaa tgctgtacca 6840 gcatctgctc cctctaccag ctggagaact actgcaacta gggcgcctaa agggcgaatt 6900 atcgcggccg ctctagacca ggcgcctgga tccagatcac ttctggctaa taaaagatca 6960 gagctctaga gatctgtgtg ttggtttttt gtggatctgc tgtgccttct agttgccagc 7020 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 7080 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 7140 tggggggtgg ggtggggcag cacagcaagg gggaggattg ggaagacaat agcaggcatg 7200 ctggggatgc ggtgggctct atgggtacct ctctctctct ctctctctct ctcactctct 7260 ctctctctcg gtacctctcc tcgagggggg gcccggtacc caattcgccc tatagtgagt 7320 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 7380 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 7440 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt 7500 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 7560 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt 7620 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 7680 agggcgaaaa accgtctatc agggcgatgg cccactactc cgggatcata tgacaagatg 7740 tgtatccacc ttaacttaat gatttttacc aaaatcatta ggggattcat cagtgctcag 7800 ggtcaacgag aattaacatt ccgtcaggaa agcttatgat gatgatgtgc ttaaaaactt 7860 actcaatggc tggttatgca tatcgcaata catgcgaaaa acctaaaaga gcttgccgat 7920 aaaaaaggcc aatttattgc tatttaccgc ggctttttat tgagcttgaa agataaataa 7980 aatagatagg ttttatttga agctaaatct tctttatcgt aaaaaatgcc ctcttgggtt 8040 atcaagaggg tcattatatt tcgcggaata acatcatttg gtgacgaaat aactaagcac 8100 ttgtctcctg tttactcccc tgagcttgag gggttaacat gaaggtcatc gatagcagga 8160 taataataca gtaaaacgct aaaccaataa tccaaatcca gccatcccaa attggtagtg 8220 aatgattata aataacagca aacagtaatg ggccaataac accggttgca ttggtaaggc 8280 tcaccaataa tccctgtaaa gcaccttgct gatgactctt tgtttggata gacatcactc 8340 cctgtaatgc aggtaaagcg atcccaccac cagccaataa aattaaaaca gggaaaacta 8400 accaaccttc agatataaac gctaaaaagg caaatgcact actatctgca ataaatccga 8460 gcagtactgc cgttttttcg cccatttagt ggctattctt cctgccacaa aggcttggaa 8520 tactgagtgt aaaagaccaa gacccgtaat gaaaagccaa ccatcatgct attcatcatc 8580 acgatttctg taatagcacc acaccgtgct ggattggcta tcaatgcgct gaaataataa 8640 tcaacaaatg gcatcgttaa ataagtgatg tataccgatc agcttttgtt ccctttagtg 8700 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 8760 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 8820 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 8880 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 8940 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 9000 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 9060 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 9120 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 9180 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 9240 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 9300 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 9360 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 9420 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 9480 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 9540 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 9600 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 9660 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 9720 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 9780 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 9840 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 9900 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 9960 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 10020 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 10080 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 10140 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 10200 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 10260 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 10320 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 10380 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 10440 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 10500 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 10560 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 10620 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 10680 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 10740 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 10800 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 10860 atttccccga aaagtgccac 10880 48 78 DNA Artificial Sequence Synthetic 48 aatttctcaa ggatattttt cttcgtgttc gctttggttc tggctttgtc aacagtttcg 60 gctgcgccag agccgaaa 78 49 93 DNA Artificial Sequence Synthetic 49 aatttctcaa ggatattttt cttcgtgttc gctttggttc tggctttgtc aacagtttcg 60 gctgcgccag agccgaaatg gaaagtcttc aag 93 50 15 DNA Artificial Sequence Synthetic 50 gcgccagagc cgaaa 15 51 30 DNA Artificial Sequence Synthetic 51 gcgccagagc cgaaatggaa agtcttcaag 30 52 7 DNA Artificial Sequence Synthetic 52 accatgt 7

Claims (44)

We claim:
1. A method of producing a transgenic animal comprising, administering to an oviduct or an ovary of an animal a composition comprising a transposon-based vector.
2. The method of claim 1, wherein the composition is injected into an artery leading to the oviduct or the ovary.
3. The method of claim 1, wherein the composition is injected into a lumen of the oviduct.
4. The method of claim 1, wherein the composition further comprises a transfection reagent.
5. The method of claim 1, wherein the animal is a bird.
6. The method of claim 5, wherein the bird is a poultry bird.
7. The method of claim 5, wherein the animal is a chicken or a quail.
8. The method of claim 7, wherein the composition is administered to a lumen of the oviduct of the chicken or the quail following an increase in proliferation of oviduct epithelial and/or tubular gland cells.
9. The method of claim 7, wherein the composition is administered to the chicken between approximately 14 and 16 weeks of age.
10. The method of claim 7, wherein the composition is administered to the quail between approximately 5 and 6 weeks of age.
11. The method of claim 1, wherein the transposon-based vector comprises:
a) a transposase gene operably linked to a first promoter, the transposase gene encoding for a transposase; and
b) one or more genes of interest operably-linked to one or more additional promoters; wherein the one or more genes of interest and their operably-linked promoters are flanked by transposase insertion sequences recognized by the transposase, and wherein the first promoter comprises a modified Kozak sequence comprising ACCATG.
12. The method of claim 11, wherein one to twenty codons at a beginning of the transposase gene are modified by changing a nucleotide at a third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon.
13. The method of claim 12, wherein the transposase gene is modified in its first ten codons.
14. The method of claim 1, wherein the transposon-based vector comprises:
a) a transposase gene operably linked to a first promoter and an avian optimized polyA sequence, the transposase gene encoding for a transposase; and
b) one or more genes of interest operably-linked to one or more additional promoters;
c) wherein the one or more genes of interest and their operably-linked promoters are flanked by transposase insertion sequences recognized by the transposase.
15. The method of claim 14, wherein the avian optimized polyA sequence comprises between approximately 40 and 100 nucleotides that precede a polyA nucleotide string.
16. The method of claim 14, wherein the transposase is a Tn10 transposase.
17. The method of claim 14, wherein the first promoter is a constitutive promoter.
18. The method of claim 14, wherein the first promoter is an an oviduct-specific promoter selected from the group consisting of ovalbumin, ovotransferrin, ovomucoid, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, and ovostatin.
19. The method of claim 14, wherein the first promoter is an ovalbumin promoter.
20. The method of claim 14, wherein the one or more genes of interest are operably-linked to a second promoter.
21. The method of claim 20, wherein the second promoter is an oviduct-specific promoter selected from the group consisting of ovalbumin, ovotransferrin, ovomucoid, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, and ovostatin.
22. The method of claim 20, wherein the second promoter is an ovalbumin promoter.
23. The method of claim 14, wherein the transposon-based vector further comprises an enhancer operably-linked to the one or more genes of interest.
24. The method of claim 23, wherein the enhancer comprises at least a portion of an ovalbumin enhancer.
25. The method of claim 14, wherein the transposon-based vector further comprises an egg directing sequence operably-linked to the one or more genes of interest.
26. The method of claim 25, wherein the egg directing sequence is an ovalbumin signal sequence as shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.
27. A method of increasing expression of a polynucleotide sequence in an avian comprising, administering to the avian a composition comprising the polynucleotide sequence operably-linked to an avian optimized polyA sequence.
28. The method of claim 27, wherein the avian optimized polyA sequence comprises between approximately 40 and 100 nucleotides that precede a polyA nucleotide string.
29. A transgenic animal made with the method of claim 1.
30. The transgenic animal of claim 29, wherein the animal is a bird.
31. The transgenic animal of claim 29, wherein the animal is a bird and the composition is administered to a lumen of the oviduct of the bird.
32. An egg produced by the transgenic bird of claim 31.
33. The egg of claim 32, wherein the egg contains a protein, a polypeptide or a peptide encoded by the transposon-based vector.
34. A method of producing proteins, polypeptides or peptides comprising:
a) administering to an oviduct of an egg-laying animal a composition comprising a transposon-based vector, wherein the transposon-based vector comprises a transposase gene operably linked to a first promoter, the transposase gene encoding for a transposase, and one or more genes of interest operably-linked to one or more additional promoters; and,
b) permitting the one or more genes of interest to be expressed into a protein, a polypeptide or a peptide.
35. The method of claim 34, further comprising
a) collecting an egg from the egg-laying animal;
b) harvesting egg white containing the protein, the polypeptide or the peptide; and,
c) purifying the protein, the polypeptide or the peptide.
36. The method of claim 35, wherein the egg-laying animal is a bird.
37. The method of claim 36, wherein the bird is a poultry bird.
38. The method of claim 36, wherein the bird is a chicken or a quail.
39. A method of producing a transgenic mammal comprising, administering to an intramammary duct system of a mammal a composition comprising a transposon-based vector.
40. Milk produced by the transgenic mammal of claim 39.
41. A transgenic mammal made with the method of claim 39.
42. The milk of claim 40, wherein the milk contains a protein, a polypeptide or a peptide encoded by the transposon-based vector.
43. A method of producing proteins, polypeptides or peptides comprising:
a) administering to an intramammary duct system of a mammal a composition comprising a transposon-based vector, wherein transposon-based vector comprises a transposase gene operably linked to a first promoter, the transposase gene encoding for a transposase, and one or more genes of interest operably-linked to one or more additional promoters; and,
b) permitting the one or more genes of interest to be expressed into a protein, a polypeptide or a peptide.
44. The method of claim 43, further comprising
a) collecting milk from the mammal; and,
b) purifying the protein, the polypeptide or the peptide.
US10/746,149 2002-06-26 2003-12-24 Administration of transposon-based vectors to reproductive organs Abandoned US20040172667A1 (en)

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US44144703P true 2003-01-21 2003-01-21
US44150203P true 2003-01-21 2003-01-21
US44139203P true 2003-01-21 2003-01-21
US10/609,019 US7527966B2 (en) 2002-06-26 2003-06-26 Gene regulation in transgenic animals using a transposon-based vector
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