EP1190042A2 - Procedes servant a manipuler le genome aviaire - Google Patents

Procedes servant a manipuler le genome aviaire

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Publication number
EP1190042A2
EP1190042A2 EP00943424A EP00943424A EP1190042A2 EP 1190042 A2 EP1190042 A2 EP 1190042A2 EP 00943424 A EP00943424 A EP 00943424A EP 00943424 A EP00943424 A EP 00943424A EP 1190042 A2 EP1190042 A2 EP 1190042A2
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EP
European Patent Office
Prior art keywords
nucleic acid
polypeptide
egg
avian
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00943424A
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German (de)
English (en)
Inventor
Paul A. Ditullio
Karl M. Ebert
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TranXenoGen Inc
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TranXenoGen Inc
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Publication date
Application filed by TranXenoGen Inc filed Critical TranXenoGen Inc
Publication of EP1190042A2 publication Critical patent/EP1190042A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A01K2267/025—Animal producing cells or organs for transplantation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/90—Vector systems having a special element relevant for transcription from vertebrates avian

Definitions

  • the invention relates to transgemc avian animals
  • the invention features methods of manipulating genomic DNA m avian species and to generate transgemc avian animals
  • a method for introducing a nucleic acid molecule into the genome of an avian species is earned out bv contacting in vivo a blastodermal cell of a fertilized hard shelled egg with the nucleic acid molecule
  • the nucleic acid molecule is not associated with a viral coat protem, e g , the nucleic acid is not delivered in a ⁇ iral particle
  • the nucleic acid molecule is introduced directly into the germinal disc of the egg To avoid disrupting the germinal disc (or blastoderm) or dispersing the blastodermal cells, the nucleic acid is delivered in a volume that is less than the volume of the germinal disc
  • the volume is greater than 1 microhter and less than 0 5 milliliters and introduction of the nucleic acid does not rupture the area opaca (membrane or sheath surrounding the germinal disc or blastoderm)
  • the nucleic acid is delivered in a volume of 5-100 microhters, more preferably 40-60 microhters. and most preferably.
  • the nucleic acid is delivered directly into the blastoderm or germinal disc and not to an area adjacent to or below the blastoderm
  • the thick albumen around the germinal disc is not removed before, du ⁇ ng, or after the delivery process
  • the nucleic acid is delivered by passing a needle or microinjection pipet directly through the shell and underlying membrane Little or no air is introduced into the egg, and the hole m the membrane left by the pipet or needle is small and self-sealing Accordingly, the method does not require deposition of an aqueous liquid o ⁇ er the opening of the egg to minimize the inadvertent introduction of air into the egg and does not require sealing the opening of the egg after nucleic acid delivery
  • the blastodermal cell is exposed to an electncal current in vivo, e g , by applying an electncal current across the egg
  • the method is useful to introduce a nucleic acid such as DNA into the genome of any avian species such as a chicken, an ostnch. an emu, a turkeys, a duck, a goose, a quail a parrot, a parakeet, a cockatoo, or a cockatiel to produce therapeutic proteins or to make an avian model for a non-avian, e g , human, disease state
  • the method is used to deliver DNA to blastodermal cells in a fertilized egg of any breed of chicken or any hyb ⁇ d breed of chicken Chicken breeds include White Leghorn, White Neighborhood Rock, Barred Neighborhood Rock, Rhode Island Red, New Hampshire and Dark Cornish
  • Nucleic acid delivery is timed to optimize uptake of the nucleic acid by blastodermal (totipotent) cells and minimize (or eliminate) uptake by cells which have begun to differentiate into vanous tissue types
  • blastodermal totipotent
  • nucleic acid dunng developmental stage X of the egg Delivery takes place at a time after oviposition but before incubation of the egg (at which time cell division and cell differentiation takes place)
  • the nucleic acid encodes a polypeptide or antisense molecule
  • the nucleic acid contains a sequence encoding an antibody or fragment thereof
  • the term antibody encompasses an mtact tetramenc antibody (e g , a monoclonal antibody) as well as an immunologically active antibody fragment, e g , a Fab or (Fab) 2 fragment, an engineered single chain F, molecule, a chime ⁇ c molecule (e g , an antibody which contains the binding specificity of one antibody, e g , of munne origin, and the remaining portions of another antibody, e g , of human o ⁇ gin
  • the antibody or fragment thereof is of human ongm
  • Other polypeptides include an insulin polypeptide (such as a human or porcine insulin polypeptide), a growth hormone polypeptide, a calcitonm polypeptide, or a serum albumin polypeptide
  • the transgemc nucleic acid encodes a porcine
  • Transcnptional regulatory sequence is a gene ⁇ c term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences to which they are operably linked
  • Transc ⁇ ption of the recombinant gene or transgene is under the control of a promoter sequence as well as other transcnptional regulatory sequences
  • an ovalbumin promoter sequence is used to direct expression of a transgene and a regulatory sequences de ⁇ ved from a chicken lysozvme gene is used to direct equal or equivalent expression of both chains of a transgemc tetrame ⁇ c antibody molecule
  • promoter is meant a minimal DNA sequence sufficient to direct transc ⁇ ption Promoters may be constitutive or inducible, and may be coupled to other regulatory sequences or "elements" which render promoter-dependent gene expression cell-type specific, tissue-specific or mducible by external signals or agents, such elements may be located in the 5' or 3' region of the native gene, or withm an intron
  • a transgene expression construct contains one or more of the following elements a chicken lysozyme or ovalbumin promoter, a chicken lysozyme enhancer, and a mat
  • nucleic acids described herein are isolated An isolated nucleic acid, e g , an isolated gene, or a fragment thereof, to be transfe ⁇ ed into a blastodermal cell of an egg is isolated by any of several methods well known to the art
  • the nucleic acid molecules are recombinant and/or have been pu ⁇ fied from the sequences which flank it in a naturally occur ⁇ ng state, I e a DNA has been removed from the sequences which are normally adjacent to the fragment, e g , the sequences adjacent to the fragment in the genome in which it naturally occurs
  • an isolated nucleic acid molecule is produced synthetically, or by treating mRNA de ⁇ ved from the transc ⁇ ption of the gene with a reverse transc ⁇ ptase so as to produce a cDNA, or by the direct isolation of the nucleic acid from cells, bacte ⁇ al clones, or from other sources
  • the invention includes sequences which hyb ⁇ dize under st ⁇ ngent conditions, with all
  • Nucleotide and ammo acid compa ⁇ sons are earned out using the Lasergene software package (DNASTAR, Inc , Madison, WI)
  • the MegA gn module used was the Clustal V method (Higgins et al , 1989, CABIOS 5(2) 151-153) The parameter used were gap penalty 10, gap length penalty 10
  • the nucleic acids descnbed herein hyb ⁇ dize at high st ⁇ ngency to a strand of DNA having the reference sequence, or the complement thereof and have transcnption regulatory activity Hyb ⁇ dization is earned out using standard techniques, such as those desc ⁇ bed in Ausubel et al (Cu ⁇ ent Protocols in Molecular Biology, John Wiley & Sons, 1989)
  • "High stnngency" refers to nucleic acid hyb ⁇ dization and wash conditions characte ⁇ zed by high temperature and low salt concentration, 1 e, hyb ⁇ dization at 42 degrees C, and in 50% formamide, a first
  • Fig 1 is a diagram of a fertilized hen's egg
  • Fig 2 is a diagram of a transgene expression cassette DETAILED DESCRIPTION OF THE INVENTION
  • the avian reproductive system is distinct from mammalian reproductive systems in that the female can store sperm and fertilize a single ovum at a time
  • the new fertilized ovum is large, fragile, and filled with yolk as it enters the reproductive track
  • Early embryonic development occurs in the oviduct as the egg is formed around the ovum
  • a protective layer of white albumen followed by an inner membrane and hardened shell before being laid (Fig 1 )
  • the ovum has matured from a single cell into a blastoderm (also known as the germinal disc) composed of 40-60,000 cells and development a ⁇ ests until the hen has laid enough eggs and beings to roost In the blastoderm state, all cells are totipotent and equally capable of cont ⁇ butmg to the germ line of the developing chick
  • Oviposition is the time at which the egg is laid In the chicken, oviposition occurs at stage X (a freshly laid egg, about 20 hours of utenne age)
  • stage X a freshly laid egg, about 20 hours of utenne age
  • DNA is therefore introduced into the blastoderm of an egg which has been incubated for 6 hours or less
  • stage X refers to a stage of development that occurs at about 20 hours ute ⁇ ne age and is charactenzed by oviposition
  • stage 10 refers to a stage of embryonic chick development (charactenzed by tissue differentiation) No tissue differentiation has occu ⁇ ed in stage X of development Accordingly, genetic manipulation occurs before differentiation of blastodermal cells into embryonic
  • the germinal disc is distinguished from the germinal crescent region in that the germinal disc contains undifferentiated blastodermal cells (at stage X or before), whereas the germinal crescent region appears in the early stages of chick embryo development (I e , stages 3-5 or 9-1 1 of chick embryo development)
  • the cells of the blastoderm are genetically manipulated both in vitro or in vivo using gene delivery techniques and then used to produce transgemc or chime ⁇ c chickens by allowing development in the egg, transfernng to a recipient unfertilized egg, or transfer ⁇ ng to the testes of a ste ⁇ le rooster for development into spermatogonia
  • the optimum time for transfection is between oviposition and several hours of activation of the egg in order to reduce the number of target cells for transfection
  • the blastoderm is accessed by cutting or d ⁇ lling a small hole in the egg shell (sitting upnght) with a scapel or dnll and gently peeling back the inner membrane to expose the white albumen
  • the blastoderm automatically onents to the top of the yolk and is visualized under light
  • the cells of the blastoderm are transfected in v ⁇ o by infusing DNA directly into the blastoderm using a synnge and small gauge needle
  • the DNA is naked or complexed with pids or other suitable compounds to facility DNA uptake (e g , DEAE-dextran) If the DNA is naked, the transfection efficiency is increased by passing an electncal current across the blastoderm or whole egg with a device such as a human heart defib ⁇ llator If a current is passed across the whole egg, two additional holes are made in the egg shell to expose the inner membrane to the cu ⁇ ent since the shell will not conduct elect ⁇ city
  • the blastoderm is removed from the egg and pooled with the cells from several eggs using a small pipet
  • DNA uptake by blastodermal cells is facilitated by such techniques as electroporation, DEAE-dextran treatment, calcium phosphate treatment, or pofection
  • the cells are transferred into the germinal disc of an unfertilized egg for development of a transgemc chick or into the testes of a ste ⁇ le rooster to induce development in spermatogonia and sperm for breeding
  • Chicks produced are tested for the presence of the transgene according to known methods, e g, by the polymerase chain reaction or southern blot analysis
  • the overall efficiency of the nucleic acid delivery procedure depends on the method and timing of gene delivery Transfection efficiency is optionally increase by methods such as
  • Prefe ⁇ ed transgene constructs include those carrying the ovalbumin promoter operatively linked to the human serum albumin gene
  • Fertilized eggs are laid and collected (day 1 )
  • the eggs are shipped at room temperature (umncubated) (day 2)
  • the eggs are allowed to site upnght for at least
  • a fertilized hen's egg was collected and placed in a humidified incubator for between 1 and 6 hours to activate the egg
  • a hole was cut in the egg shell using a dremel and the inner membrane was folded back
  • the egg was placed on a light to visualize the germinal disc which was infused with a hpid/DNA complex
  • the inner membrane was folded into place and the egg sealed with parafilm
  • the egg was placed back into the incubator and allowed to develop to term ⁇ blood sample or inner membrane sample as taken from the resulting chick and tested for the presence of the transgene by the polymerase chain reaction with transgene specific p ⁇ mers
  • a mosaic or chime ⁇ c chick is bred to a rooster to produce fully transgemc offsp ⁇ ng
  • Example 2 In vitro DNA delivery of blastodermal cells and transfer of transfected eggs to an unfertilized egg
  • a genomic sequence encoding human insulin was operatively linked to a promoter which directs transcription of a nucleic acid to which it is operably linked in avian eggs.
  • the promoter is a human lactoferrin promoter.
  • transcription regulatory elements are derived from a milk specific promoter, e.g., a mammalian lactoferrin gene promoter.
  • the expression cassette contains a promoter region derived from the human lactoferrin gene operably linked to a heterologous sequence.
  • a heterologous sequence is one that does not encode a lactoferrin polypeptide.
  • the promoter region includes at least 20 nucleotides of the nucleotide sequence of SEQ ID NO:l .
  • the promoter region contains nucleotides 1 -154 of SEQ ID NO: 1 or 2.
  • BamHl restriction site GGATCC (nucleotides 5-8) and Xhol site (nucleotides 140- 145) are italicized. These restriction sites may be altered, e.g., replaced with other restriction sites or with nucleotides that do not represent restriction enzyme recognition sites.
  • Table 2 Human Lactoferrin promoter region
  • lactoferrin-derived promoter regions described above are linked to the nucleotide sequence of SEQ ID NO:3 (GENBA KTM accession no. S52659).
  • isolated is meant a nucleic acid molecule that is free of the genes which, m the naturally-occumng genome of the organism, flank the sequence of interest
  • isolated includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a procaryote or eucaryote; or which exists as a separate molecule (e g , a cDNA or a genomic or cDNA fragment produced by PCR or rest ⁇ ction endonuclease digestion) independent of other sequences.
  • telomere sequence which is part of a hyb ⁇ d gene encoding additional polypeptide sequence
  • the term excludes large segments of genomic DNA, e g , such as those present in cosmid clones, which contain a given DNA sequence flanked by one or more other genes which naturally flank it in a naturally-occumng genome Lactofemn-de ⁇ ved transc ⁇ ption regulatory sequences, are attached to a nominal promoter (e g , the nominal lactofermn promoter or a heterologous promoter) which in turn is operably linked to a sequence to be transc ⁇ bed
  • the heterologous sequence to be transc ⁇ bed is a polypeptide-encodmg sequence or antisense sequence
  • the regulatory sequences of the invention operably linked to a polypeptide-encodmg sequence direct production of the encoded polypeptide m avian tissues
  • nucleic acid fragment is a portion of at least 20 continuous nucleotides identical to a portion of length equivalent to one of the reference nucleotide sequences or to its complement
  • promoters which direct transcnption m eggs of an avian animal are known in the art, e g , the chicken ovalbumin promoter (GENBANKTM J00895 or M24999) and the chicken lysozyme promoter (GENBANKTM J00886 or V00429)
  • An expression vector is constructed using a chicken ovalbumin promoter for expression of cloned sequences (Gannon et al , Organisation and sequences at the 5 'end of a cloned complete ovalbumin gene Nature 278 428434, Lai et al , The ovalbumin gene Structural sequence m native chicken DNA are not contiguous Proc Natl Acad Sci USA 75(5) 2205-2209, and Kaye et al , EMBO J 3 1 137-1 144)
  • Other regulatory elements which direct transcnption of transgenes include a nuclear DNA attachment element which mediates elevated and position-independent gene activity (Stief, A , et al ,
  • Protein or polypeptide products to be expressed by the transgene include human insulin (GENBANKTMV00565), human calcitomn (GENBANKTM XI 5943, Broad et al., Nucl Acids Res 17 6999-701 1 ), human serum albumin (GENBANKTM M12523, J04457), and a porcine single chain insulin
  • the nucleic acid sequence of the porcine single chain insulin sequence are shown below in Tables 4 and 5
  • GCAACTAGACTCGAG SEQ ID NO 4
  • the invention includes sequences which hyb ⁇ dize under st ⁇ ngent conditions, with all or part of the sequence reported in a reference sequence and retains transcnption regulatory function
  • the nucleic acid may contain one or more sequence modifications in relation to a reference sequence Such modifications may be obtained by mutation, deletion and or addition of one or more nucleotides compared to the reference sequence Modifications are introduced to alter the activity of the regulatory sequence, e g , to improve promoter activity, to suppress a transc ⁇ ption inhibiting region, to make a constitutive promoter regulatable or vice versa Modification are also made to introduce a rest ⁇ ction site facilitating subsequent cloning steps, or to eliminate the sequences which are not essential to the transcnptional activity
  • a modified sequence is at least 70% (more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%) identical to a reference sequence The modifications do not substantially alter the transc ⁇ ption promoter function associated with
  • a lactofer ⁇ n expression cassette was constructed using lactofer ⁇ n transcnption regulatory elements.
  • the cassette contained 3Kb of promoter and 7Kb of 3 ' flanking sequence with unique Sail and Notl rest ⁇ ction sites at the 5 ' and 3 'ends, respectively
  • the vector contained a unique Xhol site for the addition of heterologous coding sequences.
  • the human insulin gene was PCR amplified from human genomic DNA with the p ⁇ mers HINF3 (5'GCCCTCGAGGACAGGCTGCATCAGAA3 ' ; (SEQ ID NO:6)) H ⁇ NR3 (5'CTCGGTGCTCGAGGCGGCGGGTGT3 ⁇ SEQ ID NO7) cloned into the vector pCR2.1 (Invitrogen, Carlsbad,CA) according to the manufacturer's instructions The gene sequenced using the Amp cycle Sequencing kit (PE Applied Biosystems, Foster City, CA) to confirm that no base mutations had occu ⁇ ed dunng amplification.
  • the gene was excised from the vector pCR2.1 as and Xhol fragment and cloned into the Xhol site of the human lactofemn expression cassette. The o ⁇ entation of the insulin gene was confirmed by rest ⁇ ction analysis and DNA sequencing. The completed vector was designated HL31 and could be excised from the bacte ⁇ al backbone as a Sail to Notl fragment Example 6 Preparation of HL31 for Transfection
  • the eggshell was removed to expose the inner shell membrane and allow the visualization of the germinal disc. If the germinal disc could not be visualized a small opening was made in the membrane.
  • the DNA/lipid solution was injected directly into the center of the disc. Nucleic acid solutions (construct encoding human insulin) were injected in a volume of 50-100 1 with a lcc syringe and 27 gauge needle or in a volume of 10-20 1 with a microinjection needle attached to a Hamilton syringe. Table 6 shows data from representative experiments. 228 eggs were injected and 26 chicks were hatched (Table 6)
  • Chimensm of the hatched chicks was tested by PCR analysis of genomic DNA isolated from the inner shell membrane (post-hatching), or the liver, kidney, and reproductive organs upon necropsy (Table 7) Out of the 3 chicks tested in expenment 32, 2 showed a strong PCR signal in the inner shell membrane sample Upon maturation, these chicks are bred to confirm germhne transmission of the transgene PCR analysis of necropsy samples (membrane and reproductive organs) taken from 4 chicks de ⁇ ved from expenments 32 & 33 revealed that 1 out of 4 chicks had detectable levels of the transgene its reproductive organs
  • Table 7 shows results from a representative analysis of tissue expression of human insulin transgene in chime ⁇ c chickens produced as descnbed above Table 7 PCR analysis of Genomic DNA Isolated from Chimenc Chicks
  • transgenes such as MT-beta-Gal, and CMV-GFP were also transfected into blastodermal cells in vivo Chicks were produced and tested for tissue expression of the transgene as descnbed above The transgemc DNA was detected in the following tissues which were tested heart, liver, and kidney
  • the methods of the invention do not utilize retroviral particles Many of those methods use non-rephcatmg retroviruses, which contain a gene of interest, to mfect a developing egg
  • This process has several limitations which include a restnction on the size of the gene packaged in the viral coat, and instability of expression from retroviral vectors
  • retroviral gene delivery is severely limited in its ability to produce high levels of multime ⁇ c proteins since each coding region w ould optimally require its own promoter
  • IRES sequences which allow for bicistromc mRNAs, howev er the expression from these types of constructs is generally low unless a selection marker is used
  • the transgene is insulated from the effects of the surrounding chromatin structure by including elements such as mat ⁇ x attachment regions (Step 3 of Fig 2) Unlike p ⁇ or art methods which have utilized mat ⁇ x attachment region, the constructs descnbed herein result in equal expression of multimenc proteins
  • the elements are called A-elements, MAR for mat ⁇ x attachment regions, SAR, scaffolding attachment regions, DCR for dominant control region, and insulators.

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Abstract

L'invention concerne un procédé servant à introduire une molécule d'acide nucléique dans le génome d'une espèce aviaire, ce qui consiste à mettre en contact in vivo une cellule blastodermique d'un oeuf fertilisé avec la molécule d'acide nucléique, cet acide nucléique n'étant pas associé à une protéine de coque virale. Elle concerne également des animaux aviaires transgéniques et des procédés permettant d'obtenir ces animaux transgéniques.
EP00943424A 1999-06-04 2000-06-02 Procedes servant a manipuler le genome aviaire Withdrawn EP1190042A2 (fr)

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US13776199P 1999-06-04 1999-06-04
US137761P 1999-06-04
PCT/US2000/040059 WO2000075300A2 (fr) 1999-06-04 2000-06-02 Procedes servant a manipuler le genome aviaire

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EP (1) EP1190042A2 (fr)
JP (1) JP2003501083A (fr)
AU (1) AU777420B2 (fr)
CA (1) CA2375441A1 (fr)
NZ (1) NZ516173A (fr)
WO (1) WO2000075300A2 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA03001915A (es) 2000-08-03 2004-09-10 Therapeutic Human Polyclonals Produccion de anticuerpos humanizados en animales transgenicos.
US20020108132A1 (en) * 2001-02-02 2002-08-08 Avigenics Inc. Production of a monoclonal antibody by a transgenic chicken
MXPA04001333A (es) 2001-08-13 2004-05-05 Embrex Inc Metodos para inyectar huevos de ave.
WO2003025146A2 (fr) * 2001-09-18 2003-03-27 Avigenics, Inc. Production d'oiseaux transgeniques par injection cytoplasmique
AU2002341795A1 (en) * 2001-09-21 2003-04-01 Avigenics, Inc. Production of transgenic avians using sperm-mediated transfection
US7323618B2 (en) 2002-02-01 2008-01-29 Origen Therapeutics, Inc. Tissue specific expression of exogenous proteins in transgenic chickens
US7145057B2 (en) 2002-02-01 2006-12-05 Origen Therapeutics, Inc. Chimeric bird from embryonic stem cells
US20060191026A1 (en) 2005-02-18 2006-08-24 Origen Therapeutics, Inc. Tissue specific expression of antibodies in chickens
AU2003212958A1 (en) * 2002-02-08 2003-09-02 Tranxenogen, Inc. Immunoglobulin purification
EP1623035A4 (fr) * 2003-04-15 2006-08-30 Univ Georgia Res Found Transgenese aviaire utilisant une region du gene de l'ovalbumine du poulet
MXPA06000562A (es) 2003-07-15 2006-03-30 Therapeutic Human Polyclonals Loci de inmunoglobulina humanizada.
PL2064325T3 (pl) 2006-09-01 2012-05-31 Therapeutic Human Polyclonals Inc Zwiększona ekspresja ludzkiej lub humanizowanej immunoglobuliny u zwierząt transgenicznych innych niż człowiek
IN2014CN02113A (fr) 2011-09-21 2015-05-29 Fujirebio Kk

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5162215A (en) * 1988-09-22 1992-11-10 Amgen Inc. Method of gene transfer into chickens and other avian species
EP0424044A1 (fr) * 1989-10-16 1991-04-24 Merck & Co. Inc. Poule transgénique exprimant l'hormone de croissance bovine
GB2282139A (en) * 1993-09-24 1995-03-29 Univ Reading Introducing DNA into the germ line of birds
AU1812597A (en) * 1996-07-08 1998-02-02 Dnavec Research Inc. In vivo electroporation method for early animal embryo
JP4731683B2 (ja) * 1997-08-22 2011-07-27 ユニバーシティ・オブ・グエルフ 卵中でのタンパク質の産生
JP2001520009A (ja) * 1997-10-16 2001-10-30 ユニバーシティ・オブ・ジョージア・リサーチ・ファウンデイション・インコーポレイテッド 鳥の導入遺伝子についてのマグナム特異的プロモーターを含むベクター

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CARSCIENCE R.S. ET AL: "Germiline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos", DEVELOPMENT, vol. 117, 1993, pages 669 - 675, XP000998006 *
See also references of WO0075300A3 *
THORAVAL P. ET AL: "Germline transmission of exogenous genes in chicken using helper-free ecotropic avian leukosis virus-based vectors", TRANSGENIC RESEARCH, vol. 4, November 1995 (1995-11-01), pages 369 - 377, XP002095225, DOI: doi:10.1007/BF01973755 *

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AU5789800A (en) 2000-12-28
AU777420B2 (en) 2004-10-14
NZ516173A (en) 2004-02-27
CA2375441A1 (fr) 2000-12-14
WO2000075300A3 (fr) 2002-01-10
WO2000075300A2 (fr) 2000-12-14
JP2003501083A (ja) 2003-01-14

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