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WO1988001648A1 - Expression de proteines heterologues par des mammiferes transgeniques en lactation - Google Patents

Expression de proteines heterologues par des mammiferes transgeniques en lactation Download PDF

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Publication number
WO1988001648A1
WO1988001648A1 PCT/US1987/002069 US8702069W WO8801648A1 WO 1988001648 A1 WO1988001648 A1 WO 1988001648A1 US 8702069 W US8702069 W US 8702069W WO 8801648 A1 WO8801648 A1 WO 8801648A1
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WIPO (PCT)
Prior art keywords
protein
gene
sequences
lactalbumin
milk
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Ceased
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PCT/US1987/002069
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English (en)
Inventor
Thomas P. Hopp
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Immunex Corp
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Immunex Corp
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Publication of WO1988001648A1 publication Critical patent/WO1988001648A1/fr
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Classifications

    • 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
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins

Definitions

  • the present invention relates generally to transgenic animals, and particularly to methods of producing recombinant proteins as components of the milk of lactating transgenic mammals.
  • recombinant proteins are produced predominantly by inserting selected genes (cDNAs) into phage, plas id, or viral expression vectors, which direct production of the desired protein products in bacteria', yeast, or mammalian cells grown in culture. .
  • cDNAs selected genes
  • 15 expression system refers to an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, and (2) a structural genetic element or elements, comprising a coding sequence(s) which is transcribed into mRNA * and translated to
  • a cDNA library obtained from mammary tissues of lactating cows can be screened with the oligonucleotide probes to isolate a cDNA correponding to the structural sequence of the milk protein, less any introns.
  • a series of studies are undertaken to determine which regions are required to regulate expression of the milk protein gene. The essential regions are identified by assembling plasmids which contain the putative regulatory sequences joined to a suitable indicator gene, as well as sequences enabling stable transformation of mammalian host cells.
  • Mammary cells capable of induction by lactogenic hormones are then exposed to the recombinant constructs under conditions which promote uptake and incorporation of foreign DNAs. Expression of the indicator gene under lactogenic inducing conditions is then monitored by an assay specific for the indicator gene product.
  • the essential regulatory regions can be identified. If regulatory sequences are not apparent by homology to known regulatory regions of other lactogenic hormone-controlled genes, then further sequences from the gene, including 5' and 3' flanking sequences and intron sequences, can be systematically assayed for their ability to confer lactogenic hormone inducibility upon the indicator gene when linked to it.
  • a construct consisting of the »5' and 3' non-coding sequences joined to * a cDNA corresponding to the protein product can be introduced to host cells, and expression of the milk- protein gene measured using a specific antibody.
  • expression systems comprising either the milk protein gene with introns removed or heterologous structural sequences are introduced to fertilized ova of a chosen species by microinjection in vitro, and the resulting microinjected zygotes implanted in the reproductive tract of a pseudopreg ⁇ ant female. Following gestation and delivery, the transplanted offspring can be genetically screened for genomic incorporation of the expression system, using polynucleotide probes. Finally, expression of the milk protein gene or heterologous protein in the milk of mature transgenic animals is confirmed by appropriate assay.
  • probes are labelled using 32P-ATP and T4 polynucleotide kinase substantially as described by Maniatis.
  • the complete nucleotide sequence of rat ⁇ -lactalbumin gene is disclosed by Qasba et al. , Nature 308:377 (1984).
  • the probes are at least 60-mers to ensure relatively efficient trans-species hydridization to DNA fragments comprising the bovine ⁇ -lactalbumin gene.
  • Restriction mapping and DNA sequencing are employed to identify the bovine genomic fragments encoding the entire bovine ⁇ -lactalbumin' gene., including at least 1000 base pairs 5' to the start of the transcription initiation codon, the 5' transcribed but non-coding sequences, the coding or structural region, and the 3' non-coding region. These regions are identified by homology with the known sequence of the rat . ⁇ -lactalbumin gene, and also by reference to the sequence of a bovine ⁇ -lactalbumin cDNA, if available. Conventional methods for DNA chain-termination sequence determination are described in the Amersham handbook M13 Cloning and Sequencing (Blenheim Crescent, London 1983); and by Messing, Recombinant DNA Tech. Bull.
  • a bovine mammary cDNA library can be contructed from bovine mammary parenchymal tissue obtained from the mammary organs of lactating cows at slaughter. The isolated tissue is mechanically comminuted and treated with collagenas ⁇ , hyaluronidase, or other suitable dispersing aids to generate a cell suspension. Polyadenylated messenger RNA is then isolated from the parenchymal cell suspensions by methods substantially similar to the guanidium thiocyanate and guanidine hydrochloride methods disclosed by Maniatis and by Strohman et al. , Cell 5 10:265-273 (1977).
  • Double-stranded cDNA is then synthesized from the polyadenylated RNA fractions by standard techniques (Maniatis), and a cDNA library created in plasmid or phage, for example, pBR322. Oligonucleotide probes corresponding 10 to structural sequences of the rat ⁇ -lactalbumin gene can then be used to screen the library for clones bearing bovine ⁇ -lactalbumin cDNA.
  • plasmids are constructed which ⁇ ontain sequences located 5' with respect to the expected transcription initiation site, as well -as part of "20 the 5' non-coding region, fused to a selected indicator gene.
  • the bacterial -chloramphenicol 3-O-acetyltransferase gene, or CAT is particularly useful for this purpose.
  • the enyzy e encoded by this gene can be sensitively and
  • pSV2-CAT plasmid containing the CAT gene, pSV2-CAT is described by Howard et al. , Proc. Nat. Acad. Sci. USA 79:6777 (1982), and is available from the ATCC under "
  • the test construct preferably contains an additional sequence enabling RNA splicing and polyadenylation following introduction to recipient mammalian cells.
  • a Ipreferred sequence for this purpose is the simian virus 40 (SV40) small-T antigen splice donor and acceptor sites (SV40 coordinates 4,035-4656) and the SV40 polyadenylation site (SV40 coordinates 2,469-2,706).
  • SV40 simian virus 40
  • These elements can be obtained as a Bglll-BamHl fragment from plasmid pSV2-dhfr (ATCC 37145), described by Subra ani et al., Molec. Cell. Biol. 1:854 (1981).
  • the plasmid thus created is then introduced to recipient cells likely to respond to lactogenic hormones.
  • recipient cells likely to respond to lactogenic hormones.
  • primary bovine mammary cell cultures can be employed, or cultures of mouse mammary cells such as the cell line COMMA-ID described by Danielson et al. , Proc. Natl. Acad. Sci. USA 81:3756 (1984).
  • the COMMA-ID cells have been shown to induce synthesis of murine caseins in response to lactogenic hormones.
  • plasmids containing the SV40 elements and CAT indicator, but lacking inserted regulatory elements are employed in parallel transfection experiments. Effective techniques for transfection of mammalian cells in culture include those disclosed by McCutchan et al., J. Natl.
  • Transfected cells are collected by scraping and resuspended in induction medium, which consists of growth medium containing prolactin (5 ⁇ g/ml), aldosterone (5 ⁇ g/ml), and hydrocortisone (1 ⁇ g/ml).
  • induction medium which consists of growth medium containing prolactin (5 ⁇ g/ml), aldosterone (5 ⁇ g/ml), and hydrocortisone (1 ⁇ g/ml).
  • the cultures are incubated at 37 ⁇ C for four days in induction medium (changed daily) and then extracts of the treated cells are tested for chloramphenicol transferase activity by the assay previously referenced. This procedure is repeated with various combinations of the putative regulatory elements selected by hybridization, as described above, until each element required for lactogen-induced expression of the CAT geie has been identified.
  • a plasmid is then constructed, containing the interleukin—2 sequence and a signal sequence derived .from the native interleukin-2 gene or the bovine ⁇ -lactaibumrn gene, in conjunction with the ⁇ —lactalbumin regulatory elements.
  • the plasmid is amplified in cultures of appropriate recipient cells, and a preparation of purified plasmid DNA cleaved with an appropriate restriction enzyme to provide a linear fragment containing the ⁇ -lactalbumin regulatory region, ⁇ -lactalbumin (or IL-2) signal peptide, and interleukin-2 structural regions. This fragment is isolated by electrophoresis on an agarose gel and reserved for injection into fertilized mouse ova.
  • mice Six week-old female mice are induced to superovulate by injection of 5 international units of pregnant mares' serum, followed 48 hours later by 2.5 international units human chorionic gonadotropin, and placed immediately with males for mating. Approximately 14 hours following mating, those females exhibiting vaginal plugs are sacrificed and their oviducts removed and placed in Krebs-Ringer bicarbonate buffered medium, containing bovine serum albumen and hyaluronidase at 1 mg/ml. Oviducts are opened with forceps and fertilized eggs and remaining follicle cells are expressed into a culture dish. After 1-2 minutes, eggs are removed and washed with culture medium previously equilibrated with 5% C02 in air at 37 ⁇ C.
  • Eggs containing pronuclei are identified under a dissecting microscope and placed in lots of 20 in a microdrop of equilibrated medium, which is then placed in a 100 mm culture dish and covered with mineral oil. Eggs are stored' in the incubator in. this manner until microinjected.
  • Microneedles having a tip diameter of about 1-2 ⁇ m are pulled from thin-walled glass tubing using a pipette puller. Holding pipettes (for holding eggs) having a tip diameter of 60-70 ⁇ m are similarly pulled from capillary tubing, and the ends fire polished using a microforge. The tips of the microneedles are allowed to fill with a suspension of plasmid DNA by capillary action.
  • the holding pipettes and microneedle barrels are filled with an inert fluorocarbon (Fluorinert, 3M) , and each microneedle and holding pipette is then secured to polyethylene tubing of appropriate diameter, which is in turn fitted to 1 L Hamilton syringes secured in icromanipulators. Both microneedle and holding pipette apparatus are secured to the stage of a light microscope having a 1200x objective.
  • Fluorinert, 3M inert fluorocarbon
  • the culture dish containing the suspended zygotes is secured to the microscope stage in proximity to the microinjection apparatus, and a microneedle containing plasmid solution is moved close to the drop containing the zygotes.
  • a zygote is then positioned on the holding pipette such that the male pronucleus is in focus, and the microneedle slowly inserted into the pronucleus.
  • Sufficient plasmid suspension, (about 2 pi) is injected to approximately double the size of the pronucleus, and then the microneedle is slowly withdrawn. This procedure is repeated with the remaining fertilized eggs.
  • taili ⁇ tips are * taken from offspring and high molecular weight DNA isolated by the method of Blin et al., Nucleic Acids Res. 3:2302 (1976). The isolated DNA's are then screened for the presence of heterologous DNA by the dot-hybridization method of Kafatos et al., Nucleic Acids Res. 7:1541 (1979), using nick-translated fragments of the cloned plasmid DNA used in the microinjection experiments. On this basis, transgenic animals are identified and isolated. At about six weeks of age, female transgenic mice are injected with a lactogenic inducing mixture of prolactin, aldosterone and hydrocortisone. Milk is expressed from the mammary glands and tested by appropriate assay for the presence of bovine ⁇ -lactalbumin or human interleukin-2. 6. Expression in other species

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Abstract

Des mammifères capables d'exprimer des protéines recombinantes par lactation sont produits par microinjection d'ADN recombinants contenant de nouveaux systèmes d'expression dans des ovules fécondés.
PCT/US1987/002069 1986-08-28 1987-08-25 Expression de proteines heterologues par des mammiferes transgeniques en lactation Ceased WO1988001648A1 (fr)

Applications Claiming Priority (2)

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US90149086A 1986-08-28 1986-08-28
US901,490 1986-08-28

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WO1988001648A1 true WO1988001648A1 (fr) 1988-03-10

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Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4873316A (en) * 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
EP0279582A3 (fr) * 1987-02-17 1989-10-18 Pharming B.V. Séquences d'ADN pour diriger des protéines vers les glandes mammaires afin d'être sécrétées efficacement
WO1990004036A1 (fr) * 1988-10-12 1990-04-19 Medical Research Council Production d'anticorps a partir d'animaux transgeniques
WO1991003551A1 (fr) * 1989-09-11 1991-03-21 Tsi-Mason Research Institute Production d'hormone de croissance dans le lait d'animaux transgeniques
WO1991008216A1 (fr) * 1989-12-01 1991-06-13 Genpharm International, Inc. Production de polypeptides recombines par des especes bovines et procedes transgeniques
EP0331564A3 (fr) * 1988-02-26 1992-01-08 Societe De Developpements Et D'innovations Des Marches Agricoles Et Alimentaires - Sodima- Union Des Cooperatives Agricoles Polysaccharide, application comme agent épaississant et comme agent antitumoral
EP0365591A4 (en) * 1987-06-16 1992-01-22 Edison Animal Biotechnology Center Dietary and hormonal regulation of expression of exogenous genes in transgenic animals under control of the promoter of the gene for phosphoenolpyruvate carboxykinase
EP0451823A3 (en) * 1990-04-11 1992-01-22 Consortium Fuer Elektrochemische Industrie Gmbh Dna constructs for expression of proteins in the lacteal gland of transgenic mammals
WO1993025567A1 (fr) * 1992-06-15 1993-12-23 Gene Pharming Europe B.V. Production de polypeptides recombines par des especes bovines et des procedes transgeniques
US5320952A (en) * 1989-09-21 1994-06-14 W. R. Grace & Co.-Conn. Enhanced gene expression in response to lactation signals
EP0555435A4 (fr) * 1991-08-13 1995-05-24 Wisconsin Milk Marketing Board
US5464764A (en) * 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
WO1996012815A1 (fr) * 1994-10-21 1996-05-02 Pharmacia & Upjohn Ab Amelioration d'un vecteur d'expression destine a la production de proteines de recombinaison
US5589604A (en) * 1991-01-11 1996-12-31 American Red Cross Expression of human protein C in mammary tissue of transgenic mammals
US5633076A (en) * 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
US5650503A (en) * 1988-11-11 1997-07-22 Ppl Therapeutics (Scotland) Limited Genetic construct of which protein coding DNA comprises introns and is designed for protein production in transgenic animals
US5700671A (en) * 1994-03-09 1997-12-23 Abbott Laboratories Methods of making transgenic animals producing oligosaccharides and glycoproteins
US5739407A (en) * 1991-08-19 1998-04-14 Symbicom Aktiebolag Human β-casein, process for producing it and use thereof
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US5776773A (en) * 1991-09-10 1998-07-07 The Babraham Institute Yeast artificial chromosomes and their use in the control of gene expression
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US5831141A (en) * 1991-01-11 1998-11-03 United States Of America As Represented By The Department Of Health And Human Services Expression of a heterologous polypeptide in mammary tissue of transgenic non-human mammals using a long whey acidic protein promoter
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US5856178A (en) * 1993-08-30 1999-01-05 Utah State University DNA cassettes for expression of lytic peptides in mammalian cells and transgenic organisms containing same
US5861299A (en) * 1988-11-11 1999-01-19 Ppl Therapeutics (Scotland) Limited Genetic construct of which protein coding DNA comprises introns and is designed for protein production in transgenic animals
US5891698A (en) * 1994-03-09 1999-04-06 Abbott Laboratories Oligosaccharides and glycoproteins produced in milk of transgenic non-human mammals
US5895833A (en) * 1993-01-28 1999-04-20 Cohesion Technologies, Inc. Production of human recombinant collagen in the milk of transgenic animals
US5965789A (en) * 1991-01-11 1999-10-12 American Red Cross Engineering protein posttranslational modification by PACE/furin in transgenic non-human mammals
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US6204431B1 (en) 1994-03-09 2001-03-20 Abbott Laboratories Transgenic non-human mammals expressing heterologous glycosyltransferase DNA sequences produce oligosaccharides and glycoproteins in their milk
US6222094B1 (en) 1992-01-23 2001-04-24 Symbicom Aktiebolag Transgenic non-human mammal expressing the DNA sequence encoding kappa casein mammary gland and milk
US6262336B1 (en) 1991-01-11 2001-07-17 American Red Cross Expression of a heterologous protein C in mammary tissue of transgenic animals using a long whey acidic protein promoter
US6689610B1 (en) 1989-08-22 2004-02-10 University Of Utah Research Foundation Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
US6743967B2 (en) 1996-04-10 2004-06-01 Chromos Molecular Systems Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
US6984772B1 (en) 1994-02-18 2006-01-10 Virginia Tech Intellectual Properties, Inc. Transgenic non-human mammals producing fibrinogen in their milk
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US7939317B1 (en) 1986-04-09 2011-05-10 Genzyme Corporation Transgenic animals secreting desired proteins into milk
US10034921B2 (en) 2013-02-13 2018-07-31 Laboratoire Français Du Fractionnement Et Des Biotechnologies Proteins with modified glycosylation and methods of production thereof
US10174110B2 (en) 2013-02-13 2019-01-08 Laboratoire Français Du Fractionnement Et Des Biotechnologies Highly galactosylated anti-TNF-α antibodies and uses thereof
US12247076B2 (en) 2015-07-06 2025-03-11 Laboratoire Français Du Fractionnement Et Des Biotechnologies Use of modified Fc fragments in immunotherapy

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Title
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Cell, Vol. 38, issued October 1984 (Cambridge, Massachusetts, USA), (SWIFT et al.), "Tissue-Specific Expression of the Rat Pancreatic Elastase 1 Gene in Transgenic Mice", pages 639-646. *
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Nature, Vol. 308, issued 22 March 1984, (London, England), (QASBA et al.), "Similarity of the Nucleotide Sequences of Rat Alpha-Lactalbumin and Chicken Lysozyme Genes", pages 377-380. *
Nucleic Acids Research, Vol. 14, issued 25 February 1986, (Oxford, England), (YU-LEE et al.), "Evolution of the Casein Multigene Family: Conserved Sequences in the 5' Flanking and Exon Regions", pages 1883-1902. *
Proc. Natl. Acad. Sci. USA, Vol. 83, April 1986, (Washington, D.C.), (BUCCHINI et al.), "Pancreatic Expression of Human Insulin Gene in Transgenic Mice", pages 2511-2515. *

Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7939317B1 (en) 1986-04-09 2011-05-10 Genzyme Corporation Transgenic animals secreting desired proteins into milk
EP0279582A3 (fr) * 1987-02-17 1989-10-18 Pharming B.V. Séquences d'ADN pour diriger des protéines vers les glandes mammaires afin d'être sécrétées efficacement
EP0832981A1 (fr) * 1987-02-17 1998-04-01 Pharming B.V. Séquences d'ADN pour diriger des protéines vers les glandes mammaires, afin d'être secrétées efficacement
US5565362A (en) * 1987-02-17 1996-10-15 Pharming B.V. DNA sequences to target proteins to the mammary gland for efficient secretion
US5994616A (en) * 1987-02-17 1999-11-30 Pharming B.V. Targeted synthesis of protein in mammary gland of a non-human transgenic mammal
US5304489A (en) * 1987-02-17 1994-04-19 Genpharm International, Inc. DNA sequences to target proteins to the mammary gland for efficient secretion
EP0365591A4 (en) * 1987-06-16 1992-01-22 Edison Animal Biotechnology Center Dietary and hormonal regulation of expression of exogenous genes in transgenic animals under control of the promoter of the gene for phosphoenolpyruvate carboxykinase
EP0347431A4 (en) * 1987-06-23 1991-10-16 Biogen, Inc. Expression of proteins in milk
US4873316A (en) * 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
EP0331564A3 (fr) * 1988-02-26 1992-01-08 Societe De Developpements Et D'innovations Des Marches Agricoles Et Alimentaires - Sodima- Union Des Cooperatives Agricoles Polysaccharide, application comme agent épaississant et comme agent antitumoral
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