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WO1988010304A1 - Regulation dietetique et hormonale de l'expression de genes exogenes dans des animaux transgeniques sous le controle du promoteur du gene pour la phosphoenolpyruvate carboxykinase - Google Patents

Regulation dietetique et hormonale de l'expression de genes exogenes dans des animaux transgeniques sous le controle du promoteur du gene pour la phosphoenolpyruvate carboxykinase Download PDF

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Publication number
WO1988010304A1
WO1988010304A1 PCT/US1988/001943 US8801943W WO8810304A1 WO 1988010304 A1 WO1988010304 A1 WO 1988010304A1 US 8801943 W US8801943 W US 8801943W WO 8810304 A1 WO8810304 A1 WO 8810304A1
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animal
gene
pepck
promoter
expression
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PCT/US1988/001943
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English (en)
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Richard W. Hanson
Mary Mcgrane
Anthony Wynshaw-Boris
Jay Short
Maria Hatzoglou
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Edison Animal Biotechnology Center
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Priority to AU19861/88A priority Critical patent/AU618958B2/en
Publication of WO1988010304A1 publication Critical patent/WO1988010304A1/fr

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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
    • 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
    • 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
    • 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
    • A01K2267/02—Animal zootechnically ameliorated
    • 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
    • A01K2267/03—Animal model, e.g. for test or diseases
    • 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

Definitions

  • the present invention relates to the control of the expression of exogenous genes in transgenic animals and in tissue culture cells.
  • the transgene may be expressed in a transgenic animal under the control of a promoter/regulatory domain of choice.
  • the promoter/regulatory domain determines whether the gene is expressed constitutively (at a constant rate and constant level) or whether it is silent or induced depending upon different environmental stimuli.
  • the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) (EC 4.1.1.32) promoter is an example of the latter type of promoter/regulatory domain.
  • the constant expression of the corresponding protein product of this gene may have undesirable effects on the host animal. This could be especially deleterious during embryogenesis when the programmed expression of genes is necessary for orderly development. Consequently, it is desirable to control expression of the gene by means of an inducible or repressible promoter. Additionally, it is desirable to use a promoter which is controllably responsive to changes in diet, since these changes are readily effected.
  • PEPCK cytosolic form of PEPCK
  • cytosolic form of PEPCK a gluconeogenic enzyme discovered in 1954 by Utter and Kurahashi. This enzyme has a high specific activity in liver, kidney cortex, and white adipose tissue and in lesser levels in lung and jejunum. Hanson and Garber, Am. J. Clin. Nutrition, 25:1010 (1972); Utter and Kurahashi, J. Biol. Che . , 207: 287 (1954).
  • cytosolic and mitochondrial forms of PEPCK encoded by different nuclear genes. There are species-specific variations in the expression of both PEPCK forms.
  • Gluconeogenesis is a process by which non-hexose precursors are converted to glucose to support glucose ho eostasis in all vertebrate animals. It occurs only in the liver and kidney cortex. Gluconeogenesis from lactate (Cori cycle) involves 13 enzymes and includes several reactions which also play a role in the citric acid cycle, or in glycolysis, as well as other reactions which are specific for this process.
  • the major precursors for glucose synthesis, in addition to lactic acid, are pyruvic acid, amino acids (such as alanine or glutamine) and glycerol. The pathway is stimulated during periods of starvation or during diabetes, and is depressed by dietary carbohydrate.
  • gluconeogenesis The major hormonal controls of gluconeogenesis are glucagon (acting via cAMP) , which stimulates gluconeogenesis, and insulin, which represses the synthesis of glucose. It is important to distinguish 5.
  • gluconeogenesis the de novo synthesis of glucose (gluconeogenesis) from glycogenolysis, the breakdown of pre-formed glucose which is stored in the liver and muscle as glycogen.
  • PEPCK is the first committed step in the gluconeogenic pathway and is the pace-setting enzyme in this process. 0
  • the marked inducibility of the gene for this enzyme reflects the important regulatory position that PEPCK plays in maintaining glucose ho eostasis.
  • PEPCK is induced by glucagon, 5 epinephrine, norepinephrine, glucocorticoids, and thyroxine, and deinduced by insulin.
  • acidosis or glucocorticoids elevate PEPCK expression, while alkalo ' sis inhibits PEPCK synthesis.
  • norepinephrine and epinephrine boost 0 PEPCK levels while insulin and glucocorticoids decrease the levels; of the enzyme. See Table I, "Factors that alter the levels of PEPCK in rat tissues", in Tilghman, et al. , "Hormonal Regulation of Phosphoenolpyruvate
  • GTP Carboxykinase
  • PEPCK Hormonal regulation of PEPCK gene expression is tissue-specific. (See Hanson and Mehlman, cited 0 above) . However, there is a paucity of information on the sequences responsible for the tissue-specific expression- of this gene or for the differences in response to hormones in tissues such as liver and adipose tissue. 5 Dietary effects on the activity of PEPCK are known. Starvation for 24 hours produces a threefold increase in enzyme activity, which is reversed by a diet high in carbohydrate (e.g., glucose, fructose, and glycerol) or exacerbated by refeeding with a high protein diet. Shrago, et al. , J. Biol. Chem., 238: 3188 (1963).
  • carbohydrate e.g., glucose, fructose, and glycerol
  • the maternal blood supply is cut off at birth, resulting in a transient neonatal hypoglycemia. This results in a fall in the concentration of plasma insulin and a rise in the level of glucagon. This causes an increase in the concentration of hepatic cAMP, which induces the initial expression of PEPCK. The appearance of this enzyme completes the gluconeogenic pathway, and hepatic gluconeogenesis is thereby initiated.
  • PEPCK gene in nuclei from the livers of animals induced by hormones is known to be high and is comparable to that reported for the heat-shock gene. See Table 1, in
  • the PEPCK promoter has been used to control expression of both the Herpes virus thymidine kinase (TK) gene and the amino-3 '-glycosyl phosphotransferase (AGPT or neo resistance) gene in transfected hepatoma (FTO-2B) cells. Both'TK and AGPT synthesis were responsive to cAMP and dexamethasone. Id. ; Wynshaw-Boris, et al., BioTechniques, 4(2) :104(1986) .
  • TK Herpes virus thymidine kinase
  • AGPT amino-3 '-glycosyl phosphotransferase
  • yeast promoters which control genes coding for enzymes in the glycolytic pathway (hexokinase 1 and 2, phosphoglucose isomerase, phosphoglycerate kinase, triose phosphate isomerase, phosphoglycerate utase, pyruvate kinase, phosphofructokinase, enolase, fructose 1, 6-diphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase , and glycolysis regulation protein) . These are coupled to foreign genes and used to control expression of those genes in transformed yeast cells.
  • Kawasaki refers in a general way to regulating the expression by choosing the appropriate nutrient medium.
  • he is limited to yeast promoters and yeast cells for the expression of any recombinant gene and production of a given gene product.
  • the gene products may also be inappropriately glycosylated due to the fact that they are secreted by yeast cells.
  • the glycolytic yeast promoters cannot by used in intact animals and will not be expressed in organisms other than yeast.
  • Kingsman, US 4,615,974 specifically used the yeast phosphoglycerate kinase (PGK) promoter, a glycolytic pathway promoter, to control alpha interferon expression in yeast. Production was induced by introducing glucose into the culture medium. There is no discussion in either Kawasaki or
  • Konrad, US 4,499,188 relates to the expression of a heterologous gene in a transformed bacterial cell under TRP promoter control.
  • the medium is initially rich in tryptophan, thereby repressing the gene.
  • Bacterial growth consumes the tryptophan, eventually switching on the gene.
  • the trp promoter is limited to use in prokaryotic cells.
  • Palmiter, US 4,579,821 describes Herpes virus thymidine kinase (TK) gene expression in adult mice grown from embryos microinjected with a recombinant rDNA vector.
  • This vector contains the TK gene operably linked to the mouse metalothionein-I (MT-I) promoter.
  • MT-I mouse metalothionein-I
  • This promoter is regulatable by administration of heavy metals such as cadmium or steroid hormones such as dexa ethasone.
  • the induction of this promoter or of the mouse MT-II promoter by feeding heavy metals to transgenic animals is inherently limited by considerations of acute and chronic toxicity and teratogenicity. Prolonged feeding of steroid hormones may also have adverse effects.
  • the MT- I promoter unlike the PEPCK promoter, is active during fetal development, fetal expression of the linked exogenous gene may have deleterious effects upon a transgenic fetus.
  • PEPCK promoter may be induced using dexamethasone, see Wynshaw-Boris, et al. (1986), it is substantially more responsive to hepatic cAMP than to glucocorticoids. Indeed, while liver PEPCK activity may be induced in an adrenalectomized animal by starvation, injection of dexamethasone into a well-fed, adrenalectomized animal does not induce PEPCK activity. Reshef, et al., J. Biol. Chem., 244:5577-81(1969).
  • the PEPCK promoter is more strongly and rapidly induced by cAMP than is the MT-1 promoter by dexamethasone.
  • the expression of the PEPCK promoter is readily modulated by adjustment of the protein and carbohydrate content of the animal's diet.
  • PEPCK promoter has special utility in the regulation of the expression of genes other than PEPCK in selected tissues of transgenic animals.
  • PEPCK promoter is not induced significantly until immediately after birth.
  • the developing fetus need not contend with the expression during development of the heterologous structural gene which is linked to the PEPCK promoter.
  • PEPCK gene expression commences only after the maternal supply of glucose is cutoff by the severance of the umbilical cord.
  • Our PEPCK-controlled genetic expression system thus has the advantage that the developing embryo and fetus are protected from the improper expression of the linked structural gene.
  • PEPCK promoter when induced, is characterized by a very high level of expression. According to Meisner et al.
  • the transcription rate of PEPCK mRNA in the liver of starved rats was 3,500 ppm, as compared to 3,000 ppm for the heat shock gene of Drosophila, 270 ppm for bovine growth hormone mRNA in the bovine anterior pituitary, and 260 ppm for metallothionein mRNA in the liver of steroid-treated rats.
  • the mRNA for PEPCK has a half-life of about 30 minutes, so mRNA levels are largely dependent on the transcription rate.
  • PEPCK promoter has particular utility in controlling the expression of exogenous genes in transgenic animals, it also is useful for controlling the expression of genes by eukaryotic cells in cell culture.
  • its prime advantages are its ready inducibility and de-inducibility and high promoter strength.
  • the PEPCK promoter is preferably induced by cAMP, and de- induced by insulin.
  • cAMP and insulin offer a paired inducer-repressor system for finely controlling gene expression.
  • FIG. 1 Transgenic mouse bearing the PEPCK/bGH gene and litter-mate (white animal) .
  • the larger, black animal on the left contains, stably integrated into its genome, a chimeric PEPCK/bGH gene.
  • FIG. 3 Growth rate of the mouse containing the PEPCK/bGH transgene.
  • the animal shown in Figure 2 TG[OPCKbGH]9Fl expresses bovine growth hormone at 0.75 mg/ml of serum and has grown to be twice the size of its litter-mates.
  • FIG. 4 Effect of High Carbohydrate and High Protein Diets and Bt2 cAMP Administration on the Levels of bGH in the Serum in Transgenic Mice.
  • Panel A A representative transgenic mouse, expressing low levels of serum bGH was placed on a high carbohydrate diet for 1 week. The animal was then fed a high protein diet devoid of carbohydrate for 1 week. Blood was drawn from the tail vein at the time intervals indicated and the levels of bGH in the serum determined.
  • Panel C Four transgenic mice, expressing bGH at serum concentrations ranging from 2.4 ng/ml to 1.4 ug/ml, were injected with Bt 2 cAMP and theophylline (both 30 mg/kg) at 3 consecutive 30 min intervals. After 90 minutes the mice were bled from the tail vein and the concentration of bGH in the serum determined. Changes in the levels of bGH in the serum are expressed as a "fold increase.”
  • Panel D A transgenic mouse was treated as described in Panel C and the alteration in the concentration of serum bGH determined.
  • FIG. 5 Tissue specific expression of the bovine growth hormone mRNA in transgenic mice containing the PEPCK/bGH gene. Analysis of bGH mRNA by Northern blotting using as a probe a segment of DNA containing the PEPCK promoter and bGH structural gene (Eco RI/Eco RI fragment) shown in Figure 6. Each lane contains 30 ug of total RNA extracted from various tissues, taken from transgenic mice.
  • the mRNA produced by the chimeric PEPCK /bGH gene contains 73 bp of PEPCK mRNA and 1.0 kb of RNA coding for bGH.' The position of both mRNA species is indicated directly on the figure, together with molecular size markers. Panel A.
  • Transgenic mouse which expresses high levels of bGH (750 ng/ml of serum) lanes 1-7, 1, liver; 2, spleen; 3, kidney; 4, heart; 5, lung; 6, brain; 7, intestine.
  • Panel B Transgenic mouse which expresses low levels of bGH (10 ng/ml of serum) . Lanes 1-7 are the same as in Panel A. Figure 6. Construction of pPCGH. A-548 to +73 fragment of the .PEPCK promoter/gene is cloned into pGH to obtain pPCbGH.
  • FIG. 7 Expression of a chimeric gene containing the PEPCK promoter ligated to the structural gene for AGPT or neo.
  • the 710 bp fragment is a transcript from the 5'LTR of the retrovirus (viral LTR RNA) and the 375 bp fragment is protected by a RNA transcript from the PEPCK promoter (PCK-neoRNA) .
  • the retroviral vector pLJ is shown in the right of the Figure as a circle. At the bottom is the retroviral vector pLJPCKneo containing the PEPCK promoter ligated to the neo structural gene and contained within the LTRs of the retroviral vector.
  • the plasmid pLJPCKneo was used for the experiments described in Figure 7.
  • Mouse DNA extracted from tail biopsies, was digested with either Pvu II or Kpn I (as indicated) .
  • Mouse DNA was from founder #34, and offspring 34-2, 34-4, 34-6 and 34-7.
  • Plasmid pPCGH was digested with Kpn I and Pvu II for control. Twenty ug of the restriction cut DNA was separated by electrophoresis and transferred to a nitrocellulose filter. The filter was hybridized with the Eco Rl-Eco Rl PEPCK/bGH fragment labeled by nick translation.
  • FIG. 10 Pedigree Illustrating Differential Transmission and Expression of the Chimeric PEPCK/bGH Gene from an Fl Male Expressing High Levels of bGH.
  • Squares represent males, circles females, and diamonds animals which died prior to assay for integration of the transgene. Filled circles or squares represent animals which were heterozygous for the PEPCK/bGH gene as determined by dot blot analysis.
  • Transgenic male animals were either out-bred to C57BL6 x SJL females or mated with heterozygous transgenic females as indicated.
  • Serum bGH levels are expressed as ng of bGH/ l of serum.
  • Fl and F2 litters are numbered for referral to Table 1. The heterozygous female from litter #2, expressing 500 ng bGH per ml of serum, is that shown in the mating between the heterozygous Fl male and female.
  • a "transgenic animal” is any animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus.
  • the term is not intended to encompass classical cross ⁇ breeding or _Ln vitro fertilization, but rather is directed to encompass animals in which one or more cells bear a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information then they, too, are transgenic animals.
  • the information may be foreign to the species of animal to which the recipient belongs, e.g., a bovine growth hormone gene in a rat cell. It may be foreign only to the particular individual recipient, such as a gene encoding an enzyme that has been introduced into an individual who congenitally lacks the ability to synthesize that enzyme. Or it may be genetic information already possessed by the recipient. In the last case, the introduced gene may be more efficiently expressed, or expressed under conditions different than the native gene, as a result of the manipulation.
  • PEPCK promoter when used without qualification, shall include the promoters for the PEPCK gene of any animal genome, or their artificial equivalents, as well as modifications of same, which are responsive to cAMP and insulin. The term thus does not include the promoter of the gene encoding the mitochondrial isozyme of PEPCK.
  • the promoters of other genes which are expressed significantly only post-parturition (after birth) may be of value in controlling the expression of genes introduced into animals.
  • One such promoter is that of the gene which codes for tyrosine aminotransferase. It will be understood that while it is desired that the promoter be selected so that the transgenic animal be essentially incapable of expression of the gene before birth, some expression may be tolerable. The nature of the gene will dictate whether its expression before birth at a particular level is acceptable or not.
  • the ratio of adult-to-fetal expression of enzyme activity was about 60:1.
  • the promoter of this invention will provide a ratio of adult-to-fetal expression of the linked gene of at least 10:1.
  • it will also have a signal strength of at least 1000 ppm, i.e., about four times the bGH or MT promoters.
  • promoters responsive to dietary signals might include those for the gene coding the NADP-malate dehydrogenase and fatty acid synthase. They are stimulated by dietary glucose (acting via insulin) and inhibited by glucagon.
  • the PEPCK promoter may be used to control the expression of any structural gene of interest, such as those encoding bovine growth hormone, adenosine dea inase, thyrotropin-releasing hormone, beta-globin; including oncogenes, and marker genes such as herpes virus' thymidine kinase gene and the bacterial transposon AGPT gene.
  • the PEPCK promoter was linked with the bovine growth hormone gene, but it will be understood that the invention is not limited to the PEPCK/bGH expression system.
  • PEPCK/bGH The rationale for the use of PEPCK/bGH is several- fold.
  • the usefulness of GH in altering body composition in livestock has been demonstrated. Currently, however, the only method for administering GH is injection directly into the animal.
  • TRH thyroid releasing hormone
  • the bovine growth hormone genomic sequence is known.
  • a bGH genomic sequence insert was removed from a lambda Charon 28 clone of a bovine placental library.
  • An Eco RI restriction digest removed a 4.3 kb sequence containing the entire structural gene, 1.7 kb of 5'-flanking sequence, and 400 bp of 3'-flanking sequence, and this was cloned into the unique Eco RI site of the commercially available plasmid pBR322.
  • the resulting bGH transfer vector is publicly available. See Woychik, et al. , Nucl. Acids Res., 10: 7197 (1982).
  • the bGH gene or other genes of interest, need not be of genomic origin. It may be a cDNA transcript, or it may be partially or wholly synthetic. References for techniques for cloning bGH are cited in the "Background" section.
  • cytosolic PEPCK promoters may be advantageously used to control the expression of a gene of interest.
  • the itochondrial form of the PEPCK enzyme is constitutively expressed under the control of a different promoter and is encoded by a different gene.
  • the promoter associated with the gene encoding the native mitochondrial form of PEPCK is not of great value in the present invention since it is not subject to the same acute regulation as the gene for the cytosolic form.
  • deletion or substitution mutants of the native promoter may have advantages in specific situations.
  • a series of 5'-deletion mutants are described in Short, et al., (1986).
  • This and other articles cited in the "Background” section have characterized the different regulatory domains of the PEPCK promoter and therefore provide some guidance as to where to change the sequence.
  • a cAMP regulatory elements is believed to lie between -91 and -80.
  • the -61 to -416 segment can act as a hormonally active enhancer. Wynshaw-Boris, et al., (1986).
  • glucocorticoid raetallothionein
  • cAMP preproso atostatin, plas inogen activator, vasoactive intestinal polypeptide
  • PEPCK promoter-bearing unit The replaced DNA included the entire 5'-flanking sequence of the bGH gene, as is shown by the markings on the bGH sequence appearing in Fig. 6.
  • the resultant fusion gene includes 73 bp of the first exon of the PEPCK structural gene linked at the Bgl II site to the start of the first exon of bGH.
  • the translation start site of PEPCK is further "downstream" than +73, there is no interference with the normal translation of the bGH gene-derived mRNA into active bGH.
  • the ligation of the PEPCK gene promoter to other genes may be accomplished by similar means, possibly including the use of other restriction enzymes and/or linker or adapter molecules.
  • the basic considerations are that the PEPCK promoter remain functional, that the entire structural gene of interest be expressed, and that no portion of the PEPCK gene is expressed as a part of a fusion protein including the desired polypeptide.
  • a chimeric PEPCK promoter/bGH gene-bearing plasmid Any art-recognized method may be used to prepare a transgenic animal bearing the desired expression system.
  • embryonic cells of the desired host animal were transformed by microinjection, and allowed to complete development and growth.
  • single cell embryos are flushed from the oviduct of superovulated C57BL6/SJL mice 14 hours after fertilization.
  • the ova are washed with hyaluronidase to remove cumulus cells and transferred to a slide, in a salt medium containing lactate and pyruvate, for microinjection.
  • an injector pipette containing approximately 1 pi of DNA solution 200 copies of the PEPCK/bGH gene
  • retroviral genome can be manipulated to include an exogenous gene and exogenous promoter.
  • the recombinant retroviral genome can be packaged within its viral capsid and used as viable infectious virus. Embryos at the single cell stage or later stages of development can be infected with the recombinant retrovirus and the provirus containing the exogenous gene will become integrated into the host genome as a single copy. Therefore, infection with recombinant retrovirus allows for the integration of an exogenous gene under the control of its own promoter or a heterologous promoter within the embryonic genome. Infected embryos can then be transferred to pseudopregnant female mice in the same manner that microinj cted embryos are transferred, and the resultant offspring then assayed for the presence of the exogenous gene.
  • PEPCK promoter linked to the AGPT gene; these have been tested for expression by infection of fibroblast cells in culture.
  • the PEPCK promoter is active within the provirus and high levels of PEPCK/AGPT mRNA have been detected in infected cells. Transcription is initiated at the proper start site and accurate hormonal regulation of transcription is observed. Therefore, the PEPCK promoter/regulatory domain will be very useful for this alternate method of production of transgenic animals.
  • the PEPCK promoter linked to an appropriate structural gene and incorporated into a retroviral vector, may be used to infect cells of fetal animals during development.
  • the injection of a retrovirus into the peritoneal cavity of 19-day fetal animals (last trimester of development) results in the integration of the chimeric PEPCK-AGPT or neo gene into the chromosomes of the liver. While not all liver cells are infected, we can detect mRNA in the liver and the transcription of the gene is stimulated by cAMP.
  • This procedure is effective, presumably because the liver is differentiating from a hemopoietic tissue to a hepatic tissue during this stage of development.
  • This differentiation involves DNA replication and the infectious retrovirus then integrates into the liver cell genome. Since the retrovirus is a replication deficient virus, the animal will not produce further rounds of viral infection.
  • This technique has potential for effectively introducing genes into animal tissues late in development, by a relatively non- invasive technique.
  • Yet another possibility is to introduce the vector into a cell and introduce the transformed cell into the animal under conditions favoring the propagation of the cell.
  • the transgene may also be introduced into the animal after birth.
  • Five mice (three-weeks old) received an injection into its tail vein of a volume of 1 ml of tissue culture media containing 10 7 particles of a replication-incompetent murine retrovirus bearing the PEPCK/bGH transcription unit.
  • serum samples exhibited a bGH concentration of 20-50 ng/ml.
  • This invention is not limited to any particular method of introducing the transgene into the animal.
  • Homozygous transgenic mouse lines are established by mating positive founder animals (positive offspring that result from the microinjected embryos) with normal mice of the same hybrid line.
  • the Fl generation that is produced should be 50% heterozygous for the transgene if the transgene is contained in all of the germ cells of the founder.
  • the heterozygous Fl animals are interbred and the F2 generation which is produced should be 25% homozygous for the transgene, 50% heterozygous, and 25% wild type.
  • the F2 generation animals homozygous for the- transgene can be produced; when these animals are bred with other homozygous mice the following generation is 100% homozygous at the transgene locus and a homozygous line has been established.
  • mice were first screened for the presence of the exogenous PEPCK/bGH DNA by dot blot and Southern analysis. Mice which were . positive by both these criteria (i.e., which exhibited the presence of the foreign gene and which yielded restriction fragments of the predicted length) were tested for expression of bGH by ELISA assay.
  • DNA was extracted from segments of the tail (about 1 cm) , according to a modification of the procedure of Davis, et al., Meth. Enzymol. , 65: 404-411 (1980).
  • Tail sections from potentially transgenic mice were crushed to a powder in liquid nitrogen.
  • the dry powder was added to an extraction buffer which contained 100 ug.ml proteinase K, 0.5% SDS, 0.1 M NaCl, 50 mM Tris pH 7.5 and 1 mM EDTA, and then incubated overnight at 55°C.
  • RNase Tl was added at a final concentration of 10 U/ml and the samples incubated 1 hour at 37°C. After RNase treatment, the DNA was extracted with a mixture which contained equal volumes of phenol and chloroform, then extracted with an equal volume of chloroform, and then ethanol precipitated.
  • the primary screening for positive transgenic animals was by DNA dot blot analysis.
  • DNA extracted from the tails of mice was denatured in 0.1 M NaOH/2.0 M NaCl and applied to a nitrocellulose filter on a Schleicher and Hydr manifold apparatus.
  • a known amount of pPCbGH plasmid DNA was used as a standard for determining the copy number of the transgene.
  • the nitrocellulose filter was baked for 2 hours and prehybridized in a 50% formaide, 20 mM PIPES, 0.5% SDS solution containing 100 ug/ml denatured salmon testis DNA.
  • the probe utilized for hybridization is shown in Figure 6 (ECORl-BamHI PEPCK/bGH segment of pPCGH) .
  • This DNA fragment was labeled with [a- 32 P]-dCTP by nick translation according to the procedure of Rigby, et al., J. Mol. Biol., 113: 237-251 (1977) .
  • the copy number per haploid genome of the gene in positive, transgenic animals was determined by dot blot analysis. The dots were excised from the nitrocellulose after hybridization and autoradiography, and the hybridized radioactivity determined by liquid scintillation counting. The radioactivity hybridized to standard DNA samples increased linearly with amount spotted.
  • mice which were positive for both integration and expression of the chimeric PEPCK/bGH gene served as founder animals for the development of individuals transgenic lines.
  • mice which were initially screened for the presence of the transgene, 2 were positive for integration (#9 and #34) .
  • Analysis of the DNA from these transgenic mice indicated that the PEPCK/bGH gene was integrated in a tandem head-to-tail repeat at single chromosomal loci in both founder animals. Digestion of the genomic DNA with Kpn I, an enzyme which cuts once within the gene v gave a fragment of the predicted length of approximately 2,700 bp, as indicated in Figure 2, for founder #34 and offspring. Because the gene is present as a tandem, head-to-tail array, digestion with Kpn I resulted in multiple copies of a restriction fragment of the same length- as the entire PEPCK/gGH gene, hybridized to the DNA probe ( Figure 9) .
  • the first generation offspring of founder #9 exhibited different dot blot patterns; these ranged from 1-5 copies per cell to 25-50 copies per cell.
  • First generation offspring of founder #9 which contained different copy numbers of the gene, became the founders of individual lines. Matings between two heterozygous animals which are offspring of founder #9 (whose genomes contained 25 copies per cell) resulted in the death of a significant number of the offspring within a few days after birth ( Figure 10) . This may indicate insertional mutagenesis in homozygous animals at a genetic locus important in development.
  • the copy number in the founder #34 line was uniform, 25 copies per cell in the founder and Fl generation mice.
  • the ELISA enzyme-linked immunosorbent assay
  • the ELISA which we employ to determine the concentration of bGH in serum, involves the use of alkaline phosphatase streptavidin which binds to biotinylated goat anti- human IgG.
  • the activity of the bound alkaline phosphatase can be measured by a colorimetry assay and therefore does not entail the use of radio-labeled ligands.
  • Micro titer plates are initially coated with guinea pig anti-bGH, in excess such that it binds all bGH which is added in the samples to be assayed. Serum samples and purified bGH are added to the "coated" micro titer plates.
  • a second antibody, monkey anti- bGH is then added to bind quantitatively to in direct proportion to the amount of bGH bound to the first antibody.
  • a third antibody, goat anti-human IgG (which recognizes monkey IgG) is chemically coupled to biotin so that the streptavidin-enzyme conjugate can be used as a detecting reagent; in this case the conjugate is alkaline phosphatase *- streptavidin.
  • the bound alkaline phosphatase conjugate (which is bound to biotinylated goat anti-human IgG, in turn bound to monkey anti-bGH) is supplied with a substrate, and a colored product is generated. This product can then be measured spectrophotometrically.
  • This enzyme ⁇ reaction is linear over a long incubation period, which enhances the sensitivity of the assay method.
  • Microtiter plates were coated with guinea pig anti-bGH, diluted 1:100 in Na a C0 3 /NaHC0 3 (pH 9.6), incubated at 37°C for 1 hour and then at 4°C overnight. The plates were washed with 0.1% bovine serum albumin in phosphate buffered saline, (PBS) and "blocked" with 10% bovine serum albumin in PBS at 37°C for 1 hour. Serum samples and standards containing known amounts of authentic bGH, diluted in 1% bovine serum albumin in PBS, were added, incubated at 37°C for 1 hour and washed as described above.
  • PBS phosphate buffered saline
  • Monkey anti-bGH ⁇ diluted 1:10,000 in 1% bovine serum albumin in PBS, was added and incubated at 37°C for 1 hour.
  • Biotinylated goat anti-human IgG diluted 1:2,000 in 1% bovine serum albumin in PBS, was added, incubated at 37°C for 1 hour and washed as described above.
  • the alkaline phosphatase-streptavidin conjugate diluted 1:2,5000 in 1% bovine serum albumin in PBS, was added, incubated at 37°C for 1 hour and washed as described above.
  • the alkaline phosphatase-streptavidin conjugate diluted 1:2,500 in 1% bovine serum albumin in PBS, was added, incubated at 37°C for 1 hour and washed as described above.
  • the RNA samples were denatured at 80°C for 5 minutes, in the above MOPS, 5 mM Na acetate, 1 mM EDTA.
  • RNA samples were denatured at 80°C for 5 minutes, in the above MOPS, 5 mM Na acetate, 1 mM EDTA.
  • the RNA samples were denatured at 80°C for 5 minutes, in the above MOPS buffer, 3% formaldehyde and 0.1% SDS.
  • the electrophoresis buffer was 20 mM MOPS, 5 mM Na acetate, 1 mM EDTA, 8.1% formaldehyde.
  • the RNA was transferred directly to "Gene Screen Plus" in 20 x SSC. When the transfer was complete, the RNA was crosslinked to the "Gene Screen Plus” membrane by exposure to ultra violet light for approximately 3 minutes and then baked for 2 hours at 80°C.
  • RNA was hybridized to a nick translated (1 x 10 6 cpm/ml) Eco RI fragment of the pPCbGH chimeric gene, shown in Figure 6.
  • Both the prehybridization and hybridization solutions consisted of 50% formaldehyde. 0.2 M NaCl, 50 mM Tris pH 7.5, 10% dextran sulfate, 0.1 sodium pyrophosphate, 1% SDS, 0.2% bovine serum albumin, 0.2% Ficoll (mw 400,000), 0.2% PVP (mw 40,000) and 0.1 mg/ml salmon testis DNA. After hybridization for 36 hours, the filters were washed twice with 2 x SSC, 0.1% SDS for 5 minutes at room temperature, followed by two washings with 2 x SSC, 0.1% SDS for 30 minutes at 42°C.
  • RNA transcript of the PEPCK/bGH gene (including the 73 bp of the first exon of PEPCK) is approximately 1 kb , whereas the mRNA for the endogenous PEPCK gene is approximately 2.8 kb.
  • the probe utilized for Northern analysis hybridizes with both the chimeric PEPCK/bGH and endogenous PEPCK.
  • transgenic animals containing serum bGH were tested for the tissue specificity of this expression, they showed high levels of expression in liver.
  • panel A and B (lane 1), there is an intense band at 1.0 kb which hybridized to the pPCbGH probe.
  • the transgene was also expressed in the kidney of the mouse in panel A; the kidney is another tissue in which endogenous PEPCK is expressed.
  • No detectable PEPCK/bGH mRNA was found in the kidney of the animal in panel B, which expressed low levels of bGH, due possibly to limitations in the sensitivity of the Northern analysis.
  • expression of the transgene in the kidneys of low expressing animals has been detected using a bovine growth hormone cDNA probe labeled by random priming.
  • Table I Properties of Transgenic Mice Containing the PEPCK/bGH Gene.
  • the copies of PEPCK/bGH per haploid genome were determined by dot blot; the plasmid pPCGH was used for the copy number standard at copy number equivalents of 1,5,10,15,25,50 and 100.
  • dots were excised from nitrocellulose filters after hybridization and autoradiography, and the amount of the probe hybridized determined by liquid scintillation counting.
  • the concentration of bGH in the serum were determined as described in the Materials and Methods.
  • the growth ratio is calculated by dividing the weight of the transgenic mouse by the average weights of littermates of the same sex.
  • the high carbohydrate diet contained 81.5% sucrose, 12.2% casein, 0.3% DL- methionine, 4% cottonseed oil, 2% brewers yeast and a
  • the high protein diet contained 64% casein, 22% a-cell nutritive fiber, 11% vegetable oil, 2% brewers yeast and a 1% mineral mix with vitamins. The mice were fed these diets and water on a ad libitum basis.
  • Transgenic animals were fasted for 24 hours and then placed on a high carbohydrate (81.5%), minimum protein (12.5%) diet. After one week on this diet, the level of bGH in the serum of a representative animals dropped from a basal value of 320 ng bGH/ml serum to 14 ng bGH/ml serum. When the high carbohydrate diet was replaced with a high protein (61%) , carbohydrate-free diet, the serum bGH levels rose from 14 ng bGH/ml to 430 ng/ml after one week ( Figure 4) . Transgenic animals which expressed bGH at lower levels exhibited the same pattern of expression correlated with these alterations in diet.
  • transgenic animals were injected intraperitoneally at three 30 minute intervals with Bt 2 cAMP and theophylline. At 90 minutes blood was drawn from the tail vein of the animal and the serum tested for bGH. Serum levels of bGH were increased 2 to 3-fold within 90 minutes following the first cAMP injection ( Figure 4) . Animals which were positive for integration of the transgene, but negative for its expression were not induced to express bGH by the administration of Bt 2 cAMP. Thus, transgenic animals bearing the chimeric PEPCK/bGH gene, which express bGH, contain the cis acting sequences required for the regulation of its expression by cAMP.
  • the rapidity of the response of the PEPCK/bGh gene in the transgenic animals to the administration of Bt 2 cAMP is also predicted from the previous studies in which we have demonstrate that this cyclic nucleotide will induce the transcription of the PEPCK gene in the livers of rats by 8-fold within 20 minutes, Lamers, W.J., et al., Proc. Natl. Acad. Sci. U.S.A., 79:5137- 5141 (1982) .
  • the segment of the PEPCK promoter/regulatory region used to construct the chimeric PEPCK/bGH chimeric gene contains a cAMP regulatory element in the region between -109/-79, Short, J. M.
  • This element contains the core sequence CTTACGTCAGAGG which is also present in the promoter/regulatory region of the gene for cytosolic PEPCK for the chicken Hod, Y., et al., J. Biol. Chem. 259: 15609-15614 (1984).
  • This regulatory element has been shown to confer cAMP sensitivity on a heterologous gene containing its own promoter and to function in a variety of cell types into which it was introduced by either transfection. Short, J. M. , et al., Biol. Chem. 261: 9721-9726 (1986) or by infection with a retrovirus.
  • transgene is integrated into the host DNA in a manner which preservers not only its tissue-specific expression, but also the ability of the gene to be regulated by the same hormones which control the expression of the native gene in the normal chromosomal location.
  • Different patterns of integration and expression of the chimeric PEPCK/bGH gene was observed in the lines of transgenic mice.
  • Founder animal #34 which expressed bGH at approximately 25 ng/ml serum, transmitted the gene to progeny in the predicted Mendelian ratios, consistent with the presence of the transgene in all of the germ cells of the founder.
  • the progeny while containing the equivalent number of copies as the founder animal, did not express detectable levels of bGH. This has also been reported for other genes in transgenic animals.
  • Founder animal #9 which was positive for integration and expression of the PEPCK/bGH gene, was mosaic. Significantly less than 50% of the progeny of this founder animal contained the transgene and of those positive for the gene, some expressed it whereas others did not. When two Fl animals (offspring of founder #9) , heterozygous for, but not expressing the transgene, were mated, homozygous animals were produced which did express bGH
  • a segment of one transgenic line is depicted as a pedigree in Figure 10.
  • the founder animal Tg[OPCGH]9 (#9) was genetically mosaic and expressed bGH at high levels.
  • One Fl male with high levels of expression and a 2-fold increase in growth was chosen to initiate a transgenic line of animals which expressed high levels of growth hormone.
  • the PEPCK/bGH gene is transmitted from the high expressor Fl male in a Mendelian fashion to the F2 generation.
  • not all of the animals which contain the transgene express bGH and those that do, express it at varying levels although the copy numbers are the same.
  • significantly more males of the F2 generation than females (11 out of 25 transgenic males vs. 1 out of 21 transgenic females, in 12 litters tested) expressed the gene at high levels, although females have integrated within their genomes an equal number of copies of the gene.
  • the acute responsiveness of the PEPCK promoter/regulatory region to induction by diet and hormones and its tissue-specific expression in the liver and kidney make it an ideal tool for targeting various structural genes of interest to these tissues. It is also possible to modulate the level of expression of the structural gene over a broad range by altering the carbohydrate content of the diet of the transgenic animal. Since PEPCK gene is normally not expressed until birth, the developing fetus is not exposed to a high level of protein from the linked structural gene. This has clear advantages with hormones such as GH, which have the potential of interfering with the normal development of the fetus. We have noted normal reproductive capacity of the animals which were generated from this initial series of experiments. It should also be possible to use the tissue specific element in the PEPCK regulon to direct the expression of a heterologous gene, containing its own promoter, to the liver.
  • mice with both high and low levels of expression of the bGH gene were more sensitive to the administration of insulin than were mice not expressing the chimeric PEPCK/bGH gene. Mice which contained the transgene, but did not express it, were no more sensitive to insulin than normal, control animals.
  • the administration of low concentration of • insulin (0.05 u /kg) to transgenic animals was lethal in the absence of glucose gavage.
  • transgenic swine with a phenotype mimicking the performance traits of swine injected with the growth hormone protein approximately 400 copies of a 2.8 kilobase (kb) linear fragment containing the PEPCK-bGH gene•were injected into the pronuclei of fertilized swine eggs.
  • kb 2.8 kilobase
  • the approximate number of integrated PEPCK-bGH gene sequences in these animals was determined by dot hybridization and positive animals were demonstrated to contain the integrated sequences with copy numbers ranging from 1 to 200 copies per cell. Seventeen transgenic animals showed significant levels of bovine growth hormone protein in their circulating serum, with concentrations ranging from 5 ng/ml to 200 ng/ml, as determined by radioimmune and ELISA assays. A detailed analysis of the integration and expression of the bGH gene in two of these animals (#11 and #44) is presented here .
  • Genomic DNA extracted from the tails of pigs was digested with Eco RI, Kpn 1, Pst I and Pvu II.
  • the digested DNA was hybridized with the Bam HI-Bgl II fragment from -547 to +73 of the rat PEPCK gene, labeled by "random priming.”
  • Eco RI digestion of the genomic DNA yielded a 2.8 kb fragment which hybridized with the above probe, indicating that the transgene was present as a tandem repeat integrated within the host genome in pigs #11 and #44.
  • the predicted internal fragments of 740 bp and 520 bp were observed in genomic DNA from both animals.
  • a third fragment which spans the junction between the tandem repeats is the same size as the 740 bp internal fragment.
  • the transgene is present as a tandem repeat in which some of the copies of the gene are inverted, as indicated by the restriction fragments generated after digestion with Kpn I and Pst I.'
  • a head to tail, tail to head repeat of the transgene should yield a 5.0 kb fragment after digestion to head repeat of the transgene should yield a 5.0 kb. fragment after digestion with Kpn I, which should hybridize with the Bam HI-Bgl II DNA probe in two positions.
  • Genomic DNA from both pig #11 and pig #44 when digested with Kpn I, yielded a 5.0 kb fragment. Digestion with Pst I should yield a 1.2 kb fragment which hybridizes with the probe at two positions when the transgene is inverted. DNA from pig #44 yielded a 1.2 kb fragment upon Pst 1 digestion, which is consistent with the presence of adjacent head to head repeats.
  • the 1.7 kb fragment is an endogenous PEPCK fragment which hybridizes with the Bam HI-Bgl II probe.
  • RN isolated from the liver, kidney, lung, spleen and intestine of pig #44 was subjected to SI nuclease digestion after hybridization with a 5'-end labeled fragment of DNA generated by restriction endonuclease digestion of PEPCK/bGH gene with BamHl and Pst I.
  • RNA from a transgenic mouse containing the same ' PEPCK/bGH chimeric gene, and expressing bGH at levels >500 ng/ml serum was analyzed.
  • a 133 bp fragment of the. Bam Hl-Pst I probe was protected from nuclease digestion by RNA from pig liver, but not by RNA from any of the other pig tissues examined.
  • the 460 bp of 5'-flanking sequence from the PEPCK gene can direct the tissue specific expression of the linked bGH structural gene in a transgenic pig.
  • PEPCK is expressed primarily in the liver and kidney cortex in mammalian species; however, PEPCK/bGH mRNA could be detected only in the liver of pig #4 .
  • the transgenic mouse that mRNA is present in both liver and kidney, although the ratio of liver to kidney mRNA is much lower than is observed for endogenous PEPCK mRNA, Meisner, H. , et al., Biochemistry 224: 412- 425 (1985) .
  • SI nuclease analysis also indicates that the PEPCK/bGH chimeric gene in the pig uses the correct start site of transcription, since the predicted size fragment (133 bp) is protected.
  • Pig #11 shown by duplicate radioimmune and ELISA assays ⁇ to contain 200 ng/ml of bGH in his serum, was placed on a similar feed regimen compared to his non- transgenic male littermate (#12) . Feed:weight gain ratios and back fat measurements were compared. The feed:gain ratio was significantly reduced in PEPCK-bGH transgenic pig #11 as compared to the non-transgenic control (30% decrease during restricted feeding conditions. Pigs #11 and 12 were compared during two consecutive periods of approximately 45 days each. Animals were fed a 16% crude protein commercial finishing ration during both periods. Pigs were 125 - days old and weighed between 50 and 75 kg when the ad libitum feeding period began (period 1) . Animals were restricted to 2.8 kg of feed per day during the second period.

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Abstract

Dans l'animal transgénique construit, une ou plusieurs cellules contiennent un promoteur du gène pour la PEPCK cytosolique liée de manière opérative à un gène non PEPCK d'intérêt. L'expression de ce gène est régulée en modifiant les composants de protéines et d'hydrates de carbone du régime de l'animal, ou par régulation hormonale directe. Le promoteur de la PEPCK est induit par une concentration élevée de protéines et est inhibé par une concentration élevée d'hydrate de carbone ou plus directement par cAMP et de l'insuline. Le gène lié est exprimé essentiellement uniquement après la naissance et essentiellement uniquement dans des tissus particuliers. Le promoteur de PEPCK possède une résistance extrêmement élevée.
PCT/US1988/001943 1987-06-16 1988-06-13 Regulation dietetique et hormonale de l'expression de genes exogenes dans des animaux transgeniques sous le controle du promoteur du gene pour la phosphoenolpyruvate carboxykinase WO1988010304A1 (fr)

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GB2223755A (en) * 1988-09-21 1990-04-18 Cambridge Animal Biotech Tissue modification
US5320952A (en) * 1989-09-21 1994-06-14 W. R. Grace & Co.-Conn. Enhanced gene expression in response to lactation signals
WO1995011308A1 (fr) * 1993-10-18 1995-04-27 Amgen Inc. Mammifere presentant une expression hepatique activee d'un transgene
EP0665883A4 (fr) * 1992-08-26 1995-06-22 Dnx Corp Systeme de regulation binaire, a mediation par un represseur, qui utilise la tetracydine pour controler l'expression de genes chez les animaux transgeniques.
WO1995023866A1 (fr) * 1994-03-04 1995-09-08 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Vecteur pour la therapie genique specifique du foie
WO1995025169A1 (fr) * 1994-03-14 1995-09-21 Universitat Autonoma De Barcelona GENE CHIMERE UTILISANT LE GENE OU L'ADNc DE L'INSULINE, EN PARTICULIER POUR LA THERAPIE GENIQUE DU DIABETE
WO1996025487A1 (fr) * 1995-02-14 1996-08-22 Ecole Polytechnique Federale De Lausanne Substances liees a la regulation de la production de polypeptides dans des cellules et techniques correspondantes
EP0737746A3 (fr) * 1989-12-01 1996-10-23 Pharming B.V. Production de polypeptides recombinants par l'espèce bovine et procédés transgéniques
US5795752A (en) * 1991-11-20 1998-08-18 Trustees Of Dartmouth College Method for creating superinduced cDNA library & isolating ligand-stimulated genes
ES2154505A1 (es) * 1994-03-04 2001-04-01 Univ Barcelona Autonoma Gen quimerico que utiliza el gen o cdna de la 3-hidroxi-3-metilglutaril-coa sintasa mitocondrial y animal transgenico no humano que expresa dicho gen quimerico para su utilizacion en el desarrollo de aproximaciones terapeuticas para diabetes mellitus.
ES2155288A1 (es) * 1992-07-30 2001-05-01 Uni Autonomo De Barcelona Gen quimerico que utiliza el gen o cdna de la insulina, en especial para terapia genica de la diabetes.
ES2156460A1 (es) * 1994-07-07 2001-06-16 Univ Barcelona Autonoma Animal transgenico no humano que sobreexpresa el gen de la p-enolpiruvato carboxiquinasa para su utilizacion en el estudio de la diabetes mellitus y en el desarrollo de aproximaciones terapeuticas a la enfermedad.
US6333447B1 (en) 1996-03-29 2001-12-25 The General Hospital Corporation Transgenic model of heart failure

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2223755A (en) * 1988-09-21 1990-04-18 Cambridge Animal Biotech Tissue modification
US5320952A (en) * 1989-09-21 1994-06-14 W. R. Grace & Co.-Conn. Enhanced gene expression in response to lactation signals
EP0737746A3 (fr) * 1989-12-01 1996-10-23 Pharming B.V. Production de polypeptides recombinants par l'espèce bovine et procédés transgéniques
US5795752A (en) * 1991-11-20 1998-08-18 Trustees Of Dartmouth College Method for creating superinduced cDNA library & isolating ligand-stimulated genes
ES2155288A1 (es) * 1992-07-30 2001-05-01 Uni Autonomo De Barcelona Gen quimerico que utiliza el gen o cdna de la insulina, en especial para terapia genica de la diabetes.
EP0665883A4 (fr) * 1992-08-26 1995-06-22 Dnx Corp Systeme de regulation binaire, a mediation par un represseur, qui utilise la tetracydine pour controler l'expression de genes chez les animaux transgeniques.
WO1995011308A1 (fr) * 1993-10-18 1995-04-27 Amgen Inc. Mammifere presentant une expression hepatique activee d'un transgene
US6268212B1 (en) 1993-10-18 2001-07-31 Amgen Inc. Tissue specific transgene expression
WO1995023866A1 (fr) * 1994-03-04 1995-09-08 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Vecteur pour la therapie genique specifique du foie
ES2154505A1 (es) * 1994-03-04 2001-04-01 Univ Barcelona Autonoma Gen quimerico que utiliza el gen o cdna de la 3-hidroxi-3-metilglutaril-coa sintasa mitocondrial y animal transgenico no humano que expresa dicho gen quimerico para su utilizacion en el desarrollo de aproximaciones terapeuticas para diabetes mellitus.
US6137029A (en) * 1994-03-14 2000-10-24 Universitat Autonoma De Barcelona PEPCK-insulin gene construct and transgenic mouse
WO1995025169A1 (fr) * 1994-03-14 1995-09-21 Universitat Autonoma De Barcelona GENE CHIMERE UTILISANT LE GENE OU L'ADNc DE L'INSULINE, EN PARTICULIER POUR LA THERAPIE GENIQUE DU DIABETE
ES2156460A1 (es) * 1994-07-07 2001-06-16 Univ Barcelona Autonoma Animal transgenico no humano que sobreexpresa el gen de la p-enolpiruvato carboxiquinasa para su utilizacion en el estudio de la diabetes mellitus y en el desarrollo de aproximaciones terapeuticas a la enfermedad.
AU699061B2 (en) * 1995-02-14 1998-11-19 Centre Hospitalier Universitaire Vaudois Materials and methods relating to the regulation of polypeptide production in cells
US6074875A (en) * 1995-02-14 2000-06-13 Ecole Polytechnique Federale De Lausanne Materials and methods relating to the regulation of polypeptide production in cells
WO1996025487A1 (fr) * 1995-02-14 1996-08-22 Ecole Polytechnique Federale De Lausanne Substances liees a la regulation de la production de polypeptides dans des cellules et techniques correspondantes
US6333447B1 (en) 1996-03-29 2001-12-25 The General Hospital Corporation Transgenic model of heart failure

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