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WO1991002793A1 - Developpement d'une cardiomyopathie chez des mammiferes non humains transgeniques - Google Patents

Developpement d'une cardiomyopathie chez des mammiferes non humains transgeniques Download PDF

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WO1991002793A1
WO1991002793A1 PCT/CA1990/000270 CA9000270W WO9102793A1 WO 1991002793 A1 WO1991002793 A1 WO 1991002793A1 CA 9000270 W CA9000270 W CA 9000270W WO 9102793 A1 WO9102793 A1 WO 9102793A1
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human mammal
pvlt
sequence
promoter
transgenic
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Lorraine Elizabeth Chalifour
Margaret Laurin Gomes
Anne-Marie Mes-Masson
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Canada Minister of Natural Resources
National Research Council of Canada
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National Research Council of Canada
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to transgenic non-human mammals containing the sequence of the polyomavirus large T-antigen (PVLT) under the direct control of a non polyomavirus promoter. These mammals are characterized by an enhanced propensity towards the development of cardiomyopathy. BACKGROUND OF THE INVENTION
  • oncogenes may be used for the production of transgenic animals such as transgenic mice.
  • oncogenes suited for the production of transgenic mice, one may mention the remarkable usefulness of the polyomavirus antigens.
  • the early region of polyomavirus contains genetic information for three viral encoded antigens ? large (100 Kd), middle (56 Kd) and small (22 Kd) T-antigens that arise by differential splicing of a primary transcript.
  • Middle T-antigen (PVMT) is a membrane protein associated with c-src and has been clearly demonstrated as the transforming protein of polyomavirus whereas small T-antigen (PVST) has an undefined role in transformation.
  • PVLT Polyomavirus large T- antigen
  • simian virus 40 and polyomavirus have been included in the genomes of transgenic mice under the direction of their own or heterologous promoters. In most instances, tumors developed in these animals after a latent period indicating that expression of the transgene alone was insufficient to produce tumors.
  • mice containing the polyomavirus large T-antigen under the control of the polyomavirus promoter have been described and usually resulted in detection of expression in the transgenes in pituitary gland and testes.
  • Hamsters and mastomys, an African rodent when infected with polyomavirus have developed tumors in many sites including the heart. It has been demonstrated that both the structural portion of the early region as well as the non-coding elements in the promoters/enhancer of the polyomavirus contribute to oncogenicity. It has also been shown that polyomavirus is generated most easily in baby mouse kidney cells although the lytic phase has also been observed in cells cultured from heart, thymus, spleen and kidneys of adult mice.
  • MT heavy-metal responsive metallothionein promoter
  • Heart disease is one of the leading cause of death in North America. Although substantial progress has been made in understanding the exact causes of cardiac arrest, much more needs to be done especially in terms of developing efficient live models on which to study heart disease. SUMMARY OF THE INVENTION
  • a transgenic non-human mammal preferably a rodent and more preferably a mouse, whose germ cells and somatic cells contain the sequence of the polyomavirus large T-antigen under the direct control of a non-polyoma promoter, preferably an inducible promoter, and more preferably the metallothionein promoter.
  • a non-polyoma promoter preferably an inducible promoter, and more preferably the metallothionein promoter.
  • non-polyoma promoter when used in the context of the present invention, is meant to include any promoter different from the promoter normally controlling the transcription of the PVLT sequence.
  • the above-mentioned fusion gene is introduced into the mammal or into an ancestor of this animal- at an embryonic stage.
  • the mammal produced through the introduction of this fusion gene is characterized by an enhanced propensity towards the development of cardiomyopathy. Therefore, in these mammals, the expression of the polyomavirus large T-antigen is associated with a cardiac enlargement leading to premature death due to cardiac arrest.
  • a method for inducing cardiomyopathy in a non-human mammal comprises introducing into the non- human mammal or an ancestor thereof at an embryonic stage, a fusion gene comprising the sequence of the polyomavirus large T-antigen and a non-polyoma virus promoter sequence.
  • the surviving transformed embryos are implanted into pseudopregnant females and the resulting progeny is screened for the presence of transgenes.
  • the enlarged heart in the MT-PVLT affected mice retains its overall structural organization as evidenced by a relatively normal gross architecture and the absence of any obvious sites of focal growth.
  • hearts from infected mammals are proportionately enlarged unlike the specific right atrial enlargement found in atrial-natriuretic factor-SV40 T-antigen transgenic mice developed previously. Histological analysis demonstrates that PVLT was able to influence the growth of both atrial and ventrical cardiomyocytes.
  • transgenic mammals developed in the context of the present invention are useful in the development of models and methods for the study of cardiomyopathy and could be used in the study of congestive heart failure.
  • they can be used in physiological studies aimed at determining -the effects of congestive heart failure on other organs or to test the effect of various pharmaceutical compounds on individuals suffering from a precarious heart condition.
  • the mammals can also be used as one component of a kit that may be sold for those specific purposes or as an element used in one of the steps of a method or protocol to assess the effect of various conditions or compounds on the overall condition of the heart.
  • transgenic mammals obtained in the context of the present invention represent a totally unexpected finding.
  • these novel mammals are the first transgenic mammals to develop a proportionately oversized heart through the introduction of an oncogene sequence.
  • transgenic mammals of the present invention are developed using the cDNA for polyomavirus large T- antigen (PVLT), preferably under the direction of a non- polyoma promoter such as the inducible heavy-metal responsive metallothionein promoter (MT).
  • PVLT polyomavirus large T- antigen
  • MT inducible heavy-metal responsive metallothionein promoter
  • Figure 1 is a schematic representation of the procedure through which plasmid pMT-PVLT is derived
  • Figure 2a represents the acrylamide gels obtained from RNase protection analysis of RNA isolated from testes tissue of the transgenic mice of the present invention
  • Figure 2b represents the acrylamide gels obtained from RNase protection analysis of RNA isolated from heart tissue
  • Figure 2c represents the schematic diagram of the RNA probe and predicted protected fragments
  • Figure 3 represents a tissue survey of RNAs from organs of an affected MT-PVLT line 8 animal;
  • Figure 4 represents the partial pedigree of the transgenic MT-PVLT line 8 mouse lineage;
  • Figure 5a represents a comparison of the chest cavity of a non-transgenic and an affected MT-PVLT line 8 animal
  • Figure 5b represents a comparison of excised hearts of a non-transgenic and an affected MT-PVLT line 8 animal
  • Figure 6a represents a histology of coronal sections of the heart of a MT-PVLT line 8 animal
  • Figure 6b represents a histology of cardiomyocytes of the ventricles of a MT-PVLT line 8 animal
  • Figure 6c represents a histology of a magnified portion of the thrombus of a MT-PVLT line 8 animal
  • Figure 6d represents a histology of a portion of an abnormally enlarged cardiomyocyte of a MT-PVLT line 8 animal.
  • Figure 7 represents a comparison of cardiomypathy between male and female MT-PVLT mice.
  • Plasmid pPXMT shown in Figure 1 was cut to completion with Bgl II and Bel I and the large fragment (3.7 Kb) containing the 0.7 Kb Kpn I to Bgl II fragment of the mouse metallothionein promoter (MT-1 promoter), vector sequence and the Bel I to Bam HI small fragment of simiam virus large T-antigen (SV40) and poly A addition signal, purified.
  • the two purified Bgl II fragments were ligated to produce a clone containing the MT-1 promoter linked by the oligonucleotide mentioned above to the cDNA of PVLT (2.4 Kb) and SV40 poly A addition sequence (267 nt) on a pXF3 vector background to form pMT-PVLT.
  • the techniques used to construct and purify plasmid pMT-PVLT are standard techniques that are well- known to those skilled in the art. A detailed description may be found in Maniatis et al., (1982), Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Generation of an RNA probe from plasmid pPVR2
  • Plasmid pPV805 essentially a wild type polyomavirus with an engineered Hind III site at 90 nt, was cut to completion with Hind III (90) and Eco Rl (1560).
  • the present invention requires the use of the wild type sequence of the PVLT gene. This sequence is available through the American Type Culture Collection under succession number 45017.
  • RNA probe generated was 1490 nt long and contained polyomavirus early region introns not present in the cDNA for polyomavirus large T-antigen.
  • Figure 2C A schematic representation of the RNA probe and predicted protected fragments is shown in Figure 2C. It is to be noted that the cDNA for PVLT is missing sequences between 409 to 795. The RNA protected fragments are thus 235 and 765 nts long. Production of transgenic mice containing the MT-PVLT DNA seguence.
  • MT-PVLT DNA Purified MT-PVLT DNA was separated from vector sequences after digestion with Xho I and BAM HI, then prepared for micro-injection as described in DePamphilis et al., 1988.
  • the MT-PVLT DNA microinjected into mouse embryos consisted of the mouse metallothionein-1 promoter (MT) followed by the cDNA for polyomavirus large T-antigen (PVLT) shown in Figure 1.
  • mice All micro-injection procedures and oviduct transfers were performed as described in the above- mentioned DePamphilis et al reference.
  • Genomic DNA was isolated from tail biopsies of three week old mice and digested with ECOR I, the DNA fragments separated by agarose electrophoresis and blotted onto Gene Screen Plus membrane. Polyomavirus-specific probe was generated by the random primer method described in Feinberg et al. , 1983. Biochem 132 6-13, from the 1470 nt Hind III to Eco Rl fragment described above.
  • mice which retained the microinjected DNA were termed "founders" and were mated to either C57B1/6 or BALB/C mice to initiate the development of progenitor lineage of MT-PVLT lines 7 to 12. All transgenic lines are different with regard to integration patterns and copy numbers per genome. All founders, except the MT-PVLT line 8 founder, have survived 16 to 18 months of age. The founder of line 8 died of unknown causes after 101 days but had previously produced transgene-positive progeny.
  • the partial pedigree of MT-PVLT line 8 lineage was compiled from birth and death records. Animals were detected as described above. Black symbols, transgenic mice; squares, males; circles, females. Diagonals, sacrificed just before death or found dead. Each series of symbols connected from above with a horizontal line is one litter and the symbols connected from below denote matings. The numbers directly below a symbol indicate age in days at death.
  • Transgene expression was determined by examining tissue from lines of MT-PVLT DNA-positive mice. Generally, expression of metallothionein fusion genes is high in liver, kidney, heart, pancreas and testes of transgenic mice but the level of expression varies widely among different transgenic lines. The targeted animals were sacrificed by cervical dislocation at an age varying between 100 and 200 days. Organs were dissected, immediately homogenized and RNA was isolated using the method described in Chirgwin J. et al., 1979. Biochemistry 1 ., 2633-2637. The quality of the purified RNA was determined spectrophotometrically and by examination of a small quantity after electrophoresis through a 1.2% agarose gel and ethidium bromide staining.
  • RNA was evaporated to dryness then RNase protection performed using the technique described in Bautsch et al., 1987. Cell 51 529-538.
  • Radioactive RNA probe was made by T7 polymerase reaction with Hind III digested pPVR2 cDNA according to the manufacturer's instructions. The fragments of probe protected by polyomavirus RNA should be shorter than the probe because the probe contains generic polyomavirus RNA included in the introns of all three polyomavirus early antigens.
  • the PVLT RNA should protect two fragments of 259 and 765 nt long whereas the probe length was 1470 nt long as shown in Figure 2. This probe did not contain any metallothionein sequences and therefore the site of RNA initiation was not examined. About 500,000 to 700,00 cpm of probe were added to each tissue and control sample, the samples denaturated and then hybridized overnight at 45°C.
  • RNA isolated from polyomavirus transformed baby mouse kidney cells 5 to 10 ug, was employed as a positive control in RNase A protection and Northern analysis.
  • RNA isolated from tissues of transgenic mice 10 ug, was denaturated then electrophoresed through a 6% formaldehyde/1.4% agarose gel and blotted on nitro-cellulose as described in the above-mentioned Maniatis et al. reference. Radioactive RNA probe as described above was hybridized to the nitrocellulose. The washed and dried blot was exposed to Kodak XAR-5 film with an intensifying screen for 2 to 4 days.
  • RNA isolated from heart and testes from 6 lines of transgenic mice are shown in Figure 2.
  • the various lanes represent the following: lanes +: RNA from Rat-1 cells transformed by polyomavirus, lane -: E. coli rRNA; lane a: non-transgenic; lane b: MT-PVLT line 7; lane c: MT-PVLT line 8 affected; lane d: MT-PVLT line 8 unaffected; lane e-. MT-PVLT line 9; lane f: MT-PVLT line 10; and lane g: MT-PVLT line 11.
  • the various lanes represent the various lanes +: RNA from Rat-1 cells transformed by polyomavirus, lane -: E. coli rRNA; lane a: non-transgenic; lane b: MT-PVLT line 7; lane c: MT-PVLT line 8 affected; lane d: MT-PVLT line 8 un
  • lane M -P-labelled Msp-1 digest of pBR32; lane a: MT-PVLT line 7; lane b: MT-PVLT line 8 affected; lane c: MT-PVLT line 8 unaffected; lane d: MT-PVLT line
  • RNA from Rat-1 cells transformed with polyomavirus 9; lane 3: MT-PVLT line 10; lane f: MT-PVLT line 11; lane g: MT-PVLT line 12; lanes h and i: 5 and 2.5 ⁇ g RNA from Rat-1 cells transformed with polyomavirus.
  • MT-PVLT line 8 was the only line of mice to demonstrate RNA protected fragments in heart (Figure 2 panel B, lane b).
  • the longer protected band seen in brain ( Figure 3, lane e) was also noted in non-transgenic animals.
  • Other tissues including liver, kidney, brain and pituitary, skeletal muscle, lung, salivary gland, stomach and mastoid were negative in RNase protection and Northern analysis for PVLT RNA in all lines (data not shown).
  • RNA from tissues of the unaffected animal did not protect any portion of the probe ( Figure 2 panel B; heart, lane c, panel A; testes, lane d, and data not shown).
  • Non-affected animals did not express the transgene in any tissue analyzed whereas affected animals expressed the transgene. Isolation of primary cells
  • Plasmid pPVa/3g may be obtained through cloning of the wild type sequence of the PVLT gene available under accession number 45017 and the polyomavirus origin containing fragment. The procedure through which plasmid pPVa/3 ⁇ is prepared is described by Muller W. J. et al. in 1983 J. Virology Vol. 47, p. 586-599. Hirt extractions as described in 1967 (J. Mol. Biot.
  • Plasmid pPV805 linear DNA was 8292 bp long and pPVa/3c linear DNA was 3316 bp long.
  • the various lanes represent the following: lane : -P-labelled Hind III digested Lamda DNA; lane +: polyomavirus containing plasmid (12 pg); kidney cells, lane a: MT-PVLT line 12; lane b: MT-PVLT line 8; lane c: non-transgenic; heart cells, lane d: non-transgenic; lanes e and f: MT-PVLT line 8; lane g: Rat-1 cells; lanes h and i: Mop-8 cells.
  • Phenotypic expression in MT-PVLT line 8 Expansion of the MT-PVLT line 8 lineage led to the documentation of premature death and severe illness in 58% of the transgene positive animals as shown in Figure 4.
  • the affected animals first symptom was rapid respiration, followed by loss of appetite, and lethargy with the appearance of the flattened ears and ruffled fur indicating a moribund condition and imminent death.
  • the time from the first sympton to death was usually less than 5 to 7 days.
  • Examination of the organs revealed a grossly enlarged heart with all other organs macroscopically normal.
  • a comparison of the cardiac phenotype between male and female MT-PVLT line 8 mice is documented in Table 1 and Figure 4. There was no difference in the incidence or amount of cardiac enlargement or age at death between male and female mice.
  • a total of 10 female and 11 male MT-PVLT line affected animals were examined. Affected animals were sacrificed just prior to imminent death and the age noted. A total of 10 C57B1/6 X Balb/c mice (Fl) male and female mice between the ages of 50 and 100 days were examined. All animals were sacrificed, the hearts excised and weighted. The weight of the Fl hearts was averaged and designated as normal. The affected hearts were weighed and the fold increase calculated as; average weight affected heart divided by average weight Fl heart. The range of increase for individual hearts was calculated as; weight individual affected heart divided by average weight Fl heart.
  • FIG. 5 shows a comparison of the enlarged heart (panel A) from an affected individual and an age-matched non-transgenic mouse (panel B).
  • the excised affected heart (panel C) was about 1.8 fold greater in length, 2.1 fold greater in width and was found to be 4.5 fold heavier than the normal heart.
  • the left ventricular muscle was thickened ( Figure 6 panel A).
  • the left atria, but never the right atria contained white discrete ball-like masses of varying number and size which on histological analysis were determined to be organized thrombi. Histological analysis
  • portions of ventricular and atrial walls were minced in 0.1 M phosphate buffered 3% glutaraldehyde solution, processed routinely and embedded in epoxy resin.
  • One micron thick section were stained with toluidine blue and 60 to 100 nm thick sections from appropriate areas were studied by electron microscopy.
  • the analysis of non-cardiac tissues from the affected animals demonstrated only minor alterations in most organs examined but lyraphoid cells appeared increased in spleen and thymus. Skeletal muscle, including esophagus, was normal.
  • cardiomyocytes up to 50 microns in width and more than several hundred microns in length with strange large nuclei and foci of contraction bands, were found in the ventricular and septal walls ( Figure 6, panel B).
  • the walls also showed occasional foci of interstitial fibrosis with vacuolar degeneration of cardiomyocytes. No overt cell necrosis was present.
  • the cardiomyocytes of the atrial walls were generally smaller; most were less than 10 microns in width. However, some slightly enlarged forms were noted in the atria wall and intrapulmonary veins.
  • the pearl-like masses in the atria were nodular aggregations of coagulated or crystallized fibrin, platelets and intermixed partly degenerated red and white blood cells (Figure 6, panels A and C). Proliferation of endothelial and fibroblasts was present where the mass adhered to the atrial wall. Electron microscopy showed the cardiomyocytes contained markedly serrated nuclear margins and a prominent nucleolus ( Figure 6, panel D). Mitochondria were enlarged with increased and haphazardly arranged cristae. The myofibrils were deranged focally and irregularly contracted.
  • This type of abnormality is usually indicative of cardio himy from hypertension, aortic valvular disease or a variety of myocarditis, including degenerative and storage diseases such as amyloidosis or glycogen storage diseases (31).
  • the aortic values were normal and no inflammatory, storage or degenerative processes were present.
  • the thrombi present in the left atrium were relatively recent and did not contain primitive esenchymal cells sometimes observed in advanced stages of clot organizations with fibrosis (31).
  • the enlarged and unusual-shaped cardiomyocyte is most consistent with a preneoplastic process and the changes of the cardiomycytes are, therefore, best characterized as dysplasia or an atypical hyperplasia.
  • These atypical cells are also apparently somewhat dysfunctional, as evidenced by derangements of myofibrils, and probably altered further by he odynamic stresses as cellular changes were most exaggerated in the left ventricle where the pressure stress is highest. Progressive dilation and failure of the left ventricle and left atrium would explain the turbulence and stagnation of blood flow with massive thrombus formation in the left atrium. Thus the animals succumbed due to left heart failure. Functional assay of large T-antigen expression in MT- PVLT line 8 mice.
  • PVLT-antigen is the single virus encoded protein absolutely necessary for polyomavirus DNA replication and can function in trans in vi tro to promote polyomavirus DNA replication. PVLT-antigen, if synthesized and functional in the isolated cell lines, would thus stimulate the DNA replication of a transfected plasmid containing the polyomavirus origin but lacking any other polyomavirus DNA sequences.
  • methylation sensitive restriction endonuclease Dpn 1 38.
  • the cleavage activity of Dpn 1 requires that adenine residues be methylated. DNA replicated in mammalian cells is non-methylated at adenine residues whereas plasmid DNA is methylated. Thus, if the PVLT- antigen was present and functional in the MT-PVLT line 8 primary cells, the replicated transfected plasmid would appear as resistant to Dpn 1 cleavage.
  • Plasmid pdPa ⁇ c containing a Bam HI to Hind III polyomavirus origin fragment (5109 - 90 bp) and described earlier, was transfected onto primary cells isolated from the MT-PVLT line 8 heart and kidney cells. Mop-8 cells, which contain a functional polyomavirus large T-antigen, and Rat-1 cells served as positive and negative controls respectively in the transfection and replication experiments. The results of this assay are shown in Figure 7. Plasmid pdPVa ⁇ .

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Abstract

L'invention se rapporte à un mammifère non humain transgénique dont les cellules germinales et les cellules somatiques contiennent la séquence de l'antigène PVLT (grand antigène T de virus polyomes) sous le contrôle direct d'une séquence promoteur de virus non polyomes, telle que de préférence une séquence promoteur inductible. Ce gène de fusion est introduit dans le mammifère non humain ou dans un ancêtre de ce mammifère à un état embryonnaire. Le mammifère non humain qui en résulte se caractérise par une propension accrue à développer une cardiomyopathie. L'invention décrit également un procédé permettant d'induire une cardiomyopathie chez un mammifère non humain. Ce procédé consiste à introduire dans un mammifère non humain ou dans un ancêtre de ce mammifère à un état embryonnaire un gène de fusion comprenant la séquence mentionnée ci-dessus, afin de produire des embryons transformés. Les embryons transformés qui survivent sont implantés dans des femelles en état de pseudo-gestation et la progéniture qui en résulte est triée pour déterminer la présence de transgènes.
PCT/CA1990/000270 1989-08-25 1990-08-24 Developpement d'une cardiomyopathie chez des mammiferes non humains transgeniques Ceased WO1991002793A1 (fr)

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US398,499 1989-08-25

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WO1991002793A1 true WO1991002793A1 (fr) 1991-03-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6333447B1 (en) 1996-03-29 2001-12-25 The General Hospital Corporation Transgenic model of heart failure
US10494421B2 (en) 2014-02-10 2019-12-03 Univercells Nv System, apparatus and method for biomolecules production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
I.A.R.C., (International Agency for Research on Cancer, Scientific Publications, No. 96, Perinatal and Multigeneration, Carcinogenesis; Third Meeting, Leningrad, USSR; 31 May - 2 June 1988; N.P. NAPALKOV et al. (Ed.), Oxford University Press, (New York, US); International Agency for Research on Cancer, 1989, (Lyon, FR), V.L. BAUTCH et al.: "Tissue Specificity of Oncogene Action: Endothelial Cell Tumours in Polyoma Middle T Transgenic Mice", pages 255-266 see the whole article *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6333447B1 (en) 1996-03-29 2001-12-25 The General Hospital Corporation Transgenic model of heart failure
US10494421B2 (en) 2014-02-10 2019-12-03 Univercells Nv System, apparatus and method for biomolecules production

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