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WO1992002618A1 - Specimens animaux servant aux thrombopathies et cardiopathies - Google Patents

Specimens animaux servant aux thrombopathies et cardiopathies Download PDF

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Publication number
WO1992002618A1
WO1992002618A1 PCT/GB1991/001314 GB9101314W WO9202618A1 WO 1992002618 A1 WO1992002618 A1 WO 1992002618A1 GB 9101314 W GB9101314 W GB 9101314W WO 9202618 A1 WO9202618 A1 WO 9202618A1
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animal
heart
segment
nucleic acid
symptoms
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PCT/GB1991/001314
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English (en)
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Peter Francis Searle
Shing Mun Huen
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Cancer Research Campaign Technology Limited
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Priority claimed from GB909017020A external-priority patent/GB9017020D0/en
Priority claimed from GB919110760A external-priority patent/GB9110760D0/en
Application filed by Cancer Research Campaign Technology Limited filed Critical Cancer Research Campaign Technology Limited
Publication of WO1992002618A1 publication Critical patent/WO1992002618A1/fr

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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to the field of molecular biology and biotechnology, and at least from one aspect it concerns nucleic acids having nucleotide sequences providing unusual properties in respect of their ability to act as transgenes in the production of trans ⁇ genic animals, especially transgenic non-human mammalian animals such as mice or other rodents for example, that exhibit surprising and unexpected phenotypic characterist ⁇ ics.
  • the invention is also concerned with transgenic animals containing transgenes comprising such nucleic acid sequences, with the production of such transgenic animals and with uses thereof, particularly in connection with the testing and/or development of potential antithrombotic or thrombolytic drugs or other therapeutic or diagnostic agents, especially in the field of cardiac medicine.
  • Transgenic animals incorporating an exogenous gene (transgene) in the genome of their somatic and germ cells, introduced (usually by micro-injection) by human intervention at an early stage into the embryo (or zygote) of the animal or ancestor of the animal, are now well- known, as are the basic techniques for their production, and many examples have been reported in the literature during the last decade.
  • Epstein-Barr virus which is an antigen- ically distinct member of the herpesvirus group of viruses that have a DNA genome, is known to include several genes that code for and express particular viral proteins of which one is known as a nuclear antigen leader protein, commonly designated EBNA-LP (or LP).
  • EBNA-LP nuclear antigen leader protein
  • transgenic mice were produced by introducing into mice embryos a transgene consisting of a DNA sequence made up of an upstream segment comprising promoter and control sequences from the mouse metallothionein-I gene fused to a segment comprising a cDNA gene segment coding for an Epstein-Barr virus nuclear antigen leader protein (LP), downstream of which was fused a further segment comprising sequences from the human growth hormone gene (hGH) that provided splice and pol adenylation signals for controlling processing of transcribed RNA and for terminating transcription.
  • LP Epstein-Barr virus nuclear antigen leader protein
  • mice in the majority of these transgenic lines were found reproducibly to have a high probability of developing blood clotting or thrombi in their circulatory system, this being generally manifested in the form of a large organised blood clot within the left atrium of the heart.
  • the development of these atrial thrombi appeared to be closely correlated with the onset of visible respiratory distress, and in the absence of intervention would generally lead to, or be followed by, death within a few days.
  • This thrombotic effect was a completely unexpected effect, and it occurred at various ages which appeared to be a characteristic of the particular line concerned. Thus in some lines it occurred at an age of about four to six months, whereas mice in other lines remained in apparent good health for eight months to over a year before developing the symptoms.
  • this thrombotic effect and/or cardiomyopathic effect is brought about, it appears to be closely related or associated with expression in at least certain somatic cells of the viral nuclear antigen leader protein or at least of the gene DNA sequence coding therefor, either directly in its original form as when first introduced or in a slightly re-arranged form which might arise upon initial integration or chromosomal incorporation into the genome of the embryo of the original founder animal.
  • DNA constructs comprising, as hereinafter more fully described, a segment embodying a gene sequence coding for an EBNA leader protein (LP) fused to an upstream segment containing mouse metallothionein-I gene promoter and control sequences effective to direct and regulate expression of the LP coding segment, and fused to a downstream segment comprising a hGH gene sequence, have been found to provide transgenes which are especially efficient in producing the above-mentioned thrombotic and/or cardiomyopathic effect, at least in rodents such as mice.
  • LP EBNA leader protein
  • composition of such DNA constructs may be modified or varied within quite wide limits without loss activity as a transgene capable of producing transgeni animals having these characteristics, so long as the D sequence is still effective in expressing a gene coding fo the EBV leader protein, or a close analogy thereto, in t transgenic animal.
  • th downstream segment containing the hGH gene sequence may b omitted or it may be replaced by a different eukaryoti gene sequence effective in providing signals fo termination of transcription and for controlling processin of transcribed RNA.
  • different promoter and contro sequences may be used in the upstream segment and, i particular, not all the control sequences need necessaril lie upstream of the LP gene, especially if an enhance sequence is provided which could be located downstream o the LP gene, or within an intron, and still contro expression in combination with the upstream promoter.
  • the transgene may comprise a suitable LP gen containing segment of DNA cut out directly from the vira genome and incorporating viral sequences to regulat expression instead of being a recombinant construct; or for introducing the transgene into the animal embryo usin a retroviral vector instead of direct micro-injection, th nucleic acid sequence may be an RNA sequence which is con verted into the corresponding DNA sequence within th embryo cell when being incorporated into the animal genome
  • the recombinant retrovirus could be produced by standar procedures using a DNA precursor introduced into retrovirus packaging cell line such as that known as PA317
  • the invention may be applicable to a variety of different non-human mammalian species since it is quite common for the same transgene to be effective in producing transgenic animals through a range of different species and such transgenic animals may often share similar characteristics imparted by the transgene, at least when the species are of a similar kind, e.g. rodent or mammalian.
  • transgenic animals produced in accordance with the invention can be especially useful as tester animals for screening and identifying or testing drugs or potential drugs for antithrombotic, thrombolytic or cardiotherapeutic activity and/or for research in connection with thrombosis and/or heart disease with the object of developing improved therapeutic treatments or diagnostic methods applicable in human medicine.
  • the invention consists in a nucleic acid which, when introduced at an early stage into a fertilised egg or embryo of a mammalian animal, is capable of being functionally integrated into the genome thereof thereby to produce a transgenic animal, characterised in that its nucleotide sequence comprises a gene sequence coding for an Epstein-Barr virus nuclear antigen leader protein, or for a homologous protein from another herpesvirus, or for a derivative of such proteins, in combination with heterologous promoter and control sequences effective to direct and regulate expression of said viral protein gene in somatic cells of a transgenic animal produced as aforesaid, such that said transgenic animal has a substantially increased probability of spontaneously developing blood clotting or thrombi in its circulatory system, especially in the heart, and/or symptoms of dilated cardiomyopathy or similar heart dysf nction.
  • the nucleic acid is a recombinant DNA construct in which the promoter and at least some control sequences are contained in a segment upstream of the viral protein gene, and the latter is fused to a downstream segment comprising at least a fragment of a eukaryotic gene sequence effective to provide signals for terminating transcription and for controlling processing of transcribed RNA during expression of the viral protein.
  • the promoter and control sequences are provided by a mouse metallothionein-I gene sequence in a segment upstream of the viral protein gene which is fused to a downstream segment containing a human growth hormone gene sequence (hGH).
  • the invention also provides cloning vectors incorp- orating therein a nucleic acid insert as specified above.
  • the invention provides a DNA sequence consisting of the DNA insert EcoRI-Sall or EcoRI-Hindlll of plasmid p(DH63)MT-LP- hGH, as hereinafter described.
  • the invention also consists in the use of a nucleic acid comprising a gene sequence coding for an Epstein-Barr virus nuclear antigen leader protein, or for a homologous protein from another herpesvirus, or for a derivative of such proteins, for producing a transgenic non-human mammalian animal, such as a mouse or other rodent for example, having a substantially increased probability of spontaneously developing blood clotting or thrombi in its circulatory system, especially in the heart, and/or symptoms of dilated cardiomyopathy or similar heart dysfunction.
  • the invention further provides a method for producing a transgenic animal, for example a mouse or other rodent, having a substantially increased probability of spontan ⁇ eously developing blood clotting or thrombi in its circulatory system, especially in the heart, and/or symptoms of dilated cardiomyopathy or similar heart dysfunction, said method comprising incorporating a nucleic acid as specified above into the genome of a non-human mammalian animal such that it is functionally integrated therein.
  • the nucleic acid is a recombinant DNA which is introduced into a fertilised egg or embryo of the animal in order to become functionally integrated into the genome thereof, said recombinant DNA being characterised in that it contains a segment comprising a gene sequence coding for an Epstein- Barr virus nuclear antigen leader protein, or for a homol ⁇ ogous protein from another herpesvirus, or for a derivative of such proteins, which segment is fused
  • the invention further provides a transgenic non-human mammalian animal, for example a mouse or other rodent, characterised in that it contains stably integrated into the genome of at least the majority of its somatic cells and germ cells an exogenous transgene derived from a DNA sequence coding for Epstein-Barr virus nuclear antigen leader protein, or for a homologous protein from anothe herpesvirus, or for a derivative of such proteins, operatively associated with expression control an regulatory sequences, said DNA sequence having bee introduced into the animal or an ancestor of the animal a a one-cell or early embryonic stage, said animal bein further characterised in that it has a substantiall increased probability of spontaneously developing bloo clotting or thrombi in its circulatory system, especiall in the heart, and/or symptoms of dilated cardiomyopathy o similar heart dysfunction.
  • a transgenic non-human mammalian animal for example a mouse or other rodent, characterised in that it contains stably integrated into the genome of at least
  • the invention further consists in the use of transgenic animals as hereinabove specified for testing antithrombotic, thrombolytic or cardiotherapeutic properties of substances administered to said animals, or for testing the activity of such substances in controlling or inhibiting the development of heart disease.
  • a method of screening and identifying or testing a drug or other substance for antithrombotic or thrombolytic activity or for activity against the development of or in the treatment of cardiac dysfunction e.g.
  • dilation cardiomyopathy comprises treating selected transgenic animals, as hereinbefore specified, with said drug or other substance concerned and detecting or noting any reduced incidence in the development of blood clotting or thrombi, and/or of symptoms of dilated cardiomyopathy and reduction in morbidity, as compared with corresponding animals that are not treated with the drug or substance, or detecting or noting an effectiveness in maintaining, restoring or improving heart function and/or in dissolving and removing blood clots already formed.
  • FIG. 1 The Figures in the drawings are diagrammatic representations (not to scale), including indications of relevant restriction endonuclease sites, showing regions of plasmid constructs used in the construction of a particular fusion gene DNA sequence that provides the effective transgene of the embodiment herein described. More specifically:
  • FIGURE 1 represents a region of a plasmid pUC-LP used as a source of a DNA sequence (cDNA) coding for an Epstein-Barr virus nuclear antigen leader protein (LP);
  • cDNA DNA sequence
  • LP Epstein-Barr virus nuclear antigen leader protein
  • FIGURE 2 represents a region of a plasmid p(RG46) used as a source of promoter and control sequences of the mouse metallothionein-I gene (mMT-I);
  • FIGURE 3 represents a region of a plasmid p(RG48)MT-hGH containing the mMT-I sequences and also a human growth hormone gene sequence (hGH);
  • FIGURE 4 represents a region of a plasmid p(DH55)LP-hGH containing both the EBNA-LP DNA sequence (LP-cDNA) and the growth hormone gene sequence hGH; and
  • FIGURE 5 represents a region of a plasmid p(DH63)MT-LP-hGH having the required final form of the fusion gene DNA sequence that contains the mMT-I sequences, the LP-cDNA sequence and the hGH sequence.
  • plasmid constructs concerned were derivatives of the polylinker-containing plasmid pUC18 (Norrander et al, Gene 26, 101-106 - ATCC No. 37253).
  • the single line sections between the marked EcoRI and Hindlll sites represent sequences of the pUCl ⁇ polylinker whilst the single diagonal cross-hatched double line block sections represent the flanking sequences of pUC18, and the horizontal brackets in FIGS. 1, 2 and 3 indicate the sources of sequences in the final construct of p(DH63)MT- LP-hGH.
  • the pUC-LP plasmid (FIGURE 1) used as the source of the Epstein-Barr virus nuclear antigen leader protein cDNA sequence was obtained from Dr. F. Wang (Harvard, USA) and contains a fragment of the T65 cDNA clone [see Sample et al (1986) Proc. Natl. Acad. Sci. USA 83, 5096-5100 and Wang et al (1987) J. Virol. 6JL, 945-954].
  • This fragment which includes the EBNA-LP cDNA sequence, is cloned into the Smal site of the polylinker of plasmid pUC18. In this process, the Smal site is destroyed and hence the position thereof is indicated in brackets in the diagram of FIGURE 1.
  • this fragment extends from an EcoRI linker upstream of the complete EBNA-LP/EBNA-2 sequence of T65 cDNA to an FnuDII site which is downstream of the LP coding sequence but which is upstream of the EBNA-2 coding sequence.
  • the EBNA-LP DNA sequence in this fragment has an open reading frame extending in the direction EcoRI to HindiII as indicated in FIGURE 1.
  • the mouse metallothionein-I gene promoter and control sequences extend from approximately 1700 basepairs upstream of the site of transcription initiation (indicated by an angular arrow) to a Bglll site about 68 basepairs downstream past the site of transcription initiation in the full gene sequence.
  • the Bglll site was made blunt by end filling with DNA polymerase-I Klenow fragment and joined to the Smal site of the pUC18 polylinker, destroying both sites, the upstream end of this insert being joined as shown to the EcoRI site next to the adjacent pUC18 flanking sequences.
  • the p(RG48)MT-hGH plasmid (FIGURE 3) was constructed from p(RG46) by inserting a BamHI/Clal fragment containing a human growth hormone gene sequence (hGH) from a plasmid pAlb-hGH (obtained from R. Palmiter, HHM1, University of Washington, Seattle, USA) between the BamHI and Sail sites in p(RG46). In this process the cut Sail and Clal sites were endfilled prior to cutting with BamHI so as to regenerate the Sail site but not the Clal site.
  • the BamHI site which was also maintained, lies upstream of the hGH coding region in the inserted fragment but just downstream of the site of transcription initiation of the natural intact hGH gene.
  • hGH gene sequence which is in itself known from the literature [see for example Seebury, P.H. (1982), DNA 1., 239-249], extends for approximately 200 basepairs downstream of the RNA polyadenylation site; the Clal site is believed to have been introduced at this position in previous subcloning steps.
  • the p(DH55)LP-hGH plasmid (FIGURE 4) was constructed by replacing the small BamHI to Hindlll region in pUC-LP with the BamHI to HindiII fragment of p(RG48)MT-hGH which contains the human growth hormone gene sequence.
  • the p(DH63)MT-LP-hGH plasmid (FIGURE 5) was made by cloning the EcoRI/BamHI fragment containing the mMT-I sequences from p(RG46) into p(DH55)LP-hGH between the EcoRI and Asp718 sites of the latter with replacement of the small fragment represented by the intervening sequence.
  • the BamHI and Asp718 ends were both made blunt by endfilling with Klenow fragment to allow their ligation.
  • mice To produce transgenic animals, in particular mice, the MT-LP-hGH fusion gene sequences were excised from the p(DH63)MT-LP-hGH plasmid vectors, using EcoRI and either Sail or HindiII restriction endonucleases, and were then micro-injected into a pronucleus of fertilised one-cell mouse eggs (zygotes) which were subsequently transferred to pseudo-pregnant foster females, all in accordance with established techniques [see for example Wagner et al (1981) P.N.A.S, USA 78, 5016, or Hogan et al (1986) "Manipulating the Mouse Embryo - A Laboratory Manual" (Cold Spring Harbor Laboratory)] .
  • the egg donor females and the males used to achieve fertilization were Fl hybrids between CBA/Ca and C57B1/6J mice.
  • transgenic mice After allowing the embryos developing from the injected eggs to develop to term, 34 young mice were born, of which seven died before three weeks 'of age (of these, three were identified as transgenic). Three of the surviving animals were also identified as transgenic by analysis of a DNA sample obtained from the tail, using a probe for the EBNA-LP cDNA sequence. These transgenic mice were bred with normal animals, and each transmitted the transgene to a proportion of the offspring. In this particular case it appeared that each of the original transgenic animals had incorporated the transgene in at least two independent chromosomal sites, since two separate lines of transgenic mice with clearly distinct properties were established from each of the founder animals.
  • the metallothionein promoter sequence used to direct expression of the transgene is known to contain, in addition to elements which function to cause a basal level of transcription, other sequence elements which enble expression to be increased in response to various inducers such as zinc, the effects described have all been obtained without the necessity of administering inducing agents to the animals. It is therefore possible that expression of the transgene could be increased to higher levels by administration of zinc or other inducers, which may have the effecr of producing more extreme symptoms or an earlier onset.
  • transgenic mice in the lines which were established appeared initially to have normal good health; however, in the majority of lines transgenic animals were observed to develop respiratory distress, which in the absence of intervention would lead to death within a few days.
  • the age at which this occurred appeared to be a characteristic of the particular line of mice, with transgenic mice in some lines developing symptoms at about 4 to 6 months of age, and mice in other lines remaining apparently healthy for 8 months to over a year before developing this disorder.
  • the most obvious characteristic feature of the pathology of the afflicted mice was found to be the presence of a large organised blood clot in the left atrium of the heart, either in the recently dead mice or in animals killed at the first appearance of the distress symptoms.
  • dilated cardiomyopathy was evidenced not only by the typical signs of respiratory distress associated with the development of the thrombotic effect, but also by dilation and hypertrophic changes in the heart that may be seen at least in the later stages of the disorder and by some disorganisation or pathological changes in the ultra- structure of the heart muscle.
  • some ultrastructural abnormalities may exist prior to the appearance of the visible symptoms, and these may result in or be associated with some gradual impairment of heart function during the asymptomatic stage.
  • the transgene in particular the MT-LP-hGH transgene
  • the heart disorder is consistent with a detrimental effect of LP expression within the heart tissue thereby producing a local heart defect which, in turn, may be responsible for the blood clot formation.
  • expression of the leader protein elsewhere in the body might adversely affect the heart, for example via endocrine or nervous pathways, or by causing hypertension, which again could lead to the symptoms and effects observed.
  • trans ⁇ genic animals produced in accordance with the invention can be useful as a valuable model for studying congestive heart disease, for identifying early warning signals of possible diagnostic value applicable to humans, and also, most importantly, for providing much needed test animals especially for screening and identifying or testing of potential drugs or other substances for the diagnosis or treatment of dilated cardiomyopathy and/or potential drugs or other substances suspected of having antithrombotic, thrombolytic or cardiotherapeutic properties.
  • Such tests may be carried out, for example, by treating selected transgenic animals with the drug or other substance under investigarion and detecting or noting any reduced incidence in the development of heart disease symptoms or blood clotting and mortality rates as compared with animals from the same stock line that are not treated with the drug or other substance, or detecting or noting effectiveness in maintaining, restoring or improving heart function and/or in dissolving and removing blood clots already formed.
  • transgenic forms of other non-human mammalian species and certainly other rodents, exhibiting similar phenotypic effects may likewise be produced by introducing the same or a similar transgene of which the essential feature appears to be that it provides a nucleic acid sequence capable of expressing a protein corresponding to, or substantially analogous to, a herpesvirus protein such as Epstein-Barr virus nuclear antigen leader protein.
  • a herpesvirus protein such as Epstein-Barr virus nuclear antigen leader protein.
  • the hGH segment downstream of the LP coding segment is a preferable component, especially insofar as it may provide signals for termination of transcription and for controlling processing of the transcribed mRNA, it may not necessarily be absolutely essential since adequate transcription and expression of the LP coding segment may be obtainable without any downstream segment or by replacing the hGH gene segment by another different eukaryotic gene sequence, as previously indicated.
  • rhe upstream mouse metallothionein-I gene sequence segment as described, which includes a heavy metal inducible promoter sequence, gives a convenient way of providing satisfactory promoter and control sequences for directing and regulating expression of the LP coding segment, and has quite often been used in producing other transgenic animals, especially mice, alternative promoter and control sequences may also give likewise satisfactory results.
  • control sequences may not all the required control sequences need necessarily lie upstream of the complete LP coding segment since, for example, a suitable enhancer sequence downstream of this LP coding segment, or within an intron thereof, could also allow for satisfactory expression in combination with the upstream promoter sequence.
  • a bacteriophage vector for this purpose, e.g. M13 or lambda, or a phagemid or cosmid.
  • the DNA sequence concerned might even be manipulated and amplified entirely by in vitro DNA replication, e.g. by use of the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • an alternative method would be to deliver a similar LP coding sequence or LP construct by use of a retroviral vector introduced into the embryo, preferably at an early stage.
  • the sequence would thus be introduced as a virus particle, with an RNA genome rather than a DNA genome, to generate a corresponding proviral DNA equivalent to the transgene within the infected cell.
  • the transgene or LP-construct sequence is preferably introduced into the animal embryo at the one-cell stage, by injecting into a pronucleus of a fertilised one-cell egg as described, it could also be introduced somewhat later if desired, at least up to about the 8-cell stage for example, even although this may increase to some extent the risk that the LP transgene will not be present in all the somatic and germ cells of the transgenic animal.
  • Other means of introduction of the transgene nucleic acid and methods of producing the transgenic animals, apart from those already mentioned, are also possible.
  • the transgenic mice can also be produced from certain totipotent cell lines, e.g.
  • the transgene construct may be introduced into the ES cells in culture by any of a variety of techniques (e.g. precipitation of DNA with calcium phosphate; electroporation; use of viral vectors), and the ES cells which have incorporated this exogeneous DNA can then be introduced inro early embryos (usually blastocysts ) .
  • the progeny of the ⁇ S cells may then contribute to the chimeric mice which develop, and after further breeding this can lead to the establishment of stable transgenic lines.
  • transgene can be expected usually to be integrated into the chromosomal DNA of the transgenic animal, it is also possible that the transgene may be maintained extrachromosomally, i.e. on an episome incorporating its own origin of replication, while still being stably and functionally integrated into the animal's effective genome.
  • the invention comprises a number of different aspects. In general, it includes ail novel and inventive features and aspects herein disclosed, either explicitly or implicitly, and either singly or in combination with one another. Moreover, the scope of the invention is not to be construed as being limited by the illustrative examples or by the terms and expressions used herein merely in a descriptive or explanatory sense.

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Abstract

L'invention décrit un acide nucléique constitué par un ADN recombiné, comportant une séquence génétique codant une protéine principale d'antigène nucléaire du virus d'Epstein-Barr fusionnée avec un segment amont contenant une séquence génétique de métallothionéine-I de souris produisant des séquences de stimulation et de contrôle et fusionnée à un segment aval contenant une séquence génétique d'hormone de croissance humaine (hGH). Les animaux transgéniques tels que les souris obtenus en intégrant cet ADN recombiné à un stade embryonnaire peu développé dans leur génome, présentent une probabilité sensiblement accrue de développer spontanément des caillots sanguins dans leur système circulatoire, particuliièrement dans l'oreillette gauche du c÷ur et/ou des symptômes de cardiomyopathie dilatée ou dysfonctionnement cardiaque similaire. De tels animaux peuvent s'utiliser pour sélectionner et identifier ou tester des médicaments ou autres substsances possédant une activité anti-thrombotique ou thrombolytique ou également cardiothérapeutique, s'appliquant en médecine pour l'homme.
PCT/GB1991/001314 1990-08-02 1991-08-01 Specimens animaux servant aux thrombopathies et cardiopathies WO1992002618A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9017020.0 1990-08-02
GB909017020A GB9017020D0 (en) 1990-08-02 1990-08-02 Nucleic acid sequences having unusual properties and uses thereof
GB919110760A GB9110760D0 (en) 1991-05-17 1991-05-17 Nucleic acid sequences having unusual properties and uses thereof
GB9110760.7 1991-05-17

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WO1992002618A1 true WO1992002618A1 (fr) 1992-02-20

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0873423A4 (fr) * 1995-11-30 2003-01-22 Brigham & Womens Hospital Methodes pour detecter des mutations associees avec la cardiomyopathie hypertrophique
EP2139314A4 (fr) * 2007-03-27 2010-06-30 Piramal Life Sciences Ltd Modele animal, systeme, et procede pour cribler des composes pour une activite antithrombotique et/ou thrombolytique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CELLULAR BIOCHEMISTRY, Vol. 0, No. Pt. C, 31 January 1991, SEARLE P.F. & HUEN D., "Left Atrial Thrombosis in Transgenic Mice Expressing the EBV Nuclear Antigen Leader Protein", page 190. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0873423A4 (fr) * 1995-11-30 2003-01-22 Brigham & Womens Hospital Methodes pour detecter des mutations associees avec la cardiomyopathie hypertrophique
EP2139314A4 (fr) * 2007-03-27 2010-06-30 Piramal Life Sciences Ltd Modele animal, systeme, et procede pour cribler des composes pour une activite antithrombotique et/ou thrombolytique

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