WO1992002618A1 - Animal models for thrombopathies and cardiopathies - Google Patents

Abstract

A nucleic acid is disclosed consisting of recombinant DNA comprising a gene sequence coding for an Epstein-Barr virus nuclear antigen leader protein fused to an upstream segment containing a mouse metallothionein-I gene sequence providing promoter and control sequences and fused to a downstream segment containing a human growth hormone gene sequence (hGH). Transgenic animals such as mice produced by incorporating this recombinant DNA at an early embryonic stage into the genome thereof have a substantially increased probability of spontaneously developing blood clotting or thrombi in their circulatory system, especially in the left atrium of the heart, and/or symptoms of dilated cardiomyopathy or similar heart dysfuncion. Such animals can be useful for screening and identifying or testing drugs or other substances for antithrombotic or thrombolytic activity or for cardiotherapeutic activity for use in human medicine.

Description

ANIMAL MODELS FOR THROMBOPATHIES AND CARDIOPATHIES

FIELD OF THE INVENTION

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.

BACKGROUND AND SUMMARY OF THE INVENTION

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.

The present invention has arisen primarily from work on the expression of Epstein-Barr virus genes in transgenic mice. The Epstein-Barr virus (EBV), 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).

In the course of this work, a number of lines of 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. Surprisingly, 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.

From continued studies and observations, further symptoms were noticed in the transgenic animals exhibiting respiratory distress as referred to above. In particular these included dilatation of the heart, especially of the left ventricle; pulmonary oedema and right ventricular hypertropy, although in contrast hypertrophy of the left ventricle was not observed. Ultrastructural abnormalities including some degree of myofibrillar disorientation and nuclear abnormalities have also been observed in the afflicted transgenic animals. It appears that these ultrastructural abnormalities may to some extent precede the manifestation of the other symptoms described. Similar symptoms are also often observed to varying degrees in the probably heterogeneous class of human diseases referred to as congestive heart failure or dilated cardiomyopathy, and indeed it now appears likely that some impairment of heart function may be the primary pathogenic effect of the transgene expression in these affected animals, with the initially observed atrial thrombus formation constituting a secondary effect. Impairment of normal sinus rhythm, such as by the occurrence of atrial fibrillation, may also have a role in the pathogenesis.

Although the mechanism is not known by which 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. In any event, particular recombinant 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. However, it is also to be expected that the particular 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.

Thus, as herein explained, in some cases 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. Or, 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.

Also, 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

Although the preliminary work has been based on th use of a gene sequence coding for an Epstein-Barr viru nuclear antigen leader protein, certain other nucleic aci sequences coding for similar proteins, especially othe homologous proteins from gamma-1-herpesviruses or othe herpesviruses, or for derivatives of such viral proteins (particularly similar proteins having minor variations in amino acid sequence insufficient to significantly change activity), should be similarly effective in providing transgenes capable of producing transgenic animals exhibiting the characteristics herein referred to.

Also, 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.

The 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.

Accordingly, there are several different aspects to the present invention.

From one aspect 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.

Preferably, 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. In preferred embodiments, 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.

Also, in a particular preferred embodiment 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. At least in preferred embodiments 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

(i) to an upstream segment comprising promoter and at least some control sequences effective to direct and regulate expression of said viral protein coding segment, and also, preferably,

(ii) to a downstream segment comprising a eukaryotic gene sequence or fragment effective to provide signals for termination of transcription and for controlling processing of the transcribed RNA.

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.

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. Thus, according to another aspect of the invention, 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.

By way of example of τhe manner in which the invention may be carried out in relation to the production of a transgenic mouse having a high susceptibility to spontaneous development of blood clotting or thrombi in its circulatory system, especially in the heart, and symptoms of cardiac dysfunction such as dilated cardiomyopathy, an illustrative embodiment will now be described in more detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

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);

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. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENT Construction of p(DH63)MT-LP-hGH

Procedures used in constructing the plasmid designated p(DH63 )MT-LP-hGH, and intermediate constructs, all employed conventional techniques well-known in the art, as described for example in Maniatis et al (1982) "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory). In general, the plasmid constructs concerned were derivatives of the polylinker-containing plasmid pUC18 (Norrander et al, Gene 26, 101-106 - ATCC No. 37253). In the diagrammatic representations in the drawings, the single line sections between the marked EcoRI and Hindlll sites represent sequences of the pUClδ 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. A sample of the plasmid p(DH63)MT-LP-hGH itself, in an Ξ.coli (DH5α) host, has been deposited, by one of the co-inventors hereof, under the provisions of the Budapest Treaty with The National Collections of Industrial and Marine Bacteria (NCIMB) Limited, 23 St Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom, on 2 August, 1990 (Accession No. 40311).

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. It is believed that 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.

In the p(RG46) plasmid shown in FIGURE 2, 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. In constructing this plasmid, 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. The 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.

Finally, 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. In this operation, the BamHI and Asp718 ends were both made blunt by endfilling with Klenow fragment to allow their ligation.

Cloning of the plasmid vectors was carried out in E.coli.

Production of Transgenic Animals

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.

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. These lines differed in the number of copies of the transgene, in the pattern of transgene and flanking DNA fragments detected on Southern blots, in the pattern of EBNA-LP expression in different tissues, and in their susceptibility to the condition of left atrial thrombosis and congestive heart failure. Most of the lines expressed relatively high levels of EBNA-LP in the liver, and more variable levels were observed in other organs, such as kidney, lung, heart, spleen, thymus, skin, and brain.

It may be noted that whereas 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.

The 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 production of the same disorder in several lines of transgenic mice with distinct transgene integration sites, including lines derived from each of three original transgenic founder animals, indicates that the phenotype is attributable to the MT-LP- hGH transgene per se, and does not appear to be due to insertion into a specific chromosomal locus, nor to be due to a mutation introduced by chance into the mouse genome.

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.

In addition, however, the presence of 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. In fact, it appears that 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.

It was found that other tissues also showed some effects, e.g. congestion of the lungs and occasional damage to liver tissue, but these appeared to be consequential effects attributable to venous hypertension resulting from the heart dysfunction.

As previously indicated, the apparent cardiomyopathic and thrombotic effects observed were completely unexpected and were very surprising when first detected. The reasons for these effects and the mechanism involved, related apparently to expression of the EBV nuclear antigen leader protein, are not yet known. However, the specificity of the effects is very striking in that transgenic mice in the majority of lines with the MT-LP-hGH transgene have been found to develop the same disorder, albeit at somewhat variable ages.

Although the mechanism by which the transgene, in particular the MT-LP-hGH transgene, produces the defect is not known, 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. On the other hand, it is also conceivable that 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.

In any event, it will be appreciated that the 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.

Whilst the invention has been more specifically described with reference to mice, as mentioned previously it is reasonable to expect that 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.

Although the particular fusion gene DNA sequence of the plasmid p(DH63)MT-LP-hGH insert herein described has been found to provide a perfectly satisfactory transgene for producing the transgenic animals, especially mice, susceptible to these cardiomyopathic and thrombotic effects, numerous variations or modifications in the make¬ up of this transgene sequence would be expected to be possible whilst still obtaining transgenic animals showing the same phenotypic effects. For example, although 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. Also, whilst use of 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. In this respect, it may be noted that 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.

Instead of cloning and propagating the LP gene construct by using a plasmid vector, it may also be possible to use a bacteriophage vector for this purpose, e.g. M13 or lambda, or a phagemid or cosmid. Or, again, 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) .

As a further possibility already mentioned, instead of making and using a recombinant DNA construct to provide the transgene and to achieve expression of the LP coding gene, it may also be possible to use a segment of DNA containing the LP gene cut directly from the viral genome concerned.

With regard to the manner of introducing the transgene nucleic acid sequence into the animal embryos, 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. In this case, 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.

Whilst in most cases 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. For instance, the transgenic mice can also be produced from certain totipotent cell lines, e.g. by introducing the nucleic acid into embryonic stem cells (ES cells) taken from the embryo which are then replaced such that the DNA introduced becomes integrated into the genome of these cells and of other cells as the embryo develops. In this case 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 ΞS cells may then contribute to the chimeric mice which develop, and after further breeding this can lead to the establishment of stable transgenic lines.

It will be appreciated that although the 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.

Many other variations or modifications may also be possible, and it will be seen that 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.

Claims

1. A nucleic acid which, when introduced at an earl stage into a fertilised egg or embryo of a mammalia animal, is capable of being functionally integrated int the genome thereof thereby to produce a transgenic animal characterised in that its nucleotide sequence comprises gene sequence coding for an Epstein-Barr virus nuclea antigen leader protein, or for a homologous protein fro another herpesvirus, or for a derivative of such proteins in combination with heterologous promoter and contro sequences effective to direct and regulate expression o said viral protein gene in somatic cells of a transgeni animal produced as aforesaid, such that said transgeni animal has a substantially increased probability o spontaneously developing blood clotting or thrombi in it circulatory system, especially in the heart, and/o symptoms of dilated cardiomyopathy or similar hear dysfunction.

2. A nucleic acid as claimed in Claim 1 urthe characterised in that it is a recombinant DNA construct i which the promoter and at least some control sequences ar contained in a segment upstream of the viral protein gene and the latter is fused to a downstream segment comprisin at least a fragment of a eukaryotic gene sequence effectiv to provide signals for terminating transcription and fo controlling processing of transcribed RNA during expressio of the viral protein.

3. A nucleic acid as claimed in Claim 1 or 2 in which th promoter and control sequences are provided by a mous metallothionein-I gene sequence in a segment upstream o the viral protein gene which is fused to a downstrea segment containing a human growth hormone gene sequenc (hGH) .

4. A cloning vector incorporating as an insert therein nucleic acid as claimed in any of the preceding claims.

5. A DNA having a nucleotide sequence corresponding to that of the DNA insert EcoRI-Sall or EcoRI-Hindi11 of plasmid p(DH63)MT-LP-hGH (NCIMB Accession No. 40311).

6. Plasmid p(DH63)MT-LP-hGH (NCIMB Accession No. 40311).

7. 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.

8. A method for producing a transgenic non-human mammalian animal 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, said method comprising incorporating a nucleic acid as claimed in any of Claims 1, 2, 3 or 5, or a nucleic acid derived from the plasmid of Claim 6, into the genome of a non-human mammalian animal such that it is functionally integrated therein.

9. A method for producing a transgenic non-human mammalian animal 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, said method comprising introducing recombinant DNA into a non-human mammalian animal at an early embryonic stage, said recombinant DNA being characterised in that it contains a segment comprising a gene sequence coding for a Epstein-Barr virus nuclear antigen leader protein, or for homologous protein from another herpesvirus, or for derivative of such proteins, which segment is fused to a upstream segment comprising promoter and at least som control sequences effective to direct and regulat expression of said viral protein coding segment.

10. A method as claimed in Claim 9 wherein the segment o the recombinant DNA which codes for said viral protein i also fused to a downstream segment comprising a eukaryoti gene sequence or fragment effective to provide signals fo termination of transcription and for controlling processin of the transcribed RNA.

11. A transgenic non-human mammalian animal characterise in that it contains stably integrated into the genome of a least the majority of its somatic cells and germ cells a exogenous transgene derived from a nucleic acid as claime in any of Claims 1, 2, 3 or 5, or a nucleic acid from th plasmid of Claim 6, introduced into the animal or a ancestor of the animal at a one-cell or early embryoni stage, said animal being further characterised in that i has a substantially increased probability of spontaneousl developing blood clotting or thrombi in its circulator system, especially in the heart, and/or symptoms of dilate cardiomyopathy or similar heart dysfunction.

12. A transgenic non-human mammalian animal characterised in that it contains stably integrated into the genome of at least the majority of its somatic cells and germ cells a exogenous transgene derived from a DNA sequence coding for Epstein-Barr virus nuclear antigen leader protein, or for a homologous protein from another herpesvirus, or for a derivative of such proteins, operatively associated with expression control and regulatory sequences, said DNA sequence having been introduced into the animal or an ancestor of the animal at a one-cell or early embryonic stage, said animal being further characterised in that it 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 dysfunction.

13. An animal as claimed in Claim 11 or 12 which is a rodent.

14. Use of transgenic animals according to any of Claims 11, 12 or 13 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.

15. A method of screening and identifying or testing a drug or other substance for antithrombotic, thrombolytic or cardiotherapeutic activity or for activity against heart dysfunction such as dilation cardioamyopathy, characterised in that it comprises treating selected transgenic animals of any of Claims 11 to 13 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 not treated with said 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.