WO1991002793A1 - Cardiomyopathic transgenic non-human mammal - Google Patents

Abstract

A transgenic non-human mammal whose germ cells and somatic cells contain the sequence of the PVLT antigen under the direct control of a non-polyoma virus promoter sequence preferably an inducible promoter sequence. This fusion gene is introduced into the non-human mammal or an ancestor thereof at an embryonic state. The resulting non-human mammal is characterized by an enhanced propensity towards the development of cardiomyopathy. Also within the scope of the present invention is a method for inducing cardiomyopathy in a non-human mammal. This method comprises introducing into a non-human mammal or an ancestor thereof at an embryonic stage, a fusion gene comprising the above-mentioned sequence to produce transformed embryos. The surviving transformed embryos are implanted into pseudopregnant females and the resulting progeny is screened for the presence of transgenes.

Description

TITLE OF THE INVENTION

Cardiomyopathic transgenic non-human mammal. FIELD OF THE INVENTION

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

The techniques through which the development of animals carrying a gene which has been introduced into their germ line or into an ancestor of the animals at an early stage of development have been substantially perfected over the last years and are becoming more and more easily accessible. Since 1981, a wide variety of transgenic mice containing genes such as human globin genes, rabbit globin genes, chicken transferrin genes, immunoglobulin genes, rat growth hormone genes, thymidine kinase genes, and human growth hormone genes have been described in the scientific literature.

In U.S. patent 4,736,866, issued on April 12, 1988 and assigned to Harvard University, the inventors Leder and Stewart disclose a non-human transgenic mammal, preferably mice, containing an activated oncogene sequence which, when incorporated into the genome of this mamal, increases its susceptibility to develop neoplasms such as malignant tumors. Mammals of this type have been found to be useful to test materials suspected of being carcinogenic by exposure to carcinogenic substances and determination of neoplastic growth as an indicator of carcinogenity.

Many oncogenes may be used for the production of transgenic animals such as transgenic mice. Among the 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. Polyomavirus large T- antigen (PVLT) is a nuclear phosphoprotein and the only viral encoded protein absolutely required for polyomavirus DNA replication. It also regulates early and late gene expression in polyomavirus infected cells. In vi tro studies have classified PVLT as an immortalizing gene. Transgenic mouse technology has permitted the introduction of novel genes into the genomes of mice. The transforming genes of both 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.

Transgenic 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.

In order to improve expression of oncogenes in transgenic mice, the heavy-metal responsive metallothionein promoter (MT) has been widely employed. It is primarily active in heart, testes, liver, kidney and pancreas in transgenic mice but activity has also been found in numerous other tissues.

Numerous studies have indicated the scarcity of effect of fusion genes in cardiac tissues and, as the incidence of cardiac tumors is rare, cardiac tissue was tought to be relatively insensitive to the action of oncogenes. The expression of the SV40 T-antigen in mice heart caused the development of tumors. It appears therefore that very few oncogenes are able to induce a reaction in the cardiac tissues and even in cases where a reaction is observed, it usually translates into the development of tumors.

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

In accordance with the present invention, there is provided 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. The expression "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. Also within the scope of the present invention is a method for inducing cardiomyopathy in a non-human mammal. This method 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. Hence, 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. The 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. For example, 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.

The characteristics of the transgenic mammals obtained in the context of the present invention represent a totally unexpected finding. Hence, these novel mammals are the first transgenic mammals to develop a proportionately oversized heart through the introduction of an oncogene sequence. In fact, as mentioned earlier, prior art studies conducted so far demonstrated that cardiac tissues are virtually unaffected by oncogene substances and that when they are affected, tumors rather than oversized organs are developed. No macroscopic nor microscopic tumors were found in any tissue analyzed from the MT-PVLT line 8 mice.

Thus, the expression of the MT-PVLT fusion gene in the heart of the transgenic mammal is accompanied by a massive hyperplasia. Heart sizes ranging from 1.5 to 5.24 times greater in weight than normal have been observed. It has also been observed that the mammals usually die of cardiac arrest at, on average, 160 days. The transgenic mammals of the present invention, preferably transgenic mice, 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). There are several means by which an oncogene can be introduced into a mammal embryo so as to be chromosomally incorporated in an activated state.

The present invention will be readily illustrated by referring to the following description. IN THE DRAWINGS

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; and

Figure 7 represents a comparison of cardiomypathy between male and female MT-PVLT mice. DESCRIPTION OF PREFERRED EMBODIMENTS Construction of plasmids pMT-PVLT and PPVR2 a) Generation of plasmid pMT-PVLT.

In order to produce plasmid pMT-PVLT, a 2808 bp. Xho 1 to Bgl II fragment of the cDNA for polyomavirus large T-antigen available at the American Type Culture Collection under accession number 41043 was added to an oligonucleotide fragment containing sites for the restriction endonucleases Bgl II - Xba I - Hind III - Hpa I - Xho I. Both fragments are shown in Figure 1. The resulting fragment was subsequently cut to completion with Bgl II and the smallest fragment (2.4 Kb) containing the above-mentioned oligonucleotide and PVLT cDNA was subsequently purified. The Xho I site in the oligonucleotide was destroyed in cloning. 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

To generate plasmid pPVR2, plasmid pGEM-1 which may be purchased from Promega was cut to completion with ECO RI and Hind III. 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). In fact, 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. The 1470 bp fragment, containing the polyomavirus early region, was isolated and ligated to the prepared pGEM-1 DNA. DNA from the resultant plasmid, pPVR2, was isolated from transformed bacteria using techniques described in the Maniatis et al. reference referred to above. In order to prepare the pPVR2 DNA for probe generation, the pPVR2 DNA was cut to completion with Hind III. The length of RNA probe generated was 1490 nt long and contained polyomavirus early region introns not present in the cDNA for polyomavirus large T-antigen. 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.

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. MT-PVLT DNA, free of vector sequences, was microinjected into fertilized 1-cell mice embryos, the surviving embryos immediately implanted into pseudopregnant females and the results of progeny screened for the presence of transgenes. All micro-injection procedures and oviduct transfers were performed as described in the above- mentioned DePamphilis et al reference. C57B1/6, BALR/C, CD-I and vasectomized CD-I male mice used were purchased from Charles River Laboratories Inc. Isolation of transgenic mice

The presence of the integrated transgene MT-PVLT DNA was essayed by Southern Blot Analysis of Genomic DNA by hybridization to the polyomavirus specific probes described above. 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. Animals 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.

Analysis of expression of polyomavirus large T-antigen in transgenic mice

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.

RNase protection essay and Northern blot analysis were performed to determine the tissue distribution of expression of the transgene in representative animals from each line of mice. Thus, total RNA, 20 to 50 ug, 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. A small quantity of the radioactive RNA probe was checked to determine if full length RNA was generated by electrophoresis through an 8M urea/6% acrylamide gel as described in the Maniatis et al. reference referred to above. Electrophoresis was followed by exposure to Kodak XAR-5 film. 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. After RNase digestion and preparation, the samples for electrophoresed through a 8M urea/6% acrylamide gel and the gel exposed to Kodak XAR-5 film with an intensifying screen for 3 to 6 days. 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.

Total 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.

Results of RNase protection analysis of RNA isolated from heart and testes from 6 lines of transgenic mice are shown in Figure 2. In Figure 2a, 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. In Figure 2a, the various lanes represent the

32 following: 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

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 μg RNA from Rat-1 cells transformed with polyomavirus.

In Figure 3, which represents M, +, and - are the same as above; lane a.- liver; lane b: kidney; lane c: heart; lane d: thymus; lane e: brain; lane f: uterus and ovaries; lane g: mastoid; lane h: stomach. Transcripts complimentary to the probe were present in RNA isolated from tissues of all 6 lines of mice. MT- PVLT lines 7, 8, 9, 10, and 11 (Figure 2 panel A., lanes a, b, d, e and f respectively) yielded correctly sized protected fragments in testeε. Lines 10 and 12 also had protected fragments in seminal vesicles (data not shown). MT-PVLT line 8 was the only line of mice to demonstrate RNA protected fragments in heart (Figure 2 panel B, lane b). RNA isolated from MT-PVLT line 8 testes (Figure 2 panel A, lane c) as well as thymus, uterus and ovaries (Figure 3, lanes d and f respectively) contained detectable RNA protected fragments. 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). Approximately 58% of MT-PVLT line 8 transgene positive animals developed a cardiac phenotype whereas other MT-PVLT line 8 animals remained healthy. 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

Animals were sacrificed by cervical dislocation and the heart and kidneys removed. Hearts were placed into basal media (DMEM plus 50 μg/ml fungizone) and minced. The pieces were placed in digestion media (basal media plus 50 μg/ml trypsin and 20 units/ml collagenase) and digestion allowed to proceed overnight at 37 C in the cell culture incubator. The cells were then washed free of enzymes and placed in growth media (basal media plus 10% horse sera) -and plated onto gelatin-coated dishes. Kidneys were minced then placed in digestion media (50% DMEM plus 50% versine plus 2.4 mg/ml trypsin and antibiotics) and incubated overnight at 4 C. After a 30 minute incubation at 37 C, the cells were washed free of enzyme and resuspended in growth media (DMEM plus 5% FBS plus antibiotics) and plated onto dishes.

Dpn-1 assay for polyomavirus DNA replication

Heart and kidney cells from MT-PVLT line 8 animals and MOP-8 and Rat-1 cells were transfected following the procedure described in 1978 cell ___\_, 725- 731 with 20 μg of pPVa3ς DNA. 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/3ς 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. .26., 365-369) were performed 72 hours later and DNA isolated from supernatants and pellets. Plasmid DNA containing polyomavirus DNA, pPV805, 100 pg, was added to each sample and the DNAs digested with Dpn 1 and Eco Rl. The DNAs were electrophoresed through agarose, blotted to Gene Screen Plus membrane and hybridized to a random primer generated probe, specific to the polyomavirus origin containing fragment (5039 to 90) present in pPVaøg DNA and pPV805 but not the transgene MT-PVLT, as described above. Plasmid pPV805 linear DNA was 8292 bp long and pPVa/3c linear DNA was 3316 bp long. As shown in Figure 7, 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.

TABLE 1

Comparison of Cardiac Myopathy in MT-PVLT Line 8 Affected Mice

MT-PVLT line 8 mice Fl

both 50-100 152

10

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.

Macroscopically the left and right hearts were normal in proportion however their large size distorted the chest cavity and restricted the space in the chest cavity for the lungs. Figure 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. When the affected heart was opened, 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

Animals were sacrificed by cervical dislocation and organs were removed and placed in 4% buffered formaldehyde. For light microscopy the heart was sliced coronally, tissue slices processed routinely and embedded in paraffin. Thick sections of 7 microns, were stained with hematoxylin and eosin and examined microscopically. Other tissues examined included liver, kidney, lung, pancreas, thymus, salivary glands, ovary, uterus, testes, urinary bladder, brain, esophagus, stomach, intestines, and skeletal muscle. For electron microscopy, 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.

By light microscopy, large cardiomyocytes, up to 50 microns in width and more than several hundred microns in length with bizarre 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.

Histologically the hearts were found to be hyperplastic, dysplastic and display cellular .and morphological changes consistent with a failing heart. This type of abnormality is usually indicative of cardio egaly 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 bizarre-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.

Primary cells were isolated from the enlarged heart and normal kidney of MT-PVLT line 8 animals and assayed for polyomavirus large T-antigen expression. 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. In order to specifically examine plasmid DNA replication we used the 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.

The plasmid, pdPaøc, 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 pdPVaøσ_. DNA isolated from both the heart (lanes e and f) and kidney (lanes h and i), but not from the Rat-1 cells (lane g), or cells from non-transgenic mice (lanes c and d) was found to be Dpn 1 resistant. The pPV805 DNA is absent from the blot indicating that the endonuclease digestion was complete. Functional analysis appeared to be very sensitive in assessing expression of the PVLT and RNA isolated from kidney was negative in RNase protection or Northern analysis for polyomavirus-specific RNA. Post- translational modifications necessary for DNA replication activity of PVLT must have been complete in the MT-PVLT line 8 heart and kidney cells in order for pVLT-antigen to function in polyomavirus DNA replication.

Claims

CLAIMS :

1. A transgenic non-human mammal whose germ cells and somatic cells contain the sequence of the PVLT antigen under the direct control of a non-polyoma virus promoter sequence, introduced into said non-human mammal or an ancestor thereof at an embryonic stage, said non- human mammal being characterized by an enhanced propensity towards the development of cardiomyopathy.

2. A transgenic non-human mammal according to claim 1, wherein the promoter is an inducible promoter.

3. A transgenic non-human mammal according to claim 2, wherein said promoter sequence comprises the sequence of a metallothionein promoter.

4. A transgenic non-human mammal according to claim 1, wherein said mammal is a mouse and wherein said promoter comprises the sequence of a mouse metallothionein promoter.

5. A transgenic non-human mammal according to claim 1 wherein said mammal is a rodent.

6. A transgenic non-human mammal according to claim 1 wherein said mammal is a mouse.

7. A method for inducing cardiomyopathy in a non-human mammal which comprises introducing into said 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-polyma virus promoter thereby producing transformed embryos, implanting said surviving transformed embryos into pseudopregnant females and screening the resulting progeny for the presence of transgenes.