WO1998045465A1 - Transgenic, non-human animal with protein deficiency, cells derived therefrom, use of said animal and cells, and method of producing said transgenic animal - Google Patents

Abstract

The present invention relates to transgenic non-human animals containing recombinant DNA having a nucleotide sequence from the third complement component gene modified in such a way that exon 24 is deleted, as well as to cells and tissue derived from said animal. It also relates to a method for producing such an animal. Furthermore, it relates to use of said animal, cells and tissue to test the involvement of the complement system in different diseases and to screen compounds interfering with the activation of the complement system.

Description

TRANSGENIC, NON-HUMAN ANIMAL WITH PROTEIN DEFICIENCY,

CELLS DERIVED THEREFROM, USE OF SAID ANIMAL AND CELLS,

AND METHOD OF PRODUCING SAID TRANSGENIC ANIMAL

Field of the invention The present invention relates to a transgenic animal containing recombinant DNA having a modified nucleotide sequence from the third complement component (C3) gene, and also to cells derived from said animal. The transgenic animal and the cells thereof are useful for re- search and development in the fields of inflammatory reactions mediated by the complement system and for testing substances inhibiting the activation and/or the function of the complement system.

Background of the invention

Complement, or the complement system, is a system of more than 30 proteins found both in plasma and on cell membranes. Complement plays an essential role in the humoral immune response. Soluble complement components are present in the blood as inactive precursors and they need to be activated to fulfil their specific physiological roles. When activated, the system mediates the following functions: initiation of inflammation, neutralisation of pathogens, regulation of antibody responses, clearance of immune complexes and disruption of cell membranes. Under certain conditions complement can, however, act as a mediator of deleterious inflammatory reactions.

The activation of the complement cascade occurs by three pathways, the classical pathway, the lectin pathway and the alternative pathway. Regardless of the pathway, the activation results in the formation of an enzymatic complex, C3-convertase, which is capable of activating the central molecule of the cascade, namely the third component (C3). The proteolytic activation of C3 gener- ates the smaller C3a fragment with anaphylatoxic properties and the larger C3b fragment with the ability to bind to an activating surface and to trigger the terminal part of the cascade, at the end of which the terminal comple- ment complex (TCC) is assembled on the target surface. C3 consists of two polypeptide chains, the α-chain and the β-chain, which are linked by one disulphide bond and by non-covalent forces (see Janatova, J. , Biochem. J. 233, 819-825, 1986; Matsuda, T., et al., Biochem. Bio- phys. Res. Com., 127, 264-269, 1985). C3 is the central molecule of the complement cascade. Patients with C3- deficiency have an increased susceptibility to pyogenic infections, and immune complex diseases are common. Despite normal antibody response to routine immunisation an impaired switch from IgM to IgG has been reported in several C3-deficient patients.

Both an excessive and an insufficient complement activation can be associated with human diseases. Excessive complement activation has been implicated in the patho- genesis of rheumatoid arthritis and some other rheumatic disorders, it has been suggested to contribute substantially to tissue damage in some of the animal models of nephritis, in human myasthenia gravis, multiple sclerosis and Alzheimer' s disease, and also in some dermatological disorders. Complement seems also to be involved in the pathogenesis of atheroma formation in atherosclerosis and in tissue damage after myocardial infarction. Complement activation may also play a role in the pathogenesis of Crohn' s disease and ulcerative colitis (see Morgan, B. P., Complement. Clinical aspects and relevance to disease., Academic Press, London 1990) . It has been proposed that the complement system response to circulatory bubbles participates in the pathogenesis of decompression sickness (Ward, C. A., et al, J. Appl . Physiol. 62, 1160- 1166, 1987; Ward, C. A., et al, J. Appl. Physiol., 60,

1651-1656, 1986; Pekna, M. , et al . , Clin. Exp. Immunol., 91, 404-409, 1993) . A wide variety of materials used in clinical medicine induce detrimental reactions when they come into contact with tissues or body fluids. The complement system has been implicated in the pathogenesis of several important syndromes occurring as a result or a complication of medical treatments. The deleterious effects of such bioincompatibility reactions may result in e.g. an increased susceptibility to infection, cardiopulmonary, renal and neuropsychological dysfunction after cardiopul- monary bypass (see Westaby, S., Intensive Care Med. 13, 89-95, 1987; and Greeley, W. J. , et al., Ann. Thorac. Surg., 52, 417-419, 1991), impaired pulmonary function after hemodialysis (see Gardinali, M., et al., Pathol . Immunopathol . Res., 5, 352-370, 1986) or chronic inflam- matory process maintained by the presence of an implant (see Remes, A., et al., Biomaterials 17:731-743, 1992).

As implied by the central position of C3 in the complement cascade, an animal lacking a functional C3 molecule will only be able to initiate the complement activa- tion pathway by the classical or the lectin pathway, but the activation cascade will be stopped before most of the known biologically active products of the cascade, namely the products of proteolytic activation of C3, the products of proteolytic activation of the fifth complement component (C5) and TCC, are generated. Thus such a C3 deficient animal would lack most of the known functions mediated by the complement system.

Transgenic animal models are useful tools to study the functions and the physiological activities of pro- teins, and a variety of such animals have been produced for this purpose. One particular technique for producing transgenic animals involves the process of homologous recombination. In homologous recombination, all or part of a genomic sequence is replaced with another DNA contain- ing homologous sequence.

Through transgenic manipulation and homologous recombination, a gene or part of a gene in the cells of an animal can be changed. By changing the gene to encode a protein that no longer functions as the native protein one creates a null mutant or null allele (see, for example, U.S. Patent No. 5,557,032).

Summary of the invention

The present invention relates to a method for producing a transgenic, non-human animal containing modified C3 DNA, said method comprising the steps of: a) introducing a transgenic DNA into embryonic stem cells of the non-human animal, the transgenic DNA comprising a C3 DNA modified in such a way that exon 24 is deleted and having both a 5' region and 3' region of ho- mology to the genomic C3 DNA of the embryonic stem cell; b) selecting an embryonic stem cell wherein the transgenic DNA has integrated into the genomic DNA; and c) introducing the selected cell from step b) into a blastocyst of a developing animal, and allowing the blas- tocyst to develop into a transgenic animal . It also relates transgenic non-human animals containing recombinant DNA having a nucleotide sequence from the third complement component gene modified in such a way that exon 24 is deleted, produced by the method described above or by any other method, as well as to de- scendants of said animals. Furthermore it relates to tissue, cell cultures and cells from said animals.

The transgenic non-human animal according to the invention is preferably a mammal, more preferably a rodent and most preferably a mouse. The invention also relates to use of said animal, tissue, cell cultures and/or cells for testing the involvement of the complement system in the pathogenesis of atherosclerosis; reactive gliosis and neurodegenerative diseases, such as Alzheimer's disease; decompression sickness, inflammatory bowel diseases such as ulcerative colitis; rheumatoid and immune complex-mediated disease; bioincompatibility reactions; for screening a compound for treatment of complement deficiency; for testing the involvement of the complement system in mucosal immunity, per orally induced tolerance and per oral vaccination; for screening and testing a compound interfering with the activation of the complement system which regard to treatment and/or prevention of atherosclerosis; reactive gliosis and neurodegenerative diseases such as Alzheimer's disease; decompression sickness, inflammatory bowel diseases such as ulcerative colitis; rheumatoid and immune complex-mediated disease; bioincompatibility reactions; for screening and testing a compound interfering with the activation of the complement system; and for screening a compound interfering with the activation of the complement system with regard to modulation of mu- cosal immunity, per orally induced tolerance and per oral vaccination.

The characterising features of the invention will be evident from the following description and the appended claims .

Brief description of the drawings

The invention will now be described in further detail hereinafter with reference to accompanying drawings on which: Fig. 1 shows a schematic representation of a strategy for producing a null mutation of the mouse C3 gene, Fig. 1A shows a restriction map of the C3 locus, Fig. IB shows a targeting DNA construct, Fig. 1C shows the location of the DNA probe and the Southern blotting screening strategy used to identify the wild-type and mutated C3 alleles, Fig. 2 shows a Southern blotting analysis of tail DNA from the FI generation of wild-type (+/+) and het- erozygote knock out mice for the C3 locus (+/-)

Fig. 3 shows a schematic representation of the targeting construct building strategy: starting materials (constructs A-D) , intermediate steps (constructs E-F) , and final construct (construct G) .

Detailed description of the invention Detailed protocols for many of the techniques known in the art are described in Ausubel, F. M., et al., Eds. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, John Whiley & Sons, Boston, MA, 1989, and Supplements through January 1997, hereinafter referred to as "Ausubel (1989)", as well as Sambrook, J. , et al., Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989, hereinafter referred to as "Sambrook (1989)", and Manipulating the Mouse Embryo. A Laboratory Manual, edited by Hogan, B., et al., Cold Spring Harbor Laboratory Press, 1994, hereinafter referred to as "Hogan (1994)". These documents are incorporated herein as reference. These documents may be relied on to enable a person skilled in the art to practice the invention.

The phrase "biological material" used herein refers to any transgenic non-human animal according to the present invention, as well as to tissue, cell cultures and cells derived from said animal. The phrases "null allele", "null mutation", and

"null mutant" refer to gene sequences encoding an altered protein as compared to the wild-type protein. In general, the "null mutant" is altered in a manner resulting in a protein substantially incapable of its primary function or in complete lack of a protein (i.e. no production) .

For example, a C3 null mutant is a DNA sequence encoding a protein that does not exhibit any of the functions for which the internal thiolester bond is required. However, functions exhibited by the β-chain (not known) and a very short amino (N) -terminal part of the -chain may be present . In addition, the "null mutant" can refer to the animal or cell bearing a gene sequence of an altered protein, as discussed above.

Transgenic DNA refers to DNA that is capable of be- ing introduced into a cell so that the DNA is incorporated into the genome of the cell. The cell may be capable of giving rise to a transgenic animal, which contains the transgenic DNA. Generally, the transgenic DNA is constructed as a vector, a transgenic vector, for admini- stration into a particular cell.

A recombinant gene or sequence simply means the gene or sequence has been manipulated in any of a number of recombinant DNA techniques known in the art.

The process for generating transgenic animals is es- sentially the same regardless of the species involved.

Briefly, transfected cells are injected into embryos at a stage at which they are capable of integrating the transfected cells, for example, at the blastocyst stage. The embryo is then replanted into a surrogate mother, result- ing in chimeric offspring possessing the transgenic DNA. Thus, all that is required is the appropriate embryonic stem cell from an animal.

The production of embryonic stem cells from a variety of animals is well known to those or ordinary skill in the art. Embryonic stem cells are available from a number of sources. These include mice, rats, cows, pigs, sheep, and other animals. For example, Joyner, A. L. describes, in Gene Targeting, A practical approach, edited by Wood, R, and Hames, B. D. , The Practical Approach Se- ries, vol. 126, Oxford IRL Press, 1993 (specifically incorporated herein by reference) , methods for producing embryonic stem cells. Also "Hogan (1994)" describes manipulation of the mouse embryo. In addition, Couly and Le Dourain, Development, 108:543-555, 1990, describe methods for isolating and manipulating chicken and quail embryos. Kimmel and Warga describe in Nature, 327:234- 237, 1987 isolation and manipulation of zebrafish em- bryos. Ware et al., Development of Embryonic Stem Cell Lines from Farm Animals, Society for the Study of Reproduction, 38:241, 1988, discuss an embryonic stem cell culture condition amenable for many species like mouse, cattle, pig, and sheep. Accordingly, this invention is as applicable to all non-human animals, even though mice are used in the examples.

Various methods for inserting DNA into animal cells are known in the art. For example, transgenic DNA can be microinjected into appropriate cells. Viral vectors can be used to introduce the DNA into appropriate cells and the genome of those cells (see, for example, Tsukui et al., Nature Biotechnology, 14:982-985, 1996). Cells can also be manipulated in vitro through transfection and electroporation methods (see Ausubel (1989) and Hogan (1994) .

Generally, transgenic DNA incorporates into a cell genome through either random integration or homologous recombination. Persons skilled in the art are familiar with strategies for increasing the relative frequency of homologous recombination versus random integration, in embryonic stem cells and other cells, in order to identify and isolate cells wherein homologous recombination has occurred (see e.g. Ausubel (1989), U.S. Patent No. 5,557,032., and Hogan (1994)). The design of transgenic

DNA vectors may involve incorporating some portion of the cellular sequence into the transgenic DNA, or a sequence homologous to the cellular sequence. That portion must be sufficiently homologous to the cellular sequence to allow the transgenic DNA and the cellular DNA to hybridise in vivo, for homologous recombination to occur.

The transgenic DNA to be inserted into an embryonic stem cell according to the present invention preferably comprises 5' and 3' regions of homology. These regions of homology function to allow the process of homologous recombination to occur in the embryonic stem cell. Numerous publications, on which persons skilled in the art may rely, discuss how to construct regions of homology for use in homologous recombination. For example, Thomas and Capecchi, Cell, 51:503-512, 1987, Mansour et al . , Nature, 317:348-352, 1988, and Capecchi, U.S. Patent No. 5,487,992 each discuss strategies for designing regions of homology in a homologous recombination technique. Thus, methods for designing a sequence for homologous recombination are known in the art.

However, homologous recombination does_, not occur for each cell where the transgenic DNA has been introduced.

In addition, it is often the case that more than one copy of the cellular DNA sequence desired to be changed or replaced through homologous recombination exists in the cell, such as in genes with more than one allele. Thus, through homologous recombination, some of the cells may incorporate the transgenic DNA at only one cellular sequence while other cells will incorporate the transgenic DNA into more than one cellular sequence, or even into every cellular sequence. Accordingly, both homozygous and heterozygous animals may be produced from the embryonic stem cells subjected to homologous recombination with transgenic DNA. A homozygous animal may be mated with wild-type animals to produce further transgenic animals heterozygotic for the transgenic DNA. To analyse the role of the complement system in the pathogenesis of a variety of pathological conditions, such as autoimmune and immune complex diseases (e.g. rheumatoid arthritis, inflammatory bowel diseases, nephritis, and systemic lupus erythematosus) , neurodegen- erative diseases (e.g. Alzheimer's disease), atherosclerosis, decompression sickness and bioincompatibility reactions in vivo, mice carrying mutated C3 alleles were generated. One allele in the mice was functionally inactivated by the technique of homologous recombination in embryonic stem cells. Mice generated from these cells, thus, carried one mutant and one wild-type allele. Fig. 1, composed of three subparts, 1A through 1C, is a schematic representation of the strategy followed in producing a null mutation of the mouse C3 gene. Fig. 1A is a restriction map of the C3 locus in which the exon 24, coding for the functionally essential internal thi- olester bond, and the cleavage sites for a number of restriction enzymes are indicated. On the drawing the restriction enzymes are identified as follows: E = EcoRI, B = BamHI, and H = Hindlll. The transgenic DNA construct of Fig. IB was used to generate a mutant C3 allele. The construct contains a 5' homologous fragment (BamHI /BamHI, 4.8 kb) and a 3' homologous fragment (BamHI/NotI, 6.0 kb) ; the NotI site being from the multiple cloning site of the λ-phage) of the C3 gene, which flank the pPGK- neocassette (neo) . Fig. 1C shows the location of the DNA probe used to identify wild-type and mutant alleles of C3 in Southern blot analysis.

In Fig. 2 the migration of the wild-type and the recombinant Hindlll fragment after hybridisation with the DNA probe is shown.

In the following sections, detailed procedures of how animals carrying mutant C3 are produced are described. The examples below are only intended to illustrate the invention and should in no way be considered to limit the scope of the invention, and even though mice are used in all the examples any transgenic non-human animal can be used in accordance with the present invention.

Example 1: Selection of C3 sequences useful as a transgene

The DNA used to generate a transgenic animal can contain a number of different DNA sequences. The selection of an appropriate sequence depends on the desired effect. For example, if an transgenic non-human animal possessing a null mutant of C3 is desired, the transgenic DNA contains sequences encoding a protein that is incapa- ble of forming the C3 and C5 convertase, generating the C3a anaphylatoxin, binding to a target surface via a co- valent bond and binding to the complement receptors for C3b and its cleavage products. In this example, a portion of the mouse C3 gene was deleted. The deleted portion contains the internal thiol- ester bond important for the conformation of the protein and its ability to bind covalently to target surfaces.

In order to select the DNA sequences to be modified according to the invention, the gene for the mouse C3 was first cloned from a mouse λFIXII-library (Stratagene, La Jolla, CA) using pHLC3.11 (de Bruijn, M. H. L. and Fey, G. H. Proc. Natl. Acad. Sci. USA 82:708-712, 1985) as the probe. Four λ-clones denoted F, G, H, I, each carrying about 20 kb of mouse genomic DNA, were isolated and characterised. A detailed restriction map of the genomic DNA was generated by digestion of the λDNA with different restriction enzymes followed by identification of the restriction fragments by Southern blotting. This map was comparable with the map published by Weibner, K et al., Proc. Natl. Acad. Sci. USA 79:7077-7081, 1982. ³²P- labelled oligonucleotide probes obtained from the sequence of the mouse cDNA coding for the exon 24 were used in the Southern blotting. The information obtained from the restriction map of the λDNA harbouring the C3 gene was used to select a DNA for transgenesis . From the restriction map shown in Fig. 1, one way of making a targeted, transgenic construct that would introduce a null mutation in the C3 gene is by deleting a restriction fragment. Replacement of the exon 24 with flanking sequences by PGK neo creates a null mutation.

Example 2: Production of transgenic vector with C3 deletion mutant

According to one embodiment of the invention, the internal thiolester bond of C3 was removed. To do this, a 3.5 kb fragment containing exon 24 was removed and replaced by pPGK neo. The C3 protein generated from the targeted allele is non-functional as it lacks the internal thiolester bond and the allele is thus a null allele. The starting materials for the generation of a recombinant transgenic vector lacking the 3.5 kb fragment and exon 24 of C3 was a 4.8 kb BamHI/BamHI fragment from λ-clone F, subcloned into plasmid pBluescript™ (Stratagene) (construct A), a 6.0 kb BamHI/NotI fragment (used to generate the 3' region of the transgenic vector) from λ-clone F, subcloned into plasmid pBluescript™ (construct B) , a 1.7 kb pPGK neo cassette (Sibilia, M. Science 269:234-238, 1995) cloned into pBluescript™ (construct C) and a 1.6 kb pPGK dTA cassette (pPGK dTA) (see Yagi, T., et al . , Analyt. Biochem. 214:77-86, 1993) cloned in pBluescript™ (construct D) . A schematic representation of the constructs A-G is found in Figure 3.

The 4.8 kb BamHI/BamHI fragment used to generate the 5' region of the transgenic vector was isolated by cutting this fragment from construct A with BamHI. Blunt ends from the BamHI sticky ends were generated with the Klenow fragment of T4 polymerase (Klenow fragment) . The 4.8 kb fragment was isolated on agarose gel made in 0,5 x TBE (Tris Borate EDTA) buffer and ligated into Hindlll digested and Klenow fragment blunted construct B, resulting in construct E.

The pPGK neo was isolated by digesting construct C with NotI and Sail and blunting with Klenow fragment. The resulting 1.7 kb fragment was ligated into EcoRI digested and Klenow fragment blunted construct E, generating construct F.

The pPGK dTA was isolated by digesting construct D with Xhol and blunting with Klenow fragment. The resulting 1.6 kb fragment was ligated into Kpnl digested and T4 DNA polymerase blunted construct F, generating construct G. This vector, containing 10.8 kb of DNA sequence homologous to the mouse C3 gene with the pPGK neo inserted in the backward direction, was chosen as the targeting vector for transgenesis . This targeting vector differs from the targeting vector used by Wessels, M. R. , et al., Proc. Natl. Acad. Sci. USA 92:11490-11494, 1995. Wessels et al. replaced a 0.6 kb of the C3 gene, coding for the C-terminal region of the β-chain and the N-terminal region of the α-chain, with a PGK neo cassette, thus also creating a null mutation of the C3 gene.

A Southern blot assay was used to distinguish be- tween the wild-type versus the mutated alleles. Genomic DNA was digested with Hindlll and prepared for blotting. The 2.1 kb radiolabelled fragment, used as the probe in this screening procedure, was generated by subcloning the 2.1 kb EcoRI/BamHI fragment from λ-clone F into pBluescript™ and subsequently isolated by digestion with EcoRI and BamHI, separation on agarose gel and extraction. The mutated allele gives rise to a genomic Hindlll restriction fragment of 8.7 kb while the wild-type allele generated a 16 kb fragment under these conditions.

Example 3: Insertion of the transgenic C3 vector into animal cells

The vector containing the mutant C3 (construct G) was transfected into E14.1 cells derived from the 129/Ola mouse (Kuhn, R. , et al., Science 254:707-710, 1991) by electroporation. Neomycin resistant clones were isolated by standard procedures and characterised. "Hogan (1994)" describes procedures for isolating and manipulating embryonic stem cells. Genomic DNA f,rom several hundred clones were isolated, Hindlll digested, and analysed by

Southern blotting. Using these procedures, three positive clones denoted 6W, 10R, and 10S were identified by detecting the 8.7 kb band and also the wild-type 16 kb band. This shows that one allele of the two C3 alleles in the embryonic stem cells has been correctly targeted by the construct and that embryonic stem cells containing the null allele for C3 were generated. Example 4: Production of transgenic C3 animals Cells from the cell lines 6W, 10S and 10R were injected into mouse blastocysts using standard procedures. "Hogan (1994)" describes procedures for injecting cells into blastocysts. The blastocysts were implanted into pseudopregnant foster mothers. The resulting chimeric mice which were generated, i.e. those possessing the transgenic DNA, were identified by the coat colour. For example, since the embryonic stem cells were derived from the 129/Ola mouse with chinchilla (white- yellow) fur coat and carriers of the Agouti locus, whereas the host blastocysts were derived from the C57BL/6 mouse with a black coat and lacking Agouti locus, the chimera will be a mixture of chinchilla (patches of skin to which only the injected embryonic stem cells contributed) , black (patches to which only the host cells contributed) and brown or Agouti (patches of skin in which there was a mixture of 129/Ola and C57BL/6 cells) . These brown patches result from C57BL/6 hair follicle cells being stimulated by the Agouti protein, secreted from the 129/Ola cells, to process melatonin (black pigment) in such a way that the hair becomes brown.

Male chimeric mice were mated with wild-type C57BL/6 females. The presence of brown mice in the offspring from these females indicated germline transmission of the transgenic DNA.

DNA prepared from tails of these FI mice were analysed by Southern blotting as above. Generally, 0,5 cm of tail tissue was surgically removed and used to prepare DNA samples. The 8.7 kb Hindlll fragment was present in 50% of the brown (Agouti) mice indicating that there was no selection against the targeted null allele of C3. The FI mice carrying the targeted C3 locus are heterozygous as they also carry a wild-type allele for C3. The presence of normally developed Fl mice carrying one inacti- vated allele of C3 shows that gene dosage for C3 is not critical for survival.

Heterozyte FI mice were crossed to generate mice homozygous for the inactivated C3 allele.

Example 5: Use of the transgenic non-human animal according to the invention as a model to study the involvement of complement activation in the pathogenesis of human disease The transgenic non-human animal according to the invention can be used as a model to study the involvement of complement activation in the pathogenesis of human diseases, such as atherosclerosis; reactive gliosis and neurodegenerative diseases, such as Alzheimer's disease; decompression sickness, inflammatory bowel diseases, such as ulcerative colitis; rheumatoid and immune complex- mediated diseases; and bioincompatibility reactions.

Atherosclerosis : Recently, mice deficient for apo- lipoprotein E (ApoE) were generated by gene targeting and were shown to develop lesions similar to those seen in human atherosclerosis (see Plump, A. S., et al., Cell 71:343-353, 1992, and Zhang, S. H., et al . , Science 258:468-471, 1992). The C3 null allele can be crossed on the ApoE-/- background and the C3-/-ApoE-/- mice can then serve as a test model to assess the effect of the complement deficiency on the time of onset and the progression of the atherosclerotic lesions as well as any potential treatment involving interference with the complement activation. Reactive gliosis and Alzheimer's disease: Recently, a mouse model for the Alzheimer' s diseases was developed by transgenic technology (see Games, D. , et al., Nature, 373 (6514) :523-527, 1995). By breeding the C3-/- allele on this respective genetic background, an animal model will be generated to examine the affect of the absence of complement activation on the development and time course of the histological hallmarks of this disease. Subsequently, the therapeutic efficiency of any interference with the complement activation can be tested on this model.

Bioincompatibility reactions: Recently it was shown that complement activation triggered by artificial mate- rial can be substantially reduced by surface modification with immobilised heparin (see Pekna, M., et al . , Biomate- rials, 14:189-192, 1993; Pekna, M. , et al., Ann. Thorac. Surg., 58-421-424, 1994; and Pekna, M., et al., Scand. J. Thor. Cardiovasc. Surg. 28:5-11, 1994). However, it re- mains to be elucidated to which extend the complement activation per se is necessary for all the various consequences of the contact of tissues and blood with artificial materials in vivo. The C3 deficient mice can be used to assess the contribution of the complement system to the inflammatory response and bioincompatibility reactions to various artificial materials and to test the efficiency of any potential interference with the complement activation in this context.

Decompression sickness: Recently it was demonstrated that mild changes occur in the complement system after decompression which was, however, insufficient to induce any signs of decompression sickness (see Pekna, M. , et al., Undersea Hyberbar. Med., 23:31-34, 1996). Both humans and rabbits that had been identified as being more susceptible to complement activation by the alternative pathway were also found to be more susceptible to decompression sickness and it has been proposed that the complement system response to circulatory bubbles participates in the pathogenesis of decompression sickness (see Ward, C. A., et al., J. Appl. Physiol. 62:1160-1166,

1987; and Ward, C. A. et al . , J. Appl. Physiol., 60:1651- 1656, 1986) . It has earlier been shown that the shear forces at blood-gas interface lead to a conformational change of the C3 molecule and the formation of iC3 (see Nilsson Ekdahl, K. et al., Scand. J. Immunol., 35:85-91, 1992; and Pekna, M. , et al., Clin. Exp. Immunol. 91:404- 409, 1993) . It has been proposed that the generation of iC3 and its capability of forming a C3-convertase may be the triggering mechanism of complement activation by gas bubbles. The C3 deficient mice can serve as a model to evaluate the role of the complement system in the patho- genesis of decompression sickness as well as to test any potential interference with the complement system as a treatment or prevention of this condition.

Ulcerative colitis: It has been shown that Gαi2 deficient mice generated by gene targeting develop inflam- matory bowel disease that is clinically and pathologically similar to ulcerative colitis in humans (see Rudolph, U. et al., Nature Genetics, 10:143-150, 1995). By breeding the C3-/- allele on the Gαi2-/- 129SV background, an animal model would be generated which would allow the testing of the role of the complement system in the development of ulcerative colitis as well as testing of any potential treatment of this condition by interference with the complement activation.

Rheumatoid and immune complex-mediated diseases: The role of the early complement components in the pathogenesis of rheumatoid arthritis can be tested by collagen type II immunisation of C3-/- mice and evaluating the joint swelling and histology of the C3-/- and control animals. For this purpose the C3 allele must first be bred on DBA/lLAcJ background. This model offers also the possibility to test the effects of a therapeutic interference with the activation of the complement cascade.

Systemic lupus erythematosus : The C3 deficient mice can be monitored for the development of histological signs of glomerulonephritis which can ensue as a result of insufficient clearance of immune complexes from the circulation due to the inability to activate the complement cascade. The C3 null allele can be bred on the New Zealand black (NZB) and New Zealand white (NZW) back- ground and C3 deficient NZB/NZW FI females examined as to the course of the renal disease (histology, proteinuria, survival) and compared with C3+/- and C3+/+ littermates. This model offers also the possibility to test the effects of a therapeutic interference with the activation of the complement cascade.

Example 6: Use of the transgenic non-human animal according to the invention as a model to study primary complement deficiencies in human diseases

Homozygous deficiencies have been recognised for the majority of the complement components and regulatory pro- teins and are often associated with a disease. Depending on the protein missing, the patients suffer from recurrent pyogenic infections, immune complex mediated autoimmune diseases, Neisserial infections, hereditary angio- oedema or paroxysmal nocturnal hemoglobinuria . As stated above patients with C3 deficiency have an increased susceptibility to pyogenic infections, and immune complex diseases are common.

The C3 deficient mice represent an optimal animal model to test the efficiency of the treatment of the re- spective immunodeficiency.

Example 7 : Use of the transgenic non-human animal according to the invention as a model to study the role of complement activation in mucosal immunity, per orally induced tolerance and per oral vaccination

The C3 deficient mice represent a model for the testing of the importance of the complement system for mucosal immunity, per orally induced tolerance and per oral vaccination and consequently the effects of the in- terference with the complement activation in this context .

Claims

1. A method for producing a transgenic, non-human animal containing modified C3 DNA, said method comprising the steps of: a) introducing a transgenic DNA into embryonic stem cells of the non-human animal, the transgenic DNA comprising a C3 DNA modified in such a way that exon 24 is deleted and having both a 5' region and 3' region of homology to the genomic C3 DNA of the embryonic stem cell; b) selecting an embryonic stem cell wherein the transgenic DNA has integrated into the genomic DNA; and c) introducing the selected cell from step b) into a blastocyst of a developing animal, and allowing the blastocyst to develop into a transgenic animal.

2. A transgenic non-human animal containing recombinant DNA having a nucleotide sequence from the third complement component gene modified in such a way that exon 24 is deleted.

3. A transgenic non-human animal produced by the method according to claim 1.

4. A transgenic non-human animal that is a descen- dent of an animal according to claim 3.

5. An animal according to anyone of claims 2-4, wherein the animal is a mammal.

6. An animal according to claim 5, wherein the animal is a rodent.

7. An animal according to claim 6, wherein the animal is a mouse.

8. Tissue derived from an animal according to anyone of claims 2-7.

9. Cell cultures derived from an animal according to any of claim 2-7.

10. Cells derived from an animal according to any of claim 2-7.

11. Use of the biological material according to anyone of claims 2-10 for testing the involvement of the complement system in the pathogenesis of atherosclerosis; reactive gliosis and neurodegenerative diseases, such as Alzheimer's disease; decompression sickness, inflammatory bowel diseases, such as ulcerative colitis; rheumatoid and immune complex-mediated disease; and/or bioincompatibility reactions.

12. Use of the biological material according to any- one of claims 2-10 for screening a compound for treatment of complement deficiency.

13. Use of the biological material according to anyone of claims 2-10 for testing the involvement of the complement system in mucosal immunity, per orally induced tolerance and per oral vaccination.

14. Use of the biological material according to anyone of claims 2-10 for screening and testing a compound interfering with the activation of the complement system which regard to treatment and/or prevention of athero- sclerosis; reactive gliosis and neurodegenerative diseases, such as Alzheimer's disease; decompression sickness, inflammatory bowel diseases, such as ulcerative colitis; rheumatoid and immune complex-mediated disease; and/or bioincompatibility reactions.

15. Use of the biological material according to anyone of claims 2-10 for screening and testing a compound interfering with the activation of the complement system.

16. Use of the biological material according to anyone of claims 2-10 for screening a compound interfering with the activation of the complement system with regard to modulation of mucosal immunity, per orally induced tolerance and per oral vaccination.