DE10025855A1 - Producing transgenic mammal comprising reduced expression of DNAse I, useful for studying development of systemic lupus erythematosus and in drug screening - Google Patents

Abstract

Producing, (P), a transgenic, non-human mammal (A) that has at least partial reduction in activity of DNAse I (I), is new. Production of a transgenic, non-human mammal (A) that has at least partial reduction in activity of DNAse I (I), where an embryonal stem cell is transfected with a (I)-targeting vector and stable clones isolated in which the endogenous (I)-encoding gene has been (partially) replaced by other sequences or destroyed, as a result of homologous recombination with parts of the targeting vector. These clones are implanted into a suitably prepared surrogate dam and progeny selected for presence of DNA incorporation. Independent claims are also included for the following: (1) (A) produced by P; (2) recombinant (I)-targeting vector used for homologous recombination; (3) stably transfected cells in which all or part of the (I)-encoding gene has been replaced by another DNA sequence; (4) model system for analysis of either the origins of SLE (systemic lupus erythematosus) or treatments of diseases, specifically SLE, that includes at least one (A); (5) testing new pharmaceuticals for activity against SLE, using (A) or stable cell clones or organs derived from (A); and (6) testing the affects of genetic, biological, chemical and physical factors on development of SLE.

Description

The invention relates to a transgenic non-human mammal, a method for the same Production, its use, cell tissue therefrom, a process for the production of such Cell tissue, its use, a recombinant DNA targeting vector and the Using this vector. - The term non-human mammals refers to Taxonomically higher units than animal species. Transgenic animals are organisms that have a additional gene that is not of its type, i.e. foreign, or a part of it in your Bear genome, or have lost part or all of a gene (gene deficiency) and in its place a foreign gene not from this animal species or a part thereof wear. Within the scope of the invention, in particular those are genetically modified We mean animals that have the foreign gene or the gene deficiency in the germ cells that that is, the foreign gene or gene deficiency vertically, i. e. from generation to generation pass on. Recombination vectors or targeting vectors are DNA constructs that are constructed in such a way that they introduce foreign DNA into the genome or the Allow replacement of genomic sequences of cells, germ cells or organisms. There too Germ cells carrying transgenes or gene deficiencies can have other corresponding transgenes Animals obtained through breeding when a special transgenic animal has been created is. Transgenic animals are known in various forms and also different Methods for producing transgenic animals are known. This is just exemplary to the literature reference ("Gene Targeting, A Practical Approach, Edited by A. L. Joyner IRL Press Oxford University Press, New York, 1993 and R. Jaenisch, Science, Vol 240, 10: 1468 ff 1988), as well as the references cited therein.

The expression cell tissue encompasses complete organs or parts of organs of an animal, however also very specific cell lines, which are isolated and cultivated therefrom, i. e. be increased can. A recombinant DNA vector is an instrument for producing a gene altered animal, which is the foreign sequence that is introduced into the animal's cells should, carries and can contain other further sequences.

The general technological background of the special created with the invention transgenic animal is as follows. SLE is a chronic disease of unknown etiology and runs in spurts. The disease occurs in humans preferably in women between the 2nd and 5th decade of life and in the worst case is fatal, with the death rate at SLE patients are three times higher than the normal population. The most common  The causes of death are uremia, heart failure and internal bleeding. SLE is primary characterized by immune complex-induced cell and tissue damage in various organs, with antinuclear antibodies (ANA) being central in pathogenesis Play a role (<95%). Essentially it is AK against dsDNA, ssDNA, RNA, Histones, nucleosomes and ribonucleoproteins (Berden et al., 1999; Eilat and Naparstek, 1999). One way of pathogenesis is the deposition or accumulation of Immune complexes e.g. B. in the vessel walls, the glomeruli of the kidney, the serous skins (Pleura, peritoneum, pericardium), the endocardium, the joints and the skin with one subsequent type III hypersensitivity reaction. As a result, it happens various inflammations such as glomerulonephritis, arthritis, serositis, endocarditis and vasculitis. Glomerulonephritis results in proteinuria and hematuria up to end-stage renal failure (uremia). The second way of pathogenesis is through Cross-reaction of certain ANA with surface antigens of body cells and one subsequent AK-mediated cytotoxic hypersensitivity reaction (type II). In particular, erythrocytes, platelets, lymphocytes and the Mesangial cells and podocytes of the kidney affected by the cross reaction (Raz et al., 1998; Raz et al., 1993; D'Andrea et al., 1996). Symptoms like hemolytic anemia, Splenomegaly, thrombocytopenia and leukopenia are the result. To overview of the topic SLE see: Berg, 1987; Riede and Herbst, 1995; Vierbuchen and Böcker, 1997; Vanholder et al., 1998; Carroll, 1998.

So far, in order to study the effects of SLE on an organism both on naturally occurring mouse mutants and on certain ones due to genetic engineering Modifications made mouse models used for SLE. F1 generation mice, from a mating of the strains NZB and NZW (for New Zealand Black and -White) arise (Morel and Wakeland, 1998), but also animals that have the lpr allele of the Fas receptor or have the gld allele of the Fas ligand inherited, show SLE symptoms (Takahashi et al., 1994). In addition to these naturally occurring mouse mutants, genetic engineering Manipulation mice are generated that have inactivating mutations in the gene for the serum Amyloid Protein (SAP) (Bickerstaff et al., 1999) or the complement factor C1q (Botto et al., 1998). All of these animals show different degrees of SLE symptoms.

The mouse models that existed so far have several disadvantages. First is not Known whether serum amyloid protein (SAP) is part of the complement cascade in patients is reduced or even absent with SLE. The lack of the CD95 receptor in the lpr Mouse mutant and similarly the lack of the CD95 ligand in the gld mouse mutant only causes an overshoot of the immune system and only leads to SLE secondarily Symptoms. Tests of immunomodulatory drugs for the therapy of SLE is on some these models are not possible because the animal's immune system is disturbed. This is especially true for  the lpr and gld mouse mutants and for the C1q deficient mouse mutant. The NCB, NZW Mouse mutants show symptoms only in the F1 generation SLE and are not genetically clearly characterized. A model that reflects the pathophysiological conditions of human So far, SLE comprehension does not exist.

It is therefore an object of the invention to overcome these disadvantages and a new one on the provide pathophysiological conditions of SLE based model. This The object is achieved according to the invention by a method for producing a transgenic non-human mammal, an embryonic stem cell with a suitable DNA Vector (Dnase 1 targeting vector) is transfected, stable cell clones are isolated and on homologous recombination at the Dnase 1 gene location can be checked. This check shows whether a foreign gene has been inserted into the genome and the Dnase 1 gene partially or completely has replaced. This stable cell clone is expanded and individual cells from it are transformed into Blastocysts injected into a non-human mammal, then the appropriate female animals implanted by a non-human mammal for delivery. The received Descendants become homologous to the presence of DNA injection and Recombination selected at the Dnase 1 gene location is characterized in that the transgenic, non-human mammals due to the introduction of DNA, a partial or shows complete reduction of Dnase 1 activity. Embryonic stem cells are cells that still have the ability to differentiate into all cells of an organism. Therefor For example, cells with the parent line name E14 can be used. Transfection refers to the process of using a vector in the genome of a foreign DNA Smuggling in cells. Stable cell clones are descendants of a cell that are mitotic Cell division has arisen, which is a gene introduced by transfection or by Transfection introduced foreign DNA have stably integrated in the genome and to the Pass on daughter cells. As suitable female animals of a non-human mammal are referred to those that according to their natural or through hormone treatment induced reproductive status for fertilized eggs or blastocysts are receptive and are capable of carrying off developing offspring.

The method according to the invention was the first to enable a transgenic non-human Mammal can be generated that Dnase 1 is deficient. It was initially surprising that the Dnase 1 deficiency despite the presence of other forms of Dnase in those caused by the Methods according to the invention produced effects at all in non-human mammals showed. It was particularly surprising, however, that the lack of Dnase 1 activity did not as previously assumed, on the fragmentation of DNA in apoptotic processes (see called "DNA laddering"). Instead, the generated according to the invention Symptoms similar to those of SLE in mammals. Inspired by these findings  Literature research by the inventors showed that there were indeed isolated references to one possible connection between Dnase 1 activity and SLE in the past, which, however, were far behind and received little attention. So there were findings that showed that z. B. Dnase 1 activity in the serum and urine of SLE patients was significantly reduced. This was especially true for patients with acute glomerulonephritis (inflammatory response in the Kidney glomeruli) (Chitrabamrung et al., 1981). Only the provision of the by Dnase 1 deficient non-human mammals produced by the method according to the invention however, allows the classification of these isolated findings into a gradually emerging one Image from the etiology of SLE. From the requirement of effective nuclear elimination Antigens for protection against autoimmunity may thus result in the function of Dnase 1 in serum and lymph. So it can be assumed that Dnase I released genomic DNA eliminated to prevent an immune response. In some SLE patients this function seems to be negatively affected.

The method according to the invention thus points over conventional methods surprisingly has the advantage of providing transgenic non-human mammals, which not only have symptoms similar to those of SLE patients, but the also caused by the same defects.

In a preferred embodiment of the method according to the invention, a is used as the vector suitable recombination vector (targeting vector) is used and the partial or complete reduction of Dnase 1 activity is achieved by the recombination event caused. This can be achieved, for example, by expressing a dominant negative mutant of Dnase 1, a specific inhibitor of Dnase 1, or by deletion of part or all of the coding region of the Dnase 1 gene by homologous Recombination using a suitable targeting vector, or by expressing a Dnase 1 antisense construct, which expresses the expression of a Dnase 1 mRNA prevented. In such a process, the recombination event turns the whole or a part of the Dnase 1 gene was replaced by another DNA sequence. Advantageously, the method is carried out in such a way that when a sequence is exchanged Use of a Dnase 1 targeting vector for an at least partial, preferably however complete loss of Dnase 1 mRNA transcription in all tissues and Organs of the transgenic non-human mammal. The vector to be used contains among other things, the sequence of the 5 'non-coding part of the Dnase 1 gene (EMBL Database approval number AJ000062). Is advantageously used as a transgenic non-human mammal uses a rodent, preferably a mouse. The invention also involves the production of transgenic, non-human mammals in which one genetic modification a reduced Dnase 1 activity, but preferably no Dnase 1  Has activity. This deficiency in the activity of Dnase 1 is related preferably achieved with the invention on all cells and organs of the mammal by partial or complete deletion of the Dnase 1 gene from the mammalian genome. In addition, the invention extends to transgenic non-human mammals, in which a part or the entirety of the Dnase 1 gene is replaced by another, foreign gene or that the All or part of its Dnase 1 gene is replaced by a non-Dnase 1 gene. This non-Dnase 1 gene can be a gene which is resistant due to its expression against certain chemicals, other low-molecular substances or certain antibiotics mediated. The gene is preferably used for resistance to G418 (“Neo gene”). In connection with the production of gene deficient mice, the Neo gene is within the Starting vector pPNT described in Tybulewicz et al., Cell, Vol 65: 1153-1163, 1991. Im With regard to the exchange of the endogenous Dnase 1 gene in a transgenic non-human mammals, the invention relates to rodents, preferably mice. In An advantageous further development of the invention will be transgenic, non-human mammals generated in addition to the partial or complete destruction or the partial or complete exchange of the endogenous Dnase 1 gene contain further mutations that either by crossing, chemical mutagenesis, radiation or other biological chemical or physical manipulation can also be introduced. Under mutations all genetic changes are understood here that affect the integrity of the genome affect. This includes naturally occurring mutations and those caused by genetic modification have been caused. The invention also relates to a Dnase 1 targeting vector that allows the transfection of embryonic stem cells Dnase 1 gene in the genome of a non-human mammal partially or completely delete or partially or completely the Dnase 1 gene of a non-human mammal to be replaced by other DNA sequences. The production of the vector starts from one Base vector pPNT, which is described in the following literature: Tybulewicz et al., Cell, Vol 65: 1153-1163, 1991. This involves the production of a Dnase 1 targeting Vector of the sequence (AJ000062), ie parts of the 5 'region of the murine Dnase 1 gene contains, also contains the 3 'non-coding part of the Dnase 1 gene and otherwise on builds the basic structure of the pPNT vector. According to the invention, a Dnase 1 targeting Vector capable of the endogenous mouse Dnase 1 gene by homologous recombination to be replaced in whole or in part by the "neo gene" for the production of genetically modified Cells and / or genetically modified non-human mammals are used. part of The invention is also a stable cell clone whose Dnase 1 gene is wholly or partly by a other DNA sequence, preferably by DNA containing the sequence for the "neo gene" was exchanged. In this respect, the invention encompasses all eukaryotic cells with the exception of human embryonic stem cells. Clones targeting with Dnase 1 Vector obtained from mouse embryonic stem cells are preferably used for Production of a transgenic, non-human mammal or for the production of individual  transgenic, non-human organs used. Furthermore, the use of a genetically modified, transgenic non-human mammal according to one of the above definitions for testing drugs or chemicals (in particular with reference to SLE) part of the invention as well as the establishment of a model system to analyze the development of SLE or to analyze the treatment of diseases, in particular SLE, characterized in that this model system has at least one contains non-human mammals according to one of the above definitions and methods.

The invention further includes methods for testing new drugs for effectiveness against SLE, characterized in that a transgenic, non-human mammal, which according to the above method has been produced, with at least one drug (or Drug combinations) is treated and its effect on SLE is determined. In In this context, the invention also relates to tests that have the effects check genetic influences on the development of SLE, which is characterized by that in a transgenic, non-human mammal, according to the above procedures has been produced by crossing or other methods as indicated above genetic or other changes are introduced and then tests for the effects of Drugs are carried out. The invention also includes methods for testing the impact of biological, chemical or physical influences, thereby characterized that one is a transgenic, non-human mammal, which according to the above has been produced, viral, bacterial, chemical and / or exposed to physical influences.

For the first time, the invention enables the representation of a direct correlation between the height of Dnase 1 activity and the risk of developing an SLE-like Autoimmune disease in the mouse. Dnase 1 deficient mice, for example, show that both full activity deficiency and decreased activity as an SLE- Risk factor must be considered. Decreased Dnase 1 serum levels in SLE patients, especially in patients with kidney involvement (Chitrabamrung et al., 1981), suggest that this may also be the case in the human system. Besides one often too observing C1q deficiency in SLE patients (Navratil et al., 1999), Dnase 1 is now that second identified protein, which is present in both mouse and human as one potential SLE-promoting factor in the event of a reduction or deficiency.

Since the Dnase 1 stringent the half-life of DNA and DNA-protein complexes, which as crucial antigens for the expression of SLE are considered in the organism influenced, the Dnase 1 deficient mouse as an ideal model for researching the initiating and promoting events for the development of an SLE disease. Suitable due to the close correlation to the conditions in human SLE disease  this SLE mouse model works better both for studying human disease as well for the development of therapeutic approaches for humans. Such a mouse model allowed z. B. Direct testing of medicinal products based on recovery (through Target substitution) of normal Dnase 1 concentrations in the serum. Above all, it can Efficacy of such drugs should be tested before the onset of the disease the latency with which symptoms develop in Dnase 1 deficient mouse is. Other existing mouse models with intact Dnase 1 cannot do this. The test of immunomodulatory drugs for the therapy of SLE is also possible, because in the Contrary to other SLE mouse models (lpr and gld mutations) the immune system of the Animals is intact. Scientifically, the importance of individual core or -Chromatin components as crucial antigens for the initiation of an SLE with the help of Immunizations are tested.

The invention is explained below in exemplary embodiments.

example 1

Deoxyribonuclease I (Dnase 1) is a Ca ²⁺ / Mg ²⁺ dependent endonuclease with a neutral pH optimum. Their primary physiological function is seen in the degradation of food DNA to resorbable oligonucleotides, which correlates with a high expression of the enzyme in organs of the digestive tract of mammals (Lacks, 1981). An expression in the kidney and other other organs as well as an occurrence of the nuclease in serum and urine has been described, so that questions regarding further tasks of the nuclease arise from its expression pattern. Additional physiological functions associated with Dnase 1 include fragmentation of the cellular genome during programmed cell death (apoptosis) (Peitsch et al., 1994; Mannherz et al., 1995) and protection against one Autoimmune freakdon against DNA and DNA-protein complexes (Lachmann, 1961). Three other members of a Dnase 1 gene family have been described in recent years (Rodriguez et al., 1997), so that it can be assumed that the nucleases have been partially characterized in terms of expression sites and functions. This applies in particular to examinations in which indirect detection methods were used.

To clarify open questions regarding the expression and function of Dnase 1, a Dnase 1 knock-out mouse model was established and characterized beginning. For this purpose, the mouse Dnase 1 gene with its 5'- and 3'-flanking regions was isolated from a genomic phage library and the subcloned phage insert for detection of the Dnase 1 gene on chromosome 16A1-3 by FISH analysis on metaphase chromosomes Mouse cell line used. A recombination vector with Dnase 1 null allele, which was characterized by the replacement of the complete Dnase 1 gene with a neomycin resistance gene, was cloned, mouse embryonic stem cells (Es cells) were transfected with this vector and clones with a homologous recombination event were selected (Fig. 1a). Cells from these clones were injected into mouse blastocysts and used to produce chimeric mice. The chimeras were then crossed with wild-type (Wt) mice to breed heterozygous Dnase 1 knock-out mice. By crossing heterozygous knock-out mice, homozygous Dnase 1-deficient animals were ultimately created ( Fig. 1b).

Fig. 1 Dnase 1 gene targeting in the mouse

a: Schematic representation of the wild-type (Wt) -Dnase 1 allele, the targeting vector and the structure of the homologous recombination generated Dnase 1 zero alleles. Dnase 1 exons are filled, numbered boxes and the specified restriction interfaces are shown as arrows. The positions of the neomycin Resistance gene (Neo) and the herpes simplex virus thymidine kinase gene (HSV-Tk) are shown as boxes. The used DNA probes to detect correct homologous recombination (shown as crosses) in the 5 'and 3' flanking region of the Dnase 1 gene in DNA samples from cultured Dnase 1 deficient mice using the Southern blot technique are labeled A and B. b: Southern blot of genomic DNA from Progeny from matings of heterozygous Dnase 1 knock-out mice to prove their genotype Animals. The left blot shows the banded pattern of DNA cut with Hind III / Xho I and with the probe A was hybridized (detection of correct recombination in the 5 'region of the Dnase 1 gene). Homozygote Dnase 1 knock-out mice (- / -) only show the band of 5.8 kb specific for the zero allele Wild-type mice (+ / +) only have the 11 kb band specific for the wild-type allele. Heterozygote Dnase 1 knock-out mice (+/-), which have both a Dnase 1 Wt and a zero allele, both show Gangs. The right blot shows the proof of the correct homologous recombination for the 3 'region of the Dna 1 gene. The genomic DNA was cut with Xho I and the blot with probe B hybridizes. c: Northern blot analysis of RNA of the parotid, kidney and small intestine of Wt- as well as hetero- and homozygous Dnase 1 knock-out mice using a Dnase 1 specific Dnase Rat 1 cDNA probe. The amount of Dnase 1 transcript is higher than that in the heterozygous knock-out mice Wt animals reduced, which shows codominant expression of both alleles of the Dnase 1 gene in Wt animals. d-f: Zymograms (ethidium bromide-stained, renatured SDS-polyacrylamide gels with embedded DNA as Nuclease substrate, Lacks, 1981) for the detection of Dnase 1 activity in organ extracts as well as in serum and Urine from Wt, hetero- and homozygous Dnase 1 knock-out mice. The reduction in activity in the samples the heterozygous knock-out mice compared to the Wt animals again demonstrate codominant expression both Dnase 1 alleles in the Wt animals. M: Molecular weight marker (the molecular weight of the rabbits Lactate dehydrogenase is given), C: bovine pancreatic dnase 1 control.

The loss of both Dnase 1 wt alleles in these animals was associated with complete loss of Dnase 1 transcripts and protein in the highly expressive organs parotid, kidney and small intestine. Furthermore, by comparing the Dnase 1 expression of Wt and heterozygous knock-out mice, it could be shown that both alleles of the gene are codominantly expressed in Wt animals for the complete expression of Dnase 1 ( Fig. 1c-f). Furthermore, the expression pattern of Dnase 1 was clearly clarified. In addition to the salivary glands and the kidney, definitive evidence for an expression of the enzyme in the small intestine was provided for the first time. The main nuclease activity of the serum was also clearly assigned to Dnase 1 for the first time. Dnase 1 is the only nuclease activity in urine. The described Dnase 1 expressions in the seminal vesicles, in the liver, prostate, in the testicles, ovary and stomach could not be confirmed.

Dnase 1 knock-out mice show symptoms of a disease that are similar to systemic lupus erythematosus in humans. This is an autoimmune disease in which self-tolerance to nuclear antigens, in particular DNA and DNA-protein complexes, is breached. As a result of the formation of antinuclear AK (ANA) there are essentially immune complex-induced hypersensitivity reactions (type III) in various tissues, such as. B. the kidney. In a large-scale study, 37 Wt mice, 78 hetero- and 69 homozygous Dnase 1 knock-out mice of the same genetic background at the age of eight months were examined in Dnase 1-deficient animals to prove the SLE symptoms. It turned out that Dnase 1-deficient animals form ANA to a high percentage and in high titers. This was shown by indirect immunostaining of fixed cells with the sera of the animals examined ( Fig. 2). The prevalence rate of ANA rose to 55.1% in the heterozygous and 55.3% in the homozygous Dnase 1 knock-out population compared to 24.3% in the Wt population (both sexes together).

Figure 2 Antinuclear antibodies (ANA) in sera Dnase 1 deficient mice

Left: Prevalence of ANA in sera from Wt- (+ / +) and hetero- (+/-) and homozygous (- / -) dnase 1-knock-out mice. ANA were determined by indirect immunostaining with the appropriate NIH 3T3 cells Sera detected using a FITC-labeled second antibody. All serums, one Detectable immunoreactivity at or above a 1/100 dilution were found to be ANA positive rated. The ANA titers were divided into four levels of intentions based on the immunostaining: negative, low (+), medium (++) and high (+++). Below the bars is the number of ANA-positive animals in relation to Total number of animals examined. Right: ANA detection by indirect immunofluorescence in sera an ANA-negative Wt mouse (a) and three ANA-positive Dnase 1-deficient mice with low (b),  medium (c) and high (d) titer. The immunostaining shows the reactivity of the ANA u. a. against Interphase chromatin and metaphase chromosomes (e).

Furthermore, a subclassification of the ANA with respect to the antigen was carried out using the ELISA Technique (Enzyme-Linked Immunosorbent Assay) performed, whereby it was shown that the antinuclear autoantibodies Dnase 1-deficient animals essentially against nucleosomes and single-stranded (ss) DNA are directed (Tab. 1).

Table 1 Subclassification of ANA in sera Dnase 1 deficient mice

Subclassification of ANA in sera with high ANA titers of 17 male and 16 female doses 1-deficient mice using the enzyme-linked immunosorbent assay (ELISA) test commercially acquired, pre-plated microtiter plates with single-strand (ss) DNA, double-strand (ds) DNA, Histones, nucleosomes and the non-nuclear, lupus-associated antigens ribosomal P proteins (Elkon et al., 1985) and Sm antigens (Moore et al., 1981). All sera whose test value in the ELISA was over one Background values (cut-off) were rated as positive. The cut off was the average of 20 sera negative in immunostaining for ANA plus three times the standard deviation. The The significance of the results (p-value) was determined using the Fisher's exact test (n / a = not significant).

In addition to ANA, some of the animals showed the development of immune complex-induced glomerulonephritis (GN), which was due to immunostaining on cryosections against mouse IgG and the complement component C3 as well as light microscopic examinations of PAS-stained (periodic acid Schiff reagent staining) paraffin sections of the kidney was found ( Fig. 3). The prevalence rate of a GN was 5.4% in the Wt animals. It increased to 16.7% in the heterozygous and to 23.2% in the homozygous knock-out mice (Table 2). A preference of female animals for the training of ANA and a GN could be shown. This also reflects the situation in human SLE disease.

Fig. 3 Immune complex-induced glomerulonephritis (GN) in Dnase 1-deficient mice

a: Histological sections of Carnoy-fixed and paraffin-embedded kidneys. Top left: PAS staining (Periodic acid Schiff's reagent) of a healthy kidney body. Top right: PAS staining one Kidney body of a Dnase 1-deficient mouse, in which an increased cellularity and an incipient mesangial proliferation can be seen (typical for GN stages II-III). Bottom left: PAS staining a sclerotic kidney body of a Dnase 1-deficient mouse. A crescent moon as a result of proliferation of the capsular epithelium has developed. The condition represents an end-stage glomerulonephritic kidney (GN stage IV). Bottom right: HE staining (hematoxylin / eosin) of a kidney section that partially massive perivascular infiltrates of leukocytes in glomerulonephritic kidneys Dnase 1-deficient animals clarifies. b: Representative confocal microscopy of immunostaining on cryosections of the kidney  Dnase 1-deficient mouse with GN stage 11 (top row) and a healthy Wt mouse (bottom row). Above left: direct immunostaining with FITC-labeled antibodies (AK) against mouse immunoglobulin G (IgG) shows a granular deposition of IgG in the glomerulus capillary walls. Top middle: the indirect one Immunostaining with AK against the complement factor C3 and TRITC-labeled secondary AK also shows one granular deposition of C3 in the capillary walls. Top right: the overlay of both Fluorescence images show the colocalization of the IgG and C3 deposits. This indicates one Correlation between glomerulonephritis and a deposition of immune complexes with subsequent Complement-mediated inflammatory response. Bottom row: The immunostaining on kidney sections of the healthy Wt mouse show no significant colocalized deposition of IgG and C3 in the glomerular Capillaries.

Table 2 Prevalence rate of glomerulonephritis dnase 1 deficient animals

Hetero- (+/-) and homozygous (- / -) Dnase 1 knock-out mice were compared to wild-type animals (+ / +) by histopathological studies related to the prevalence rate of an immune complex-induced Glomerulonephritis (GN) examined. The number of affected and unaffected animals is related to that Gender (m = male, w = female) and the genotype. The severity of the GN was rated in five Stages divided: stage 0: no pathological changes, stage I-III: glomerular changes in 0% -25%, 25% -50% and 50% -75% of the total glomeruli. Stage IV: more than 90% of the glomeruli were sclerotic and / or showed crescents (proliferation of the outer capsular epithelium). The significance of the Results (p-value) were determined using the Fisher's exact test.

Some of the animals died during the study period mentioned. Unless an autopsy is still taking place was possible, a terminal glomerulonephritic kidney could be the cause of death be diagnosed. The mortality of the heterozygotes was 12.1% and that of the homozygous knock-out mice at 13.7%. No Wt mice died during the same period.  

Other SLE-associated symptoms such as splenomegaly, perivascular infiltration of leukocytes ( Fig. 3a), and leukopenia and anemia were often observed in these animals.

Fig. 4 Dnase 1 activity compared in sera from patients with nephropathy Control persons

Dnase 1 activity in sera from 10 patients with different forms of nephropathy (squares) were determined using the Single Radial Diffusion Method (SRED) (Nadano et al., 1993). 4 of these patients suffered from lupus nephritis (black squares). These patients showed a clearly low nose 1- Serum activity (7 ± 0 ng / ml) compared to healthy male (16 ± 5.5 ng / ml, black diamonds) and female (14.2 ± 6.5 ng / ml, black circles) control subjects. Because not all patients are humiliated Dnase 1 activity can be ruled out proteinuria as the cause of the finding. The simple one The standard deviation of the control persons is given.

The prevalence rate of the development of an SLE-like disease in Dnase 1 knock-out mice correlates with the Dnase 1 concentration in the animals, ie it is higher in Dnase 1-deficient animals than in the heterozygous knock-out animals ( Fig . 2, Tab. 2). This illustrates that both a reduction and a loss of Dnase 1 activity in the mice can be seen as a risk factor for an SLE-like disease. This in turn correlates with a reduced Dnase 1 activity in the serum of SLE patients with kidney involvement, as described in the literature (Chitrabamrung et al., 1981) and could be confirmed by our own analyzes ( Fig. 4). It can therefore be said that Dnase 1 has a function in maintaining self-tolerance towards nuclear, DNA-containing antigens. This function fits into the evolving picture of the causes of SLE disease. It is assumed that impaired elimination or increased release of apoptotic material initiates or promotes the disease (Rosen and Casciola-Rosen, 1999).

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Raz E., Brezis M., Rosenmann E. and Eilat D. (1989): Anti-DNA antibodies bind directly to renal antigens and induce kidney dysfunction in the isolated perfused rat kidney. J. Immunol. 142, 3076-3082.
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Example 2

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over 1 million people in the United States. Typical for SLE is the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune-complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III which manifests as glomerulonephritis (GN), arthritis and general vasculitis. The aetiology of SLE remains obscure but several studies suggest that increased liberation or a disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease ^1-6 . Consequently, Dnase 1 which is the major nuclease present in serum, urine and secreta may be held responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE ^7-11 . To test this hypothesis we have generated Dnase 1 deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE namely the presence of ANA, the deposition of immune-complexes in glomeruli and full blown glomerulonephritis in a Dnase 1 dose dependent manner. Furthermore, in agreement with earlier reports ¹⁰ we find Dnase 1 activities in serum to be lower in SLE patients than in normal subjects. Both findings strongly suggest that lack or reduction of Dnase 1 is a critical factor in the initiation of human SLE.

The Dnase 1 gene locus comprises 9 exons ¹² which were entirely removed in a targeting vector based on the plasmid pPNT and replaced by the neomycin resistance gene (Neo). Genomic sequences flanking the Dnase 1 coding region were inserted 3 'and 5' of the Neo cassette followed at the 5 'end by the gene for the Herpes Simplex Virus thymidine kinase (HSV-Tk). Both the Neo and Tk genes were driven by the mouse pGK1 promotor. Transfection in E14 embryonic stem (ES) cells yielded 90 clones after selection in G418 and counterselection in gancyclovir. Seven of these 90 clones had undergone homologous recombination at the Dnase 1 locus as verified by Southern blotting. The correct recombination of the targeted allele was checked at the 3 'and the 5' end with the indicated probes ( Fig. 1). Four of the targeted ES cell clones were injected into blastocysts of C57BL / 6 mice and produced in every case chimeras that were able to transmit the targeted allele. Two lines of Dnase 1 deficient animals were established from two independently generated ES cell clones by backcrossing the chimeras with C57BL / 6 wild type (wt) mice. Dnase 1 heterozygous mice were obtained and again crossed with C57BL / 6 wt animals. All mice used in this study resulted from intercrosses of these F2 Dnase 1 heterozygous mice. Dnase 1 ^+/- mice were fertile and healthy and produced homozygous Dnase 1 deficient mice in a mendelian fashion. Mice from both lines showed the same characteristics in all aspects and thus were not further distinguished.

The absence of Dnase 1 specific mRNA in Dnase 1 ^{- / -} mice could be demonstrated by Northern blotting of RNA from parotids, kidney and small intestine. Significantly, the level of Dnase 1 mRNA was reduced to about 50% of wt levels in Dnase 1 heterozygous animals ( Fig. 1). Further, Dnase 1 activity was absent in all organs of animals homozygous for the disrupted allele, but was reduced in small intestine, kidney, serum and urine of heterozygous animals compared to wt mice ( Fig. 1). This demonstrated that the Dnase 1 expression level depends on both alleles of the gene and that in the presence of a single Dnase 1 allele only reduced expression levels are reached. Young mice that lacked both Dnase 1 alleles or were heterozygous were healthy. However, at the age of 6-8 months several animals showed symptoms of disease and eventually died. An increased mortality of 12% in Dnase 1 ^+/- and 14% in Dnase 1 ^{- / -} deficient mice in comparison to 0% in the wt population was observed and a first analysis of both Dnase 1 ^+/- and Dnase 1 ^{- / -} animals revealed the presence of ANA and signs of GN. This suggested a correlation between the activity of Dnase 1 and an SLE like disease and prompted us to perform a more detailed analysis.

We sacrificed 78 Dnase 1 ^+/- , 69 Dnase 1 ^{- / -} animals and 37 wt controls at the age of 8 months and analyzed their sera for the presence of ANA and the severity of GN. The percentage of male mice positive for ANA rose from 15% found in the wt population to 48% (p: 0.008, Fisher's exact test) in Dnase 1 ^+/- and to 56% (p: 0.0018) among Dnase 1 ^{- / -} mice (Table 1). In the female population this difference was less striking. The percentage of ANA positive wt females was at 35% and rose to 65% (p: 0.031) in Dnase 1 ^+/- female animals and to 73% (p: 0.013) in Dnase 1 ^{- / -} female mice (Table 1) . Immunofluorescence confirmed that the ANA found in Dnase 1 deficient mice were directed against chromatin structures ( Fig. 2). Quantification of ANA was made by end point titration in an indirect immunostaining of NIH 3T3 cells and three titer ranges were defined with a cut off rate at 1/100 (low: 1 / 100-1 / 640, medium: 1 / 1280-1 / 2560, high: 1 / 5120-1 / 20480). We found that significantly more Dnase 1 ^+/- and Dnase 1 ^{- / -} females had high titer ANA than wt controls (Table 1). In the male Dnase 1 deficient population, this was also true for the medium titer range (Table 1). Next, we further subclassified ANA using high titer sera from Dnase 1 ^+/- and Dnase 1 ^{- / -} mice by ELISA with respect to their reactivity against ssDNA, dsDNA, histones, nucleosomes and the lupus-associated antigens ribosomal P-proteins and Sm -antigen. A high percentage of sera from male and female Dnase 1 deficient mice contained antibodies against nucleosomes and ssDNA (Table 2). Female Dnase 1 deficient mice contained also antibodies against dsDNA (Table 2). The frequency of antibodies against histones alone was low and against ribosomal P-protein not significant (Table 2) suggesting that ANA found in Dnase 1 deficient animals are mostly directed against DNA-protein complexes.

The prevalence of GN in the wt population was 0% in male mice and rose to 15% (p: 0.080, Fisher's exact test) in Dnase 1 ^+/- and to 19% (p: 0.037) in Dnase 1 ^{- / -} male animals (Table 3, Fig. 3a). In female mice the prevalence of GN was at 12% in the wt control population and rose to 19% (p: 0.26) in Dnase 1 ^+/- and to 31% (p: 0.11) in Dnase 1 ^{- / -} animals (Table 3). This suggested that the prevalence of GN is significantly increased in Dnase 1 deficient animals over wt controls. It appeared that GN frequency was higher in male than in female Dnase 1 deficient mice. However, we suspect that the overall frequency of female Dnase 1 deficient animals suffering from GN is higher than the calculated 19% or 31%, respectively, because we found a high number of dead animals among the female Dnase 1 deficient population most of which could not be analyzed due to post mortem autolysis. But all those dead animals that still could be analyzed showed symptoms of GN stage IV (data not shown). In addition, we found that GN coincided with the deposition of IgG and the complement component C3 at identical locations of the glomerular basement membrane (GBM) in all 22 Dnase 1 ^{- / -} and Dnase 1 ^+/- animals with GN that were tested ( FIG. 3b). Deposition of IgG and C3 increased with the severity of GN being marginal at stage I (not shown). A high prevalence of ANA in connection with immune-complex GN are strong indicators for an SLE like disease in Dnase 1 deficient animals. As additional symptoms of SLE, 24 of 66 Dnase 1 ^+/- and 21 of 49 Dnase 1 ^{- / -} animals tested showed perivascular leukocyte infiltration in the kidney. 10 of 66 Dnase 1 ^+/- and 17 of 49 Dnase 1 ^{- / -} animals tested suffered from splenomegaly (not shown).

The fact that Dnase 1 ^+/- mice exhibited a 50% lower Dnase 1 expression in comparison to wt mice ( Fig. 1) might explain the observation that these animals developed the same phenotype as Dnase 1 ^{- / -} animals albeit with lower prevalence. The lower Dnase 1 expression correlated with decreased enzyme activities which in turn may have been insufficient to overcome the effect of natural occurring Dnase 1 inhibitors in the serum ^11,13 . These data support the hypothesis that Dnase 1 is a crucial factor in the aetiology of SLE being responsible for the clearance of DNA and DNA-protein complexes accidentally present in the serum or at sites of high cell turnover as potential autoantigen.

The features described here for Dnase 1 deficient animals did not occur in all animals at the same time. The Dnase 1 deficient mice showing SLE like symptoms could be divided into three groups. Almost all Dnase 1 ^{- / -} animals with few exceptions were positive for ANA, the 1 ^st group showed in addition to GN perivascular kidney infiltration and splenomegaly, the 2 ^nd group lacked GN but still had a high prevalence of perivascular kidney infiltration and splenomegaly and the 3 ^rd group only displayed serum ANA without other significant signs of SLE or renal disease (Table 4). This heterogeneity of symptoms is characteristic of a multifactorial disease and argues for the requirement of additional events.

To further test the possible link between Dnase 1 and SLE, we analyzed the serum of 10 patients which had undergone renal biopsy because of nephropathic diseases. Among these 10 patients, 4 patients suffered from SLE with GN and had a significantly lower Dnase 1 activity (7 ± 0 ng / ml) with a p-value (student's T-test) of 0.007 when compared to healthy male controls (16 ± 5.5 ng / ml) and a p-value of 0.054 when compared to healthy female controls (14.2 ± 6.5 ng / ml) than patients suffering nephritis for reasons other than SLE (n = 6) ( Fig. 4). Only one patient with diabetic nephropathy had a lower Dnase 1 activity (5 ng / ml) than the SLE patients ( Fig. 4). All 10 patients showed proteinuria. Since the level of Dnase 1 activity was not generally lower in all 10 patients, a decrease of Dnase 1 due to general proteinuria could be excluded. These results are in agreement with earlier data reporting low Dnase 1 levels in SLE patients ¹⁰ and SLE-prone NZB / NZW (F1) mice ¹¹ and provide additional support for a causative link between low Dnase 1 activities and SLE.

Our data suggest that Dnase 1, besides its digestive role in the alimentary tract, may have an additional protective task, which comprises the removal of DNA from soluble or deposited autoantigenic nucleoprotein complexes in order to prevent immune stimulation and to reduce inflammation in target tissues. It is evident that Dnase 1 is expressed at sites where high cell turnover makes the removal of residual nuclear debris necessary such as the gastrointestinal and genitourinary tract, mammary gland, skin and haematopoetic system ^7,8,14,15 . Apparently, reduction or loss of Dnase 1 activity could result in a high risk to produce ANA as a potential prerequisite to develop a SLE-like disease in connection with environmental factors. The fact that ANA in SLE patients are directed against own cellular DNA or DNA-protein complexes favors this hypothesis ^3,16 . In support of this suggested novel role of Dnase 1 are reports showing that application of Dnase 1 to SLE-prone NZB / NZW (F1) mice results in a dose dependent reduction of anti-DNA IgG antibodies ^17,18 .

Further indications of the necessity for "cleaning up" after cell death in order to prevent SLE-like diseases come from mutant mice deficient for the complement component C1q ¹⁹ or serum amyloid P component (SAP) ²⁰ . Mice targeted for Dnase 1 (this study), SAP and C1q genes show a striking similarity in prevalence and gender distribution of ANA ^19.20 . The prevalence of GN was also similar among the three types of mice with the exception of male SAP deficient animals ²⁰ . These mice do not show a predisposition for GN. It has been suggested that both SAP and C1q prevent the emergence of DNA-protein complexes as autoimmunogens. C1q mediates its effect by supporting the elimination of apoptotic bodies as a potential source of DNA autoantigens. In contrast, SAP binds to chromatin and possibly prevents its recognition by antigen receptors. The strikingly similar outcome of deficiency for SAP, C1q and Dnase 1 suggests that the elimination of DNA-protein complexes is a crucial process for an organism to prevent autoimmune disease of the SLE-type and if any of these fails, SLE-development becomes a likely event. In this respect, our results warrant further evaluation of Dnase 1 in therapeutic strategies ²¹ or as a prognostic indicator for the development of SLE.

Materials and Methods Dnase 1 activity

For the zymograms, 12% SDS polyacrylamide gels containing 10 µg / ml calf thymus DNA were run with protein extracts from parotids (0.5 µg), small intestine (30 µg), kidney (20 µg) or with 1 µl of urine or 2 µl of serum. After electrophoresis, proteins in the gel were renaturated in reactivation buffer (20 mM TrisfHCl, pH 7.3, 5 mM CaCl ₂ , 5 mM MgCl ₂ , 5% skim milk powder) for 16 hours, stained with ethidium bromide and photographed ⁷ . Dnase 1 activity manifests as lack of ethidium bromide staining at distinct locations in the gel due to the hydrolysis of the co-polymerized calf thymus DNA. The positive control (C) was 0.5 u bovine pancreas Dnase 1 (Stratagene GmbH, Germany). For the measurement of Dnase 1 activity in patient sera, the single radial enzyme-diffusion method (SRED) was used as previously described ²² . The positive control here was human recombinant Dnase 1 (Dornase alfa, Hoffmann-La Roche AG, Germany).

Detection, quantification and subclassification of ANA

Anti-nuclear antibodies (ANA) were revealed by indirect immunofluorescence on methanol acetone fixed NIH 3T3 cells using a FITC conjugated goat anti-mouse Ig secondary antibody (DAKO Diagnostika GmbH, Germany). All sera showing detectable reactivity at or above a dilution of 1/100 were considered ANA positive. The staining was evaluated in three different titer ranges: low (1 / 100-1 / 640), medium (1 / 1280-1 / 2560) and high (1 / 5120-1 / 20480) (see Table 1, Fig. 2 ). Subclassification was performed by ELISA using precoated plates for ssDNA, dsDNA, histones, ribosomal P-proteins ²³ and Sm-antigens ²⁴ according to instructions given by the manufacturer (Euroimmun GmbH, Germany). Plates precoated with nucleosomes (reconstituted with histone H1) from Medipan Diagnostika GmbH (Germany) were used according to their instructions. As a secondary antibody a horseradish peroxidase labeled goat anti-mouse Ig was used (Sigma Aldrich, Germany). To determine the cut off for the ANA subclassification we used 20 ANA negative sera from wt mice and determined their reactivity in the ELISA. The mean of these values plus 3 standard deviations was set as the lower limit for positive results.

Histopathology and immunofluorescence

Organs were removed from autopsied animals, fixed in Carnoy solution (60% ethanol, 30% chloroform, 10% acetic acid), embedded in parafin and stained with either hematoxylin / eosin (HE) or according to the Periodic Acid-Schiff (PAS) procedure. For cryosections organs were frozen in liquid nitrogen and sectioned on a cryotome, dried and stained with the appropriate antibodies. Glomerular damages were scored by a blinded observer as follows ¹⁹ : stage 0: no involvement, stage I to III: glomerular changes in 0% -25%, 25% -50% and 50% -75% of total glomeruli. Stage IV: greater than 90% of glomeruli were sclerotic and / or formed crescents.

Figure Legends

Fig. 1 Deletion of the nose 1 gene ¹² by gene targeting. a, Schematic representation of the Dnase 1 allele, the targeting vector and the structure of the targeted gene. Dnase 1 exons are depicted as filled boxes, the neomycin resistance gene (Neo) and the Herpes Simplex Virus thymidine kinase gene (HSV-Tk) are indicated. The probes used to verify homologous recombination at the 3 'and 5' end were denoted A and B. The restriction fragments generated by the diagnostic digests are shown under the scheme of the targeted Dnase 1 allele; the wt DNA fragment lengths are depicted above the scheme of the wt Dnase 1 allele. Restriction sites are indicated. b, Southern blot of genomic DNA derived from offspring generated from intercrosses between Dnase 1 ^+/- animals. Left panel: Hind III / Xho I digests hybridized with probe A show the presence of the wt allele of 11 kb and the targeted allele of 5.8 kb in heterozygous animals and the absence of the wt allele in homozygous mice. Right panel: Xho I digests hybridized with probe B show the presence of the wt allele of 13.8 kb, the targeted allele of 6.8 kb in heterozygous animals and the absence of the wt allele in homozygous Dnase 1 ^{- / -} mice. c, Northern blot analysis of RNA from parotids, kidney and small intestine of wt (+ / +), heterozygous (+/-) and homozygous (- / -) Dnase 1 deficient animals hybridized with a labeled rat Dnase 1 cDNA ²⁵ as a sample. The size of the Dnase 1 specific transcript is indicated as 1.2 kb ⁸ . Uniform loading of the gel was controlled by staining the filter after the transfer with Ponceau S. df, Zymograms showing Dnase 1 activity were prepared using cell extracts from parotids, small intestine and kidney as well as serum and urine of wt (+ / +) , heterozygous (+/-) and homozygous (- / -) Dnase 1 deficient animals. The molecular weight of the glycosylation isoforms of Dnase 1 is between 32 and 38 kD. M: molecular weight marker (the size of rabbit lactic dehydrogenase is indicated), C: bovine pancreas Dnase 1 control. All data shown in cf are representative for 10 tested animals of each genotype.

Fig. 2 Anti-nuclear antibodies (ANA) in Dnase 1 deficient mice. Immunofluorescence staining demonstrating that ANA in Dnase 1 deficient mice are directed against nuclear structures and chromatin. Upper left panel: staining with serum from a wt control animal showing no reaction against nuclear structures. Upper right panel: staining with serum from a C57BL / 6-Fas ^{lpr / lpr} animal positive for ANA (medium titer range), lower left panel: staining with serum from a Dnase 1 ^{- / -} animal positive for ANA (high titer range) . Lower right panel: staining with serum from a Dnase 1 ^{- / -} animal at higher magnification showing the reaction of ANA against metaphase chromosomes.

Fig. 3 Immune-complex nephritis in Dnase 1 deficient mice. a, paraffin embedded kidney sections after Carnoy fixation. Upper left panel: PAS staining of a normal glomerulus from a wt control animal. Upper right panel: PAS staining of a glomerulus from a Dnase 1 ^{- / -} animal showing increased cellularity and beginning mesangioproliferation. Lower left panel: PAS staining of a sclerotic glomerulus forming a crescent from a Dnase 1 ^{- / -} mouse representative for GN stage IV. Lower right panel: HE staining demonstrating a perivascular leukocyte infiltration of a kidney vessel. b, representative confocal micrographs of immunostainings from cryosections of a diseased kidney with GN stage II from a Dnase 1 ^+/- mouse (upper row) and of a normal wt kidney (lower row). Upper leftmost panel: staining with FITC conjugated goat anti mouse IgG antibodies (Rockland Inc., USA) shows granular deposits of IgG in the GBM. Upper middle panel: staining with a polyclonal sheep anti-mouse C3 antibody (Biotrend GmbH, Germany) and a TRITC conjugated rabbit anti-sheep Ig secondary antibody (Rockland Inc., USA). Upper rightmost panel: merge of both FITC and TRITC signals demonstrates the colocalization of IgG deposits and the C3 component of the complement system. Lower row: micrographs of a normal wt kidney treated identically as the diseased Dnase 1 ^+/- kidney (upper row) . Significant deposition or colocalization of IgG and C3 are missing in wt kidneys.

Fig. 4 Dnase 1 activity in sera of patients with nephropathic diseases. Activities of 10 patients which had undergone renal biopsies for various reasons (squares) were measured with the single radial enzyme-diffusion method (SRED). 4 of these 10 patients suffered from SLE (filled squares). The Dnase 1 activity was calculated from a reference series using human recombinant Dnase 1. The levels of patients were compared to the levels found in healthy male (n = 12, black diamonds) or female (n = 9, black dots) subjects. For the controls a mean value ± 1 standard deviation is given.

Table Legends

Table 1 The number of animals positive for ANA is given and is specified according to gender, genotype and titer range. Percentages of positive mice are given in parentheses. Statistical evaluation was performed by using the Fisher's exact test (p values). n. s., not significant.

Table 2 Sera with high ANA titers from 17 male and 16 female Dnase 1 deficient mice (Dnase 1 ^+/- and Dnase 1 ^{- / -} ) were tested for their reactivity against different nuclear components as ssDNA, dsDNA, histones, nucleosomes and Sm- antigens as well as against ribosomal P proteins. Given are the percentages of sera that were positive for a specific antigen. Statistical evaluation was performed by using the Fisher's exact test (p values). ns, not significant.

Table 3 Animals that were homozygous and heterozygous for the targeted Dnase 1 allele were compared to wt controls with regard to prevalence and grade of GN by histopathological evaluation ¹⁹ . The number of mice afflicted with GN is specified according to genotype and gender. Percentages and significance levels (p values, Fisher's exact test) are given underneath.

Table 4 Summary of phenotypic abnormalities observed in homozygous Dnase 1 ^{- / -} deficient mice. The presence and titer range of ANA was determined by immunofluorescence, the diagnosis of GN and perivascular leukocyte infiltration in the kidney was based on the inspection of histopathological sections ¹⁹ , splenomegaly was determined by comparing the size of the spleen to wt controls. Kidney infiltration was judged absent (-) or of low (+), medium (++) and high (+++) degree. Splenomegaly was either absent (-) or present (+).

References

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Table 1

Anti-nuclear antbodies (ANA) in Dnase 1 deficient and wt mice

Table 2

Sub-classification of ANA in high titer sera of Dnase 1 +/- and - / - animals

Table 3

Histopathology of Dnase 1 deficient animals (kidney)

Table 4

Phenotypic characterization of representative Dnase 1 deficient mice

Claims (24)

1. A method for producing a transgenic, non-human mammal, wherein an embryonic stem cell is transfected with a suitable Dnase 1 targeting vector, stable cell clones are isolated in which the endogenous Dnase 1 gene is partially or completely by homologous recombination with parts of the targeting vector other sequences have been replaced or partially or completely destroyed, are implanted in suitably prepared female animals of a non-human mammal and the progeny obtained are selected for the presence of the DNA introduction are characterized in that the genetically modified, non-human mammal has a partial consequence of the DNA introduction or complete reduction of Dnase 1 activity. 2. The method according to claim 1, characterized in that a suitable vector Recombination vector (Dnase 1 targeting vector) is used and the partial or complete reduction of Dnase 1 activity caused by the recombination event becomes. 3. The method according to claim 2, by the by a suitable recombination vector Expression of transdominant negative Dnase 1 mutants is achieved which inhibits the activity of Reduce or switch off nose 1. 4. The method according to claim 2, characterized in that by the Recombination event all or part of the Dnase 1 gene by another DNA sequence is exchanged. 5. The method according to claim 4, characterized in that the sequence exchange to one at least partial but preferably complete loss of transcription from Dnase 1 mRNA in all organs and cells of the transgenic non-human concerned Leads mammal. 6. The method according to any one of the preceding claims with a vector containing the Sequence deposited with the approval number AJ000062 in the EMBL gene bank and simultaneously contain sequences of the pPNT vector (see above) 7. The method according to any one of the preceding claims, characterized in that the non-human mammal is a rodent, preferably a mouse.   8. A non-human mammal according to any one of claims 1-7. 9. Transgenic, non-human mammal characterized in that it is characterized by the genetic modification a reduced Dnase 1 activity, but preferably none Dnase has 1 more activity. 10. Transgenic non-human mammal according to claim 9, characterized in that the Reduction or deficiency of Dnase 1 activity in all mammalian cells and organs is present. 11. Transgenic, non-human mammal according to claim 9 or 10, characterized in that that all or part of its Dnase 1 gene is from another DNA sequence is exchanged. 12. Transgenic, non-human mammal according to claim 11, characterized in that the the Dnase 1 gene completely or partially replacing DNA the sequence the "Neo Gen ", specified and identified in the reference Tybulewicz et al., Cell, Vol 65: 1153-1163, 1991 and the references cited therein. 13. Transgenic, non-human mammal according to one of claims 9-12 characterized in that it is a rodent, preferably a mouse. 14. Mammal according to one of the above claims (1-14), characterized in that he additionally natural and / or genetic modifications, preferably additional Transgenic, partial or complete deletion of other non-Dnase 1 genes, partial or complete replacement or replacement of non-Dnase 1 genes with other DNA Sequences, especially a C1Q or a SAP deficiency. 15. Recombination vector (Dnase 1 targeting vector) for homologous recombination in Method according to the above claims (1-7) 16. recombination vector, characterized in that it has sequence which with the Approval number AJ000062 has been deposited in the EMBL gene bank and at the same time Contains sequences of the pPNT vector (see above). 17. Use of a recombination vector according to one of claims 15-16 for Production of genetically modified cells and / or genetically modified, transgenic non-human mammals.   18. Stable transfected cell clone, characterized in that all or part of the Dnase 1 gene by another DNA sequence, preferably by DNA containing the Sequence was exchanged as described in claim 16. 19. Use of a stably transfected cell clone according to claim 18 for the production genetically modified, transgenic non-human mammals or for production individual organs. 20. Use of a transgenic, non-human mammal according to one of claims 1-14, for testing drugs or chemicals, especially with regard to SLE. 21. Model system for analyzing the development of SLE or for analyzing the treatment of Diseases, especially SLE, characterized in that there is at least one contains non-human mammals according to any one of claims 1-14. 22. A method for testing new drugs for efficacy against SLE, thereby characterized in that a non-human mammal according to one of claims 1-14 or a stable cell clone according to claim 18 or an organ according to claim 19 treated at least one drug (also drug combinations) and its Effect on SLE determined. 23. Procedure for testing the impact of genetic influences on the development of SLE, characterized in that in a non-human mammal according to one of the Claims 1-14 by crossing or by other methods of further genetic Changes in particular the C1Q and SAP mutations or naturally occurring ones Mutations, especially those of the lpr, gld and the NZB / NZW mouse variants underlying mutations. 24. Method for testing the effects of biological, chemical or physical influences on SLE, characterized in that a non-human mammal according to one of the Claims 1-14 viral, bacterial, chemical, physical or mutagenic Exposures.