Transgenic animals for studying regulation of genes

Abstract

A transgenic non-human animal (e.g., a transgenic mouse) comprising a polynucleotide encoding a soluble marker protein functionally linked to a regulatory sequence of an endogenous gene encoding E-selectin. Also described are methods for detecting modulators of the E-selectin gene using said transgenic non-human animal, and cultured cells obtained therefrom.

Description

BACKGROUND OF THE INVENTION

[0001] Vascular endothelial cell (EC) activation is one of the first events in inflammatory conditions and transplant rejection. A specific marker of EC activation is E-selectin. Expression of this gene is generally absent under resting conditions and induced by inflammatory stimuli, as well as being NF-kB dependent. Expression is restricted to the endothelium.

[0002] Immunohistochemistry is a standard technique by which signalling events involved in disorders associated with inflammatory, thrombotic, ischaemic or neoplastic conditions or transplant rejection, can be assessed; and, for example, E-selectin expression can be readily studied by this method in vitro. However, monitoring of E-selectin expression in vivo has been limited to immunohistochemistry of biopsies or post-mortem specimens. There has been a need by workers in the art for an in vivo model allowing assessment of E-selectin expression in a live subject.

SUMMARY OF THE INVENTION

[0003] The present invention relates to an animal model useful for screening potential therapeutic agents for the treatment of disorders associated with inflammatory, thrombotic, ischaemic or neoplastic conditions or prevention of transplant rejection. In particular, the present invention provides a novel model in which endothelial cell activation can be assessed in vivo by a simple and sensitive enzyme assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 (top panel) schematically illustrates the strategy which is pursued in Examples 1 and 2 for targeted replacement of the native mouse E-selectin allele with secreted alkaline phosphatase (SEAP). The SEAP-1 gene, or the SEAP-2 gene fused to the SV40 late mRNA polyadenylation site, is inserted into the mutagenized start site (ATG) of the E-selectin gene, together with a Neo R/loxP fragment. FIG. 1 (bottom panel) schematically depicts the SEAP-1 and SEAP-2 messenger RNAs. In all of the figures, genetic constructs are conventionally depicted with the 5′ terminus at left.

[0005] FIG. 2(a) (“Endogenous E-selectin locus.”) schematically depicts the E-selectin targeting constructs prepared in Examples 1 and 2 for carrying out homologous recombination in murine embryonic stem cells. Said constructs comprise the SEAP-1 gene (or the SEAP-2 gene fused to the SV40 late mRNA polyadenylation site) inserted into the mutagenized start site (ATG) of the E-selectin gene, together with a NeoR gene flanked by loxP sites. FIG. 2(b) (“Targeted locus.”) depicts the heterologous E-selectin allele (the “targeted locus”) (also referred to as the “knock in allele”) resulting from integration of the targeted construct into the murine chromosomal E-selectin allele. FIG. 2(c) (“Targeted locus after Cre-mediated neo cassette removal.”) shows the targeted locus following Cre-mediated removal of the neo casette and one of the LoxP sites.

[0006] FIG. 3 (“Induction of SEAP by cytokines or LPS”) depicts elevated plasma levels of SEAP-1 (left panel) or SEAP-2 (right panel) in TNF- or LPS-induced heterozygous lines, with maximum levels of plama SEAP attained at 6 hours (SEAP-1) or 24 hours (SEAP-2) post-stimulation; and by comparison the absence of induction in NaCl or IFN controls.

[0007] FIG. 4 (“Analysis of SEAP expression in the two lines”) is a bar chart representing pooled data from n experiments which shows elevated plasma levels of SEAP-1- and SEAP-2 (left panel) and fold stimulation (right panel) in transgenic heterozygous (±) mice at 6 hours following LPS or TNF induction, compared to baseline levels.

[0008] FIG. 5 (“Comparison of heterozygous and homozygous animals.”) is a bar chart representing pooled data from n experiments which shows elevated plasma levels of SEAP-1 (left panel) and SEAP-2 (right panel) in transgenic homozygous TNF-induced lines relative to transgenic heterozygous lines.

[0009] FIG. 6 shows the localization of SEAP expression by alkaline phosphatase histochemistry performed on cryosections from tongues of SEAP-2 mice 6 hours after saline (“SEAP-2 control”) or LPS injection (“SEAP-2 LPS”). The panels marked “LPS” show the immunolocalization of E-selectin in LPS treated mice. Arrows point to stained vessels.

[0010] FIG. 7 charts the elimination half-life of SEAP in TNF- or LPS- treated SEAP-1 animals in relation to the elimination curve of i.v. administered alkaline phosphatase.

[0011] FIG. 8 (“Inhibition of SEAP expression by proteasome inhibitor.”) illustrates inhibition of SEAP expression in an SEAP-1 line by a proteasome inhibitor, clasto-lactacystine-&bgr;-lactone, as compared to placebo, and placebo and TNF.

DETAILED DESCRIPTION

[0012] The present invention provides in a first aspect a transgenic non-human animal wherein expression of an exogenous soluble marker specifically by endothelial cells, e.g., vascular endothelial cells, is regulatable by a chemical stimulus, including a cytokine, or a physical stimulus including light or temperature.

[0013] In particular, the invention comprises a transgenic non-human animal comprising a polynucleotide encoding a soluble marker protein functionally linked to a regulatory sequence of an endogenous gene encoding E-selectin.

[0014] The soluble marker protein is preferably an exogenous protein, i.e. which is not normally produced by the animal. Alternatively, the protein may be produced by the animal but is not normally under the control of a regulatory sequence of an endogenous gene encoding E-selectin. The protein is selected to be detectable in plasma or blood samples by standard experimental means.

[0015] Transgenic non-human animals include animals, e.g., mammals, e.g., pigs, cows, goats, as well as non-human primates, such as monkeys, and rodents, such as mice and rats, into which a suitable DNA construct has been introduced directly, as well as progeny of such animals still retaining said construct. Examples for useful animal lines include any animal line normally kept as laboratory animals. This invention shall also be understood to include in all its aspects somatic recombinant non-human animals. The term “transgenic” refers in general to animals having an exogenous gene (i.e. non-native gene, i.e. trans-gene), such as a marker gene, in the chromosomal DNA of their germ line; and the term “somatic recombinant” refers to animals having an exogenous gene, such as for example a marker gene, in the DNA of at least a portion of their somatic cells.

[0016] Soluble marker proteins of the invention include proteins with or without enzymatic activity which are conveniently secreted either naturally or as a result of genetic manipulation into the extracellular space. Genes encoding suitable plasma-soluble, non-toxic marker proteins are known (e.g., human growth hormone), commercially available (e.g., a modified, secreted heat stable alkaline phosphatase), or may be prepared following established routes of gene identification, isolation, amplification and modification, e.g., ligation.

[0017] In preferred embodiments, the soluble marker transgene of the invention is a secreted form of heat-stable human placental alkaline phosphatase gene lacking the membrane anchoring domain, referred to herein as “SEAP”(for “secreted alkaline phosphatase”). Of course, the introduced marker gene need not be limited to the SEAP gene, but may also comprise one or more soluble marker proteins having additional reporter functions.

[0018] In its more particular aspects, the invention contemplates a transgenic (or somatic recombinant) non-human animal having inserted in its genome a soluble reporter gene, wherein the soluble reporter gene is under the control of the promoter of an E-selectin gene of a chromosomal E-selectin allele of said animal.

[0019] The inserted soluble marker gene can be placed at a variety of positions within the genomic E-selectin sequence, provided that its expression remains under the control of the endogenous E-selectin promoter.

[0020] The exogenous soluble marker gene is preferably inserted into the genome of the subject animal by means of a “targeting construct” comprising the marker gene under the control of the native E-selectin promoter of the animal to be rendered transgenic or a somatic recombinant, and optional additional transgenes.

[0021] Preferably, the targeting construct is also constructed so that the marker gene or other transgene is inserted in a manner to decrease or prevent (i.e. “knock out”) expression of the native E-selectin structural gene.

[0022] Most preferably, the genetic construct is inserted at the E-selectin allele transcription initiation site, at the E-selectin allele translation initiation site, or at any position in between.

[0023] The targeting construct is generally inserted into the host animal genome by homologous recombination into the wild-type chromosomal E-selectin allele, and therefore can be engineered to recombine at any of a variety of positions in the E-selectin allele so long as the inserted marker gene is positioned such that expression of the soluble marker gene is under the control of the E-selectin promoter.

[0024] In a preferred embodiment, the E-selectin gene of the non-human animal is modified, as a result of the homologous recombination, by insertion of the genetic construct at a site which is between the transcription and translation initiation sites of the native E-selectin structural gene of the animal, thereby placing expression of the soluble marker gene under the control of the native E-selectin promotor, while “knocking out” normal expression of the E-selectin structural gene.

[0025] The resulting allele, comprising the inserted soluble marker sequence under the control of the native E-selectin promoter, and optional additional transgenes, is often referred to as a “knock-in” allele.

[0026] The invention contemplates a transgenic animal that is either homozygous or heterozygous for the knock-in allele.

[0027] In its most preferred embodiment, the invention consists of a transgenic mouse that is homozygous or heterozygous for an E-selectin allele having inserted therein a genetic construct comprising a soluble marker gene under the control of the E-selectin promoter, wherein said inserted genetic construct diminishes or prevents expression of the native E-selectin structural gene of that allele. Such a mouse is commonly referred to in the art as a “knock out” mouse.

[0028] The targeting construct is preferably constructed so as to assist in homologous recombination of the synthetic sequence with the endogenous gene; and accordingly, will also preferably comprise at least elements of the native structural E-selectin gene.

[0029] As indicated above, the targeting construct may comprise additional components such as one or more additional reporters, selection markers such as a neo resistance cassette, and the like. (Upon insertion into the cellular genome of the subject animal, certain selection markers may be excised, for example, by Cre-mediated neo cassette removal.)

[0030] Recombinant DNA constructs of the present invention may be prepared according to procedures known in the art, starting from, em a genomic clone. A gene encoding E-selectin and its regulatory sequence may be identified and amplified following procedures known in the art, e g..by using appropriate primer pairs. Whether one or the other of the methods known in the art for the preparation of the DNA constructs is applied may depend on their intended further use.

[0031] The targeting construct of the invention is preferably prepared from an isolated genomic clone of the native E-selectin gene of the subject animal by inserting the marker gene at a suitable position (1) to be under the control of the E-selectin promoter, and (2) to reduce or prevent normal expression of the native structural E-selectin gene.

[0032] Preferably, the marker gene itself is inserted between the transcription and translation initiation sites of the isolated genomic E-selectin clone so as to “knock out” normal expression of the endogenous functional E-selectin gene.

[0033] In a particular embodiment which is illustrated in Example 1, step (a)(v), the targeting construct comprises, in order from 5′ to 3′: the native murine E-selectin promoter; a fragment of the native murine structural E-selectin gene comprising exon I; an SEAP-encoding component (comprising a Kozak consensus translation initiating signal, the SEAP coding sequence, and an SV40 late mRNA polyadenylation signal) cloned into the mutated translational start site, Mfe I, of the murine E-selectin gene in exon II; a neoR cassette (comprising a loxP site; a neoR cassette, and a second LoxP site) cloned into intron II; and an additional fragment of the structural E-selectin gene comprising exons III, IV and V. A species of such a targeting construct has SEQ. ID. NO: 9.

[0034] In another embodiment of a murine SEAP-targeting construct which is illustrated in Example 2, step (a)(ii), the targeting construct comprises, in order from 5′ to 3′: the native murine E-selectin promoter; a fragment of the native murine structural E-selectin gene comprising exon I; an SEAP-encoding sequence (comprising a Kozak consensus translation initiating signal and an SEAP coding sequence) cloned into the mutated translational start site, Mfe I, of the murine E-selectin gene in exon II, a neoR cassette (comprising a loxP site; a neoR cassette; and a second loxP site) cloned into intron II; and and exons III, IV and V. A species of such a targeting construct has SEQ. ID. NO: 10.

[0035] Thus this invention provides a recombinant DNA construct comprising a polynucleotide having a nucleotide sequence as illustrated in SEQ ID NO: 9 or SEQ ID NO: 10.

[0036] Well-established methods for germ-line or somatic insertion of a DNA construct include viral or non-viral vector-mediated gene transfer into fertilized eggs, zygotes or early embryos and/or a specific tissue (such as brain) in the adult animal, e.g. by gene transfer into embryonic stem cells, retroviral infection of early embryos or pronuclear microinjection.

[0037] For example, gene transfer into embryonic stem cells may be carried out using a “knock-in” strategy performed, e.g., in two sequential steps: (1) by first generating a recombinant DNA construct (the “targeting construct”) comprising a genomic clone of the E-selectin gene having inserted therein an exogenous genetic construct comprising the soluble marker gene of interest; and (2) by transfecting or otherwise inserting the targeting construct into the embryonic stem cells under conditions suitable to effectuate homologous recombination of the targeting construct with the endogenous, chromosomal E-selectin allele.

[0038] Identification of successful embryonic stem cell clones may be by, e.g., using the Neo-loxP approach, i.e. by insertion of a Neo resistance cassette carrying the thymidine kinase promoter and Neo cDNA flanked by loxP sites, into an intron of the E-selectin gene. Successfully targeted ES-cell clones may then be transfected with a plasmid expressing the Cre-recombinase, thereby removing the neo-selection cassette (as well as one of the loxP sites in intron II).

[0039] Using standard technology for the preparation of a transgenic animal (e.g., a mouse), the insertion of the targeting construct is made in a single allele of the native E-selectin gene, and through genetic crossing, there can thereby be obtained a fertilized egg with the insertion in either a single E-selectin allele, i.e. heterozygous, or in both E-selectin alleles, i.e. homozygous. Further manipulation of resulting fertilized eggs, zygotes or early embryos and breeding of resulting transgenic founder animals follows established routes of breeding transgenic animals. Successful transgenic non-human animals may be identified, e.g., by fur colour, e.g., by the absence of fur colour.

[0040] Accordingly, the targeted E-selectin gene (“targeted locus”) of Example 1, following Cre-mediated neo cassette removal, is schematically depicted in FIG. 2(c) hereof, and comprises, in order from 5′ to 3′: the native murine E-selectin promoter; a fragment of the native murine structural E-selectin gene comprising exon I; an SEAP-encoding component (comprising a Kozak consensus translation initiating signal, the SEAP coding sequence, and an SV-40 late mRNA polyadenylation signal) cloned into the mutated translational start site, Mfe I, of the murine E-selectin gene in exon II; a single loxP site in intron II, and an additional fragments of the structural E-selectin gene comprising exons III, IV and V. A species of such a targeting construct has SEQ. ID. NO: 11.

[0041] Likewise, the targeted E-selectin gene (“targeted locus”) of Example 2, following Cre-mediated neo-cassette removal, is also schematically depicted in FIG. 2(c), and comprises, in order from 5′ to 3′: the native murine E-selectin promoter; a fragment of the native murine structural E-selectin gene comprising exon I; an SEAP-encoding component (comprising a Kozak consensus translation initiating signal and, the SEAP coding sequence) cloned into the mutated translational start site, Mfe I, of the murine E-selectin gene in exon II; a single loxP site in intron II, and an additional fragments of the structural E-selectin gene comprising exons III, IV and V. A species of such a targeting construct has SEQ. ID. NO: 12.

[0042] Thus this invention also provides a recombinant DNA construct comprising a polynucleotide having SEQ ID NO: 11 or SEQ ID NO: 12, and a transgenic (or somatic recombinant) non-human animal comprising in its genome a nucleotide sequence having SEQ ID NO: 11 or SEQ ID NO: 12. Most preferably, the invention consists of a transgenic knockout mouse comprising in its genome SEQ. ID. NO: 11 or SEQ. ID. NO;12.

[0043] The transgenic or somatic recombinant non-human animals of the invention are useful for readily assessing and non-invasively monitoring in a body fluid, e.g., in plasma, the expression of a marker for E-selectin. An advantage of this model is that the expression of a marker for an inflammatory, thrombotic, ischaemic or neoplastic condition can be followed in an easy and experimentally reproducible system, and time course analyses are possible in this model without killing the animals. For example, this model offers the possibility to assess the specific activation of endothelium in different disease models of e.g., inflammation, angiogenesis, atherosclerosis, thrombosis, and acute or chronic transplant rejection. The model can be used for profiling of compounds inhibiting specific signaling events related to the expression of E-selectin. For example, the model can be used to study the specific effect of inhibitors of the NFKB pathway.

[0044] Models based on the transgenic or somatic recombinant non-human animals of the invention may be used for example to identify and assess the efficacy of potential therapeutic agents in disorders associated with inflammatory, thrombotic, ischaemic or neoplastic conditions. In particular such models may be used in screening or characterization assays for detecting agents likely to, e.g., prevent or treat inflammation, angiogenesis, thrombosis and acute or chronic transplant rejection.

[0045] Accordingly in a further aspect the invention comprises a method for testing a potential therapeutic agent for a specified condition, in particular a disorder associated with inflammatory, thrombotic, ischaemic or neoplastic conditions or transplant rejection, wherein the agent is administered to the transgenic non-human animal. Moreover the invention comprises a screening or characterization assay consisting in or including such a method.

[0046] Methods for testing potential therapeutic agents using animals are well known in the art. The transgenic non-human animals of the invention may be used in analogous manner.

[0047] The effects of the potential therapeutic agent may be determined by administering the agent to a transgenic non-human animal, monitoring marker concentration in a body fluid, e.g., plasma, and comparing the result with a result obtained from an untreated transgenic or somatic recombinant non-human animal.

[0048] In a further embodiment the present invention is directed to a novel modulator of a regulatable protein identified by a screening assay comprising administering the potential modulator to a transgenic or somatic recombinant non-human animal, monitoring marker concentration in a body fluid, e.g., plasma, and comparing the result with a result obtained from an untreated such animal.

[0049] Methods for monitoring marker concentration in a body fluid are well known in the art. For example, concentration of a marker enzyme may be determined by quantifying the enzymatic activity. Marker proteins without enzymatic activity may be quantified by other means e q., by immunological methods.

[0050] The transgenic non-human animals are further useful to monitor disease development and progression in established models of disease involving spontanously developing or induced disease conditions in wild-type or transgenic animals. For this, the transgenic non-human animals may be crossed with animal strains known for their use as animal models. Examples for suitable animal strains include mouse strains, e.g., atherosclerosis prone mice, e.g., targeted disruption of e.g., LDL-R or Apo E; mouse models for inflammation, e.g., mouse strains showing defects in NF&kgr;B signalling, defects in TNF and TNF-R, IL-1 and IL-1-R, or mutations such as the MRL-lpr mouse; mouse strains with immune disorders, e.g., cytokine transgenic or knockout mice; Thrombosis-prone mice, e.g., plasminogen or plasminogen activator deficient mice; and mouse strains which develop tumors. Disease progression may be monitored by e.g., following marker gene expression in the F1 animals or in offsprings resulting from crossing in of the transgenic non-human animals for several generations into the respective disease model strain.

[0051] The transgenic animals of the invention can also be used to study tissue distribution of E-selectin expression by various analytical methods including in situ histological analysis of the expression pattern of the reporter gene, and in vitro detection of the reporter gene in various tissues, tissue sections and/or cell types.

[0052] The invention further contemplates isolated cells or a cell culture derived from a transgenic animal (e.g., mouse) of the invention. The cells can be obtained directly from the animal, from a descendent animal, or can be a progeny of a primary culture of one or more cells of the animal. The isolated cell can be in the form of a single cell or cell line, or a composition of mixed cells. The cell can be obtained from an animal which is the descendant of a transgenic animal of this invention, such as by a cross with another animal having either the same or a different genetic background. Thus an isolated cell can be either homozygous or heterozygous for a reporter gene in the E-selectin allele. The isolated cell is preferably an endothelial cell.

[0053] In accordance with the foregoing the present invention thus provides

[0054] (1) A transgenic or somatic recombinant non-human animal, e.g., a mammal, eg a pig, a primate, such as a monkey, or a rodent, such as a mouse or a rat, wherein expression of an exogenous soluble marker, eg human growth hormone or a modified, secreted heat stable alkaline phosphatase, specifically by endothelial cells, e.g., vascular endothelial cells, is regulatable by a chemical stimulus, including a cytokine, or a physical stimulus including light or temperature, e.g., a transgenic non-human animal comprising a polynucleotide encoding a soluble marker protein functionally linked to a regulatory sequence of an endogenous gene encoding E-selectin; and isolated cells of said animal.

[0055] (2) A transgenic non-human animal wherein in the presence of a stimulus, e.g., an inflammatory cytokine, a soluble marker is specifically produced by endothelial cells.

[0056] (3) A transgenic non-human animal comprising a recombinant DNA construct comprising a polynucleotide having a nucleotide sequence as illustrated in SEQ ID NO: 1 1 or SEQ ID NO: 12.

[0057] (4) A non-human animal being an offspring of a transgenic non-human animal as under (1), (2) or (3) crossed with a non-human animal showing a disease condition and/or its sequellae, e.g., symptoms, resulting from an altered genetic background, either spontaneously developed or following genetic manipulation.

[0058] (5) A transgenic non-human animal cell comprising a recombinant DNA construct comprising a polynucleotide having a nucleotide sequence as illustrated in SEQ ID NO: 11 or SEQ ID NO: 12.

[0059] (6) A recombinant DNA construct comprising a polynucleotide having a nucleotide sequence as illustrated in SEQ ID NO: 9 or SEQ ID NO: 10.

[0060] (7) A method for testing a potential therapeutic agent for a specified condition, in particular a disorder associated with inflammatory, thrombotic, ischaemic or neoplastic conditions or transplant rejection, wherein the agent is administered to a transgenic non-human animal as under (1), (2) or (3), monitoring marker concentration in a body fluid, e.g., plasma, and comparing the result with the result obtained from an untreated transgenic or somatic recombinant non-human animal as under (1), (2) or (3), an elevated level being indicative of the potential of the agent.

[0061] (8) A method for screening of modulators of E-selectin expression comprising the steps of

[0062] (a) administering the potential modulator to a transgenic or somatic recombinant non-human animal as under (1), (2) or (3),

[0063] (b) monitoring marker concentration in a body fluid, e.g., plasma, and

[0064] (c) comparing the result with the result obtained from an untreated transgenic or somatic recombinant non-human animal as under (1), (2) or (3), a modulated level being indicative of the therapeutic potential of the agent.

[0065] (9) A novel modulator of E-selectin expression identified by a method as under (6) or (7).

[0066] (10) A method for treating a patient suffering from an inflammatory, thrombotic, ischaemic or neoplastic condition or from transplant rejection comprising administering to the patient a pharmaceutically effective amount of a modulator as under (8).

[0067] (11) A method to monitor disease development and progression in established models of disease involving spontanously developing or induced disease conditions in wild-type or transgenic animals comprising

[0068] (a) crossing of the transgenic non-human animals as under (1), (2) or (3) with animal strains known for their use as animal models and

[0069] (b) monitoring disease progression in offsprings in relation to marker concentration in a body fluid.

[0070] The following examples illustrate the invention without limitation.

EXAMPLE 1

[0071] Preparation of Transgenic Mice Expressing a Secreted Heat Stable Alkaline Phosphatase Gene Under the Control of the E-selectin Regulatory Sequence Without the 3′UTR of E-selectin Containing the mRNA Destabilizing AUUUA Repeats

[0072] (a) Preparation of DNA Construct

[0073] (i) Mutation of the ATG Start Codon

[0074] The ATG start site of E-selectin is mutated from . . . GTC ATG MT . . . to . . . GTC AAT TG MT . . . to introduce a unique Mfe I restriction site (underlined) within the genomic E-selectin fragment for cloning of SEAP, as follows:

[0075] A ˜6 kb Xbal genomic mouse E-selectin clone (D3-ES cells) in the pBS-KS(−) vector (Stratagene) is used to modify the ATG start codon by PCR. The 5′-fragment is amplified by PCR using the primerpair SEQ ID NO: 1 and SEQ ID NO: 2 [50 &mgr;l standard reactions are prepared using 400 nM primer, 200 nM dNTP's, PCR buffer with 1.5 mM MgCl2, 1.5 U Taq DNA polymerase in a PTC-200 thermocycler]. The 3′-fragment is amplified with the primerpair SEQ ID NO: 3 and SEQ ID NO: 4. The two fragments are purified using agarose gel electrophoresis. 200 fmol of each partial fragment is used for a PCR pre-amplification without primers to generate the full-length fragment (4×94° 60″; 40° 120″; 72° 120″). The full-length fragment is amplified using primers SEQ ID NO: 1 and SEQ ID NO: 2 in a standard PCR reaction. The PCR fragment is gel purified, digested with Afl-2, the DNA precipitated using sodium acetate/ethanol, cut with BamH1 and purified by agarose electrophoresis.

[0076] (ii) mE-selectin With Mutated ATG (mE-Sel/mutATG).

[0077] Genomic Xbal/mE-Sel/pBS-KS(-) clone (is partially cut with BamH1, the 8.5 kb fragment isolated using gel electrophoresis and digested with Afl-2. The 8 kb fragment is purified and ligated with the 520 bp PCR fragment obtained in (i) above carrying the mutated ATG start codon. The ligation mixture is precipitated (sodium acetate/ethanol) and redissolved in TE Buffer pH 8.0. 2 &mgr;l (20%) of the ligation is electroporated into E.coli XL1-blue electrocompetent cells and clones screened by PCR followed by precipitation and digestion. Several positive clones are verified by sequencing using standard protocol.

[0078] (iii) mE-selectin With Mutated ATG/Neo Cassette (mE-Sel/mutATG/Neo).

[0079] 2.3 &mgr;g plasmid mE-Sel/mutATG is linearised with Afl-2 and blunt ended with Klenow DNA polymerase using standard protocol and the gel purified vector is dephosphorylated with Shrimp alkaline phosphatase (SAP). The Neo cassette is isolated by digesting 5 &mgr;g plasmid pRay-2 [Storck et al., Nucleic Acids Res. 24:4594-4596 (1996)] with Xho-1, the DNA precipitated cut with Xma-1. The 1400 bp NeoloxP fragment is purified by gel electrophoresis and 0.5 &mgr;g of the isolated fragment is blunt ended using Klenow. 7 fmol Vector and 20 fmol NeoloxP fragment are ligated und electroporated into E.coli XL1-blue, clones selected by PCR (SEQ ID NO: 1 and SEQ ID NO: 2). Several isolated plasmids are controlled by restriction digest (Sph1, Xba1, Nco1, BamH1) and for a 3′-5′ orientation of the NeoloxP insert.

[0080] (iv) mE-Selectin With SEAP-2 in Mutated ATG Site (mE-Sel/SEAP-2/Neo).

[0081] 5 &mgr;g plasmid mE-Sel/mutATG/Neo is linearized with Mfe-1, purified by gel electrophoresis and the isolated vector dephosphorylated with SAP. The SEAP-2 fragment is isolated from 5 &mgr;g plasmid “pSEAP2”(Accession No: U09660; Clontech) by EcoR1 and Mfe-1 digestion. The 1703 bp fragment is purified using gel electrophoresis. 5 fmol vector and 20 fmol SEAP-2 are ligated and electroporated into E. coli XL1-blue as described above. Clones are selected by plasmid digestion using Nde-1; BamH1; Xba-1 and the sequence of several positive clones is verified by DNA sequencing. FIG. 1 schematically depicts the insertion of the SEAP-2 fragment into linearized mE-Sel/mutATG/Neo.

[0082] v) Generation of Targeting Construct SEQ ID NO: 9.

[0083] 6.6 &mgr;g plasmid mE-Sel/SEAP-2/Neo is linearized with Bpu10l and the 6370 bp fragment isolated by gel electrophoresis and blunt ended with Klenow. 2 &mgr;g plasmid pBS2-SK(−) (Stratagene) is linearised with EcoRV, purified and dephosphorylated with SAP. 30 fmol Bpu10l fragment are ligated with 15 fmol vector pBS2SK and electroporated into E.coli XL1-blue. Clones are selected by Xba-1, BamH1, and EcoR1 digestion. 120 &mgr;g endotoxin-free plasmid Tar-mE-Sel SEAP-2/Neo is linearised with Not-1, heat inactivated, precipitated and redissolved in 70 &mgr;l TE Buffer low EDTA (0.1 mmolar). The resulting targeting construct is a polynucleotide having SEQ. ID. NO: 9. FIG. 2(a) depicts the SEAP-2 targeting construct and its insertion site in the endogenous E-selectin locus.

[0084] (b) Generation of E-selectin-SEAP Knock-in BALB/c ES Cell Lines.

[0085] 5×106 BALB/c ES cells [Noben-Trauth et al.,Transgenic Res. 5:487491 (1996)] are electroporated with 30 &mgr;g of the linearized targeting construct. Transfected cells are selected with G418 (200 &mgr;g/ml). FIG. 2(b) schematically depicts the targeting construct as inserted into the targeted locus of the endogenous E-selectin gene. G418-resistant clones are screened for homologous recombination events by PCR [the ES cells are lysed 1 h/37° with 20 F1 Lysis buffer (PCR buffer 1×; SDS 1.7 &mgr;M; Proteinase K 50 &mgr;g/ml) heat inactivated 85°/15 Min. and cleared by centrifugation. 1.3 &mgr;l lysed solution is used in for a 50 &mgr;l nested PCR. PCR (1): primer pair SEQ ID NO: 5 and SEQ ID NO: 6 (20 cycles) followed by PCR (2): 1.3 ul PCR (1) and primer pair SEQ ID NO: 7 and SEQ ID NO: 8 using 25 cycles.] and positive clones are further verified by Southern analysis.

[0086] Targeted ES lines are subjected to a further electroporation with a plasmid carrying the Cre gene under the transcriptional control of the HSV tk-promoter. Individual clones are grown in duplicate 96-well plates in the presence or absence of G418. The targeted locus after Cre-mediated neo cassette removal (leaving one loxP locus) is depicted in FIG. 2(c). The absence of the neo selection cassette is verified by Southern blot analysis of individual G418 sensitive clones.

[0087] (c) Generation of E-selectin-SEAP Knock-in Mice

[0088] BALB/c-ES cell clones carrying one E-selectin-SEAP allele are injected into BU6-III host blastocysts and transferred into pseudopregnant foster mothers according to standard protocols. Chimeras are mated with BALB/c females and albino offspring (indicative for germ line transmission) are analyzed by PCR for target integration [200 ng genomic DNA are used in the same nested PCR as described in (vi) but in 30 &mgr;l reactions] and Southern analysis. Heterozygous animals are generated by back-crossing of F1 animals to Balb/c wild type animals and Southern analysis of the F2 animals. The homozygous lines are established by mating heterozygous F1 animals.

Example 2

[0089] Preparation of Transgenic Mice Expressing a Secreted Heat Stable Alkaline Phosphatase Gene Under the Control of an E-selectin Regulatory Sequence Maintaining the 3′UTR of E-selectin Containing the mRNA Destabilizing AUUUA Repeats

[0090] (a) Preparation of DNA Construct.

[0091] (i) mE-Selectin With SEAP-1 in Mutated ATG Site (mE-Sel/SEAP-1/Neo).

[0092] 5 &mgr;g plasmid obtained in Example 1 (a)(ii) is cut with EcoR1 and Bsm-1 and the 1615 bp fragment purified by gel electrophoresis. 45 fmol SEAP-1 fragment is ligated into 5 fmol mE-Sel/mutATG/Neo, linearized with Mfe-1 and dephosphorylated, using 22 fmol Linker Bsm-1/Mfe-1 (GTTTAA). FIG. 1 schematically depicts the insertion of the SEAP-1 fragment into linearized mE-Sel/mutATG/Neo. Clone selection and sequencing are done as described above. The selected clone has a deletion of two base pairs (CA) in the Bsml site located 40 bp 3′ of the Stop codon.

[0093] (ii) Generation of Targeting Construct SEQ ID NO: 10.

[0094] 5 &mgr;g plasmid mE-Sel/SEAP-1/Neo are treated as described above. 120 &mgr;g endotoxin-free plasmid Tar-mE-Sel SEAP-1/Neo is linearised with Not-1, heat inactivated, precipitated and redissolved in TE buffer (0.1 mM EDTA). The resulting targeting construct is a polynucleotide having SEQ. ID. NO: 10. FIG. 2(a) depicts the SEAP-1 targeting construct and its insertion site in the endogenous E-selectin locus.

[0095] (b) Generation of E-selectin-SEAP Knock-in BALB/c ES Cell Lines and Mice.

[0096] Cell lines and mice are generated following the protocol as described in Example 1 (b) and (c) above.

[0097] Test 1: LPS and Cytokine Induction of SEAP Expression in Heterozygous Mice.

[0098] Heterozygous mice of Example 1 and Example 2 are injected with LPS (Sigma, Serotype 0:111B4 Cat. L4130 ) at a dose of 25 &mgr;g per mouse. Murine recombinant TNF&agr; (R&D Systems, Cat No. 410-MT) is diluted in sterile saline and administered at a dose of 500 ng/mouse. Administration is intravenously, e.g., into the tail vein, subcutaneously or intraperitoneally.

[0099] Blood for analysis is taken from a superficial vein of the mouse into heparinized glass hematocrit capillaries. The blood is centrifuged in a table-top Eppendorff centrifuge at maximal speed for 5 min to prepare plasma.

[0100] A standard curve is created with human placental alkaline phosphatase (PAP) (Sigma Cat. P-3895). A stock solution of 2 mg/ml dissolved in buffer (Tris/HCl 50 mM pH 7.4; NaCl 150 mM; BSA 1%) is stored at −80° C. in aliquots. The standard curve is diluted in mouse plasma containing Heparin. The range of the standard curve is between 0.5 and 50000 ng/mL PAP.

[0101] For the enzyme assay the Tropix Kit (Tropix, Cat. No BP300) is used. The analysis of heat stable alkaline phosphatase activity in standards and samples is performed in duplicates according to the manufacturer's suggestion.

[0102] The results are plotted in FIGS. 3 and 4. FIG. 3 shows that animals of Example 1 and Example 2 display significantly increased levels of circulating SEAP 6 hours after the administration of LPS as well as TNF&agr;. The maximal levels of plasma SEAP are reached at 6 hours post-stimulation in animals of Example 2, the maximum reached in animals of Example 1 is not seen until 24 hours post stimulation. SEAP-2 mice express about 1000-fold more marker enzyme than SEAourP-1 animals. NaCl and IFNg (which are known not to induce E-selectin in vitro) have no effect on SEAP induction in either line, demonstrating that SEAP expression follows the same stimulation pattern as E-selectin. FIG. 4 shows pooled data obtained at the 6 hour time point from n experiments. The plasma levels and the stimulation factors are shown (means ± SEM). In the SEAP-1 line, LPS-or TNF-stimulated SEAP expression at 6 hours is higher than in the SEAP-2 line (about 10-fold vs. about 3-fold).

[0103] Test 2: LPS and Cytokine Induction of SEAP Expression in Homozygous Mice.

[0104] The baseline plasma levels of SEAP is consistently and significantly higher in the homozygous compared to the heterozygous animals. The level of expression 6 hours after TNF&agr; challenge is about 3-fold higher in the homozygous mice compared to the heterozygous animals of Example 1 and Example 2 while no change is seen in either line in saline-injected control animals. FIG. 5 depicts a comparison of blood samples taken from heterozygous and homozygous mice treated with murine TNFa (500 ng/mouse) at 6 hours after stimulation (means ± SEM of n animals per group).

[0105] Test 3: Analysis of the Site of SEAP Expression.

[0106] Animals from Example 1 and Example 2 are treated with either LPS or saline, killed after 6 hours and organs removed for histology. Wild-type animals serve as controls.

[0107] After fixation for 3 hours in 4% PBS-buffered paraformaldehyde, tissues are infused with sucrose over night, embedded in OCT compound and snap frozen. The following results are obtained with tongue tissue. Cryosections (8 &mgr;m) are taken on polylysine coated slides, briefly dried and then stored at −80° C. For enzyme histochemistry, slides are brought to room temperature and then incubated for 45 min at 65° C. in 50 mM Tris/100 mM NaCl. The sections are cooled to room temperature and further incubated in alkaline phosphatase substrate mixture prepared as follows: 100 mg nitro tetrazolium blue and 30 mg 5-bromo4-chloro-3-indolyl phosphate are each dissolved in 5 ml N,N-dimethylformamide and then added to 90 ml 200 mM Tris buffer pH 9.2. The sections are incubated at 37° C. for 3-4 h, briefly washed, counter-stained and inspected under a microscope. Immunohistochemistry is performed with anti CD31 and anti E-selectin antibody.

[0108] In the non-heat inactivated tissue sections from mouse tongue very strong alkaline phosphatase activity is observed in several cell types. In the sections which were heat-inactivated, the background alkaline phosphatase activity is absent in the wild type control animals as well as in the SEAP-1 animals of Example 2 and, as shown in FIG. 6, in the unstimulated SEAP-2 animal of Example 1 (boxes marked “SEAP-2 control”). In the LPS-treated animal of Example 1 (FIG. 6, boxes marked “SEAP-2 LPS”) a specific staining is seen in the endothelium of some but not all vessels. The stained vessels are the same as those which express E-selectin after LPS stimulation (se FIG. 6, boxes marked “LPS”). Thus heat stable alkaline phosphatase enzyme activity is detected upon LPS stimulation in SEAP-2 mice in the same vessels as those which in wild type animals express E-selectin.

[0109] Test 4: Elimination Kinetics of SEAP. The elimination kinetics of SEAP are investigated in an experiment in which wild-type mice are injected i.v. with human alkaline phosphatase, and plasma samples are analyzed. A conventional PK method is employed to analyze plasma concentration data and to estimate amount of SEAP expressed after stimulation, yielding the following data: 1 TNF LPS Animal Group Parameters mean SD mean SD SeAP-1 AUC (ng · h/mL) 3250 1040 3990 660 Expression (ng) 321 102 393 65 SeAP-2 AUC (ng · h/mL) 2740 700 3930 160 Expression (? g) 270 69 388 16

[0110] Endogenously produced SEAP is found to distribute mainly into the blood stream. As depicted in FIG. 7, the elimination half-life of SEAP is about 10 hours.

[0111] Test 5: Effect of Inhibitory Compounds on SEAP Expression.

[0112] Clasto-lactacystine-&bgr;-lactone, a commercially available proteasome inhibitor is dissolved in DMSO (10 &mgr;l/100 &mgr;g) and further diluted with 90 &mgr;l Peg300. This mixture is further diluted 1:10 in saline and injected i.v. to animals of Example 2 at a dose of 1 mg/kg. Ten to 15 min later TNF is administered i.p. to compound-treated or untreated animals of Example 2. Blood samples are taken from the animals at baseline, 6 hours and 24 hours and SEAP levels analyzed. A significant inhibition of marker enzyme expression is observed 6 hours after TNF administration in the animals which received clasto-lactacystine-&bgr;-lactone and TNF compared to the animals which received TNF alone (p=0.045, two-way ANOVA). FIG. 8 shows that in SEAP-1 animals, the proteasome inhibitor significantly inhibits marker enzyme expression after TNF stimulation at 6 hours, but shows no effect at 24 hours in this mouse line. In SEAP-2 animals the compound does not modify SEAP expression at 6 hours or 24 hours after TNF simulation

[0113] Test 6: Time Course of Endothelial Activation During Atherosclerotic Lesion Development.

[0114] Homozygous mice derived from animals from Example 1 or Example 2 are mated to homozygous mouse strains prone to atherosclerosis, e.g., LDL-R −/− or apoE −/−. The offspring of these animals are fed a high cholesterol diet. At weekly intervals blood samples are taken and marker enzyme is quantified. Mice show an increase in marker enzyme prior to fatty lesion development.

[0115] Test 7: Endothelial Cell Activation During Progression of Inflammatory Disease.

[0116] Animals from Example 1 or Example 2 are crossed with mouse strains predisposed to spontaneous development of inflammatory disorders e.g., the MRL-lpr mouse strain. Blood samples are taken from offspring at daily-to weekly time intervals and marker enzyme expression is monitored. The level of marker enzyme expression is correlated with disease onset.

Claims

1. A transgenic or somatic recombinant non-human animal comprising a polynucleotide encoding a soluble marker protein functionally linked to a regulatory sequence of an endogenous gene encoding E-selectin.

2. A transgenic or somatic recombinant non-human animal according to claim 1 having the polynucleotide encoding a soluble marker protein inserted into a region of an E-selectin gene of a chromosomal E-selectin allele of said animal, which is between a transcription start site and a translation start site of said E-selectin gene.

3. A transgenic or somatic recombinant non-human animal according to claim 1 wherein the soluble marker protein is a secreted alkaline phosphatase.

4. A transgenic or somatic recombinant non-human animal according to claim 2 wherein the soluble marker protein is a secreted alkaline phosphatase.

5. A transgenic or somatic recombinant non-human animal according to claim 1 wherein the animal is a transgenic animal.

6. A transgenic or somatic recombinant non-human animal according to claim 1 wherein the animal is a transgenic mouse.

7. A transgenic mouse having in its genome a soluble reporter transgene under the control of the promoter of an E-selectin gene of a chromosomal E-selectin allele.

8. A transgenic mouse according to claim 7 wherein the soluble marker protein is a secreted alkaline phosphatase.

9. A transgenic knockout mouse which is homozyous or heterozygous for a chromosomal E-selectin allele comprising a genetic construct comprising a soluble reporter transgene, said soluble reporter transgene being under the control of the promoter of the E-selectin gene of said chromosomal E-selectin allele, and said genetic construct being inserted into a region which is between a transcription start site and a translation start site of the E-selectin gene of the chromosomal E-selectin allele.

10. A transgenic mouse according to claim 9 wherein the soluble marker protein is a secreted alkaline phosphatase.

11. A transgenic mouse according to claim 10 wherein the genetic construct comprises SEQ ID NO: 11 or SEQ ID NO: 12.

12. An isolated cell derived from the transgenic mouse according to claim 9.

13. DNA having SEQ ID NO: 9 or SEQ ID NO: 10.

14. A method of screening for an agent having therapeutic utility, comprising

(a) administering the agent to a transgenic or somatic recombinant non-human animal according to claim 1

(b) monitoring marker concentration in a body fluid of said animal, and

(c) comparing said marker concentration to the concentration of marker in an untreated transgenic or somatic recombinant non-human animal, an elevated level being indicative of the therapeutic utility of the agent.

15. A method of screening for an agent which is a modulator of E-selectin expression, comprising:

(a) administering the agent to a transgenic or somatic recombinant non-human animal according to claim 1,

(b) monitoring marker concentration in a body fluid, and

(c) comparing said marker concentration to the concentration of marker in an untreated transgenic or somatic recombinant non-human animal, an modulated level being indicative of the capability of said agent of modulating E-selectin expression.

16. A novel modulator of E-selectin expression identified by a method according to claim 15.

17. A method for treating a patient suffering from an inflammatory, thrombotic, ischaemic or neoplastic condition or from transplant rejection comprising administering to the patient a pharmaceutically effective amount of a modulator acording to claim 16.

18. A method of monitoring disease progression comprising

(a) crossing a transgenic non-human animals according to claim 1 with an animal strain used as an animal model,

(b) recovering at least one offspring, and

(c) monitoring at least one offspring for disease progression in relation to marker concentration in a body fluid.