WO1998009510A1

WO1998009510A1 - Methods of diagnosing and treating cancer

Info

Publication number: WO1998009510A1
Application number: PCT/AU1997/000564
Authority: WO
Inventors: Felix Beck; Robert James; Kallayanee Chawengsaksophak
Original assignee: Howard Florey Institute Of Experimental Physiology And Medicine
Priority date: 1996-09-04
Filing date: 1997-09-01
Publication date: 1998-03-12
Also published as: AU4003597A

Abstract

The present invention relates generally to methods of diagnosing and treating cancer and more particularly colon cancer. Even more particularly, the present invention provides a genetically manipulated live animal model comprising a mutation in a Cdx2 Drosophila caudal gene homologue useful for developing diagnostic and treatment protocols for colon cancer. The present invention further provides agents useful for diagnosing and treating colon cancer in animals such as mammals including humans.

Description

METHODS OF DIAGNOSING AND

TREATING CANCER

The present invention relates generally to methods of diagnosing and treating cancer and more particularly colon cancer. Even more particularly, the present invention provides a genetically manipulated live animal model useful for developing diagnostic and treatment protocols for colon cancer. The present invention further provides agents useful for diagnosing and treating colon cancer in animals such as mammals including humans.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. A particularly important field is research into the development of cancers, such as colon cancer. An understanding of the molecular and genetic mechanisms leading to the development of colon cancers is required to facilitate rational design of diagnostic and treatment protocols.

Colon cancer and more specifically colorectal carcinoma develops from genetically directed stepwise phenotypic alterations of the epithelium (1). The genetic basis behind the phenot pic alterations are diverse and affects tumour suppressor genes, oncogenes and mismatch repair genes and leads to invasive carcinoma. The homeobox is a conserved DNA sequence encoding a sequence-specific DNA-binding homeodomain having transcription factor properties (2). Cdx2 is one of three murine homologues of the Drosophila homeobox gene, caudal (3). The others are Cdxl (4) and Cdx4 (5). In Drosophila, disturbance of caudal expression causes severe disruption in body segmentation and posterior structures (6). In the mouse, Cdx2 is expressed extra-embryonically at 3.5 day post coitum (d.p.c.) in the trophectoderm and later in some trophectodermally derived placental tissues. Embryonic expression of Cdx2 begins at 8.5 d.p.c. in the posterior gut, the tailbud and the posterior part of the neural tube (7).

In work leading up to the present invention, the inventors sought to investigate the functional role of Cdxl during mouse embryogenesis using genetically altered mice. In the course of this investigation, it was surprisingly observed that mice heterozygous for Cdx2 developed at high frequency intestinal adenomatous polyps. The genetically altered mice provides a model for colon carcinoma in animals such as mammals including humans and provides a basis for the development of a range of diagnostic and therapeutic agents for colon carcinoma.

Accordingly, one aspect of the present invention contemplates a genetically altered animal or progeny of said animal having a predisposition to develop growth of neoplastic cells in intestinal epithelium.

More particularly, the present invention contemplates a genetically altered animal or progeny of said animal comprising a mutation in at least one allele of a homologue of the Drosophila caudal gene.

Even more particularly, the present invention is directed to a genetically altered animal or progeny of said animal comprising a mutation in at least one allele of Cdx2 or equivalent thereof and which is a homologue of the Drosophila caudal gene and wherein said genetically altered animal has a predisposition to develop growth of neoplastic cells in intestinal epithelium.

The animal may be a laboratory test animal such as a mouse, rat, rabbit or guinea pig; a livestock animal such as a pig, sheep, cow, horse, donkey or goat; a companion animal such as a cat or dog or a captive wild animal such as a kangaroo, deer or fox. Preferably, the animal is a laboratory test animal and is most preferably a mouse.

A "predisposition" to develop growth of neoplastic cells includes at least 1 in 100, more preferably at least 1 in 50, even more preferably at least 1 in 10 and still even more preferably between 2 in l0 to 9 in 10 animals develop neoplastic cell growth over from about 3 months to about 2 years.

The genetically altered animal contains a disruption to the expression or expression product of a Drosophila homologue of caudal. Preferably, the homologue is Cdx2 although the present invention extends to equivalents of this gene or homologues of this gene. Most preferably, the gene is murine Cdx2. The mutant Cdx2 gene generally contains a single or multiple nucleotide substitution, addition and/or deletion such that the gene is no longer transcribed or is transcribed into a non-full length transcript or is incapable of directing a normal functional Cdx2 protein or is incapable of directing sufficient production of normal Cdx2 protein. In one embodiment, the Cdx2 alleles are deleted or substantially deleted by homologous recombination. However, any of a range of genetic alterations may be induced to effectively render the mouse or other genetically altered animal homozygous Cdx2 mutants or heterozygous Cdxl mutants.

The growth of neoplastic cells is generally in the form of intestinal adenomatous polyps and this is considered a form of, or a preliminary form of, colon carcinoma. Tumour growth in a heterozygous Cdx2 mouse may be characterised by at least one of the following: (i) single or multiple polyps mainly in proximal large intestine; and/or (ii) large bowel masses having the appearance of tubulovillous adenomata at the microscopic level; and/or

(iii) crypt architecture grossly disturbed with abnormally situated mitoses in dysplastic epithelial cells; and/or (iv) absence of Cdxl expression in epithelial cells of colonic tumours; and/or (v) areas of metaplasia or heterotopia including gut epithelium.

The genetically altered animals of the present invention provide an animal model for carcinoma of the colon or at least a precursor stage thereof. More particularly, the animal model is for human carcinoma of the colon.

Accordingly, another aspect of the present invention provides an animal model for carcinoma of the colon, or a precursor stage thereof, said animal model comprising an animal or its progeny having a predisposition to develop growth of neoplastic cells in intestinal epithelium.

More particularly, the present invention contemplates an animal model for carcinoma of the colon or a precursor stage thereof, said animal model comprising a genetically altered animal or its progeny comprising a mutation in at least one allele or homologue of the Drosophila caudal gene.

Even more particularly, the present invention is directed to an animal model for carcinoma of the colon or a precursor stage thereof, said animal model comprising an animal or its progeny having a mutation in at least one allele of Cdxl or equivalent thereof and which is a homologue of the Drosophila caudal gene and wherein said animal has a predisposition to develop growth of neoplastic cells in intestinal epithelium.

Most preferably, the present invention provides an animal model for carcinoma of the colon or a precursor stage thereof, said animal model comprising a mouse or its progeny having a mutation in at least one allele of the Cdxl gene or its equivalent and which is a homologue of a Drosophila caudal gene.

With respect to the latter preferred embodiment of the present invention, the genetically altered mouse is generally a Cdxl heterozygous mutant. However, after tumour development, the neoplastic cells may be homozygous Cdxl mutant, referred to herein as a null mutation or -/- mice. Heterozygous mice are referred to as +/- mice. Normal mice are referred to as +/+ mice.

In accordance with the present invention, -/- may be rescued which may provide an alternative or complementary mouse model for colon cancer.

The genetically altered animals of the present invention permit the development of a range of diagnostic tests for carcinoma of the colon or a precursor thereof or a predisposition to develop carcinoma of the colon. The present invention is predicated in part on the high rate of tumour development in Cdxl +/- mice. Accordingly, screening for individuals such as humans for a mutation in a Cdxl allele will provide an indication on the likelihood of cancer development in the colon. For example, where a subject such as a human subject has relatives with a history of colon cancer, the subject to be tested can be screened for a mutation in an allele of Cdxl. This will provide an indication of whether familial cancer is likely to develop.

According to this embodiment, there is provided a method of determining a subject's predisposition to developing familial carcinoma of the colon, said method comprising obtaining a biological sample from said subject containing cells and screening for the presence of a mutation in a homologue of the Drosophila caudal gene wherein the presence of a mutation in at least one allele is indicative that the subject is likely to develop carcinoma of the colon.

Preferably, the gene to be screened is Cdxl or its equivalent.

Subjects having mutant Cdxl alleles are more likely to develop colon cancer.

The development or likelihood of development of sporadic carcinoma of the colon may be tested by obtaining a biopsy from the intestine and screening for the presence of a mutation in the Cdxl gene. Again, the presence of a mutation in at least one allele is indicative of a strong likelihood that a tumour will develop in the colon.

According to this embodiment, there is provided a method of diagnosing colon cancer or a likelihood of developing colon cancer in a subject, such as a human subject, said method comprising obtaining a sample such as a biopsy sample or a sample obtained following surgery of the subject's intestine and screening for the presence of a mutation in an allele of a homologue of a Drosophila caudal gene such as a Cdxl wherein the presence of a mutation in at least one allele is indicative that colon cancer has or will develop.

The screening may be accomplished in any number of ways such as using oligonucleotide probes or primers, polymerase chain reaction (PCR) analysis such as RT-PCR, differential hybridisation or truncated protein analysis.

Alternatively, an immunological test may be conducted of intestinal tissue. Accordingly, the present invention extends to antibodies to all or part of Cdx2 for use in screening for the presence or absence of Cdx2 expression. The antibodies may be monoclonal or polyclonal and may be fragments of antibodies (e.g. Fab fragments). The antibodies may also be synthetic or recombinant or the antibodies may be hybrid antibodies.

The antibodies may be directed to recombinant Cdx2 or to a derivative thereof A derivative includes a truncated form thereof, a fragment or part thereof or a fusion molecule comprising all or part of Cdx2 and another molecule such as maltose binding protein or glutathione S- transferase.

The antibodies may be labelled with a reporter molecule capable of providing an identifiable signal. Antibody binding is then detected by screening for the reporter molecule. Alternatively, antibody binding is detected by binding of a second antibody directed to said first antibody and labelled with a reporter molecule. Accordingly, the present invention is directed to both mentioned antibodies.

Both polyclonal and monoclonal antibodies are obtainable by immunization with Cdx2 or its derivatives or with synthetic fragments of the protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of Cdx2 or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting Cdx2 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for Cdx2 or its derivatives or homologues for a time and under conditions sufficient for an antibody-Cdx2 complex to form, and then detecting said complex.

The presence of Cdx2 may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These assays also include direct binding of a labelled antibody to a target.

In accordance with the present invention, the sample is one which might contain Cdx2 such as cells or cell extract and tissue biopsies. The choice of sample will also depend on whether detection of sporadic or familial cancer is being sought. Sporadic cancer would require analysis of biopsy samples whereas familial cancer may be screened using any cells.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen- bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β- galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluoro genie substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In one method, the enzyme-labelled antibody is added to the first antibody-Cdx2 complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with fight of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in an enzyme immunoassay, the fluorescent labelled antibody is allowed to bind to the first antibody-Cdx2 complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and enzyme immunoassay techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention also contemplates genetic assays such as involving, for example, PCR analysis to detect Cdxl alleles or their derivatives. Alternative methods or methods used in conjunction with PCR analysis include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphous analysis (SSCP), specific oligonucleotide hybridisation or methods such as direct protein truncation tests.

The present invention is further directed to an isolated nucleic acid molecule comprising a mammalian homologue of the Drosophila caudal gene. A mammal as contemplated above is as hereinbefore defined but does not extend to a mouse homologue when the homologue is Cdxl. This aspect of the present invention is described hereinafter with reference to the human homologue of Cdxl. This is done, however, with the understanding that homologues in other non-murine animals are contemplated by the present invention.

Accordingly, another aspect of the present invention contemplates an isolated nucleic acid molecule corresponding to a human homologue of the Drosophila caudal gene, Cdxl.

The nucleic acid molecule of this aspect of the present invention encodes Cdx2 and comprises a sequence of nucleotides having at least about 60% similarity to the nucleotide sequence set forth in SEQ ID NO: 1 and which is capable of hybridising under low stringency conditions, at 42 °C to the nucleotide sequence set forth in SEQ ID NO: 1.

The present invention also provides a nucleic acid molecule which encodes Cdx2 and comprises a sequence of nucleotides having at least about 60% similarity to the nucleotide sequence set forth in SEQ ID NO:6 and which is capable of hybridising under low stringency conditions, at 42^CC to the nucleotide sequence set forth in SEQ ID NO:6. The sequence set forth in SEQ ID NO:6 is a genomic sequence for murine Cdxl.

The nucleotide molecule is preferably derivable from the human genome but genomes and nucleotide sequences from non-murine animals are also encompassed by the present invention.

Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.

Accordingly, another aspect of the present invention provides a method for cloning a nucleic acid molecule encoding human Cdxl or a derivative thereof or a part thereof, said method comprising screening a nucleic acid library with mouse Cdxl DNA or mRNA or an oligonucleotide probe based on mouse Cdxl DNA or mRNA. The method of this aspect of the present invention may also use PCR to clone the target nucleotide sequence.

A further aspect of the present invention contemplates a method for cloning a nucleotide sequence encoding a human Cdxl, said method comprising searching a nucleotide data base for a sequence which encodes a molecule having at least 60% amino acid similarity to mouse Cdxl or where the nucleotide sequence is at least 60% similar to the mouse Cdxl gene, designing one or more oligonucleotide primers based on a nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said human Cdxl or part thereof.

Preferably, the nucleic acid library is a cDNA, genomic or mRNA library.

Preferably, the nucleic acid library is a cDNA expression library.

Another aspect of the invention is directed to the human genomic Cdxl gene and to 3 ' and 5 ' regions thereof as well as fusion molecules between Cdxl and other molecules such as maltose binding protein and glutathione-S-transferase.

Still another embodiment contemplates the promoter or a functional part thereof of the human genomic Cdxl gene. The promoter may readily be obtained by, for example, "chromosome walking".

Another aspect of the present invention is directed to human Cdx2 protein including a recombinant form thereof having an amino acid sequence of at least 60% similarity to mouse Cdx2, set forth in SEQ ID NO:2.

Other percentage similarities at the nucleotide or amino acid levels include about 70%, about 80%, about 90% and about 95%.

The present invention further contemplates a range of derivatives of human Cdx2 protein and its genetic sequences. Derivatives include fragments, parts, portions, mutants, homologues and analogues of the human Cdx2 polypeptide and corresponding genetic sequence. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to Cdx2 or single or multiple nucleotide substitutions, deletions and/or additions to the Cdxl genetic sequence. "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences. Reference herein to "human Cdx2" includes reference to all derivatives thereof including functional derivatives or Cdx2 immunologically interactive derivatives.

Analogues of Cdx2 contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH^ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBH

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the i idazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not Umited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)_n spacer groups with n= 1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific- reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and 1^ - me thylamino acids, introduction of double bonds between C_α and C_p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

These types of modifications may be important to stabilise Cdx2 if administered to an individual or for use as a diagnostic reagent.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

The present invention further contemplates chemical analogues of Cdx2 capable of acting as antagonists or agonists of Cdx2 or which can act as functional analogues of Cdx2. Chemical analogues may not necessarily be derived from Cdx2 but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of Cdx2. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.

The identification of Cdx2 permits the generation of a range of therapeutic molecules capable of modulating expression of Cdxl or modulating the activity of Cdx2. Modulators contemplated by the present invention include agonists and antagonists of Cdxl expression. Antagonists of Cdxl expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms. Agonists of Cdxl include molecules which overcome any negative regulatory mechanism. Antagonists of Cdx2 include antibodies and inhibitor peptide fragments. TABLE 1

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan Nmtφ

D-orni thine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D- α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D- α-methylmethionine Dm et N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D- α-methy lproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreomne Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtφ N-cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3-dipheny Ipropy l)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3 -indolylyethy l)glycine Nhtφ D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtφ N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(/. -hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtφ L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2 ,2-diphenyl- Nmbc ethylamino)cyclopropane

Another embodiment of the present invention contemplates a method for modulating expression of Cdxl in a human, said method comprising contacting the Cdxl gene encoding Cdx2 with an effective amount of a modulator of Cdxl expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of Cdxl. For example, a nucleic acid molecule encoding Cdx2 or a derivative thereof may be introduced into a cell. Conversely, Cdxl antisense sequences such as oligonucleotides may be introduced to the cell.

Another aspect of the present invention contemplates a method of modulating activity of Cdx2 in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease Cdx2 activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of Cdx2 or its receptor or a chemical analogue or truncation mutant of Cdx2. The Cdx2 molecule may require modification to permit transport of the molecule through the various membranes in the cell.

In a particularly preferred embodiment, the Cdxl gene or a functional encoding nucleotide sequence is introduced to a cell via a variety of genetic means such as by viral vector.

The identification of the role of Cdxl in colon cancer development permits a range of diagnostic applications as hereinbefore described as well as opportunity for gene therapy.

Another embodiment of the present invention contemplates an animal model for carcinoma of the colon or a precursor stage thereof, said animal model comprising a genetically altered animal or its progeny comprising a mutation in at least one allele of the Drosophila caudal gene or a homologue of said gene.

The present invention is further described by the following non-limiting figures and examples.

In the figures:

Figure 1 is a representation showing the generation of Cdxl mutant mice.

(A) Schematic represents the endogenous mouse Cdxl locus (top), Cdxl targeting vector (middle) and mutant allele (bottom). Cdxl coding exons are shown in black boxes PGK-neo is indicated by the stippled box. The 5' external and internal neo probes used for genomic Southern analysis of ES cell colonies (see below) are shown in the black lines. Arrows indicate the primers (pi, p2 and p3) used for genotyping progeny of heterozygote matings by the polymerase chain reaction (PCR)- see Table 2. Restriction enzyme sites: B, BamHl, E, EcoRl, H, HindSl, N, Ncol, S Sαcl. The Xba* restriction site was introduced during construction of Cdxl targeting vector. The bacterial neo and selection cassette driven by a phosphoglycertekinase promoter (PGK-neo), together with 4 kilo-base-pair (kb) 5' and 5.4 kb 3' flanking sequence served to replace 5.9 kb of the native gene. This involved the whole of the open reading frame (including the homeobox motif), except for the first 136 base-pair (bp). G418 was used to identify neomycin resistant clones.

The targeting vector was linearlised and electroporated into W 9.5 ES cell line. After 11 days of G418 selection, 912 drug resistant colonies were picked and subsequently analysed by Southern analysis. Nine clones yielded the expected restriction fragments. Two were selected to produce chimeric mice by micro-injection into C57BL/ 6xC57BL/10ScSn host blastocysts. Male chimeric animals were mated with C57BIV6 to obtain germ line transmission of the mutant allele.

(B) Expected restriction fragment lengths identified in wild type (WT) and mutant (MT).

(C) Southern analysis of five out of nine Cdxl targeted lines. The left lane shows a wild-type W 9.5 cell line and remainder show targeted cell lines. Top figure left is HindlU/Xba digested DNA and right is EcoRl digested DNA hybrised with the 5 ' external probe. Bottom is the same membrane hybridised with neo probe.

(D) PCR assay used to genotype 3.5 d.p.c. progeny of heterozygote intercrosses showing three possible genotypes: wild type (+/+), heterozygote (+/-) and homozygote (-/-).

Blastocysts were flushed from the uterus and conceptuses older than 5.5 d.p.c. were dissected free of maternal tissues. To isolate DNA for PCR, both embryonic and neonatal tissues were lysed in a non-ionic lysis buffer such as PNDB lysis buffer (PNDB buffer contains KCI, Tris- HCI, MgCl₂.6H₂O, gelatin, Nonident P40 and Tween 20) with proteinase K (100 μg/ml) at 56 °C. PCR mixture contained three primers shown in Figure 1 A: pi (5'- TAAAAGTCAACTGTGTTCGGAATCC -3' [SEQ ID NO:3]), p2 (5'- ATATTGCTGAAGAGCTTGGCGGC -3' [SEQ ID NO:4]), p3 (5'- GGGACTATTCAAAGTACAGGAG -3' [SEQ ID NO:5]).

Reaction conditions were 96 °C for 30 sec, 65° C for 1 minute and 72 °C for 3 minutes for 35 cycles in a 50 μl mixture containing 0.2 μM of each primer and 0.2 mM of each of dNTP and lxPCR buffer (Gibco). A 636 bp amplification product is generated from the mutant allele between the neo primer p2 and the Cdxl endogenous primer pi, while a 424-bp product is amplified from the wild type allele between primers pi and p3.

Figure 2 is a representation showing:

(A) growth retarded heterozygote Cdxl +/- new born mouse (left) compared to normal Cdx2 +/+ litter mate (right). (B) four heterozygote mice at 3 weeks of age (left) compared to a wild type animal (right). The phenotypic effect varies in severity.

Figure 3a is a presentation showing moφhology of the cervical and upper thoracic vertebrae in Cdxl +/- heterozygotes (Het) and a wild type (WT) litter mate. The vertebral number is shown on the left side of the panel. Vertebrae 3, 6 and 7 and 10 from heterozygous animals show morphological features characteristic of the immediately cranial vertebrae in WT litter mate control. This is best seen in expression of anterior tubercles in heterozygote vertebrae 7, absence of these tubercles in heterozygote vertebrae 6 and the presence of a prominent spinous process in heterozygote vertebrae 10. Abbreviations: FT, foramen transversum; TA, tuberculi anterior; PS, spinous process.

Figure 3b is a representation showing malformation of the ribs. Sternum and ribs from three Cdxl +/- heterozygotes (2, 3 and 4) and one wild type litter mate (1) showing an anterior homeotic shift in the heterozygote animals. Heterozygote (2) shows the eighth rib attached to the sternum on the left side. In heterozygote (3) the second rib is attached to the sternum at the top of the manubrium together with the first rib. The latter is incomplete and partially attached to the second rib (as is often seen in a cervical rib). The eighth rib is attached to the sternum bilaterally. Heterozygote (4) shows a similar arrangement on the left, while the second rib has retained its normal sternal attachment on the right.

Figure 4 is a representation showing tumours of the large intestine in Cdxl +/- animals:

(a) gross appearance of tumour at junction of caecum and proximal colon;

(b) lower power section of preduculated tubular adenoma from proximal colon showing abnormal crypt architecture Haematoxylin and Eosin (H&E) stain.

(c) multiple mitoses in abnormal situations and dysplastic cells from a tubulovillous adenoma of the proximal colon. H&E stain.

(d) giant cells and dysplastic epithelial cells from a tubulovillous adenoma of the proximal colon. H&E stain.

(e) metaplastic region of keratinising epithelium within a large bowel adenoma. H&E stain.

(f) tumour cells appear to be penetrating a blood vessel. H&E stain. (g) absence of Cdxl staining in the epithelial cells of a tubulovillous adenoma from the proximal colon with clear staining of adjacent normal tissue, (h) section through a duodenal tumour. H & E stain, (i) section of small intestine showing abnormal villi and crypts. H&E stain. EXAMPLE 1 GENERATION OF Cdx2 MUTANT MICE

ES cell lines were generated which have one non-functional allele of Cdxl by conventional gene targeting. A Cdxl targeting vector, which replaced most of the first and all of the subsequent coding exons with a PGK-neo cassette was electroporated into a W 9.5 embryonic stem (ES) cell line (Figure 1). Nine Cdxl knockout cell lines were isolated with a targeting efficiency of approximately 1%. Three of these cell lines were selected for the generation of chimeric mice by blastocyst injection. Two produced chimeric mice which transmitted the mutation to their progeny. Heterozygote offspring were viable and fertile, although many had visible growth defects and abnormal tails.

EXAMPLE 2 GENOTYPING OF Cdxl MUTANT MICE

Genotyping of post-implantation embryos at various stages and of post-natal animals showed that offspring were either heterozygotes or wild type (Table 2). Furthermore, the ratio between these differed at birth from the expected Mendelian 2:1 (χ² = 13.05 > P0.01 where df = 1). No post-natal deaths were recorded among 181 live births followed for 28 days. Immediately before implantation, homozygous negative blastocysts were demonstrated by PCR and, although numbers are small, embryos approximate to the expected 1:2: 1 ratio (Table 2). These results indicate that homozygous null mutants do not survive the per-implantation period and that there is probably some preferential loss of heterozygote embryos during gestation. Most of the heterozygous loss seem to occur early in gestation, since an abnormally high number of demonstratable resoφtion sites were not seen. The earliest expression of Cdxl during development occurs in the trophectoderm at 3.5 days; this may explain why null mutant blastocysts and possibly some heterozygotes fail to implant successfully. EXAMPLE 3 ANALYSIS OF Cdxl MUTANT MICE

Cdxl heterozygotes show a range of abnormalities. Many animals are growth retarded at birth and this is often associated with a shortened or kinky tail (Figure 2). The gene is normally expressed in the spongy cytotrophoblastic cells of the placenta (descendants of the trophectoderm). These cells constitute the generative layer of the trophoblast. Disturbance of their growth would result in placental insufficiency leading to fetal death or growth retardation. Examination of heterozygote placentae at full term indicated a relative paucity of the spongiotrophoblastic layer in a number of specimens from growth retarded fetuses

Skeletal analysis of heterozygote animals showed an anterior homeotic shift of the cervical and thoracic spine in 5 of the 5 animals examined (Figure 3a). Rib abnormalities, in keeping with this phenotype, were also apparent (Figure 3b). These results indicate that the Cdxl is haplo- insufficient and that loss of function leads to an anterior homeotic shift, a situation similar to that described for other homeobox mutations

EXAMPLE 4 DETECTION AND ANALYSIS OF TUMOURS

Examination of the gut of 20 heterozygote animals between 12 weeks and 28 weeks of age showed multiple adenomatous polyps mainly located in the proximal large intestine of 18 out of 20 animals. Macroscopically detectable tumours varied between 1 and 10 in number and were occasionally present in the small intestine, as well as in the large intestine. No tumours were seen in wild type litter mates of the heterozygote juveniles. Tumours varied from those just visible to the naked eye to ones up to 8 mm in length and 4 mm in width (Figure 4a). Frequently, the largest were pedunculated (Figures 4a, b). Microscopically the large bowel masses had the appearance of tubulovillous adenomata. Crypt architecture was grossly disturbed (Figure 4c) many abnormally situated mitoses were seen in dysplastic epithelial cells and occasional giant cells were present (Figures 4c, d). Areas of metaplasia were also seen (Figure 4e). Some tumours appeared to be infiltrating the muscularis mucosa and very occasionally it appeared as if blood vessels were penetrated (Figure 4f). The epithelium surrounding the tumours was occasionally abnormal, showing loss of mucous secreting cells in intestinal crypts with an otherwise generally normal architecture. When colonic tumours were stained for Cdxl, it was found that the epithelial cells had completely ceased to express the gene in contrast to clear Cdxl staining in the surrounding epithelium (Figure 4g). In the small intestine, macroscopically visible tumours (Figure 4h) were less frequent but extensive regions in which the crypts and villi had an area found architecture were apparent (Figure 4i).

EXAMPLE 5 FUNCTION OF Cdxl IN MICE

Cdxl has multiple functions in the mouse, including axial pattern formation and the regulation of growth and differentiation in the intestine. In the extraembryonic membranes, homozygous loss of expression in the trophoblast stops the process of implantation, while heterozygous inactivation may inhibit this process or else lead to placental insufficiency.

EXAMPLE 6 IMMUNOHISTOCHEMICAL PROCEDURES

Tissues were processed and analysed as described by James et al. (18). Tissue was fixed in methacarn (60% v/v methanol, 30% w/v chloroform, 10% v/v acetic acid) for 1 hour at room temperature, embedded in parafin wax, and cut into sections (3 μm). Polyclonal antibodies raised in rabbits against a bacterially produced fusion protein containing the amino-terminal 109 amino acids of murine Cdx2 were then used to detect the protein in these sections. The specificity of the antibody was established both by Western blot analysis (8) and on tissue sections by preincubating the antisera either with 5 μg of purified fusion protein (Cdx2-maltose- binding protein) or the same amount of bacterial protein (maltose-binding protein). Antigen- antibody complexes were visualised with a peroxidase-based detection system and all sections were counterstained with haematoxylin. EXAMPLE 7 DEVELOPMENT OF -/- MICE

Embryonic cells at the early Morella stage (i.e. above 6-8 cells) from normal mice are subject to electrical treatment to generate a tetraploid embryo. These are permitted to proceed to the blastocyst stage. At the approximate time that the inner cells begin to die or just prior to this time, -/- cells are infected into the remaining viable extra embryonic membrane. The injection of the -/- cells effectively replace the inner cell mass and -/- embryos continue to grow on a functioning placenta.

The generation of -/- mice provides a further useful model for cancer development.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

TABLE 2 Genotyping of the offspring from the Cdxl heterozygote intercrosses

Age No. of No. of No. of +/+ +/- -/- litters resorptions embryos typed

3.5 d.p.c. 4 0 34 10 17 7

4.5 d.p.c. 1 0 5 4 1

5.5 d.p.c. 2 0 11 11 0

6.5 d.p.c. 3 0 16 6 10 0

7.5 d.p.c. 1 10 3 6 0

9.5 d.p.c. 1 5 2 3 0

10.5 d.p.c. 3 1 27 8 18 0

1 1.5 d.p.c. 0 9 2 7 0

12.5 d.p.c. 0 3 1 2 0

13.5 d.p.c. 3 4 1 3 0

14.5 d.p.c. 0 4 2 2 0

16.5 d.p.c. 2 2 14 8 6 0

17.5 d.p.c. 1 7 3 4 0

18.5 d.p.c. 0 7 3 4 0

New born 9 55 22 33 0

28 days 32 181 82 99 0

Genotyping was performed by PCR except for * where the genotype was determined by immunostaining. In these animals, it is not possible to distinguish wild type embryos from heterozygotes. Wild type, +/+, heterozygote, +/-, homozygote, -/-. d.p.c. days post coitum. BIBLIOGRAPHY:

1. Fearon, ER, Vogelstein B, Cell 61: 759-767, 1990.

2. McGinnis, W et al Cell 68: 283-302, 1992.

3. James, R & Kazenwadel, J J Biol Chem 166: 3246-3251 , 1991.

4. Duprey, P et al Genes & Develop 1: 1647-1654, 1988.

5. Gamer, L & Wright, CVE Mech Develop 43: 71 -81 , 1993.

6. Macdonald, PM & Struhl, G Nature 314: 537-545, 1986.

7. Beck, F Erler, T & James, R Develop 1 Dynam 104: 217-229, 1995.

8. James, R, et al J. Biol. Chem. 269: 15237, 1994.

SEQUENCE LISTING

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(i) APPLICANT: HOWARD FLOREY INSTITUTE OF EXPERIMENTAL PHYSIOLOGY

AND MEDICINE

(ii) TITLE OF INVENTION: METHODS OF DIAGNOSING AND TREATING CANCER

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(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: HUGHES DR, E JOHN L

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +61 3 9254 2777

(B) TELEFAX: +61 3 9254 2770 (2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 937 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..934

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

ATG TAC GTG AGC TAC CTT CTG GAC AAG GAC GTG AGC ATG TAT CCT AGC 48 Met Tyr Val Ser Tyr Leu Leu Asp Lys Asp Val Ser Met Tyr Pro Ser 1 5 10 15

TCC GTG CGC CAC TCC GGC GGC CTG AAC CTG GCT CCG CAG AAC TTT GTC 96 Ser Val Arg His Ser Gly Gly Leu Asn Leu Ala Pro Gin Asn Phe Val 20 25 30

AGT CCT CCG CAG TAC CCG GAC TAC GGT GGT TAC CAC GTG GCG GCC GCG 144 Ser Pro Pro Gin Tyr Pro Asp Tyr Gly Gly Tyr His Val Ala Ala Ala 35 40 45

GCG GCT GCT ACG GCG AAC TTG GAC AGC GCT CAG TCC CCA GGG CCA TCC 192 Ala Ala Ala Thr Ala Asn Leu Asp Ser Ala Gin Ser Pro Gly Pro Ser 50 55 60

TGG CCC ACC GCG TAC GGC GCC CCT CTC CGC GAG GAC TGG AAT GGC TAC 240 Trp Pro Thr Ala Tyr Gly Ala Pro Leu Arg Glu Asp Trp Asn Gly Tyr 65 70 75 80

GCA CCC GGG GGC GCT GCG GCA GCC AAC GCG GTA GCC CAC GGT CTC AAT 288 Ala Pro Gly Gly Ala Ala Ala Ala Asn Ala Val Ala His Gly Leu Asn 85 90 95

GGT GGC TCC CCG GCC GCC GCT ATG GGC TAC AGC AGC CCC GCC GAA TAC 336 Gly Gly Ser Pro Ala Ala Ala Met Gly Tyr Ser Ser Pro Ala Glu Tyr 100 105 110

CAC GCG CAC CAT CAC CCG CAT CAT CAC CCG CAC CAT CCG GCC GCC TCG 384 His Ala His His His Pro His His His Pro His His Pro Ala Ala Ser 115 120 125

CCG TCC TGC GCC TCC GGC TTG CTG CAG ACG CTC AAC CTC GGC CCC CCG 432 Pro Ser Cys Ala Ser Gly Leu Leu Gin Thr Leu Asn Leu Gly Pro Pro 130 135 140

GGG CCC GCA GCC ACC GCC GCC GCC GAA CAG CTG TCC CCC AGC GGC CAG 480 Gly Pro Ala Ala Thr Ala Ala Ala Glu Gin Leu Ser Pro Ser Gly Gin 145 150 155 160

CGG CGA AAC CTG TGC GAG TGG ATG CGG AAG CCC GCG CAG CAG TCC CTA 528 Arg Arg Asn Leu Cys Glu Trp Met Arg Lys Pro Ala Gin Gin Ser Leu 165 170 175

GGA AGC CAA GTG AAA ACC AGG ACA AAA GAC AAA TAC CGG GTG GTG TAC 576 Gly Ser Gin Val Lys Thr Arg Thr Lys Asp Lys Tyr Arg Val Val Tyr 180 185 190

ACA GAC CAT CAG CGG CTG GAG CTG GAG AAG GAG TTT CAC TTT AGT CGA 624 Thr Asp His Gin Arg Leu Glu Leu Glu Lys Glu Phe His Phe Ser Arg 195 200 205

TAC ATC ACC ATC AGG AGG AAA AGT GAG CTG GCT GCC ACA CTT GGG CTC 672 Tyr lie Thr lie Arg Arg Lys Ser Glu Leu Ala Ala Thr Leu Gly Leu 210 215 220

TCC GAG AGG CAG GTG AAA ATT TGG TTT CAG AAC CGC AGA GCC AAG GAG 720 Ser Glu Arg Gin Val Lys lie Trp Phe Gin Asn Arg Arg Ala Lys Glu 225 230 235 240

AGG AAA ATC AAG AAG AAG CAG CAG CAG CAA CAG CAG CAG CAG CAA CAA 768 Arg Lys lie Lys Lys Lys Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 245 250 255

CAG CCT CCA CAG CCG CCG CCA CAA CCT TCC CAG CCT CAG CCG GGT GCC 816 Gin Pro Pro Gin Pro Pro Pro Gin Pro Ser Gin Pro Gin Pro Gly Ala 260 265 270

CTG CGG AGC GTG CCG GAG CCC TTG AGT CCT GTG ACC TCC TTG CAA GGC 864 Leu Arg Ser Val Pro Glu Pro Leu Ser Pro Val Thr Ser Leu Gin Gly 275 280 285

TCA GTG CCT GGT TCT GTC CCT GGG GTT CTG GGG CCA GCT GGA GGG GTT 912 Ser Val Pro Gly Ser Val Pro Gly Val Leu Gly Pro Ala Gly Gly Val 290 295 300

TTA AAC TCC ACT GTC ACC CAG TGA 937

Leu Asn Ser Thr Val Thr Gin 305 310

( 2 ) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 311 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Tyr Val Ser Tyr Leu Leu Asp Lys Asp Val Ser Met Tyr Pro Ser

1 5 10 15 Ser Val Arg His Ser Gly Gly Leu Asn Leu Ala Pro Gin Asn Phe Val 20 25 30

Ser Pro Pro Gin Tyr Pro Asp Tyr Gly Gly Tyr His Val Ala Ala Ala 35 40 45

Ala Ala Ala Thr Ala Asn Leu Asp Ser Ala Gin Ser Pro Gly Pro Ser 50 55 60

Trp Pro Thr Ala Tyr Gly Ala Pro Leu Arg Glu Asp Trp Asn Gly Tyr 65 70 75 80

Ala Pro Gly Gly Ala Ala Ala Ala Asn Ala Val Ala His Gly Leu Asn 85 90 95

Gly Gly Ser Pro Ala Ala Ala Met Gly Tyr Ser Ser Pro Ala Glu Tyr 100 105 110

His Ala His His His Pro His His His Pro His His Pro Ala Ala Ser 115 120 125

Pro Ser Cys Ala Ser Gly Leu Leu Gin Thr Leu Asn Leu Gly Pro Pro 130 135 140

Gly Pro Ala Ala Thr Ala Ala Ala Glu Gin Leu Ser Pro Ser Gly Gin 145 150 155 160

Arg Arg Asn Leu Cys Glu Trp Met Arg Lys Pro Ala Gin Gin Ser Leu 165 170 175

Gly Ser Gin Val Lys Thr Arg Thr Lys Asp Lys Tyr Arg Val Val Tyr 180 185 190

Thr Asp His Gin Arg Leu Glu Leu Glu Lys Glu Phe His Phe Ser Arg 195 200 205

Tyr lie Thr lie Arg Arg Lys Ser Glu Leu Ala Ala Thr Leu Gly Leu 210 215 220

Ser Glu Arg Gin Val Lys lie Trp Phe Gin Asn Arg Arg Ala Lys Glu 225 230 235 240

Arg Lys lie Lys Lys Lys Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 245 250 255

Gin Pro Pro Gin Pro Pro Pro Gin Pro Ser Gin Pro Gin Pro Gly Ala 260 265 270

Leu Arg Ser Val Pro Glu Pro Leu Ser Pro Val Thr Ser Leu Gin Gly 275 280 285

Ser Val Pro Gly Ser Val Pro Gly Val Leu Gly Pro Ala Gly Gly Val 290 295 300

Leu Asn Ser Thr Val Thr Gin 305 310 (2) INFORMATION FOR SEQ ID NO : 3 :

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : TAAAAGTCAA CTGTGTTCGG AATCC 25

(2) INFORMATION FOR SEQ ID NO : :

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : ATATTGCTGA AGAGCTTGGC GGC 23

(2) INFORMATION FOR SEQ ID NO: 5:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGGACTATTC AAAGTACAGG AG 22

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7162 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GGATCCCAGC CATCCACTAA TTACTGCCTT CCATATCGTG GGTCCAGTTG TTGGCTTTGA 60 AGAGGGTTTC GATTGTTTAA TGTAATAGTT TTTAAAAATG AATTGCTTTC CTTGAACTTA 120

AATCTTAACT GTAAGACAAA TCCTGGGGTG GGGGGCGCAA CCTGGAGGTA GAATCGTCTT 180

TTCTCTTTTG TAGAGGCCGA GTGGTTTCCT TTGACAAGGA AA AAATGTA TTGCTGAGGG 240

TTGTCCGACC GTCCGTCCGT CAGTCTCCTC CTGGTTCTCA GAGCGGGAAG GAATAGCCTT 300

CACCGCAGCT CTACAAGCCA CCTACCTCCT GGGTTGGTCA GAAAGCTCTT CAAAGCAATG 360

ACTTCTCCCC CCCCCCCCCG CCCCCTTTCT TGACCACCTT CTGCCTGAGA ATGTACGAAA 420

GGCTGCGCCC GCCCGTTTCC AAACCCAGCT TCCCACCAAG CCTGCCTTTC TGGACTTCCC 480

AAGGCGTTTG CAAGTCTCTT CATTTCAGTC TTTGAACCTG TGATTGGAGG TTAAAGTGCA 540

CCCAGGTTGG AAGGAGGAAG CTCGTAGCTA GCAAGAAGGG CCTGAATATT CAGCTGTGAT 600

CTGGTCGCTG CCCTCTCAGG AGCGCCCCCC TCCCCAACTT TTAAAATGCA AATCGTGTTT 660

CTGGGGGTGG TGGTGGTGGG GACTGTGTGC GTAGCGCGCT GCGCCTCGAC GTCTCCAGCC 720

ATTGGTGTCT GTGTCATTAC TAATAGAGTC TTGTAAACAC TCGTTAATCA CGTAAGGCCG 780

CTGGCCTGGG ACTCCGCGAG CCAACCTGCG GCGGGTCATC CCCGCCTCTA CAGCTTACTG 840

GCAAGGAGGT GGGAGGAAAG AAGGAAGAGA GGGGAGGAGG CAGGACGGAG GGAGGGACTG 900

CCCGGGAGGC AGAAGCTCTG CAAGGAGCCG ACGGAGCACC GTGGGCTGAG GTGCAGCCAG 960

CTACCTTTAT CTCTAGCCCC CTGCGCCTCG CGCCTCTGGC AGCCTTCAAC GTTTGTCCCC 1020

AGACAGCATG GTGAGGTCTG CTCTGGGTCC CTCGCCACCA TGTACGTGAG CTACCTTCTG 1080

GACAAGGACG TGAGCATGTA TCCTAGCTCC GTGCGCCACT CCGGCGGCCT GAACCTGGCT 1140

CCGCAGAACT TTGTCAGTCC TCCGCAGTAC CCGGACTACG GTGGTTACCA CGTGGCGGCC 1200

GCGGCGGCTG CTACGGCGAA CTTGGACAGC GCTCAGTCCC CAGGGCCATC CTGGCCCACC 1260

GCGTACGGCG CCCCTCTCCG CGAGGACTGG AATGGCTACG CACCCGGGGG CGCTGCGGCA 1320

GCCAACGCGG TAGCCCACGG TCTCAATGGT GGCTCCCCGG CCGCCGCTAT GGGCTACAGC 1380

AGCCCCGCCG AATACCACGC GCACCATCAC CCGCATCATC ACCCGCACCA TCCGGCCGCC 1440

TCGCCGTCCT GCGCCTCCGG CTTGCTGCAG ACGCTCAACC TCGGCCCCCC GGGGCCCGCA 1500

GCCACCGCCG CCGCCGAACA GCTGTCCCCC AGCGGCCAGC GGCGAAACCT GTGCGAGTGG 1560

ATGCGGAAGC CCGCGCAGCA GTCCCTAGGA AGCCAAGGTA AGCGGTGCCG GGGCGCGCCC 1620

CAGGTCCCGC GTGCGTTGGG GCTGTCGCGG GCTGTCTGCC AGCTCCTAGC CAGGACAGGA 1680

GAAGGGGCAA AGGGGGAAAT ACAGGTGGAT TCATCCCAGG AGTGTATATT TGTGTCTCGC 1740 TCCCCCAGGT TTGTTTAGGG CAGTACCCCA TTGGCAACCT CGGGACGCCT TCGGAAGCTC 1800

CCGGTAGTGT CCTAAGCACT GTCGCCCGCC GCGAGCTGAA TCGTGCCCTT GAGTTGGCTG 1860

CCTCCCGCAT TCCTTACACC GTTCCTGGCC GAGAGGGTCG GGCCGCTTGG ATTCTCTTTG 1920

CCGTGCCTAC TCAACTTAGC TACCTGCTTT TCTTCTTCCA TCTGAGGTAC CCCCCCCGAA 1980

AGGCCAGGGA GTGTGGAGGC TGCGCCAGGA TCAGTTTGGC CATTAGGGTC TACTGAAGGG 2040

GTCGGTGCTG CTGCGAGCAC AGACGCCGCG GTGCGCCCCT GGAGAGAAGG CGGAGCCCCC 2100

AGAAGCCTCG GAGGAAGTGG GGGAACTCCA GGGTCCCTGA CTCTTCCCTC TCTCTGCCAA 2160

GCAAAGTCTC TCATAGAAAT CCTCTCTTAG CATCACAGCG GACTCGGCTT CTTAAGAAGG 2220

TTTCTTCCTG ACCCTTCCCT TCTAGAATCT CGTGTGTCTG AGAGTGTTGT GTGTTGGATT 2280

CGGGAGATTT TTCACGAAAG ACCCGAGATT GCCGGTCTGT GGGACTCACA AGCTCTGTTT 2340

GCCCTTTCTG ATGTCCGGAT GTGTATAGGA GTGGGAAGGA AGTGAGCAAA GTGTGACAGG 2400

GAGTGGTACT AAGAGTCCCC CCTTTTTTGT TGTTCCGTGA AGCCTTCTCC AGCTCTTTGA 2460

GCTTCCGCTT CTCTGGATTT GCTCCTTGTT GGCACAGCCA GGCCACATGC CCGGGTTATG 2520

GCCACTTTGA GGAGGTTTAT AGCCAATTAC AGCCGTATAA ATCAGAGCGG CACTTGGGGG 2580

CCTGTCGCTG GACACTTCTG CGTCACTGCG CACACCCCGT CAACATCTTG GTGGGCAATG 2640

CTGCCGGAGT GAAGTCTCCA GATTAGCCTC CAGTGGGCTT TGACATTGTC ATTTCCAGGA 2700

GGACTTACTC CTAAACCGGT TCTCCGCATA AACCCTACTT TCCCAAGCCT GCCTGAAAGG 2760

TAGTGAGCGG AGGATCGCCG GTAGGGGTCT TAGCCCTCCG AAAGAACTTT TGCGTTCAGG 2820

GGGTGGGTGG TCCGAGACTA GGGTCCTGGG AGGGGGAGGA GAACCTCAGG AGCCCAGGCC 2880

GCGGGGTCAG CAGATGAGAG GTCGCTGTGA AGTCCCGCGC CGAGGTGGCA AGACCGCTGT 2940

CGGCGCCGGG CGCCCTTGTG ATGTTAATGT AGGGAAGCTG TGGCCCGCAG TGCCGGAGCC 3000

CGACTCTTCT GCAGAGCGTC ATCTGTCAAG GGGCAGAGGC GTGTACGCCG CCCGGCCGGC 3060

TTTGATGTAC ACCTTTCAGC CCTGCCATTG TCTCTTTTAG GGCCCGGAAA GAGGGTGGTG 3120

ACTTTCGCAG TCAATAATGA ATTCTGACAA CTCCTTCTCA TCCCTCCCCT CTTCCAGCCC 3180

CCCTCCTTGG TTCCCAGGGG GTGCGGGGCG CCTGCAAGCC CTTGGGGAGC GGACCGCAGG 3240

GCTCACGGAC CATCTTTGCG TTCCTCAGCC AGCGCGTGGT GCTCTAAGAG CAGCATCCGT 3300

TCTAGGGCGA GGACCGCTGT GGGAAATTGT TCTGCGCCTC GCTTCCCAGG TCTGATAGTT 3360

TTCTTTAGTA CTACAAGCTC TTTGCTATTC GGTCTGCACA CTTAAGATTT GGTTCTGTCG 3420 TCTTCTGTGG AGTGGGGCAC TAGCGAGTGT GTTACAGGCT CCAGTGAGCC CGCTCCGGAC 3480

CGGTTGGATC CCTTTACTTC CAAAGCAGGC GCGGGGTCGG GGCCGCAGCG CCCATTCAAA 3540

TGCGCCTGCG GGGCTGTGTT TATGTTAATG CTCCTGGCGG GGAAGGGGAT GAAAGCTGAG 3600

GCCGCCATTT GCTCAGTAGT GGTAATTCAA ACAGAATGCG GTTGTTATTA AGGCATTGAA 3660

AGCGCTTTTC TTTGATAAGA TCGCCTTGTC TTGCATTGTT TGCGACTGTG TTCCCCTTTC 3720

CAGCGGCGGG GGGACCTCTC TCTGATCCCT CCTGTATCTT CCCAGGACAC TTTTGTAGTG 3780

GTTTGCAAAG GTTTTTACCT GAACAAAGTC AGCCTGCGGG GCCTTAAACA CCCCTGGGCA 3840

ACTCCCACCC CTTAGATCTC CTTGTTCAAC TAGGCACTGA GCACTAAAAT CTATCTGTTT 3900

TGATGTGAGA CCTCTCCAAC TTTGGACGTG GAACAGTTCC TGGCCAGCAT TCTGTCCATT 3960

GAAGCATCCC CGACTTAACC GGGTTGAATA GCCAGACTCC TGCCTGATCC AGCAGCCCCG 4020

CGCTATTTGT CAAGGTCCTT CAGTAAAACA ATCAATCCAA GATTTAATAT GTTGACTCAA 4080

TCCAATGATC ACAGGTCACT TGGTCAGGAT AGTTGCCTCG GGGGATAAAT GGGAAATTAA 140

AAAAGAAAGA ATGCCCCAGG CCAAACCTAG CTGGGCAGGT GAGACGCGTT CCAAGTAGAA 4200

GGATTTTCCT CCTGACAACA TGGAGATAGC ACTCAGAATT CCAGGGCAGG ACTTTGTGTG 4260

TGGTACTGGT GACTGGTGGG GATCTGCCAG CCCATGCATC CATCTTTCCA CTATATTGAA 4320

ACAAAAGGTA ACTACCCGCT TACCCAGACG CCACATCCCA GGTATTTTCA AAGTGGCCTG 4380

CTGTTTCATT TCTAACAAGT CTGAAAATGT GGATATTGAT ATTCCTGGGG ACTCTGGTCA 4440

CTTTCCCACC GCCCTTTTTT CTGATCCTAA ATATACTATA TTTGATTTAT GGAAAGGAGG 4500

GGGGCTGTGT GAGAGGTGTT CCTTTCAGGT TAGTTGTGCC TCCAGGCTTA CACCCCCCTT 4560

CCCCAGGATC CTGTGGGTGG ATATTGTCAA CCTGGGTAGT AGACACCCCC CTCCATCAGT 4620

GGATGGAGAG AAATGACTCC TGGGTTAGGG AGGTTTGTCA TTACCTATGA CTGATGGATT 4680

GTAGTTCTTA GCCAAAACTT CTCCTTCCTC CACAGTGAAA ACCAGGACAA AAGACAAATA 4740

CCGGGTGGTG TACACAGACC ATCAGCGGCT GGAGCTGGAG AAGGAGTTTC ACTTTAGTCG 4800

ATACATCACC ATCAGGAGGA AAAGTGAGCT GGCTGCCACA CTTGGGCTCT CCGAGAGGCA 4860

GGTGGGAATC GGCTTTCCTG GGTCTGGTTA TTATGGCCTG GGGCAATCTT AATGGGTAGC 4920

ATTAGAAAGT TTGGTCGAGG GAGCTGTTGC CTTTTGAGAA TCCTTGAGTG TCCAGTTGTT 4980

TTTGCCGCTC TTTCAATGGA TCAGAGAGCT GTAAGGGAAA AGCCAAGTCG AGGCTACAGG 5040

GTGGAACTGC ACTTGGACAG AGAAAGAGCG ATTGGAACCA TGGTTCTCAA CCTGTGGGTC 5100 GCGACCCCTC AGCTTGCCAC TCAAACACGC TGTGCTTTAT AATAGCAGCA AAATTATAGT 5160

TATGAAATAG CAATTAAAAT AATTTGTGAT TGGGGGTGTC ACCACAACAT GAAGAACTGT 5220

CCTAAAAAGA AAAACAAAGG TCCAGTCTAC GCATACAAAC TAAAGTCTGC TCATACAAAT 5280

TAAAGAGGCA GGGGCTGGAG AGGTGATGCT TAAGAACACA CACTCTTGCA GAGGATCGGA 5340

GTTCTGTTTC CAGCATCCAC GTCAGGTGGC TCACAGTGTC ACAGTGTTGG CTAACTCTGA 5400

TTGCAGGGGA CACCCAGTCG CTCTGGCTTC TGTGGATATA TATGAATGTA TACAGACATA 5460

CACATATCAA AGATTTAAAA ATCCTTGAAC TTTTTTTTTC ATTAACACAA AAGAGGCCCT 5520

ACTTGCACAC CAACACTTTC TGTAGTCTAG GGAAAAGGGA AGGAACAAAC AAGAGAAATT 5580

CATAGCCAGG AAGTTTTGGA CAAAGTTTCA TTGTCTGTGC CAAGGTGAAG AACATCGCAG 5640

TACCAATGAG CTGTGGGAGC CCAAGGGTGA CCCACGAGGC ACACTGAGCT GGCCAACCAT 5700

TTGTGTAGGG AATAGCTTTC GTCATGGTTC CGTTCCCTGG TTCTGAGGTT CTGTTGCTAG 5760

TAAAGGGTTT TTTGTTTGTT TGTTTTTTGT TTTTTGAAAG CAACTTGGGG AGGGGGCAGG 5820

TAAGTAAAGA GGACGAGGCT GGCCTGGCTT TGGGAACATT TCCCAAACTC AGTGAAAGAA 5880

GGATGCTCCC GGGGTGGCGC CATTTCTCTG TTCTTATCTT CTTCTACCTT CTATTTTGTC 5940

ATCTCTAGGT TAAAATTTGG TTTCAGAACC GCAGAGCCAA GGAGAGGAAA ATCAAGAAGA 6000

AGCAGCAGCA GCAACAGCAG CAGCAGCAAC AACAGCCTCC ACAGCCGCCG CCACAACCTT 6060

CCCAGCCTCA GCCGGGTGCC CTGCGGAGCG TGCCGGAGCC CTTGAGTCCT GTGACCTCCT 6120

TGCAAGGCTC AGTGCCTGGT TCTGTCCCTG GGGTTCTGGG GCCAGCTGGA GGGGTTTTAA 6180

ACTCCACTGT CACCCAGTGA CCCCTCCCGT GGTCTGAAGC GGCGGCGGCA CAGCAATCCC 6240

AGGCTGAGCC ATGAGGAGTA TGGACGCTGC GAGAATCCTC AGAAGAGATT CCTCTCCTCC 6300

TACCCACGAA CAGCATCTAC TGATGGAGAT TGAGGACAGA AGATGAGTGG AATTATGGAC 6360

CTCAGGGGAA GACATGGTTT AGATTTTTTT TTTCTTTTTA ACTTTTCCCA TTCCGACTCT 6420

TCCTGCCAGC AACGACAAAC GAAGTGATTC CTGGGGCTTC TTCGTTCATG CTCTTTGCCA 6480

GGACTGACTA CCGACATGAA GCTTTCAGCC TCTTTTGCCC CAGCTCTTTG CCTCTCTGTA 6540

TTTCTGTGTG GAGCTGAGGA GAGAGTGAGA CTGGATGGGG TGGGGGTAGC AATACTTGAG 6600

CCAAGGTGGC TGTTTCCTGC TGACTGCTTT CTGAGAACCA GCTGGCCGTC CTGCCTCCGG 6660

GCCAGGGACT ATTCAAACTA CAGGAGCCAG AGGCAGCTAA GATAGCTGGA CTGACCGAAG 6720

TCTGCAGAAC CTCCCCCACC AGGTGGTCTG GGCTTTCTTC TCCACAAATC AGGAAGGGGT 6780 GGTGGGTTCA GGGGCTGCGG TGAGAGGGGG TTGGTTAGCC AACGCCAGGC CCCTGCGACA 6840

AGGGCTTGTT TAGAAAGCCT GTCACCAGAG CTGCTGTAGG CGGAATGTAT GTCTGTGTTG 6900

TAAATGCCAG AGCCAACCTG GACTTCCTGT CCCTTCCCTC GTCTTTGGCT GAAGAAGACC 6960

GGAATTGTTT GCTGCTGTTC GAGTCACTGA TCTGTGTAAC GAGCCAAACA AGCCTTTTAA 7020

AAAGCCTTCT TGATCCATGG GTAGAGAAGT TGTATGGTGA AGGGAAGTCG GGAGGGGGGG 7080

AAGGGGATCC GAACACAGTT GACTTTTATT TTGTAAAAAG ACAAAGATAA ACGAACTTTA 7140

ACAGAAAAAA AAAAAAAAAA AA 7162

Claims

CLAIMS:

1. A genetically altered animal or progeny of said animal having a predisposition to develop growth of neoplastic cells in intestinal epithelium.

2. A genetically altered animal according to claim 1 wherein said animal comprises a mutation in at least one allele of a homologue of the Drosophila caudal gene.

3. A genetically altered animal according to claim 2 wherein the animal is a laboratory test animal, a livestock animal, a companion animal or a captive wild animal.

4. A genetically altered animal according to claim 3 wherein said animal is a laboratory test animal.

5. A genetically altered animal according to claim 4 wherein said animal is a mouse.

6. A genetically altered animal according to claim 2 or 5 wherein the homologue of the Drosophila caudal gene is Cdx2.

7. A genetically altered animal according to claim 6 wherein said animal carries a heterozygous mutation for Cdx2.

8. A genetically altered mouse or progeny thereof carrying a mutation in one allele of murine Cdx2 and exhibiting or having a predisposition to exhibit:

(i) single or multiple polyps mainly in proximal large intestine; and/or

(ii) large bowel masses having the appearance of tubulovillous adenomata at the microscopic level; and/or (iϋ) crypt architecture grossly disturbed with abnormally situated mitoses in dysplastic epithelial cells; and/or (iv) absence of Cdxl expression in epithelial cells of colonic tumours; and/or (v) areas of metaplasia or heterotopia including gut epithelium.

9. An animal model for carcinoma of the colon or a precursor stage thereof, said animal model comprising a genetically altered animal or its progeny comprising a mutation in at least one allele of the Drosophila caudal gene or a homologue of said gene.

10. An animal model according to claim 9 wherein said animal carries a mutation in at least one allele of Cdx2 or its equivalent.

11. An animal model according to claim 10 carrying a heterozygous mutation in Cdx2 or its equivalent and exhibiting or having a predisposition to exhibit:

(i) single or multiple polyps mainly in proximal large intestine; and/or

(ii) large bowel masses having the appearance of tubulovillous adenomata at the microscopic level; and/or (iii) crypt architecture grossly disturbed with abnormally situated mitoses in dysplastic epithelial cells; and/or (iv) absence of Cdx2 expression in epithelial cells of colonic tumours; and/or (v) areas of metaplasia or heterotopia including gut epithelium.

12. An animal model according to claim 9 or 10 or 11 wherein said genetically altered animal is a mouse.

13. A method for determining a subject's predisposition to developing familial carcinoma of the colon, said method comprising obtaining a biological sample from said subject containing cells and screening for the presence of a mutation in a homologue of the Drosophila caudal gene wherein the presence of a mutation in at least one allele is indicative that the subject is likely to develop carcinoma of the colon.

14. A method according to claim 13 wherein the gene to be screened is Cdxl or its equivalent.

15. A method for diagnosing colon cancer or a likelihood of developing colon cancer in a subject, said method comprising obtaining a tissue sample from said subject and screening for the presence of mutation in at least one allele for Cdxl wherein the presence of such a mutation is indicative that colon cancer has or will develop.

16. A method of diagnosing colon cancer or a likelihood of developing colon cancer in a subject, such as a human subject, said method comprising obtaining a sample such as a biopsy sample or a sample obtained following surgery of the subject's intestine and screening for the presence of a mutation in an allele of a homologue of a Drosophila caudal gene such as a Cdxl wherein the presence of a mutation in at least one allele is indicative that colon cancer has or will develop.

17. An antibody to all or part of Cdx2 for use in screening for the presence or absence of Cdx2 expression.

18. A method for detecting Cdx2 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for Cdx2 or its derivatives or homologues for a time and under conditions sufficient for an antibody-Cdx2 complex to form, and then detecting said complex.

19. An isolated nucleic acid molecule comprising a sequence of nucleotides corresponding to a human homologue of the Drosophila caudal gene Cdxl.

20. An isolated nucleic acid molecule according to claim 16 wherein the nucleotide sequence has at least about 60% similarity to the nucleotide sequence set forth in SEQ ID NO: 1 or its complementary form and which is capable of hybridizing to SEQ ID NO: 1 at 42°C under low stringency conditions.

21. An isolated nucleic acid molecule according to claim 16 wherein the nucleotide sequence has at least about 60% similarity to the nucleotide sequence set forth in SEQ ID NO: 6 or its complementary form and which is capable of hybridizing to SEQ ID NO: 6 at 42°C under low stringency conditions.

22. A method for cloning a nucleotide sequence encoding a human Cdxl, said method comprising searching a nucleotide data base for a sequence which encodes a molecule having at least 60% amino acid similarity to mouse Cdxl or where the nucleotide sequence is at least 60% similar to the mouse Cdxl gene, designing one or more oligonucleotide primers based on a nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said human Cdxl or part thereof.

23. An isolated human Cdx2 protein including a recombinant form thereof having an amino acid sequence of at least 60% similarity to the amino acid sequence set forth in SEQ ID NO:2.

24. A method for modulating expression of Cdxl in a human, said method comprising contacting the Cdxl gene encoding Cdx2 with an effective amount of a modulator of Cdxl expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of Cdxl.

25. A method of modulating activity of Cdx2 in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease Cdx2 activity.

26. A method of reducing the likelihood of development of colon cancer or reducing the spread of colon cancer in a subject, said method comprising removing colon tissue, introducing into said tissue a nucleic acid molecule encoding a non-mutated Cdxl gene or part thereof and returning said colon tissue to said subject.