WO2004044197A1

WO2004044197A1 - Knock-in mouse prostate cancer model

Info

Publication number: WO2004044197A1
Application number: PCT/CA2003/001750
Authority: WO
Inventors: Jim W. Xuan; Chandra J. Panchal
Original assignee: Procyon Biopharma Inc.
Priority date: 2002-11-13
Filing date: 2003-11-13
Publication date: 2004-05-27
Also published as: AU2003301986A1; CA2411729A1

Abstract

A number of transgenic mouse models of adenocarcinoma of prostate cancer have been established. However, all previous models use a histological grading system which is not clinically applicable. In view of these limitations a knock-in mouse adenocarcinoma prostate model (KIMAP) was developed. This model was established by targeting the PSP94 gene with a tumor-inducer gene (SV40 Tag). The KIMAP model shows the applicability of the Gleason histological grading system, which is widely used in the clinical diagnosis and prognosis of human prostate cancer. Our evaluation indicates that the KIMAP model meets requirements for a new standard murine model for both basic and clinical studies of prostate cancer.

Description

KNOCK-IN MOUSE PROSTATE CANCER MODEL

FIELD OF THE INVENTION

This invention relates to DNA constructs, isolated cells and transgenic non-human mammals (e.g., animals) carrying such construct. More particularly, transgenic mice with a heterologous gene inserted inside the PSP^'94 gene exon/intron region were generated herein.

BACKGROUND OF THE INVENTION

Prostate cancer (CaP) is the most frequently diagnosed malignancy among adult males in North America. Since prostate cancer is unique to human, the mouse prostate does not develop neoplasm spontaneously, progress toward understanding the biology of CaP, molecular profiling and development of new therapies for this disease has been dependent on the establishment of an appropriate in vivo model system that adequately recapitulates the spectrum of latent, steady growing and metastatic forms of the human disease with all the histopathological clinical categories .

Currently there are several animal models of prostate cancer development, including a spontaneous canine prostatic adenocarcinoma model, rat Noble prostate cancer models (Leav, I., et al.,. J.Natl. Cancer Inst., 80: 1045-1053, 1988), a prostate organ reconstitute model (Thompson, T.et al., Urol Oncol, 2: 99-128, 1996), and transgenic mice models (Spence, Scheppard, P. C, et al., Proc.Natl .Acad.Sci .U.S.A. , 86: 7843-7847, 1989; Gingrich, J. R., et al., Cancer Res, 56 : 4096- 4102, 1996; Greenberg, N. M. , et al . , Pr^'oc. atl. cad.Sci .U.S. A. , 92 : 3439-3443, 1995; Green, J. E., et al . , Prostate, 36: 59-63, 1998; Shibata, M. A., et al . , EMBO J. , 18 : 2692-2701, 1999; Rodriguez, R. , et al., Cancer Res, 57: 2559-2563, 1997; Wei, C.,^' et al . , Proc. atl.Acad.Sci.U.S.A. , 94 : 6369-6374., 1997; Perez-Stable, C, et al., Cancer Res, 57: 900-906, 1997,-Garabedian, E. M., et al . , Proc. atl.Acad.Sci.U.S.A. , 62 : 227-237, 1998) for review see (DiGiovanni, J. , et al., Proc.Natl. cad.Sci.U.S.A. , 97 : 3455-3460, 2000,-Watabe, T., et al., Proc. atl.Acad.Sci.U.S.A. , 99 : 401-6., 2002). Many of these models have shown limitations .

Only a few targeting vectors have been successfully used to express heterologous genes in the prostate epithelium of transgenic mice. These include regulatory elements derived from the rat prostate steroid-binding protein (PSBP or C3 (1) ) , the human prostatic specific antigen (PSA) , the human foetal Gγ globin/SV40Tag, Cryptdin-2^' (CR- 2)/SV40Tag, bovine, keratin 5 promoter (BK5) promoter/insulin growth factor 1, prostate stem cell antigen (PSCA) and an osteocalcin-based co-targeting vector (Matsubara, S., et al., Cancer Res, 61 : 6012-9., 2001) . However most of these CaP models also lead to targeting of non- prostate tissues. Currently, only one transgenic model has been widely utilized as a mouse CaP model. The transgenic adenocarcinoma mouse prostate (TRAMP) and the LPB-Tag (Kasper, S., et al., Lab Invest, 78 : 319-333, 1998; Masumori , N. , et al . , Cancer Res, 61 : 2239-2249, 2001) models are both based on a prostate-specific rat probasin ,(rPB) gene for targeting to the prostate and for directing the SV40 Tag (SV40 T and t antigens) expression. However, targeting to non-prostate tissues has also been reported (Wu, X., et al., Mech.Dev., 101 : 61-9., 2001).

All aforementioned transgenic mouse prostate cancer models were only characterized by utilizing a non-clinical histological grading system, which has not gained widespread clinical acceptance and has not shown to be reproducible or prognostically reliable in clinical practice. All have limitations in providing the whole gamut of heterogeneity in mimicking the architectural patterns of human prostate cancer for the applicability to the Gleason grading system, a system which has enjoyed the greatest . application worldwide . These shortcomings may be at least due to the transgenic technique adopted, since the transgenic technique is very empirical. By utilizing a short DNA unit to obtain forced exogenous gene expression, only a limited length of transgenic DNA could be tested, and it is often affected by insertion sites and the copy number of transgene. Moreover, there is a requirement for breeding and selection of founder lines.

In view of the limitations of the transgenic technique, alternative models have been sought, utilizing knock-out technique and newly discovered genes . A number of knock-out mice with a tumor suppressor gene PTEN/Mmacl were generated. Invariably the resultant homozygous mutant generated were fatal in early embryonic development, and in heterozygous form, mice developed neoplasia in multiple organ systems (breast and prostate) (Podsypanina, K. , et al., Proc.Natl. Acad. Sci. U.S.A., 96: 1563-6, 1999; Di Cristofano, A., et al . , Nat. Genet., 27 : 222-4., 2001). Mice lacking Mxil mice (belonging to Myc oncop_roteins) exhibited progressive, multisystem (kidney, spleen, prrostate) abnormalities, and had increased susceptibility to tumorigenesis with the cancer-prone phenotype (Schreiber-Agus, N. , et al . , Nature, 393 : 483-7., 1998). A homeobox gene Pax-2 is considered important in the regulation of the reproductive system development, and the Pax-2 knock-out mouse showed absence of kidney, ureters and the genital tracts including the seminal^' vesicles, although the prostate was normal (Miyamoto, N. , et al. Development, 124 : 1653-1664, 1997) . Mice deficient in another homeobox gene, such as Nkx3.1 (Kim, M. J. , et al . , Cancer Res, 62 : 2999-3004, 2002), developed prostatic intraepithelial neoplasia ^' (PIN) -like lesions resembling human PIN. However, as with all experimental mice loss-of-function, the knock-out prostate cancer models established so far have rarely developed prostate carcinoma (review see: Abate-Shen C et al., Trends Genet., 18 : S1-S5, 2002).

In this study, we report the establishment of the first knock-in (of PSP94 gene) mouse prostate cancer model (PSP-KIMAP) , generated using gene targeting techniques, with close resemblance to clinical histopathological features observed in humans . We demonstrated that the PSP-KIMAP model behaves as an endogenous mutation ^" (cancer development) model and may be used as a standard mouse prostate cancer model. Thus, the present invention discloses transgenic non-human mammals (e.g., mice) susceptible to prostate tumor formation. More particularly, in this application the exon/intron of mouse PSP94 gene was modified by inserting a heterologous gene allowing the development of prostate cancer with similarities to the development of CaP observed in humans .

SUMMARY OF THE INVENTION

A number of transgenic mouse models of adenocarcinoma of prostate cancer have been established. However, all previous models use a histological grading system which is not clinically applicable. In view of these limitations a knock-in mouse adenocarcinoma prostate model (KIMAP) was developed. This model was established by targeting the PSP94 gene with a tumor-inducer gene (SV40 Tag; i.e., simian virus 40 Tag) . The KIMAP model shows the applicability of the Gleason histological grading system, which is widely used in the clinical diagnosis and prognosis of human prostate cancer. Our evaluation indicates that the KIMAP model meets requirements for a new standard murine model for both basic and clinical studies of prostate cancer. CaP in the KIMAP model showed the same prevalent range of Gleason grades and scores as those observed in human prostate cancer cases.

In accordance with one aspect, the present invention relates to a (isolated) DNA construct comprising a) a first PSP94 gene segment, b) an insert and; c) a second PSP94 gene segment, said first and second PSP94 gene segments being different and said insert being located between the first and second PSP94 gene segment.

For example, one of the PSP94 , gene segment mentioned above may be a functional PSP94 promoter (either chimeric or comprising wild type sequences) . Depending on the recombination technique used, the DNA construct may have a PSP94 promoter that is at the 5' or 3' end relative to the insert. A functional PSP94 promoter is a sequence that is derived from the PSP94 gene (promoter) and that is able to drive the expression of a protein, for example, a protein encoded by an insert. The_.- DNA construct may comprise sequence facilitating recombination (a gene targeting plasmid) . The DNA construct may also comprise a sequence enabling selection of cells having incorporated (carrying) such DNA construct . These sequences may include for example the neomycin gene .

The DNA construct of the present invention may be used for different purpose than to study prostate cancer development, e.g., to follow development of an embryo (prostate tissue) , to study prostate targeting, etc. Therefore, in accordance with the present invention, the insert may be selected from the group consisting of, a reporter gene, a gene encoding a therapeutic protein. The insert may also be a gene able to be transcribed into a polynucleotide selected from the group consisting of an antisense RNA and a ribozyme, the polynucleotide may target for example, a gene capable of initiating tumor formation. It is to be understood herein that more than one insert (different insert or repeat of the same insert) may be included in the DNA construct (transgenic non-human mammal) of the present invention.

It is to be understood herein that a specific gene encoding a protein may fall in more that one of the category listed in the above mentioned group .

Further in accordance with the present invention, the gene capable of initiating tumor formation may be, for example, a SV40 T antigen or any other suitable gene capable of tumor formation (for example see table 1) . Also in accordance with the present invention, the SV40 T antigen may be selected from the group consisting of the SV40 large T antigen, the SV40 small t antigen and combination thereof.

A gene capable of initiating (promoting, inducing) tumor formation may be, for example, an oncogene. Any oncogene or effective sequence thereof may be used to produce the transgenic mouse of the invention. Table 1 below, lists some known^' viral and cellular oncogenes, many of which are homologous to DNA sequence endogenous to mice and/or humans, as indicated. The term "oncogene" encompasses both the viral sequences and the homologous endogenous sequences'. Any other oncogene, that are not part of table 1 may be suitable for the present invention (e.g., Bcl2, mutated p53, cbl, etc.), more particularly in the generation of transgenic non-human mammals . Those are especially of use when studying prostate tumor development and metastasis in a transgenic non- human mammal .

In accordance with the present invention, the therapeutic protein may be selected,^' for example, from the group consisting of a cytotoxic protein, a protein causing apoptosis, an anti-oncoprotein, a protease, a suicide protein, a cytokine, a chemokine, a costimulatory molecule and an antigen or any other desired therapeutic protein.

Also in accordance with the present invention, the reporter protein may be selected, for example, from the group consisting of beta- galactosidase, luciferase, red fluorescent protein, green fluorescent protein, alkaline phosphatase, chloramphenicol acetyl transferase, and horseradish peroxidase. In accordance with the present invention, the first PSP94 gene segment may comprise at least a part of the^" promoter/enhancer region and the second PSP94 gene segment may comprises at least a part of the PSP94 gene exon/intron region.

In another aspect, the present invention provides an isolated cell having incorporated (carrying) a DNA construct mentioned herein.

The cell having incorporated (carrying) (incorporated means that the DNA construct is inside the cells, either integrated in the genome or not) the DNA construct described herein are generated using techniques (e.g., micro-injection, recombination vectors, etc.) known in the art.

In yet another aspect, the present invention provides a transgenic non- human mammal susceptible to prostate tumor formation (susceptible to prostate neoplasia) , having genomically-integrated in a non-human mammal cell (s) , an insert (located) inside the PSP94 gene exon/intron region.

It is also to be understood herein that a suitable cell or suitable non-human mammal used to generate a cell having an integrated insert or to generate a transgenic non-human mammal may be one having in its genome, at least part of a PSP94 gene. The presence of the PSP94 gene (in the cell or non-human mammal genome) may be necessary for the recombination event to occur.

It is, to be understood herein that the insert may or may not functionally inactivate the PSP94 gene, for example the presence of the insert inside the PSP94 gene exon/intron region may disturb (disrupt) partially or totally its transcription or the presence of the insert may alter the PSP94 gene exon/intron region in a way to produce a deleted (shorter) form of the protein. On the other hand, the presence of the insert may lead to the production of a fusion protein comprising the (polypeptide coded by the) insert and the whole or some parts of the PSP94 protein. In some circumstances, it may be useful to totally inhibit the expression of the PSP94 protein while in other circumstances it may be useful to preserve its expression and/or generate fusion proteins. Using a DNA construct as described herein (having an insert between two PSP94 gene segments) may allow the fusion of a tag (e.g., fluorescent) to PSP94 (either in the 5' end or 3' end or in the middle of the protein) to follow the localization of PSP94 in a non-human mammal.

One object of the present invention being that the insert may be under the control of a functional PSP94 promoter (or a chimeric PSP94 promoter) . The insert may be integrated outside of the promoter region of the PSP94 gene. It may be integrated, for example, at the junction of the promoter and exon/intron region or inside the exon/intron region.

A transgenic non-human mammal may be a homozygote, a heterozygote, a non-human-mammal , of a founder line, a non-human mammal of a breeding line, and a non-human mammal of a subsequent generation. A transgenic non-human mammal also includes a non-human mammal before its delivery (before its birth) and at different development stage (2 cell-, 4 cell, 8 cell- embryo, multinucleated embryo, morula, blastocyst, foetus, etc. ) .

In a further aspect, the present invention relates to a transgenic non- human mammal, susceptible to prostate tumor formation (susceptible to prostate neoplasia) , having genomically-integrated in non-human mammal cells, and insert replacing at least a part of the PSP94 gene exon/intron region.

In accordance with the present invention the insert (genomically integrated in a non-human mammal cell(s)) may be a gene (expressing a protein) capable of initiating tumor formation.

In accordance with the present invention, prostate tumor development in the transgenic non-human mammal may be monitored using a Gleason grading system.

Further in accordance with ^' the present invention, the kinetic of prostate tumor development in the transgenic non-human mammal may substantially correlate with the kinetic of prostate tumor development in a human.

Also in accordance with the present invention, a tumor marker which may be selected from the group consisting of Hepsin, Mapsin and spermine binding protein may be expressed in the transgenic non-human mammal. In yet a further aspect, the present invention relates to the use of the transgenic non-human mammal described herein for evaluating the efficacy of drug candidates in inhibiting (partial or total, /decreasing) growth of prostate related neoplasia (e.g., prostate cancer at different stages, prostatic adenocarcinoma, benign prostate hyperplasia (BPH) , etc.).

Without being limited to the following, a suicide protein may be selected, for example, from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase or any

^• other desired suicide protein. A cytotoxic protein may be selected, for example, from the group consisting of the A chain of diphteria toxin, ricin, and abrin or any other desired cytotoxic protein. A protein causing apoptosis may be selected, for example, from the group consisting of caspases, Fas-Ligand, Bax and TRAIL or any other desired protein causing apopotosis. An anti-oncoprotein may be selected, for example, from the group consisting of p53, p21, and Rb (retinoblastoma) or any other desired anti-oncoprotein.. A protease may be selected, for example, from the group consisting of awsin, papain, proteinase K, and carboxypeptidase or any other desired (suitable) protease. A cytokine may be selected, for example, from the group consisting of IL-1 (IL means interleukine) , IL-2, IL-6, IL-12, GM-CSF (Granulocyte macrophage colony stimulating factor) , G-CSF, M-CSF, IFN-alpha, IFN-beta, _FN- gamma (interferon gamma) , TNF-alpha (TNF mans tumor necrosis factor) , and TNF-beta or any other desired cytokine. A chemokine may be selected, for example, .from the group consisting of Mig-lalpha, Mig- Ibeta, IP-10, and MCP-1 or any other desired chemokine.

In accordance with the present invention, the (transgenic) non-human mammal may be, for example, a (transgenic) mouse. Further in accordance with the present invention, the non-human mammal of the

■ present invention may also be, for example, without being limited to a dog, a cat, a monkey (of any desired species) , a sheep, a cow, a pig, a horse, etc.

It is to be understood herein that the sequence of mouse PSP94 promoter may be found in Genebank accession no. AF087140, sequences of mouse

PSP94 exon 1 and intronic sequences may be found in Genebank accession no. AF033264, the sequence of mouse PSP94 exon 2 and intronic sequences may be found in Genebank accession no. AF033265, sequence of mouse PSP94 exon 3 and intronic sequences, may be found in Genebank accession no. AF039596 and in AF033266, sequence of mouse PSP94 exon 4 and intronic sequences may be found in Genebank accession no.AF136292.

Unless otherwise indicated, the recombinant DNA techniques utilized in the present invention are standard procedures, known to those skilled in the art . Example of such techniques are explained in the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al ., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) , T.A. Brown (editor) , Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al . (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) and are incorporated herein by reference .

Those skilled in the art of molecular cloning will know that new DNA construct (s) may be made from known DNA sequence by deleting or recovering some DNA- fragments using restriction endonuclease (i.e., enzyme) that will specifically recognize a DNA sequence comprised in the desired gene or DNA sequence. For example, using a Kpnl (i.e., K) enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GGTACC-3' -(coding sequence shown only)) will be cut using suitable conditions. When using HindiII (i.e., H) enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-AAGCTT-3' (coding sequence shown only)) will be cut using suitable conditions. When using BamHI (i.e., B) enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GGATCC-3' (coding sequence shown only)) will be cut using suitable conditions. When using Bgr_.II (i.e., Bg) enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-AGATCT-3' (coding sequence shown only)) will be cut using suitable conditions. When using EcoRV enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GATATC-3' (coding sequence shown' only)) will be cut using suitable conditions (i.e., suitable buffer, temperature and volume described by the manufacturer). When using Pstl (i.e., P) enzyme, any double stranded DNA^' comprising the sequence recognized by this enzyme (i.e., 5'-CTGCAG- 3' (coding sequence shown only)) will be cut using suitable conditions. When using Sail enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GTCGAC-3' (coding' sequence shown only)) will be cut using suitable conditions. When using Stul enzyme,' any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-AGGCCT-3' (coding sequence shown only)) will be cut using suitable conditions. When using Xbaϊ enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5' -TCTAGA- 3' (coding sequence shown only)) will be cut using suitable conditions. When using Hindi enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5' -GT-pyrimidine-purine-AC-3 ' (coding sequence shown only)) will be cut using suitable conditions. When using Clal enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-ATCGAT-3' (coding sequence shown only)) will be cut using suitable conditions. When using EcoRI enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5' -GAATTC-3' (coding sequence shown only)) will be cut using suitable conditions. When using BsaAI enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'- pyrimidine-ACGT-purine-3' (coding sequence shown only)) will be cut using -suitable conditions.

Suitable conditions for restriction enzymes include, for example, suitable buffer, temperature and volume. Suitable conditions are described by manufacturers (e.g.,- New England Biolab, Pharmacia) .

Those skilled in molecular cloning will also know that following digestion with restriction enzymes, the desired DNA may be ligated, for example, into a linearized plasmid (i.e., vector, DNA construct) or to' another linear DNA molecule having matching ends. Alternatively, following digestion with restriction enzymes, the cohesive ends of DNA may be transformed to blunt ends using any suitable DNA Polymerase (e.g., T4 DNA Polymerase) or any suitable Nucleases (e.g., Mung Bean

Nuclease) and the desired DNA may be ligated, for example, into a linearized plasmid (i.e., vector) or to another linear DNA molecule having suitable ends (e.g. blunt ends). Ligases (e.g., T4 DNA Ligase) will catalyze the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme (when used in suitable conditions described by the manufacturer)_, will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA, or DNA/RNA hybrids .

In order for ligation to occur, the DNA molecules (e.g., desired DNA, linearized plasmid) . the 5' end of the DNA molecule must be phosphorylated. Such phosphorylation may occur for example by using Polynucleotide kinase. Suitable Polynucleotide kinase (e.g., T4 Polynuleotiude kinase) will catalyze (when used in suitable conditions described by the manufacturer) the transfer and exchange of Pi (i.e., inorganic phosphorus) from the gamma position of ATP (i.e., adenosine triphosphate) to the 5' hydroxyl terminus of polynucleotides (double- and single-stranded DNA and RNA) and nucleoside 3' -monophosphates .

Following ligation, DNA may be transformed in bacteria for amplification, and may be purified from lyzed bacteria. Following purification, the DNA construct (linearized or not) may be transferred

(e.g. transfected, transformed, electroporated,- micro-injected, lipofected etc.) into a desired host (e.g., a eukaryotic cell, an oocyte, an embryonic cell, a bacteria, a yeast, etc.) t As used herein the expression "DNA construct" includes without limitation; a vector, a plasmid (e.g., linearized or not) and a DNA fragment that may be used to transfer DNA sequences from one organism to another.

As used herein the expression "PSP94 gene" relates to coding and non- coding regions of said gene.

As used herein, the expression "vector" refers to an autonomously replicating DNA or RNA molecule into which foreign DNA or RNA fragments are inserted and then propagated in a host cell for either expression or amplification of the foreign DNA or RNA molecule. The term "vector" comprises and is not limited to a plasmid, (e.g., linearized or not), or a DNA construct that may be used to transfer DNA sequences from one organism to another. The term "vector" includes viral and non-viral vector. Viral vetors may be derived, for example, from a retrovirus . a herpes virus, an adenovirus, an adeno-associated virus, Sindbis virus, poxvirus, etc. Non-viral vector includes, but are not limited to, bacterial plasmids. As used herein the term "transgene" refers to a DNA construct (e.g., DNA fragment) that has been incorporated into the genome of an organism.

As used herein the expression "operatively linked" refers to two or more distinguishable DNA sequences of a transgene which are linked according to recombinant technology techniques so that they may act together to control and express a protein encoded RNA in a suitable tissue or cell type. An example would be the operatively linking of a promoter/tissue-specific enhancer to a DNA sequence coding for the desired protein (s) so as to permit and control expression of the DNA sequence and the production of the encoded protein (s) .

As used herein the "PSP94 gene exon/intron region" relates to the transcriptional region of the PSP94 gene, i.e., the region of the gene which is transcribed into a mRNA (mRNA precursor, i.e., comprising the corresponding exon/intron RNA sequence) this exon/intron region being distinct from the "regulatory region" or promoter region.

As used herein the term "regulatory region (s) " refers to region having an effect on the transcriptional control of a gene, the level of expression of a gene or on its specific expression in a given cell type or tissue type. The term "regulatory region(s) " includes tissue specific elements, promoter, enhancer, polyadenylation signal, or any regions of a gene, either upstream (5') or downstream (3') having an influence on the transcriptional control of a gene or on the level of expression of a gene. The "regulatory region (s)" may be isolated from existing DNA sequence (s) or may be man-made by known techniques of molecular biology. Existing DNA sequence may be derived, for example, without being limited to, from virus, bacteria, yeast, or higher eukaryotes .

Transcription control sequences are sequences, which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as, but not limited to, promoter, enhancer, operator and repressor sequences. It is to be understood herein that a gene is transcribed (expressed) into a messenger RNA (spliced or unspliced) . In turn a mRNA (spliced when required) is translated (expressed) into a protein.

As used herein the term "polynucleotide" refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, "polynucleotide" refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells . "Polynucleotide" includes but is not limited to linear and end-closed molecules. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

As used herein, the term "tumor" relates to solid or non-solid tumors, metastatic or non-metastatic tumors, tumors of different tissue origin including, but not limited to, tumors originating in the liver, lung, brain, lymph node, bone marrow, adrenal gland, breast, colon, pancreas, prostate, stomach, or reproductive tract (cervix, ovaries, endometrium etc.). The term "tumor" as used herein, refers also to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

It is to be understood herein that a polynucleotide or polynucleotide region that has a certain percentage (for example 75%, 80%, 85%, 90% or 95%) of sequence identity (homology) to another sequence may function in an equivalent or sufficient manner. A certain percentage (for example 75%, 80%, 85%, 90% or 95%) of sequence identity to another sequence (over the active region of said sequence) means that, when aligned, that percentage of bases (in the active region) is the same in comparing the two sequences. This alignment and the percent homology or sequence identity may be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds, 1987) supp.30, section 7.7.18, Table 7.7.1. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pensylvania) ,. Thus any DNA construct having significant homology to regulatory regions described herein are encompassed by the present invention.

It is to be understood herein, that if a "range", "group of substances" or particular characteristic (e.g., temperature, concentration, time and the like) is mentioned, the present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges or sub-groups therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein. Thus, for example, with respect to a temperature greater than 100° C, this is to be understood as specifically incorporating herein each and every individual temperature state, as well as sub-range, above 100° C, such as for example 101° C, 105° C and up, 110° c and up, 115° C and up, 110 to 135° C, 115° c to 135° C, 102° c to 150° C, up to 210° C, etc.;

with respect to a temperature lower than 100° C, this is to be understood as specifically incorporating herein each and every individual temperature state, as well as sub-range, below 100° C, such as for example 15° C and up, 15° C to 40° C, 65° C to 95° C, 95° C and lower, etc.;

- with respect to reaction time, a time of 1 minute or more is to be understood as specifically incorporating herein each and every individual time, as well as sub-range, above 1 minute, such as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hours to 20 hours etc.; Table 1

Abbreviation Virus

src Rous Sarcoma Virus (Chicken) yes Y73 Sarcoma Virus (Chicken) fps Fujinami Sarcoma Virus (Chicken, Cat) abl Abelson Murine Leukemia Virus (Mouse) ros Rochester-2 Sarcoma Virus (Chicken) fgr Gardner-Rasheed Feline Sarcoma Virus (Cat) erbB Avian Erythroblastosis Virus (Chicken) fms McDonough Feline Sarcoma Virus (Cat) mos Moloney Murine Sarcoma Virus (Mouse) raf. 3611 Murine Sarcoma. sup . + Virus (Mouse) Ha-ras-1 Harvey Murine Sarcoma Virus (Rat)

Balb/c mouse; 2 loci

Ki-ras 2 Kirsten Murine Sarcoma Virus (Rat)

Ki-ras 1 Kirsten Murine Sarcoma Virus (Rat) myc Avian MC29 Myelocytomatosis Virus (Chicken) myt Avian Myelo Blastomas (Chicken) fos FBJ Osteosarcoma Virus (Mouse) ski Avian SKV T10 Virus (Chicken) rel Reticuloendotheliosis Virus (Turkey) sis Simian Sarcoma Virus (Woolly Monkey)

N-myc Neuroblastomas (Human)

N-ras Neuroblastoma, Leukemia Sarcoma Virus (Human)

Blym Bursal Lymphomas (Chicken) mam Mammary Careionoma (Human) neu Neuro, Glioblastoma(Rat) ertAl Chicken AEV (Chicken) ra-ras Rasheed Sarcoma Virus (Rat) mnt-myc Carcinoma Virus MH2 (Chicken) myc Myelocytomatosis OK10 (Chicken) myb-ets Avian myeloblastosis/erythroblastosis Virus

E26 (Chicken) raf-2 3611-MSV (Mouse) raf-1 3611-MSV (Mouse) Ha-ras-2 Ki-MSV (Rat) erbB Erythroblastosis virus (Chicken) BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A is a schematic illustrating the genomic organization of the mouse PSP94 alleles (Wt: wild type 129 Sv mice) showing the four exons -three- introns. Structure of pPNTIσxP derived recombinant targeting plasmid is shown in the second line. The suppositional homologous double crossing over is in the 3.84 kb promoter/enhancer region and intron I. The structure of knock-in (KI) mutant (Mut) allele was inserted with a 2.7 kb SV40 Tag structural gene and neomycin gene (1.9 kb) . The location of the probe used for screening homologous targeting at mouse PSP94 gene in ES cell clones is shown at the bottom, and is located 3' downstream of 3 'arm of the targeting vector. Two Xba 1 restriction fragments of Wt and Mut are shown when detected by the probe in Southern blotting experiments. Primer pairs for PCR genotyping were shown for Wt and Mut alleles .

Fig. IB is a photograph of a Southern blotting experiments demonstrating screening of G418 resistant ES cell clones . Three positive mutants (KI) and a number of wild type ES clones were indicated with Xba 1 fragments of 13 kb and 8.5 kb separately.

Fig. 1C is a photograph of a Southern blotting experiments characterizing heterozygous (+/-) and homozygous knock-in (KI) mice.

Fig. ID is a photograph of a 1.5 % agarose gel electrophoresis of the fast PCR genotyping of KIMAP mice, Three types of mice Wt, heterozygous (+/-) and homozygous (-/-) were shown by two pairs of primers.

Fig. 2A is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Sections were stained with hematoxylin/ eosin (H&E). HGPIN of 7 weeks, H&E, x 25.

Fig. 2B is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. HGPIN with microinvasion (10 weeks), H &E x25. Arrow indicates a microinvasion site.

Fig. 2C is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Gleason grade 1, H&E, x 25. Inlet: x 40. Fig. 2D is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Gleason grade 2, H&E x 25. Inlet: x 40.

Fig. 2E is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Gleason grade 3, H&E of x 25. Inlet: x 40.

Fig. 2F is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Gleason grade 4; H&E of x 25; Up inlet: cribriform pattern of adenocarcinoma, x 40. Lower inlet: local metastasis in to stroma indicated by arrows, x 40.

Fig. 2G is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Gleason grade 4 showing multiple foci with two solid rounded tumor masses. Gleason scores 9 (4 + 5) . Left: grade 5; right: grade 4; H&E, x 40.

Fig. 2H is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Gleason grade 5 tumor foci showing (by arrow) one typical comedocarcinoma (right inlet, x40) . Masses of cribriform tumor with central necrosis were indicated by arrow on the bottom.

Fig. 21 is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. Grade 5 tumor showing anaplastic, raggedly infiltration (inlet x40) .

Fig. 2 is a photograph showing histological analysis of the prostate samples in PSP-KIMAP mice. IHC with a SV40 Tag antibody of HGPIN (hematoxylin counterstaining) showing nucleus staining in atypia area only, while normal glands are negative, x 40.

Fig. 3A is a graph showing correlation of age with tumor development of KIMAP mice. Percentages of cancer (all histological patterns higher than PIN) in total testing mice incidence were plotted against different age groups .

Fig. 3B is a graph showing correlation of age with tumor development of KIMAP mice. Plotting similar to Fig. 3A was observed for percentage of well differentiated and moderately differentiated CaP (WD +MD CaP) ; Fig. 3C is a graph showing correlation of age with tumor development of KIMAP mice. Plotting similar to Fig. 3A was observed for poorly differentiated CaP (PDCaP) ;

Fig. 3D is a graph showing correlation of age with tumor development of KIMAP mice. Plotting similar to Fig. 3A was observed for PINs (low and high PIN, including high PIN with micro-invasion) .

Fig.4A is a graph showing correlation of Gleason scores in KIMAP mice with age group. Gleason scores distribution in KIMAP correlated with different ages groups .

Fig.4B is a graph showing correlation of Gleason scores in KIMAP mice with age group. Linear correlation of Gleason scores in KIMAP with age. Scatted dots represent the distribution of scores at different ages. Regression line was drawn by the Sigma Plot program. Numbers of mice analyzed in each group are indicated. Error bar: mean + SD .

Fig. 5 is a graph illustrating prostate targeting of SV40 Tag induced tumorigenesis and developments in KIMAP mice. The rate of non- prostate cancer (NPT) incidence (in both males ^• and females) was indicated as percentage of mice showing prostate cancer only, i.e. prostate targeting mice as 100 %. Numbers refer the numbers of NPT mice.

Fig. 6 is a graph evaluating the genomic and phenotypic stabilities in KIMAP mice: In KIMAP mice (n is the numbers of mice tested) , histological grades were compared by percentages in total mice tested for three breeding generations (FI, F2, F3, n = 9, 24, 4 separately) in the same age groups 20-27weeks respectively.

Fig. 7 is a graph evaluating the phenotypic variations of founder (F₀) breeding lines of KIMAP. Only two age groups mice were tested, as they represented the age groups with most frequent tumor incidence. Percentages of cancer incidence (CaP %) in total mice tested were plotted for six independently generated founders of KIMAP. DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES METHODOLOGY

Establishment of knock-in mouse of adenocarcinoma

A targeting vector pPNTloxP was used to construct a PSP94 gene- targeting plasmid (Fong, G. H. , et al. Nature, 376: 66-70, 1995; Peng, J., et al., Proc.Natl.Acad.Sci.U.S.A. , 97 : 8386-8391, 2000). As shown in Fig. 1A, the right and left arms for homologous crossing-over are the 3.842 kb PSP94 promoter/enhancer region and part of exon 1 and the 5.5 kb intron 1 sequences (Xuan, J. W., et al., DNA Cell Biol, 18 : 11- 26, 1999). Following double crossing-over in the mouse genome in the 5' end of the PSP94 gene, the SV40 Tag (2.7 kb, both large T and small t antigens) and the neomycin gene (1.9kb) were inserted at the middle of the first exon (Kpnl site) of the mouse PSP94 gene. The recombinant targeting vector DNA was introduced by electroporation into the embryo stem (ES) cell line Rl. More than 500 G418-resistant individual clones were screened and eight positive ES clones were characterized by Southern blotting. The probe used for PSP94 gene targeting by Southern blotting experiments is a 500 bp (Stul/Kpnl) located downstream from the right arm of the vector and was comprising of the complete exon 2 and the flanking intron area (Fig. 1A) . Fig. IB illustrates the results of Southern blotting, showing the wild type (-12 kb) and mutant (~9 kb for knocked in) genomic fragments. Negative screening of GCV (ganciclovir) for TK (Herpes virus) expression was also performed at the same time. Production of chimeras by aggregation with. 8-cell embryo cells of diploid CD1 strain and implantation into pseudo- pregnant female hosts were performed according to techniques known in the art. From two positive ES clones with PSP94 gene targeting, 20 chimeras (11 male and 9 females) with different fur coat colors were obtained. The chimeras were all tested for germline transmitability by breeding with CD1 and 129 Sv strains, which were screened for germline progenies firstly for black coat color and by PCR genotyping.

PCR- genotyping , Identification and Breeding of Knock-in mice by PCR and Southern Blotting All male chimeras were found to be partly fertile in breeding within the CD1 background. The majority of fertile males produced either no germlines, or the germlines proved not genetically transmittable to the progenies in breeding within either the CDl or 129Sv background. Only two female chimeras produced knock-in mice with mutation of the PSP94 gene transmittable to germlines. The founder line was designated as the germline mouse produced from chimeras mating with CDl or 129Sv with full fertility and with a transmittable mutant genotype to the next generation. Because germline transmittance through female chimeras is rare, some of the founder line mice and their breeding progenies (FI, F2, F3, either heterozygous or homozygous) were further characterized by Southern blotting experiments (Fig. IC) as well as by PCR genotyping (Fig. ID) . Primer pairs used for screening for germline progenies from chimeras by PCR genotyping were: Pr36-Prl6 for wild type genome testing Pr36 5 ' -GGC AAC AGC GTG TCA AAG-3 ' (promoter region near exon 1) and Prl6 5'-CTA GCT CTG TCCAAG GA-3 ' (5' end of intronl) ; Pr36-PrSVTag (5 ' - CTA GCT CTG TCCAAG GA-3', located at the 5 ' end of the SV40Tag region) .

Southern blotting: High molecular weight genomic DNA from Rl embryo stem cell culture and mouse tail and liver was purified as previously reported. Southern blots were prepared using - 5μg mouse tail chromosomal DNA for each lane digested by restriction enzyme and separated in a 0.5 % agarose gel. For KIMAP mice, the probes utilized were the same as for ES cell colony screening.

Mouse anatomy: prostate and non-prostate targeting (NPT) testing The prostate along with the male accessory glands, i.e. the ventral and

. dorsolateral prostate lobes (VP, DLP respectively) , seminal vesicles

(SV) and coagulation gland (CG, or anterior gland) , were dissected out separately as per the description and definition reported (Imasato, Y. , et al., Endocrinol, 242: 2138-2146, 2001). Surgical castration was performed under anesthesia via the scrotal route . All organs and tissue samples, (as indicated in the text) were subjected to gross pathological inspection, and any suspicious or abnormal looking tissue was sampled for immediate histological slide processing. In performing these procedures, non-prostate targeting (NPT) was defined as _ neoplastic changes (the maximum not beyond the PIN) undetectable in the prostate, but detectable in non prostate tissues. Histopathological characterization and definitions of various degrees of Cap in Knock- in mice

Study of tumor development in the knock-in (KIMAP) model was performed. Mice sacrificed at ^' different ages (weeks) were classified into the following five histological patterns: Hyperplasia (Hyp), Low grade PIN (LGPIN) , High grade PIN (HGPIN) (Fig. 2A) , HGPIN with microinvasion (MI) (Fig. 2B, 2H for immunohistochemistry) and adenocarcinoma. The extent and intensity were determined according to standards previously reported (Imasato, Y. , et al., J.Urol., 164 : 1819-1824, 2000).

Gleason grading and Gleason scoring in the histological classification of KIMAP mice were performed as per the standards (Deshmumukh, N. and Foster, C. S. Grading prostate cancer. In C. S. Foster and D. G. Bostwick (eds.), Pathology of the prostate cancer, pp. 191-227. Philadelphia: W.B Saunders, 1998; Mostofi, F. K. , et al., Histological Typing of Prostate Tumours, Second Edition ed. World Health Organiztion, international Histological Classification of Tumours, Springer, 2002) : The architectural patterns observed were assessed by five different grades: Grade 1 (very well-differentiated): single, separate, uniform glands closely packed, with definite edge (Fig. 2C) ; Grade 2 (well-differentiated) : single, separate uniform glands loosely packed, with .irregular edges (Fig. 2D) ; Grade 3 (glands with variable and distorted architecture) : single, separate, uniform scattered glands and smoothly circumscribed papillary/ cribriform masses (Fig. 2E) ; Grade 4 (poorly differentiated) : cribriform masses with ragged and invading edges (Fig. 2F) ; Grade 5: no glandular differentiation, anaplastic tumors growing in non-glandular solid masses of cells (Fig. 2F, G. H, I) . Based on the most prevalent pattern ("the primary pattern") and the second most prevalent pattern ("secondary pattern") , the Gleason score was derived by adding the primary pattern grade number to the secondary grade number (Fig. 2G) . If only one pattern is seen throughout, the Gleason score is derived by the doubling "grade" number. Fig. 2G shows the Gleason score of 9 (4+5) . According to the Gleason score system, adenocarcinoma and carcinoma in KIMAP mice were classified into three histological grading groups: (1) scores 2-4 as well differentiated adenocarcinoma (WDCaP) ; (2) scores 5- 7 as moderately differentiated adenocarcinoma (MDCaP) ; (3) scores 8-10 as poorly differentiated adenocarcinoma (PDCaP) . All ^'histopathological grading was determined by three scientists independently and blind analysis was performed

Immunohistochemistry (IHC) After fixation, samples were washed with 70 % ethanol and embedded in paraffin. Slides of 4 μm thick were then cut, deparaffinized and rehydrated as we previously reported. Monoclonal antibodies against SV40 TAg oncogene (Calbiochem, CA) and a polyclonal antibody against recombinant pTrcHis-mouse PSP94 were used for immunohistochemistry with an ABC kit (StreptABC complex kit, DAKO, Mississauga, Ont.) at 1: loo and 1:400 dilution separately. All IHC slides were counterstained with hematoxylin.

Statistical Analysis Statistical software packages of SPSS (Version 10.0) and Sigma Plot

2000 (Version6.1, SPSS Scientific, Chicago, IL) were used for statistical analysis and production of the graphs. Based on the normality of data (by a Kolmogorov Smirnov Z test, SPSS) , one way ANOVA test of variances was used for statistical analysis. Pearson's test (SPSS) was used for linear correlation analysis.

RESULTS

Evaluation of CaP development in Knock in KIMAP model KIMAP mice were first characterized for the correlation of tumorigenesis and CaP development with age (7-52 weeks, Fig. 3A-D) . According to the standard of histological patterns as previously reported (Greenberg, N. M. , et al., Proc.Natl.Acad.Sci.U.S. A. , 92 : 3439-3443, 199S ;Mostofi, F. K. , Sesterhenn, I. A., and Davis, C. J. E. Histological Typing of Prostate Tumours, Second Edition ed. World Health Organization, international Histological Classification of Tumours, Springer, 2002) , percentages (% of mice analyzed) of categories of cancer (CaP, Fig. 3A) , well and moderately differentiate CaP (WD+MD CaP, Fig 3B) , poorly differentiated CaP (PDCaP, Fig. 3C) and PINs (including high grade PIN with microinvasion, Fig. 3D) were determined. Fig. 3 shows a steadily prolonged cancer development in KIMAP mice which correlated with age. Majority (50/64) of CaP in KIMAP were well and moderately differentiated CaP in almost all age groups (80-100 %, Fig. 3B) . CaP incidences in KIMAP mice were shown to be synchronous as evidenced by: (1) High rate in KIMAP (Fig. 3A) ; (2) in KIMAP mice, PINs (Fig. 3D) and poorly differentiated Cei ₍pig. 3C) were found only at the early, and later (after 37-50 wee_j-_{s) age} groups separately (for a summary see Table 2) .

Since the process of ^' adenocarcinoma development in KIMAP tt_tice is steadily prolonged, and extended multiple foci of the adenoc£s._rc;i__noma were observed (shown in Fig. 2G) , the cancer architecture in Klli^ _mi_ce was further characterized by the Gleason grading system and sul_._seq_Uent Gleason scoring. By age group of 12 to 19 weeks, the Gleason _SCOres 2, 5 and 6 were detected at 37 % (3/8), 12 % (1/8) and 50 % ₍ /₈₎ separately (Fig. 4A) . ^' By age group 24 to 27 weeks, Gleason grade ₃ increased to 80 % (8/10) . By age group 28 to 31 weeks, Gleason g_ra_e 4 was detected at approximately 25 % (3/12) . By age of 52 e =]^_{s a}]_]_ mice (n=5, Fig. 3A and 4A) synchronously developed visible cancer (grade 4-5) in the lateral prostate lobe. Fig. 2 G, H, I showe_d. _soi d tumor mass, typical comedocacinoma structure (Grade 5, Fig. 2 ij_{) ancj} ragged infiltration to the stromal tissues.

Fig. 4B shows a linear correlation of Gleason scores with age i^ K_IMAP mice, and a correlation coefficient of 0.71 (P<0.01, by Pearson's correlation test) . The average Gleason grade is at 3-4 arid the subsequently average Gleason scores were at 5-7 (Table 1) , which. i_s the same average range as in humans .

The KIMAP mice (n>252) were monitored for 15 months and no non- rogt te targeting was observed (Fig. 5) .

Genotypic and phenotypic stability

Genotype and phenotype stability were evaluated with regard to e cop_y number, rate of CaP incidence and tumor development in at least three breeding generations in KIMAP mice. Fig. 6 shows that three generations (F1-F3) of KIMAP mice followed the same tumor devei₀p_ment pattern. This phenotypic stability was also maintained in other strains CDl and 129Sv (data not shown) . Fig. 1 C also shows the knock- in genotype remained stable for targeting at the PSP94 gene.

The founder line of knock-in mice was designed as a fertile germii_ne of mice with a transmittable mutant genotype . Most of the mutant phenotypes were tested in breeding progenies with a CDl background. As shown in Fig. 7, six breeding founder lines of KIMAP were tested and all showed a similar tumor development. In the process of KIMAP mouse breeding, along with their three generations of progenies, no variation (P> 0.05) of tumor development or growth rate amongst various families was observed and separated.

We report herein the first knock-in mouse prostate cancer model resulting from a single endogenous single mutation under the control of a prostate specific gene promoter / enhancer of PSP94. One of the unique features of this KIMAP model is the applicability of the Gleason histological grading and scoring system, a system widely used clinically in grading prostate cancer. The most prevalent range of Gleason grades (3-4) and Gleason scores (5-7) were the same in KIMAP mice as in human CaP cases, and a linear correlation of Gleason grades and scores with the age of the animal was observed.

The Gleason grading system has been advocated as a way to improve the pathologist' s ability to accurately predict the biological behavior of a particular tumor, as this system seems to deliberately recapitulate every single step of CaP developments specific to the human situation. To establish such an experimental paradigm has proven to be difficult, since none of the previous models have demonstrated eatures , which would qualify for classification by the Gleason grading system.

The KIMAP model has many advantages over previous models. The KIMAP model has been shown to be stable and to have no non-specific prostate targeting. Furthermore, the insertion site of the heterologous gene (insert) is predictable and the copy number of the insert is also controllable. A steadily prolonged tumor growth starting after puberty in all breeding lines in KIMAP mice is the most notable feature. This feature permits the application of Gleason grading system.

The KIMAP revealed fundamental similarity in the histopathological characteristics as well as underlying molecular pathways. The simplest theoretical explanation for the different histopathological characteristic is that none of the previously described transgenic CaP models is fully regulatable as a result of an endogenous mutation by a prostate specific gene. The knocked insertion at the PSP94 gene endorses KIMAP model as being a highly regulatory CaP model, as it only knocks in a SV40 Tag in the PSP94 structure gene and no regulatory region (cis, trans) will be affected. The PSP94 gene promoter is a strong promoter, and the SV40 Tag is coupled with the full capacit_y a promoter of the most abundant prostate secretory gene.

We have observed PSP94 suppression in the early puberty in heterozy-, KIMAP mice while SV40 expressed was initiated, and this suppression _was maintained at a low, but not completely shutting down level _Qf expression by the PSP94 promoter/enhancer (data not shown) . The s-^ _4Q Tag induced tumorigenesis of the prostate gland along with _t^ secretion of PSP94 protein, starting from puberty (5-7 weeks of a.__je) The SV40 Tag expression is in turn controlled by the prostate, s _nce when normal prostate function is disturbed by tumor growth, SV40 _TAq expression will be suppressed by the PSP94 promoter/enhancer reg_j__on We reported this apparent suppressive mechanism of PSP94 in human prostate cancer biopsy samples (Imasato, Y. , et al., Endocrinol, _j.₄2 - 2138-2146, 2001; Imasato, Y. , et al., J.Urol., 164 : 1819-1824=, 2oo_θ) and this suppression was also observed with another abundant secretor_y protein, i.e., PSA (Stege, R., et al., Clin. Cancer Res., 6: _.60~_]_₆₅ 2000.). The balance of positive (tumorigenesis) and negative ₍the suppression) regulatory effects to the SV40 Tag structural gene ena.hι_es the tumor to remain stable for most of the post-puberty period.

The KIMAP model is a highly predictable model. Although tumor genes_¬ is a stochastic event inside the acini of the gland, statisticall_y significant majority of KIMAP mice undergo steady cancer growth af er puberty. The CaP in KIMAP mice show some desirable features, such _as . (1) a homogenous and synchronous growth (Fig. 3, 4 and 6) ; (2) _]__ow variation and high stability of both phenotype and genotype in _aτ-ι breeding lines and in progenies of several generations; (3) selective prostate targeting with no incidence of non-prostate tumor inducti_on (4) KIMAP is an invasive cancer model, since it showed early occurr_ence and a high incidence of PIN with microinvasion. A highly invasi_Ve human CaP specific form of comedocarcinoma was also identified in Kli^ mice of 50 weeks age. We have characterized that KIMAP mice _are responsive to castration (androgen deprivation, data not shown) .

It is interesting that transmittance of PSP-KIMAP mutant introduced _]-_y gene targeting in embryo stem cell culture was through female c imeras In addition to this evidence of infertility of male chimeras, _we observed that the depressed PSP94 expression in homozygous KIMAP _tni_ce resulted in the imbalance gender distribution in the progen._es (unpublished data) . Since the primary biological function of PSP94 is still .unknown, we may postulate that the original function of PSP94 is related to male fertility.^' PSP94 was hypothesized as a prostatic inhibin protein (PIP) to β-FSH (Follicle stimulation hormone) , although this hypothesis is highly contentious . In the present experiment on the knock- in of PSP94 function, we observed no support for a "PIP" theory: (l)We observed a close correlation with SV40Tag expression with tumorigenesis and progression (Fig. 2J) ; (2) In KIMAP mice, we observed suppression of PSP94 expression, which is the result of disrupting normal PSP94 secretion function by CaP as with all other secretory proteins from the prostate, but not vice versa; (3) We observed no significant differences in CaP development between homozygous and heterozygous KIMAP mice, as in heterozygous, PSP94 expression level should exist at a certain level comparable to the wild type mice. Further experiments are required to clarify the real biological function of PSP94, one of the most abundant proteins from the human semen. Table 3 summarizes the advantages and characteristics of the KIMAP mouse model.

Table 2

Table 3

Gene Profiling

The basic concept behind the use of GeneChip arrays (e.g. ,Affymetrix, Santa-Clara, California) for gene expression may be performed as follows: labeled cDNA or cRNA targets derived from the mRNA of an experimental sample are hybridized to nucleic acid probes attached to the solid support. By monitoring the amount of label associated with each DNA location, it is possible to infer the abundance of each mRNA species represented. Although hybridization has been used for decades to detect and quantify nucleic acids, the combination of the miniaturization of the technology and the large and growing amounts of sequence information, have enormously expanded the scale at which gene expression can be studied.

Results of gene profiling indicate that the KIMAP mouse model show an increase in the expression of genes which are well studied clinically for prostate cancer. These includes, B-cell linker (BLNK) which is part of the T-cell activation family, PDZ domain containing 1 (Pdzkl) which is part of the cell communication family, Prostate specific ets transcription factor and serine proteinase inhibitor-clade B-member 5 (Serpin or MASPIN) , which are part of the cell growth, motility and signal transduction family and transmembrane protease, serine 2 (Hepsin) which is part of the metabolism, cellular biological process family. Expression of these genes is not associated with current transgenic prostate cancer models. The KIMAP model represents therefore a significant improvement over current models. In fact, this model shows a good correlation with CaP clinical data observed in humans as illustrated in Table 4 below.

Table .

KIMAP model CaP in humans

Kinetics of tumor development Slow Slow

Well and moderately Majority Majority differentiated CaP

Poorly

Minority Minority differentiated CaP

Heterogeneity High High

Multifocality (tumor extent) High High

Correlation with age Correlation Correlation Lymph node metastasis Late stage, >80% 2-5% Neuroendocrine carcinoma Rare Rare DNA ploidy by flow cytometry Diploid (%) 89 75 Nondiploid (%) 11 25 DNA proliferation by flow cytometry %S phase 3.6 3.1

%S phase + G2M phase 10.7 10.5 Molecular profiling Tumor markers : PSP94 (serum) Exist Exist Spermine binding protein Exist Exist Hepsin/maspin Exist Exist Mucin 10 Lost Lost I muno -defense system Active Active

Claims

Claims :

1. A DNA construct comprising a) a first PSP94 gene segment, b) an insert and; ^' c) a second PSP94 gene segment, said first and second PSP94 gene segments being different and said insert being located between the first and second PSP94 gene segment.

2. The DNA construct of claim 1, wherein said insert is a gene capable of initiating tumor formation.

3. The DNA construct of claim 1, wherein said insert is selected from the group consisting of a gene encoding a therapeutic protein and a gene encoding a reporter protein.

4. The DNA construct of claim 1, wherein said insert is selected from the group consisting of a gene able to be transcribed into a polynucleotide selected from the group consisting of an antisense RNA and a ribozyme, said polynucleotide targeting a gene capable of initiating tumor formation.

5. The DNA construct of claim 2, wherein said gene capable of initiating tumor formation is a SV40 T antigen.

6. The DNA construct of claim 5, wherein said SV40 T antigen is selected from the group consisting of the SV40 large T antigen, the SV40 small t antigen and combination thereof.

7. The DNA construct of claim 3 , wherein said therapeutic protein is selected from the group consisting of a cytotoxic protein, a protein causing apoptosis, an anti-oncoprotein, a protease, a suicide protein, a cytokine, a chemokine, a costimulatory . molecule and an antigen.

8. The DNA construct of claim 7, wherein said suicide protein is selected from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase.

9. The DNA construct of claim 7, wherein said cytotoxic protein is selected from the group consisting of the A chain of diphteria toxin, ricin, and abrin.

10. The DNA construct of claim 7, wherein said protein causing apoptosis is selected from the group consisting of caspases, Fas-Ligand, Bax and TRAIL.

11. The DNA construct of claim 7, wherein said anti- oncoprotein is selected from the group consisting of p53, p21, and Rb.

12. The DNA construct of claim 7, wherein said protease is selected from the group consisting of awsin, papain, proteinase K, and carboxypeptidase .

13. The DNA construct of claim 7, wherein said cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, GM-CSF, G-CSF, M-CSF, IFN-alpha, IFN-beta, IFN-gamma, TNF- alpha, and TNF-beta.

14. The DNA construct of claim 7, wherein said chemokine is selected from the group consisting of Mig-lalpha, Mig-lbeta, IP-10, and MCP-1.

15. The DNA construct of claim 3, wherein said reporter protein is selected from the group consisting of beta- galactosidase, luciferase, red fluorescent protein, green fluorescent protein, alkaline phosphatase, chloramphenicol acetyl transferase, and horseradish peroxidase.

16. The DNA construct of claim 1 wherein said first PSP94 gene segment comprises at least a part of the promoter/enhancer region and wherein said second PSP94 gene segment comprises at least a part of the PSP94 gene exon/intron region.

17. An isolated cell having incorporated the DNA construct of claim 1.

I 18. A transgenic non-human mammal susceptible to prostate tumor formation, having genomically-integrated in non-human mammal ceils, an insert inside the PSP94 gene exon/intron region.

19. The transgenic non-human mammal of claim 18, wherein said insert is selected from the group consisting of a gene capable of initiating tumor formation, a reporter gene, a gene encoding a therapeutic protein, a gene able to be transcribed into a polynucleotide selected from the group consisting of an antisense RNA and a ribozyme said polynucleotide targeting a gene capable of initiating tumor formation.

20. The transgenic non-human mammal of claim 19, wherein said gene capable of initiating tumor formation is a SV40 T antigen.

21. The transgenic non-human mammal of claim 20, wherein said SV40 T antigen is selected from the group consisting of the SV40 large T antigen, the SV40 small t antigen and combination thereof .

22. The transgenic non-human mammal of claim 19, wherein said therapeutic protein is selected from the group consisting of a cytotoxic protein, a protein causing apoptosis, an anti- oncoprotein, a protease, a suicide protein, a cytokine, a chemokine, a costimulatory molecule and an antigen.

23. The transgenic non-human mammal of claim 22, wherein said suicide protein is selected from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase .

24. The transgenic non-human mammal of claim 22, wherein said cytotoxic protein is selected from the group consisting of the A chain of diphteria toxin, ricin, and abrin.

25. The transgenic non-human mammal of claim 22, wherein said protein causing apoptosis is selected from the group consisting of caspases, Fas-Ligand, Bax and TRAIL.

26. The transgenic non-human mammal of claim 22, wherein said anti-oncoprotein is selected from the group consisting of p53, p21, and Rb.

1?

27.. The transgenic non-human mammal of claim 22, wherein said protease is selected from the group consisting of awsin, papain, proteinase K, and carboxypeptidase.

28. The transgenic non-human mammal of claim 22, wherein said cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, GM-CSF, G-CSF, M-CSF, IFN-alpha, IFN-beta, IFN- gamma, TNF-alpha, and TNF-beta.

29.- The transgenic non-human mammal of claim 22, wherein said chemokine is selected from the group consisting of Mig-lalpha, Mig-lbeta, IP-10, and MCP-1.

30. The transgenic non-human mammal of claim 22, wherein said reporter protein is selected from the group consisting of heta- galactosidase, * luciferase, red fluorescent protein, . green fluorescent protein, alkaline phosphatase, chloramphenicol acetyl transferase, and horseradish peroxidase.

31. A transgenic non-human mammal, susceptible to prostate tumor formation, having genomically-integrated in non-human mammal cells, an insert replacing at least a part of the PSP94 gene exon/intron region.

32. The transgenic non-human mammal of claim 31 wherein said insert is selected from the group consisting of a gene capable of initiating tumor formation, a reporter gene, a gene encoding a therapeutic protein, a gene able to be transcribed into a polynucleotide selected from the group consisting of an antisense RNA and a ribozyme said polynucleotide targeting a gene capable of initiating tumor formation.

33. The transgenic non-human mammal of claim 32, wherein said gene capable of initiating tumor formation is a SV40 T antigen.

34. The transgenic non-human mammal of claim 33, wherein said SV40 T antigen is selected from the group consisting of the SV40 large T antigen, the SV40 small t antigen and combination thereof .

35. The transgenic non-human mammal of claim 32, wherein said therapeutic protein is selected from the group consisting of a cytotoxic protein, a protein causing apoptosis, an anti- oncoprotein, a protease, a suicide protein, a cytokine, a chemokine, a costimulatory molecule and an antigen.

36. The transgenic non-human- mammal of claim 35, wherein said suicide protein is selected from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase.

37. ^' The transgenic non-human mammal of claim 35, wherein said cytotoxic protein is selected from the group consisting of the A chain of diphteria toxin, ricin, and abrin.

38. The transgenic non-human mammal of claim 35, wherein said protein causing apoptosis is selected from the group consisting of caspases, Fas-Ligand, Bax and TRAIL.

39. The transgenic non-human mammal of claim 35, wherein said anti-oncoprotein is selected from the group consisting of p53, p21, and Rb.

40. The transgenic non-human mammal of claim 35, wherein said protease is selected from the group consisting of awsin, papain, proteinase K, and carboxypeptidase .

41. The transgenic non-human mammal of claim 35, wherein said cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, GM-CSF, G-CSF, M-CSF, IFN-alpha, IFN-beta, IFN- gamma, TNF-alpha, and TNF-beta.

42. The transgenic non-human mammal of claim 35, wherein said chemokine is selected from the group consisting of Mig-lalpha, Mig-lbeta, IP-10, and MCP-1.

43. The transgenic non-human^' mammal of claim 34, wherein said reporter protein is selected from the group consisting of beta- galactosidase, luciferase, red fluorescent protein, green fluorescent protein, alkaline phosphatase, chloramphenicol acetyl transferase, and horseradish peroxidase.

44. The transgenic non-human mammal of claims 18 or 31, wherein said transgenic non-human mammal is a transgenic mouse .

45. The transgenic non-human mammal of any one of claims 18 or 31, wherein prostate tumor development is monitored using a Gleason grading system.

46. The transgenic non-humaή mammal of any one of claims 18 or 31, wherein the kinetic of prostate tumor development substantially correlates with the kinetic of prostate tumor development in a human.

47. The transgenic non-human mammal of any one of claims 18 or 31, wherein a tumor marker selected from the group consisting of Hepsin, Mapsin and spermine binding protein is expressed.

48. The use of the transgenic non-human mammal of claims 18 or 31 for evaluating the efficacy of a drug candidate in inhibiting the growth of prostate related neoplasia.