CA2411729A1 - Knock-in mouse prostate cancer model - Google Patents

Abstract

A number of transgenic mouse adenocarcinoma prostate cancer models 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

TITLE: KNOCK-IN MOUSE PROSTATE CANCER MODEL
S FIELD OF THE INVENTION
'Phis invention relates to DNA constructs, isolated cells and transgenic non-human mammals te.g., animals) carrying such construct. More particularly, transgenic mice with a hetero~ogous gene inserted inside the PSP94 gene exon/intron region were gener~,teci herein.
BACKGROUND OF THE INVENTION
IS Prostate cancer (CaP) is the must 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 histopat~hological clinical categories.
Currently there are several animal models of prostate cancer development, 2S including a spontaneous canine prostatir adenocarcinoma model, rat Noble prostate cancer models (Leav, I., et al., .,l,Natl.Cancer Inst., 80: 1045-1053, 1988), a prostate organ reconstitute model (Thompson, T.et al., Urol Oncol, 2: 99-128, 1996), and transgeriic rnice models (Spence, Scheppard, P. C., et al., Proc.Natl.Acad.Sci.U.S.A., 86: 7843-784'7, ~~989; Gingrich, J. R., et al., Cancer Res, 56: 4096-41.0'~~, 1996; G-eenberg, N. M., et al., Proc.Natl.Acad.Sci.U.S.A., 9~: :3439-3443, 1995; Green, J. E., et al., Prostate, 36: 59-63, 1998; ~h:ibata, M. r~., et al., EMBO J., 18: 2692-2701, 1999; Rodriguez, R., et al., Cancer Res, ~7: 2559-2563, 1997; Wei, C., et al., Proc.Natl.Acad.Sci.U.S..A., 94: 6369-6374, 1997; Perez-Stable, C., et 3S al., Cancer Res, 57: 900-906, 1997;Garabedian, E. M., et al., Proc.Natl.ACad.Sci.U.~.A., 5<': 227-237, 1998) for review see (DiGiovanni, J., et al., Proc.Natl.Acad.::~ci.tl.S.A., 9?: 3455-3460, 2000;Watabe, T., et al., Proc.Natl.Acad.Sci.U.S.la., 99: 407-6., 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 o.f transgenic mice. These include regulatory elements dervved from t:he rat prostate steroid-binding protein (PSBP or C3(1)), the hurnan prostatic specific antigen (PSA), the human foetal Gy globin/SV40Tag, Cryptdin-2 (CR-2)/SV40'rag, bovine keratin 5 promoter (BK5) promoter/insul.irz growth factor 1., prostate stem cell 2.ntigen (PSCA) and an osteoca~..c:in--based co-t:argeting vector (Matsubara, S., et al., Cancer Res, 61: 6012--9., 2001). However most of these CaP
models also leads to targeting of non-prostate tissues. Currently, only one transgenic model has been widely util~~zeci as a mouse CaP model. The t:ransgenic adenocarcinoma mouse prostate (TRAMP) and the LPB-Tag (Kasper, et al., Lab Invest, 78: 319-333, 1998; Masumori, N., et al., Cancer Res, 61: 2239-2249, 2001) models are bot~u 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-clinic<31 histological grading system, which has not gained widespread clinical acceptance and has not shown to be reproducible or prognostic:ally reliable in clinical practice. All have limitations in providing the whole gamut of reterogene~.ty 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 t.ransgenic technique is very empirical. By utilizing a s:hcirt DNA unit to obtain forced exogenous gene expression, only a limited .length of transgenic DNA could be tested, and it is often affected by insert.i.on site: and the copy number of transgene.
Moreover, there is a requirement for breeding and selection of founder lines.
In view of the limitations of t=he transgenic technique, alternative models have been sought, utilizing knock-out t~wchnique and newly discovered. genes.
A number of knock-out mice ~~~ith a tumor Auppressor gene PTEN/Mmac1 were generated. Invariably the resultant hoznc~zyc~ous 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., Prc>c.Natl. Acad. Sci. tJ.S.A., 96: 1563-8., 1999;

I~i Cristofano, A., et al.., Nat. Genet., 27: 2,22-4., 2001). Mice lacking Irtxil mice (belonging to Myc oncoproteins) exhibited progressive, multisystem (kidney, spleen, prostate) abnormalities, and had increased ;susceptibility to t:umorigenes.is 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 i.ncludincr the seminal vesicles, although the prostate was normal (Miyamc:~to, N., et: al-. '~~evelopment, 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, t:he knock-out prostate cancer models established so far have rarely developed prostate carcinoma (review see: Abate--Shen C et al., Trends Genet:., I8: 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 c'~ose resemblance t.o clinical histopathological features observed in humans. "uVe demonstrated that the PSP-KIMAP model behaves as an endogenous mutation (cancer developmenr_) model and can be used as a standard mouse proat:ate 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 cance:c with si.milarit:i.es to the development of CaP
observed in humans.

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A number of transgenic mouse adenocarcino:ma prostate cancer models have been established. However, a',.1. previous models use a histological grading system which is not clinically applicable. In view of these limitations a knock-in mouse adenocarcinoma prostate model. (KINIAP) 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 prognosi:> of r~uman prostate cancer. Our evaluation indicates that the :r~INIAP model. meets requirements for a new standard murine model for both basic and clinical studies of prostate cancer. CaP in th~a KIMAP model. showed the same prevalent range of Gleason grades and scores as those observed in huma:: prostate cancer cases.
In accordance with one aspect, t=he present invention relates to a (isolated) DNA construct comprising a) a first PSP94 gene segment, b) an inser t 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 (p:romoter) and that: is able ~o cix-ive the expression of a protein, for example, a protein encoded by an insert. The DNA construct may comprise sequence facilitat=ng recorcu~ination (a gene targeting plasmid). The DNA construct may also comprises sequence enabeling selection of cells having incorporated (ca.rrying) such DNA construct. These sequence may include for example the neomycin gems.
In accordance with the present invention, the insert may be selected, for example, from the group consisting of a gene (expressing/encoding a protein) capable of initiating tumor formation, a reporter gene, a c_~ene encoding a therapeutic protein, a gene able t:o be transcribed into a polynucleotide selected from the group COIISl:itlng of an antisense RPdA and a ribozyme said polynacleotide targeting a gene (expressing/encoding a protein) capable of initiating tumor formation or any other suitable gene having a desired effect.
Lt is to be understood herein that a spec:if_ic gene encoding a protein may fall in more that one of the car_egory lis=ed in the above mentioned group.
Further in accordance with the' present invention, the gene capable of initiating tumor formation mar be, for example, a SV4U 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.
i~ gene capable of initiating (promoting, inducing) tumor formation may be, Eor example, an onc:ogene. Any oncogene or effective sequence thereof can be used to produce the transgenic mouse of the invention. Table 1 be7_ow, Lists some known viral .=_~nd cei:l.ular oncogenes, nuany of which are homo7_ogous to DNA sequence endogenous to mice and/or humans, as indicated. The germ "oncogene" encompasses both the viral sequence: 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 tv.mor development and metastasis in a transgenic non-human mammal.
In accordance with the present invention; the therapeutic protein may be selected, for example, from t.he> group consisting of a cytotoxic protein, a protein causing apoptosis, an anti--oncop:.otein, a protease, a suicide protein, a cytokine, a chemokine, a cost:imulatory 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, 3S luciferase, red fluorescent i;~x-otein, green fluorescent protein, alkaline phosphatase, chloramphenicol acetyl transferase, and horseradish peroxidase.
In accordance with the present invention, the first PSP94 gene segment may 4~ comprise at least a part of hie promotex/enhanc:er 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 :.ncorporated (carrying) the DNA construct mentioned herein.
'Che cell having incorporated (carrying) (incorporated means that the DNA
r_onstruct is inside the cells, either integrated in the genome or not) the 'ANA construct described herein are generated using techniques (e. g., micro-IO injection, recombination vectors, etc.) k.riown ire the art.
In yet another asps~ct, the present invention provides a transgenic non-human mammal susceptible to prostate tumor f.orir.ation (susceptible to prostate neoplasia), having gea:tomically-integrated in a non-human mammal cell(s), an insert (located) inside the I?SP94 gene exon/intron region.
It is also to be understood herein that a suitable cell or suitable non-human mammal used to generate: ~~ cell. having an integrated insert or to generate a transgenic non-human mammal is one having in Its genome, at least part of a PSP94 gene. The presence oY the PSP94 gene (in the cell or non-human mammal genome) may be necessary for the recombination event to occur.
It is to be under~;tood herein that, the insert may or may not functionally inactivate the PSP94 gene, fox' example the presence of the insert inside the PSP94 gene exon/int;ron 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 t.o produce a deleted (shorter) form of the protein. On the other hand, t:he presence o~. the insert may lead to the production of a fusion proteii:a comprising f~lnP insert and the whole or some parts of the PSP94 protein. 1:n some circumstances, it may be useful to totally inhibit the expression of the PSP94 protein while in other circumstances it may be useful. to prese~~:ve its expression and/or generate fusion proteins. Using a DNA construct as de.>cribed herein (having an insert between two PSP94 gene segments) to may allow the fusion of a tag (e.g., fluorescent) to PSP 99 (either i;~ the 5' end or 3' end or in the middle of the protein) to follow the localisation of PSP94 in a non-human mammal.

One of the object of the presnent: invention beir_g that the insert is preferably under the control of a functional PSP94 promoter (or a chi.meric PSP94 promoter), it is p:referab:l.e that the insert being integrated outside of the promoter region of the PSP94 gene. It. may be integrated, for $ example, at the jun~~tion of the promoter and exoniintron region or inside the exon/intron region.
A transgenic non-human mammal may be a hornozygot.e, a heterozygote, a non-human mammal of a founder line, breeding __ine, and subsequent generations.
A. transgenic non-hu::nan mammal al.:~o ,includes a nc>n--human mammal before its delivery (before its birth) and at different development stage In accordance with the present invention, the insert (genomically integrated in a non-human mamrraa:l cell(s)) ma,~ be selected, for example, 1$ from the group consisting of a gene (expreessingiencoding a protein) capable cf initiating tumor formation, ::l reporter gene, a gene encoding a therapeutic protein, a gene able r_o be transcribed into a polynucleotide selected from the group consisting of an <inti.sense RNA and a ribozyme said polynucleotide targeting a gene (expressing/encodi.ng a protein) capable of 2~ initiating tumor formation or any ot=her suitable cJene having a desired effect.
In a further aspect, the present :invention relates to a t:ransgenic non-r.uman mammal, suscepti.bl.e to prostate tumor f_ormati.on (susceptible to 25 prostate neoplasia) , having ge~nomically-imtegrat:ed in non.--human mammal cells, an insert replacing at least a par= of the PSP94 gene exon/intron region.
I:n accordance with the present: invention she inse:rt (genomically integrated 30 i.n a non-human mammal cell(s)) may be selected from the group consisting of a gene (expressing a protein) capable of initiating tumor formation, a reporter gene, a gene encoding a therapeutic protein, a gene able to be transcribed into a polynucleoti.de selected from the group consisting of an antisense RNA and a ribozyme said polynucleotice targeting a gene capable 35 of initiating tumor formation.
7.n yet a further aspect, the present invention relates to the use of the t:ransgenic non-human mammal described herein for evaluating the efficacy of drug candidates in inhibiting (partial or total, /decreasing) growth of g prostate related neoplas:i.a (e. g., prostate cancer at different stages, p:rostatic adenocarcinoma, benign prostate hyperplasia (BPH), etc.).
Without being limited to the fo:l.lowing, a suicide protein may be selected, S for example, from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia cali cytcasine 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,. ric:in, and abrin or any other desired cytotoxic protein. A proteir causing apr~pt.osis may be selected,, for 1~ 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 de:si.red anti--onc:opx~atein. A protease may be selected, for example, from the group con:~i.s=-ing of awsin, papain, 15 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 st:'~mmlatinc~ factor), G-CSF', M-CSF, IFN-alpha, IFN-beta, IF:N-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-lbeta, IP-10, and MCP-1 or any other designed chemokine.
I:n accordance with the present invention, the transgenic non-human mammal 2S 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 beincl :Limited to a dog, a cat., a monkey (of any desired species) , a sheep, a c::ov~, a pig, a horse, 30 Unless otherwise indicated, tine recombinant DNA techniques utilized ir_ the present invention are standard procedures, known; to those skilled in the <~rt. Example of such technique's are explained in the literature in sc>urces such as J. Perbal, A Practical. Guide to Molecu7-,ar Cloning, John Wiley and :ions (1984), J. Sambrook et al ., Molecular Cloning: A Laboratory Manual, 35 Cold Spring Harbor Laboratory Press (1989 ) , '1'.J~. Brovm (editor) , Essential I~IOlecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (7.991), :7.M. Glover and B.D. Hames (edi.tors), DNA Ci.on_~ng: A Practical Approach, 'Jolumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols >n 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
$ constructs) can be made from .~:nown DNA sequence. by deleting or recovering some DNA fragments using restriction endoruclease (i.e., enzyme) that will specifically recognize a DNA sequence comprised ir_ the desired gene or DNA
sequence. For example, using a KpnI (i.e., K) a_nzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GGTACC-3' (coding sequence shown only)) wi-1'. 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 condi-tions. When using BamHI
(.i.e., B) enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GGA'rC:'-3' (coding sequence shown only)) will be cut using suitable conczitions. Where using Bg.iII (i.e>., Bg) er.;zyme, any double stranded DNA compri:.;ing the sequencE: recognized by this enzyme (i.e., 5'--AGATCT-3' (coding secxuence shown only)1 will be cut using suitable conditions. When using EcoRV enzyme, any double stranded DNA
comprising the sequence recognized by this enz~,rme (i.e., 5'-GATATC-3' (coding sequence shown only)) will be cut. using suitable conditions (i.e., suitable buffer, temperature and volume descriued by the manufacturer).
When using PstI (i.e., P) enzy~ue, any double stranded DNA comprising the sequence recognized by this enzyrne (i.e.: 5'-CTGCAG-3' (coding sequence shown only)) will be cut using suitable r_onditi.ans. When using SalI
enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-GTCGAC-3' (wading sequence shoum only)) will be cut using suitable conditions. When using StuI enzyme, any double stranded DNA
comprising the sequence reco<~nized by this enzyme (i.e., 5'-AGGCCT-3' (coding sequence shown only)) will be cut using suitable conditions. When using XbaI enzyme, any doubly stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-TCTF.GA--3' (coding sequence shown only)) will be cut using suitable conditions. Whir. using HincII enzyme, any double stranded DiJA 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. Vdhen using ~:laI enzyme, any double stranded DNA comprising the sequence recognized by this enzyme (i.e., 5'-ATCGAT-3' (coding sequence shown only)) wil:L be cut using suitable conditions. When using EcoRI enzyme, any double stranded DNA comprising the sequence 4U recognized by this enzyme (i..e., '~'-GAA'rTC--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'-p:yrimidine-ACGT-purine-3° (coding sequences 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 Eiiolab, Pharmac.ia).
Those skilled in molecular cloning will also know that following digestion with restriction enzymes, the desired DNA may be: ligat.ed, for example, into a. linearized plasmid (i.e., vector, DNA construc:tl or to another linear DNA
molecule having matching ends. .!~lternativc>ly, following digestion with restriction enzymes, the cohesive ends of DNA may be transformed to blunt 1$ ends using any suitable DNA Pc:>Lymerase (e.g., T4 DNA Polymerase) or any suitable Nucleases (e.g., Mung Bean Nuclease) axed the desired DNA may be '_igated, for example, into a linearized plasmid (i.e., vector) or to another linear DNA molecule having suitable ends (e. g. b:Lunt ends). Ligases (e. g., T4 DNA Liga~~e) will catalyze the fornuati.on 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 Dh-A, RNA, or DNA/RNA hybrids.
In order for ligat:ion 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 examp~.e py using Polynucleotide kinase.
Suitable Polynucleotide kinase (e. d., T4 Poiynuleotiude k:i.nase) will catalyze (when used in suitable conditions described by the manufacturer) the transfer and exchange of P. (i.e., inorgaic phosphorus) from the game position of ATP (i.e., adenosine triphosphate) to the 5' hydroxyl terminus of polynucleotides (double- a.n,~i single-strandec'i DNA and RNA) and nucleoside 3' -monophosphates.
3$ Following ligation, DNA may be transformed in bacteria for amplification, and may be purified from lyse>d 'bacteria. Following purification, the DNA
construct (linearized or not) may be transferred (e. g. t:ransfected, transformed, electroporated, micro-injected, li.pofected etc.) into a desired host (e.g., a eukaryotic cell, a oocyte, an embryonic cell, a bacteria, a yeast, etc.) P,s used herein the expression "DNi~ construct" includes without limitation;
~. vector, a plasmid (e.g. , lir:cearizc=_d or uot) and a DNA f ragment that can be used to transfer DNA sequences from one organism to another.
F,s used herein the expression "PSP9~~ gene" relates to coding and non-coding regions of said gene.
F,s used herein, the expression, "vector" refers t:o an autonomously 1~ replicating DNA or RNA molecule into wh:icli foreign DNA or RNA fragments are inserted and then propagated i.n a host cell for either expression or 2.mplification of the foreign a>NA or RNA molecule. The term "vector"
comprises and is not limited t:o a p:lasmid, (e.g., linearized or not), or a DNA construct that can be used to transfer DNA ;sequences from one organism 1$ t.o another. The term "vector" includes viral and non-viral vector. Viral vetors may be derived, f:or exa.m~.'~e, from ~x retrov;:rus, a herpes virus, an a.denovirus, an adeno-associated virus, Sindbis virus, poxvirus. Non-viral vector includes, but area not limited to, hacteri_a:L plasmids.
P,s used herein the term "trans.gene" refer: to a DNA construct (e.g., DNA
fragment) that has been incorporated into the genome of an organism.
P.s used herein the expression "operativel~~ linked" refers to two or more distinguishable DNA sequences of a transgf>ne whi.cln are linked according to 2$ recombinant technology technic:~ues so that they may act. together to control and express a protein encoded R_'VA in a suitable tissue or cell type. An example would be the operativEl-y :Linking of. a px.-omoter/tissue-specific enhancer to a DNA sequence coc~i:lg for the desired prot:ein(s) so as to L~ermit and control expression of the DNA :_equenc:e and the production of the 30 encoded protein(s).
P.s used herein the "PSP94 genEexon/intron regior_" relates to the transcriptional region of the PSP94 gene, i.e., the region of the gene ~~hich is transcribed into a mP:NA (mRNA precursor, i..e., comprising the 3$ corresponding exon/intron RNA sequence) tl:~is exonfintron region being distinct from the "regulatory region" or promoter region.
P,s used herein the term "regulatory region(s1" refers to region having an effect on the transcript:ional c:mtrol of a gene, the level of expression of 2. gene or on its specific expression in a given cell type or tissue type.

The term "regulatory region(s)" includes t:issue specific elements, promoter, enhancer, polyadenylat:ion signa'_, 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)" can '~e isolated from existing DNA sequences) or can be man-made by known techniques of mo-_ecular k~iology. Existing DN.
sequence can be derived, for example, without being limited to, from virus, bacteria, yeast, or higher eukaryotes.
Transcription control sequences are sequences, vlhich control the initiation, elongation, and terminat:ion oi= transcription. Particularly important transcription control sequences are tr,.o;~e which control transcription initiation, such as, but not= limited to, promoter, enhanc er, operator and repressor sequence;a.
1$
It is to be understood herein that: a gene is transcribed (expressed) into a messenger RNA (spliced or unspl.~-ced). In turn a mRNA (spliced when required) is translated (expres:~ed) into a pr-otein.
As used herein the term "polynucleot:ide" refers to any polyribonucleot.ide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotides" include, w~_t.hout 1-imitation single- and double-stranded DNA, DNA that i:a a mixture of single- and double-stranded regions, single- and double-str<~ntied RNA, anti Rya that is a mixture of single- and double-atranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more t:yp~calL~~, double-stranded or a mixture of single- and double--stranded rec~:ions. In addition, "polynucleotide" refers to trip_Le--st:randec~ regions comprising RNA or DNA
or both RNA and DNA. 'Phe term polsrr~ucl.eotide also :iricludes DNAs or RNAs containing one or more modified bases and DNAs ~~_ RNAs with backbones modified for stability or for or_her reasons. °'Moc~ified" bases include, for example, tritylated bases and unusual basE:s such. as inosine. A variety of modifications has b~°en made to CoNA and RNA; thus "polynucleotide"
embraces chemically, enzymatically or met~abol_.ica7_ly modified. forms of polynucleotides as typically found in nature, as vaell as the chemical forms of DNA and RNA characteristic o:w viruses and cell-s. "POlynucleotide"
includes but is not limited to 1-inear and end-closed molecules.
"Polynucleotide" also embraces xelat:ively shoat polynucleotides, often referred to as oligonucleotides.

As used herein, the term "tumoi:~'" relates to solid or non-solid tumors, me~tastatic or non-metastatic tumors, tumors of clif_ferent 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 reproduc~ave 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 pol.ynucleotide or polynucleotide region that has a cc>rtain 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'x, 80~, 85~, 90~ or 95~) of sequence identity to another sequence (over the active region of said sequence) means that., when aligned, that percentage cf bases (in the active region) is the same i.n comparing the two sequences.
This alignment and the percent: homology o.= sequence identity can be determined using software programs known in the art, for example, those described in Current Protoco l; :in Molecul~~r ~3iology (Ausubel et al., eds, 7.987) supp.30, section 7.7.18, 'table= 7.7.1.. A preferred alignment program is ALIGN Plus (Scientific and Edu~:ational Software, Pensylvania). Thus any DNA construct having significant homology to regulatory regions described herein are encompassed by the present invention.
:Ct is to be understood herein, that if a "range", "group of substances" or particular characteristic (e.9., t:emperature~, concentration, time and the like) is mentioned, the present invention rc;lates to and explicitly incorporates herein each and every specific member and combination oi= 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 tlnerei.n. Tritzs, for example, - with respect to a temperature greater than 100° C, this is to be understood as specific::af.ly incorporating herein each and every individual temperature :;torte, a:; well as sub-range, above 100° C, such as for example 101" C, 105° C and up, 110° f. and up, 115° C
and up, 110 to 135° C, 115° c to 135° C, 102° C to 150° C, up to 210° C, et.c. ;

- with respect to a tenuperature lower than 100° C, this is to be understood as specifically incorporating herein each and every individual temperature state, as wel:L 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 vre:Ll as sub-:range, above 1 minute, such as for example 1 minute, 3 to 15 minute , 2 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hc:rurs to 20 hours etc.;

Table 1 Abbreviation Virus src Rous Sarcoma Virus (Chicken) yes Y'73 Sarc~.oma G'irus (Chic:ken) fps Fujinami Sarcoma Virus (Chicken, Cat) abl Abelson Murine Leukemia 'J:irus (Mouse) ros Rochester-2 Sarcoma Virus (Chicken) fgr Gardner-Rasheed Feline Sarcoma Virus (Cat) erbB Avian Erythroblastosis Virus (Chicken) fms McDonougtn Fel ine Sarcoma V:.rus (Cat ) mos Moloney Murine Sarcoma Virus (Mouse) raf 3611 Murine Sarcoma + Virus (Mouse) 1$ Ha-ras-1 Harvey Mur~ne Sarcoma Virus (Rat) Balb/c mouse: 2 loci Ki-ras 2 Kirsten..Murine Sarcoma Virus (Rat) Ki-ras 1 Kirster: Murine Sarcoma Virus (Rat) myc Avian MC29 Myelocy~omatosis Virus (Chicken) myt Avian Myelo Blastomas (Chi.cken) fos FBJ Ost:eosarcoma Virus (Mouse) ski Avian 5KV 'P10 Virus(Chicken) re) Reticuloendotheliosis Virus (Turkey) sis Simian Sarcoma Virus(Woolly Monkey) 2$ N-myc Neurob7_astomas (Human) N-ras Neurob7.astoma, Leukemia Sarcoma Virus (Human) Blym Bursal L~ymphomas(Chicken) mam Mammary Carcionoma(Human) neu I~leuro, Glioblastoma(Rat) ertAl C.'hickem AEV (Chicken) ra-ras Rasheec:I Sarcoma Virus(.Rat) mnt-myc Carcinoma Virus MH2(Chicken) myc _Myelocytomatosis QK10(Chicken) myb-ets Avian myeloblastosis~erythroblastosis Virus 3$ F26 (C-hi.cken) raf-2 3611-MSV (Mouse) raf-1 3611-MS\,r (Mouse) Ha-ras-2 Ki-MSV (RGt) erbB Lrythro.~lastosis virus(Chi.cken) BRTEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic illustrating tre generation of PSP94-knock- in mice. (A) Genomic organization of the mouse PSP94 alleles (Wt: wild type 129 Sv mice) showing the four exons -three- introns. Structure of pPNTIoxP 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 :C. The structure of knock-in (KI) 1~ 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 ~~n ES cell clones is shown at the bottom, and is located 3' down stream of ;i'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. Primers pairs for PCR
genotyping were shown for Wt and Mut alleles. (8) Southern blotting experiments demonstrating screening of 64:18 resistant ES cell clones.
Three positive mutant (KI) and a number of wild type ES clones were indicated with Xba 1 fragments of 1:3 kb azd 8.5 kb separately. (C) :>outhern blotting experiments characterizing heterozygous (+;-) and homozygous knock-in (KI) mice. ~. 1.5 ~ agarose gel electrophoresis of the fast PCR genotyping of KIDZAP mice. 'three types of mice GVt, heterozygous (+/-) and homozygous (-/-) were shown by two pairs of primers.
Fig. 2 represents photographs of histological analysis of the prostate samples in PSP-KIMAP mice. Sections were stained with hematoxylin/ eosin (H&E). (A) HGPIN of 7 weeks, H&E, x 25. (8) HGPIN with microinvasion (10 weeks), H &E x25. Arrow indicates microinvasion site. (C) Gleason grade 1, H&E, x 2'i. Inlet: x 40. (D) Gleasor~ grade 2, H&E x 25.
3~ Inlet: x 40. (E) Gleason grade 3, H&E of x 25. Inlet: x 40. (F) Gleason grade 4. HkE of x 25. Up inlet: cribr:if_arm pattern of adenocarcinoma, x 40. Lower inlet: local metastasis in to stroma indicated by arrows. x 40. (G) Gleason grade 4 showing multiple foci with two solid rounded tumor masses . Gleason <scores 9 ( ~~ + 5 ) , left grade 5 ; right, 3$ grade 4; H&E, x 40. (H) Gleawon grade _'. tvzmo:r foci showing (by arrow) one typical comedocarcinoma (right i.nle:t, x60) . '"tosses of cribriform tumor with central necrosis were ind:i.cated by arr~aw ~m the bottom. (I) Grade 5 tumor showing anaplastic, raggedly infilt:ratvion ;inlet x40). (J). IHC with a SV40 Tag antibody of HGPIN ()zemat:oxylin cc.~unterstaining) showing nucleus 40 staining in atypia area only, while normal glands are negative. x 40.

F:ig 3 are graphs showing correlation of age with tumor development of KIMAP
mice. (A) Percentages of: cancer (all histologic:al patterns higher than PIN) in total testing mice inc:i dence were plot:t:ed against different ache groups. Similar p~.otting was srown in (B) for percentage of well differentiated and rnoderately d~:.iferentiated <:a1? (WD +MD CaP); in (C) for poorly differentiated CaP (PDCaT.y ; and in (D6 for PINS (low and high PIN, including high PTN with micro-invasion).
1~ Fig.4 are graphs showing correl<~tion of C)leason scores in KIMAP mice with age group. (A) Gleason scores distribution in i~cIMAP correlated with different ages groups. (8) Linear corre7.ation of Gleason scores in K:LMAP
with age. Scatted dots represent. the dist:ribur.ior~~ of scores at different ages. Regression line was drawn by the S~.gma Plot program. Numbers of 1S mice analyzed in each group are indicated. F'rror bar: mean ~ SD.
Fig. 5 is a graph illustrating prostate targeting of SV40 Tag induced tumorigenesis and developments in K-IMAP mice. The rate of non-prostate cancer (NPT) incidence (in both males and fema:lE:s) was indicated as 2~ percentage of mice showing prostate cance~ only, i.e. prostate targeting mice as 100 ~. Numbers refer the nurnbers of NPT mire.
F'ig. 6 is a graph evaluating the genomic and phenotypic stabilities in R:IMAP mice: In KIMAP mice (n is the numbers of mice tested), histological 2S grades were compared by percerutages in total mire tested for three breeding generations (F1, F2, F3, n = 9, 24, 4 se~arate:Lyi in the same age groups 20-27weeks respectively.
Fig. 7 is a grapgh evaluating the phenotypic variations of founder (FD) 3~ breeding lines of KIMAP. Only two age groups mice were tested, as they represented the age groups wit:l: 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
rrsTxovoLOCY
EXAMPLES
Establishment of knock-in mouse of adenocarcinoma A targeting vector pPNTIoxP was used to construct a PSP94 gene-targeting plasmid (Fong, G. H., et al. Nature, 376: 6Ez-7=0, 1995; Peng, J., et a:1., Proc.Natl.Acad.Sci.IJ.S.A., 97: 8386--8397., 200iJ). 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 Cel7~ F3i.o', 18: 11-26, 1999). After double crossing-over in the mo~.z~;e 3enome in t:he~ 5' end of the PSP94 gene, the SV40 Tag (2.7 kb, both large "' and small t antigens) and the neomycin gene (l.9kb) were inserted at. the middle of the first exon (Kpn1 site) of the mouse PSP94 gene. 'fhe recornb.nant t:ai:geting erector DNA was introduced by electroporation into the emb::ryo stem (FMS) cel.I line R1. More than 500 6418-resistant individual clones were scr<~ened and eight positive ES
clones were characterized by Southern blotting. The probe used for PSP94 gene targeting by Southern blotting experiments was a 500 by (Stu1/Kpnl) located down-stream from the righr_ arm of the vector and was comprising of the complete exon 2 and the flanking intron area (Fig. 1A). Fig. 1B
illustrates the results of Southern blotting, showing the wild type (~12 k:b) and mutant (~9 kb for knocked in) genc,rnic fragments. Negative screening of GCV (ganciclovir) fo.r TK (He:rpes virus) expression was also performed at the same time. Production of chimeras by aggregation with 8-cell embryo cells of diploid C'.D1 strain and implantation into pseudo-pregnant female hosts were performed according to techniques known in the art. From two positive FS clones with PSP94 gene targeting, 20 chimeras (11 male and 9 females) with different fur coat colours were obtained. The chimeras were all tested for c:~ermline transmitability by breeding with. CD1 and 129 Sv strains, which were screened fox germline progenies firstly for black coat color and by the PCR genotyping.
PCR-genotyping, Identification and Breeding of Knock-in mice by PCR and Southern Blotting X111 male chimeras were found to be partly fertile in breeding within the (~D1 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 CD1 or ~_29Sv background . Only two female chimeras produced knock-in mice with mutation of the PSP94 gene transmittable to germlines. ThE.3 founder l.i.ne was designated as the S g~armline mouse produced from c:h:i.rneras mating with CD1 or 129Sv with fuel fertility and with a transmittaable mutant genoi~ype to the next generation.
Because germline transmittance through female chimeras is rare, some of the founder line mice and their breeding progenies (F1, F2, F3, either heterozygous or homozygous) were further c:harac°~erized by Southern blotting experiments (Fig. 1C) as well a:~ by PCR genot:yping (Fig. 1D) . Primer pairs used for screening for germlin~~ prc>gen.iee; from chimeras by PCR
genotyping were: Pr36-Prl6 for wild type c~enome testing Pr36 5' GGC AAC
AGC GTG TCA AAG 3' (promoter regi..on 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 molecul;~r weight. c~enom:ic DNA from R1 embryo stem cell culture and mouse tail and lever was puz-ifi.ed as previously reported.
Southern blots were prepared using -- 5 ~,g mouse tail chromosomal DNA for each lane digested by restriction enzyme and separated in a 0.5 ~ agarose c~el. For KIMAP mice, the probes utilized were the same as for ES cell colony screening.
DTouae anatomy: prostate and non-prostate targeting (NPT) test3agr 7'he prostate along with the male aco.essory glands, i.e. the ventral and dorsolateral prostate lobes (VP, DLP res'pectivel'y), seminal vesicles (SV) and coagulation gland (CG, or anterior gland), were dissected out separately as per the description and definition reported (Imasato, Y., et ~~1., Endocrinol, 1~!2: 2138-2146, 2001). Surgical castration was performed under anaesthesia via the scrotal route. R11 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 neopl.astic changes (the maximum not beyond the PIN) undetectal.~7.e _in the prostate, but detectable in non a~rostate tissues.

xistoyatholoQical characterization aad definitions of various degrees of Cay~ is Knock-in mice T?ze study of tumor development :in the knock-in sKIMAP) models was p~=rformed. Mice sacrificed at c)ifferent 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 (:~I)(Fig. 2B, 2H for immunohistc>Chemistry) and adenocarcin.oma. The extent and intensity were determined according to standards previously reported (Imasato, Y., et al., J.Urol., :6~: 1819--1.824, :?000), Gleason grading and Gleason scoring in the h:istological classification of KIMAP mice were performed as per the standard (Deshmumukh, N. and Foster, C. S. Grading prostate cancer. In C. S. Foster and D. G. Bostwick (eds.), Pathology of the prostates cancer, pp. 191--22'7. Philadelphia: W.B Saunders, 1998; Mostofi, F. K., et al., H:i.stologica=_ Typing of Prostate Tumours, Second Edition ed. World Health Organiztion, international. Histological Classification of Tumours, Springer; 2002;: 'The architectural patterns observed were assessed by fi~ae different grades: Grade 1 (very well-cifferentiated): single, separate, uniform glands closely packed, with definited edge (Fig. 2C); Gracie 2 (well--d:ifferentiated): single, separate uniform glands loosely packed, with irregular edges (E'ig. 2D); Grade 3 (glands with variable and distorted archi~:ect.urE:): single, separate, uniform scattered glands and ~omoo~h:Ly cir~_umscr;abed papillary/ cribriform masses (Fig. 2E); Grade 4 (po<:~rly differe~~tiated): cribri.form masses with ragged and invading edges (Fig. 2F); Grade 5: n.o glandular differentiation, anaplastic tumors growing in non-glandular solid masses of cells (Fig. 2F, Ci. H, I). Based on the most prevalent patterr_ (" the primary pattern.") and the second most. prevalent X:~attern ("secondary pattern"), the Glea~~on score was derived x>y adding the primary pattern grade number to the secondary grade number (Fig, a::C~). If only one pattern .is seen throughout, r_he Gleason score i.s 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 :?-4 as well differentiated adenocarcinoma (WDC:aP); (2) ;scores 5-7 as moderately differentiated ~~denocarcinoma (MD(:aP) , (3) scores 8-10 as poorly differentiated adenocarcinoma (PDCaP). All. histopathological grading was determined by three scientists :independently and blind analysis was performed zmmuaohistochemistry (IxC) After fixation, samples were washed with ;'0 '~ ethanol and embedded in paraffin. Slides of 4 ~.m thick were then cut:, deparaffinized and rehydrated as we previously reported. Monoclonal. antibodies against SV40 Tag oncogene (Calbiochem, CA) and a polyc~_onal ,~.nt:ibod.y against recombinant pTrcHis-mouse PSP94 were used .:or immunohistocr_eraistry with an ABC kit (StreptABC complex :kit, DAKO, Mississauga, Ont ) at 1: 100 and 1:400 dilution separately. All IHC ;al.ides were count.erstained with hematoxylin.
1~ Statistical Analysis Statistical software packages of SPSS (Version 10.0) and Sigma Plot :2000 (Version6.l, SPSS Scientific, ~::'.h~_cago, Ilf) were used for statistical analysis and production of the graphs. Based on the normality of data (by a Kolmogorov Smirnov Z test, SP.3:s), one way ANOVA test of variances w,as 15 used for statistical analysis. hearson"s test (SPSS) was used for linear correlation analysis.
RESULTS
20 Evaluation of CaP development in Knock in IcIMAP model K.IMAP mice were first characterized for the corrv.iation of: tumorigenesis and CaP development with age (7-52 weeks, Fig. 3A-D). According to the standard of histological patterns as pr_e~riousl~.~ reported (Greenberg, a~.
N:., et al., Proc.Natl.Acad.Sc:i..iJ.S.A., 92: 3439-3443, 1995;Mostofi, F. K., 25 Sesterhenn, I. A., and Davis, C. ,T. E. Histological Typing of Prostate 'tumours, Second Edition ed. Wora.d Health Organizt:ion, international F:istological Classification of: 'fomours, Sp~ringer, 2002), percentages (~ of mice analyzed) of categories of cancer (CaP, Fig. 3A), well and moderately differentiate CaP (WD+MD CaP, Fig 313), poorly differentiated CaP (PDCaP, fig. 3C) and PINS (including Y°~igh grade PIN with microinvasion, Fig.
3D) were analyzed. Fig. 3A shows a steadily prolonged cancer development i.n KIMAP mice which correlated. with age. Majority (50iE>4) of CaP in k:IMAP were well and moderately differenr_i<xte<i Ca P in almost all age groups (80-100 ~, Fig. 3B). CaP incidences in KIMAP mice were shown to be 35 synchronous as evidenced by: (L) High .rate in F;IMAP (Fi.g. 3A); (2) in F;IMAP mice, PINS (Fig. 3D) and poorly dif:ferent:iated CaP (Fig. 3C) were found only at the early and later (after 37-50 weeks) age groups separately Ifor a summary see Table 2).

Since the process of: adenocarc:i-noma development in KIMAP mice is steadily p:_olonged, and extended multip:Le foci. of the ade:nocarc.inoma were observed (shown in Fig. 2G), the cancer architecture in F;.IMAP mace was further characterized by the Gleason grading system and subsequent Gleason scoring.
By age group of 12 t:ol9 weeks, t:he Gleason sr_ores 2, 5 and 6 were detected at 37 ~ (3/8), 12 ~ (1/8) and .5C~ ~ (4/8) separately (Fig. 4A). By age group 24 to 27 weeks, Gieason grade 3 increased to 80 ~ (8/10). By age group 28 to 31 weeks, Gleason grade 4 was detected at approximately 25 ~
(3/12). By age of 52 weeks all mice (n=5, Fig. 3A and 4A) synchronously developed visible cancer (grade 4-5) in the lateral prostate lobe. Fig. 2 G, H, I showed solid tumor mast, typical comedocacinoma structure (Grade 5, Fig. 2 H ), and ragged inf.,:l.t.ration t:o the stromal tissues.
Fig. 4B shows a linear correlation of Gleason scores with age in KIMAP
I$ mice, and correlation coefficient: is 0.71 (P<0.01, by Pearson's correlation test). The average Gleason grade i:> at 3--4 and the subsequently average Gleason scores were at 5-7 (Table 1), which is the same average range as in humans.
The KIMAP mice (n>252) were monitored for 15 months and no non-prostate targeting was observed (Fig. 5).
Giersotypic assd phenotypic stability Genotype and phenotype stability were evaluated with regards to the copy number, rate of CaP incidence and tumor d~sveloprnent in at least three breeding generations in KIMAP mice. Fig. 6 shows that three generations ~;F1-F3) of KIMAP mice followed the same tumor development: pattern. This phenotypic stability was also maintained in other strains CD1 and 129Sv ;data not shown). Fig 1. C also shows the knock-in genotype remained stable for targeting at the PSP94 gene.
'Che founder line of knock-in mice was designed as a fertile germline of mice with a transmittable mutant genotype. Most of the mutant phenotypes were tested in breE>ding progenies with a CD1 background. As shown i.n :gig. 7, six breeding fc>under J.ines of KTMAP were tested and all showed a similar tumor deve~_opment. In the process of Y~IMAP 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.

W~~ 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 / enhancc:r of PSP94. Une of the unique features of this KIMAP model is the applic~ak:~ility of the Gleason histological grading and scoring system, a system widely used <~1111ically 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 animal age was observed.
The Gleason grading system has been advocated as a way to improve the pathologist's ability to accurately predi<:t the biological behavior of a particular tumor, as this system seems to deliberately recapitulate every single step of CaP developments specific t:o the human situation. To 1S establish such an experimental paradigm has proven to be difficult, since none of the previous models have demonstrated features, which would qualify for classification by the Gleasorr 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 heterologaus gene (insert) is predictable and the copy number of the in:aert is also controllable. A.
steadily prolonged tumor growth starting after puberty i.n all breeding 1_ines in KIMAP mice is the most. notable fdature. This feature permits the application of Gleason grading system.
~,he KIMAP revealed fundamenta=', similarity in the histopathological characteristics as well as underlying molecular pathways. The simplest i:heoretical explanation for the different histopathological characteristic :is that none of the previously described transcJenic CaP znodels is fully :_egulatable as a rEault of an e:~ndogenous mutation by a prostate specific ~~ene. 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 nc> regulatory region (cis, trans) will be affected. The PSP94 gene promoter is a strong promoter, and the SV40 Tag is coupled with the full capac°:ty cf a promoter of the most abundant prostate secretory gene, We have observed PSP94 suppression in the early puberty i.n heterozygous KIMAP mice while SV40 expressed wa~~ initi.ate~d , and this suppression was maintained at a low, but not completely shutting down level of expression b~,r the PSP94 promoter/enhancer (data not shown). The SV 40 Tag induced t~.imorigenesis started from puberty (5-7 weeks oi'_ age) of the prostate gland along with the secretion of PSP94 protein. The SV40 Tag expression is in S turn under control by the prostate, since when normal. prostate function is disturbed by tumor growth, SV40 expression, will be suppressed by PSP94 promoter/enhancer region. We reported this apparent suppressive mechanism of PSP94 in human pYostate cancer biopsy :samples (Imasato, Y., et al., Endocrinol, 142: 2138-2146, 200;; Imasato, Y., a_t al., J.Lsrol., 164: 1819-1824, 2000) and this suppression also observed '~~it.h another abundant secretory protein of PSA (Stege, k., et a~w., Clin.Cancer Res., 6: 160-165, 2000.). The balance of positive stumorigenesis) and negative (the suppression) regulatory effects t.~:o t:he SV~1C) Tag structural. gene that enable the tumor to remain stable for most of the> pos t--puberty period.
The KIMAP model is a highly pr°e3ictable model. Although tumorigenesis is a stochastic event inside the acini of the gland, statist:Lcally a significant majority of KIMAP mice underg~~ steady cancer growth after puberty. The CaP in KIMAP mice show some desirable features, such as: (1) 2~ a homogenous and synchronous growth (Fig. 3, 4 and 6); (2) low variation and high stability of both phenotype and genotype in all breeding lines and ._n progenies of several generations; (3) selective prostate targeting with no incidence of nor.-prostate t:.umor induction:., (4) KIMAP is an invasive cancer model, since it showed early occurrence and a high incidence c>f PIN
with microinvasion. A highly invasive, human. CaP specific form of ~~omedocarcinoma was also identified in KIMAP mice of 50 weeks age. We have characterized that KIMAP mice are responsive to castration (andl-ogen deprivation, data not shown).
3~ It is interesting t:hat transmi.t::tance of PSP--KIt~IAP mutant introduced by gene targeting in embryo stem cell culture wa;: through female chimeras. IT1 addition to this evidence of infertility of male chimeras of KIMAP mice, we observed that the depressed PSP94 expression in homozygous K.IMAP mice resulted in the imbalance genc:ier distribution in the progenies (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 inhibi.n protein (PIP) to (3-FSH (Follicle stimulation hormone), although this hypothesis is highly contentious. In the present experiment on the knock-in of PSP94 function, we obse>rved no support. for the "PIP" theory: (1)We observed a close correlation with SV40Tag expres:;ion with tumorigenesis and progression (Fig. 2J') ; (J) In E;I:MAP mice, we observed suppression of PSlP94 e;cpression, which ie~ 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 :(tIMAP mice, as in hs>terozygous, PSP94 expression level should exist at a certain level comparable to the wild type mic~e:~. Further experiments are required to c:Larify the real biological function of PSP94, one of the most abundant 1~ protein from the human semen. Table 3 summarizes the advantages and characteristics of the KIMAP mouse model.
Table 2 Mouse CaP model KIMAP
Steadily prolonged ~~.ro~;~~t ri ~~~-3 5 weeks or late synchronous among Tumorigenesis and cancer growth I indi.v~iduals Extent (Foci ~ Multiioci, evenly distribution) dist:ri.but:ed Intensi.t.y (Variation of scores ) DetE:ctable Gleasox~. grades majority 3-4 Adenocarci:noma -~ - --foci Gleason scores Majority 5-7 Majority (Gleason Glandular differentiation sccres 5-?) Poorly differentiated.
adenocarcinoma i Slowly developed - --r-~COrrelation with tumor SV 40 tag expression ~ devs=;.opment Metastasis r~onunon in early stage Table 3 ~IM_AP_ ransgene structure:

nlimited length, Promoter /enhancer region ith full regulatory and apacities Tissue specific elements (TSE) Copy of transgenes ingle copy Transgene insertion lane targeting Insertion of mutation ndogenous ode of transgene expression Cis-traps oth ~~is and traps action Transgene expression .onstitutional atural precise gene targeting, xperimental procedures and epeatable Results Mostly adenocarcinoma umor development Synchrcmous Scheduled and predictable Breeding procedures Stable phenotype g Founder line independent Genetic transmittance_ Mend_el Segregation y-Sensitivity of Prostate Targeting%~ NPT
~
incidence of non-prostate targeting) tility potential Standard

Claims (43)

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 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.

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

4. The DNA construct of claim 3, 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.

5. The DNA construct of claim 2, 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.

6. The DNA construct of claim 5, wherein said suicide protein is selected from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase.

7. The DNA construct of claim 5, wherein said cytotoxic protein is selected from the group consisting of the A chain of diphteria toxin, ricin, and abrin.

8. The DNA construct of claim 5, wherein said protein causing apoptosis is selected from the group consisting of caspases, Fas-Ligand, Bax and TRAIL.

9. The DNA construct of claim 5, wherein said anti-oncoprotein is selected from the group consisting of p53, p21, and Rb.

10. The DNA construct of claim 5, wherein said protease is selected from the group consisting of awsin, papain, proteinase K, and carboxypeptidase.

11. The DNA construct of claim 5, 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.

12. The DNA construct of claim 5, wherein said chemokine is selected from the group consisting of Mig-1alpha, Mig-1beta, IP-10, and MCP-1.

13. The DNA construct of claim 2, 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.

14. 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.

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

16. A transgenic nor-human mammal susceptible to prostate tumor formation, having genomically-integrated in non-human mammal cells, an insert inside the PSP94 gene exon/intron region.

17. The transgenic non-human mammal of claim 16, 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.

18. The transgenic non-human mammal of claim 17, wherein said gene capable of initiating tumor formation is a SV40 T antigen.

19. The transgenic non-human mammal of claim 18, 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.

20. The transgenic non-human mammal of claim 17, 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.

21. The transgenic non-human mammal of claim 20, wherein said suicide protein is selected from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase.

22. The transgenic non-human mammal of claim 20, wherein said cytotoxic protein is selected from the group consisting of the A chain of diphteria toxin, ricin, and abrin.

23. The transgenic non-human mammal of claim 20, wherein said protein causing apoptosis is selected from the group consisting of caspases, Fas-Ligand, Bax and TRAIL.

24. The transgenic non-human mammal of claim 20, wherein said anti-oncoprotein is selected from the group consisting of p53, p21, and Rb.

25. The transgenic non-human mammal of claim 20, wherein said protease is selected from the group consisting of awsin, papain, proteinase K, and carboxypeptidase.

26. The transgenic non-human mammal of claim 20, 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.

27. The transgenic non-human mammal of claim 20, wherein said chemokine is selected from the group consisting of Mig-lalpha, Mig-lbeta, IP-10, and MCP-1.

28. The transgenic mom-human mammal of claim 20, 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.

29. 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.

30. The transgenic non-human mammal of claim 29 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.

31. The transgenic non-human mammal of claim 30, wherein said gene capable of initiating tumor formation is a SV40 T antigen.

32. The transgenic norm-human mammal of claim 31, 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.

33. The transgenic non-human mammal of claim 30, 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.

34. The transgenic non-human mammal of claim 33, wherein said suicide protein is selected from the group consisting of herpes simplex virus-1 thymidine kinase and Escherichia coli cytosine deaminase.

35. The transgenic non-human mammal of claim 33, wherein said cytotoxic protein is selected from the group consisting of the A chain of diphteria toxin, ricin, and abrin.

36. The transgenic non-human mammal of claim 33, wherein said protein causing apoptosis is selected from the group consisting of caspases, Fas-Ligand, Bax and TRAIL.

37. The transgenic non-human mammal of claim 33, wherein said anti-oncoprotein is selected from the group consisting of p53, p21, and Rb.

38. The transgenic non-human mammal of claim 33, wherein said protease is selected from the group consisting of awsin, papain, proteinase K, and carboxypeptidase.

39. The transgenic non-human mammal of claim 33, wherein said cytokine is selected from the group consisting of IL-1, IL-2, IL-6, IL-12, GM-CSF, G-CSF, M-CSF, TFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta.

40. The transgenic non-human mammal of claim 33, wherein said chemokine is selected from the group consisting of Mig-lalpha, Mig-1beta, IP-10, and MCP-1.

41. The transgenic non-human mammal of claim 33, 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.

42. The transgenic non-human mammal of claims 16 or 29, wherein said transgenic non-human mammal is a transgenic mouse.

43. The use of the transgenic non-human mammal of claims 16 or 29 for evaluating the efficacy of drug candidates in inhibiting the growth of prostate related neoplasia.