EP1007560A1

EP1007560A1 - Method for screening and transgenic model

Info

Publication number: EP1007560A1
Application number: EP98904502A
Authority: EP
Inventors: Jan TÖRNELL; Jon Kindblom; Hakan Wennbo; Olle Isaksson; Gunnar Norstedt
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-02-14
Filing date: 1998-02-13
Publication date: 2000-06-14
Also published as: AU6235398A; WO1998035989A1

Abstract

A method for the screening and identification of low molecular weight compounds which interact with lactogenic/somatogenic receptors is claimed. The method is characterized by the use of isolated tissues for in vitro cultivation as organ culture, primary cells, immortalized or transformed cells from transgenic animal that over-express hormones/receptors belonging to the somatic or lactogenic family. Also a transgenic non-human animal that over-express prolactin which cause phenotypic alternations notably in the prostate and/or mammary gland and use thereof is claimed.

Description

METHOD FOR SCREENING AND TRANSGENIC MODEL

Technical field of the invention The present invention relates to a method for the screening and identification of low molecular weight compounds which interact with lactogenic/somatogenic recep- tors and which is characterized by the use of isolated tissues for in vitro cultivation as organ culture, primary cells, immortalized or transformed cells from a transgenic non-human animal that over-express hormones/receptors belonging to the somatogenic or lacto- genie family, e.g. prolactin (Prl) and growth hormone (GH) receptors.

The invention also relates to a transgenic non-human animal that over-express prolactin which cause phenotypic alterations notably in the prostate and mammary gland and human e.g. growth hormone (GH) receptors and the use of the transgenic animal in the screening and identification method.

Background Transgenic animals represent an important scientific tool to explore functions of specific genes in a physiological environment. Several examples exist where transgenic animals have been made that serve as useful models for human disease. The use of transgenic animals also in- elude research in endocrinology and it has been well es^¬ tablished that over-expression of growth hormone provides a model for acromegaly. In the creation of transgenic animals one can sometimes reveal more unexpected findings .

Description of the invention The bases for the present invention is the unexpected finding that over-expression of prolactin (Prl) cause a specific phenotype in prostatic and breast tis-

sues. According to the present invention this finding can be used to establish a system where one can seek to find compounds that would circumvent the phenotype caused by the transgene. In the extension of this invention one can extrapolate into human systems and utilize the invention to find drugs that would function in humans.

As specified below, according to the present invention it has been found that a transgene that encode rat prolactin (rPrl) cause a specific phenotype to occur when expressed m transgenic mice. The phenotypic alterations include increased weight of thymus, spleen, Kidney, tes- tis, seminal vesicula and prostate without, affecting body weight (see Abstract Vennbo et al; Growth Hormone Research Society, November 1996) . The results are important for the understanding of the pathophysiology of Prl. According to the present invention it is possible to use the animals as discussed above to screen for new drugs. The present invention is based on the use of transgenic animals where the inventors of the present mven- tion unexpectedly have found that the use of specific DNA constructs allows the generation of transgenic animals that exhibit a specific phenotype, these ani als can be used as tools for screening and identification of low molecular weight compounds which interact with ..acto- genic/somatogenic receptors or in other ways mnibit or facilitate signals derived from lactogenic/somatogenic receptors and such use has not to our knowledge been reported earlier. According to the present invention it is also possible to grow cells derived from transgenic ani- als that are characterized by the expression of the transgene in question and that these cells subsequently can be used to screen compound libraries. The endpomt measurement may vary but need to be relevant for the effects of the transgene. Examples of cellular endpomt measurements per se is know in the art and include receptor bindmg/mternalization assays, activation of mtra cellular signals and proliferation assays. The attached claims define the present invention. Examples, not intended to restrict the invention, are given below in order to illustrate the invention. The examples given are focused on the utility of rat Prl as a transgene of relevance but it is inferred that also human Prl and corresponding human Prl receptors may be used. The latter is relevant because of the pharmaceutical need for drugs that act on human receptors. In a similar type of experimental protocol the inventors also disclose the use of a human growth hormone receptor cDNA construction that is particularly suitable for the generation of an animal model that responds to human GH or analogues thereof.

Brief description of the drawings

In the examples below reference is made to the accompanying drawings on which: Figure 1 shows the Mt-rPRL- B02 plasmid. Figure 2 illustrates the correlation between the pros- tate wet weight and the serum testosterone levels ^• in the PRL transgenic mice.

Examples

Example 1 - Generation of an animal that over-express rat Prl

An expression vector was constructed as outlined below. The key components in this vector is a metal- lothionein promoter followed by the rat Prl gene. This construct was injected into fertilized eggs using conventional techniques and offspring was analyzed using Southern blotting techniques. Positive off-spring were bred and later used to analyze phenotypic changes. It was shown that transgenic expression resulted in an increase in serum Prl. It was also evident that certain tissues, notably prostate, increase in weight. Materials and methods

Construction of the metallothionem promoter- rat prolactin plasmid

The rat PRL expression vector, Mt-rPRL- B02 was based on the pRPRL-Hmdlll A and B plasmids described earlier and the methallothιoneιn-1 (Mt-1) promoter from MtbGH 2016 plasmid. The Mt-1 promoter was subcloned as a 650 bp fragment into a BsmFl site 5' of the start coαon in the rat PRL gene inserted in a pGEM-7Z vector (Promega) resulting in the Mt-rPRL- B02 plasmid (Figure 1). The metallothionem promoter, the rat prolactin gene and tne in ection fragment are indicated in figure 1. The Mt-1-rPRL fragment was excised by digestion with BstEII, located m the Mt-1 promoter, and BamHI located 3' m tne polylmker of pGEM-7Z. Transgenic mice were generated in C57BL/6JxCBA-f2 embryos by standard micromjection procedures. The DNA fragment to be injected was excised from the plasmid Mt-rPRL-WB02 by restriction enzyme cleavage with BstEII and BamHI, separated by gel electrophoresis through a 0.7 % agarose gel, cut out, isolated using ISO- tachophoresis and precipitated with ethanol.

The oovme GH (bGH) DNA fragment was isolated from the plasmid MtbGH 2016 (generously provided by Dr. P. C. Palmiter¹ as a BstEII-EcoRI fragment, separated by gel electropnoresis through a 1% agarose gel, cut out, isolated using Genclean II kit (Bio 101) . To identify transgenic animals DNA was extracted from 0.5 cm sections of tails from 3 weeks old mice by digestion with 400 mg of protemase K 0.6 ml of 1M urea, 100 mM NaCl, 50 M Tris HCl (pH 8.0), 10 mM EDTA, 0.5% sodium dodecyl sul- fate (SDS) at 55°C for 16 h. The digested tails were freezed for 2 h in -70°C, then precipitated with lsopro- panol and washed with ethanol. The presence of the Mt-1 rPRL transgene was detected with PCR (94°C for 5 mm and 30 cycles of sequential incubations at 94°C for 30 s, 54°C for 30 s and 72°C for 120 s) using one primer located in the Mt-promoter (5' -GCGAATGGGTTTACGGA-3' ) and one m the rPRL gene (5' CCATGAAGCTCCTGATGCT-3' ) . Mice that had integrated the bGH transgene were identified witn PCR (the same incubation conditions as for rPRL) using the same Mt-promoter primer and one primer located in the bGH gene (5 CTCCAGGGACTGAGAACA-3) . Tap water and pelleted food were freely available.

RNA analysis Total RNA was isolated from frozen tissues by acid guanidmium thiocyanate-phenol-cnlorophorm extraction described by Chomczynski and Sacchi. Specific RNA was analyzed using a reverse transcriptase (RT)-PCR assay. The RT reaction was performed with 0.5 mg RNA as a template in the presence of 0.25 mg olιgo-(dt) primer (Promega), 5 units AMV-RT (Promega), 20 units PNAsin (Promega) and dNTP (Pharmacia) at a final concentration of 1 mM per nucleotide. RT buffer (50 mM Tris-HCl; pH

8.3, 50 mM KC1, 10 mM MgCl₂, 0.5 mM spermidme and 10 mM DTT) was added to a total volume of 20 ml. After oenatu- ration at 70°C for 5 mm and annealing in room temperature for 10 mm the elongation was carried out for 60 mm at 42°C. The RT reaction was terminated by heat mactiva- tion (95°C for 7.5 minutes). Rat PRL specific RNA was analyzed by amplifying an aliquot of cDNA by PCR (94°C for 5 mm and 30 cycles of sequential incubations at 94°C for 30 s, 60°C for 30 s and 72°C for 120 s.) using a sense primer located in exon 4 (5-TCCATGAAGCTCCTGATGCT-3') and an antisense primer located in exon 5

(5-GGATGGAAGTTGTGACCA-3') specific for rat PRL (Figure 2) . The PCR products were analyzed by electrophoresis in 1% agarose gel. The size of the fragment amplified from spliced RNA should be 152 bp and that from unspliced RNA or contaminating DNA 1252 bp . The fragments were transferred to Hybond-N nylon membranes (Amersham) and the membranes were baked m 80°C for 2 h and prehyoridized in hybridization buffer (0.2 M NaH₂P0₄, pH 7.4, 8c SDS, 1 mM EDTA, 1% BSA fraction V) at 60°C for 2 h. As probe a 823 bp PstI fragment containing the rat PRL cDNA was used, labeled with a random priming kit (Amersham) and P32dCTP. The hybridization was carried out in the same ouffer at 60°C for 12-16 h and washed with 2 x SSC, 0.5 % SDS at 60°C for 1-2 h and with 0.1 x SSC, 0.1 % SDS at 60°C for 0.5-2h. Mouse PRL specific RNA was analyzed by amplifying cDNA by PCR (94°C for 5 mm and 30 cycles of sequential incubations at 94°C for 30 s, 56°C for 30 s and 72°C for 120 s.) using a sense primer located in exon 1 (5-GTCACCATGACCATGAAC-3) and an antisense primer located in exon 5 (5-GGATGGAAGTTGTG ACCA-3) . The size of the fragment amplified from spliced RNA should be 558 bp . The transfer, hybridization, probe labeling and washing were carried out as above. The mouse PRL cDNA was amplified from mouse pituitary cDNA by the same protocol and primers used for detection of mouse PRL expression in pros- tate. The PCR fragment was subcloned into a pCRTMII vector (Invitrogen) and identified as mouse PRL by digestion with restriction enzymes. As probe the entire subcloned fragment from the vector was used. Specific RNA for the long form of the mouse PRL receptor was amplified by PCR (94°C for 5 mm and 30 cycles of sequential incubations at 94°C for 30 s, 56°C for 30 s and 72°C for 120 s.. using a sense primer located in the extracellular part of the receptor (5 GACTCGCTGCAAGCCAGACC-3) and an antisense primer located in the mtracellular part of the long rorm of the receptor ( 5-TGACCAGAGTCACTGTCAGG-3) . The size of the fragment amplified from spliced RNA should be 440 bp . The transfer, hybridization, probe labeling and washing were carried out as above. An EcoRI-XhoI fragment of the plasmid 4A314 (R. Ball, Basel, Switzerland, unpublished data) containing the long form of the mouse PRL receptor cDNA was used as a probe. DNA content analysis. Total nucleic acids (TNA) were extracted by homogenization of frozen tissues m 1 % sodiumdodesulphate (SDS) , 20 mM Tris-HCl (pH 7.5) and 4 mM EDTA, followed by a 45 mm di- gestion with protemase-K in 45°C and extraction with phenol-chloroform. The DNA content in the TNA preparations were measured with a fluorescence spectrophotometer (450 nm excitation and 555 nm emission) after addition of Hoecht s dye H 33258 (0.2 eeg/ml in 2 M NaCl, 1 mM EDTA and 10 mM Tris pH 7.4) .

Measurement of rat prolactin: Serum levels of rat PRL were measured by rat prolactin RIA (Amersham U.K.) according to a protocol from the manufacturer. Mouse PRL does not cross react with the antibody raised against rat PRL according to the manufacturer. Serum was either col- lected from the mice tails m heparin coated glass capillaries or by heart puncture in heparin coated syringes when sacrificed. All samples were analyzed in duplicates.

Measurement of IGF-I: The IGF-I concentration m serum was determined by a radioimmunoassay after acid ethanol extraction according to the manufactures protocol (Nicols Institute Diagnostics, San Juan Capistrano CA, USA) in a single assay.

Measurement of testosterone: Serum testosterone was measured by radioimmunoassay according to the manufactures protocol (ICN Biomedicals, Inc. Costa Mesa CA, USA) Histology Tissue pieces were fixed in 4% paraformaldehyde in pnospnate buffered saline (PBS; pH 7.4) overnight or longer, dehydrated and embedded in paraffin. Sections were stained n hematoxylm/eosm. Statistics Statistical differences were calculated using the Wilcoxon rank sum test. Significance levels less than 0.05 were considered significant .

Results

Generation of rat prolactin transgenic mice

The rat RPL gene has been described and contains 5 exons . The entire gene is approximately 11 kb and the cDNA 800 bp . In the Mt-rPRL- B02 plasmid used to generate rPRL transgenic mice the metallothιonem-1 (Mt-1) promoter was inserted 33 basepairs upstream of tne first exon into a BsmFl site in the rPRL gene. The lmerized fragment from the plasmid outlined in figure 1 was micro- injected into 250 mouse zygotes obtained after superovu- lation and implanted into 9 foster mothers resulting in 40 newborn mice. Three mice were identified as carrying the Mt-rPRL construct using PCR analysis. The founder animals, two females and one male were mated for establishing lines of transgenic mice. Transgenic lines were successfully established from the female founder animals mated to normal male mice. Male offspring from a bGH transgenic founder were used. Male mice from this line have serum levels of bGH higher than 5 times the normal peak values in mice.

The transgenic mice had elevated serum levels of rPRL The rPRL levels in the founder animals were evaluated by RIA analysis at 35 days of age. One female founder (LI) had very high levels of PRL (470 ng/ml) and the other female founder (L2) expressed the transgene at lower levels (11 ng/ml) . The rPRL levels m the male founder (L3) was 32 ng/ml. Rat PRL levels were also measured in all the animals included in the study when sacrificed (Table 1) . Expression levels of rPRL protein were stable over the life span of the animals. Offspring generated from transgenic line LI snowed consistently hign serum rPRL-levels while offspring generated from L2-lme expressed the transgene at lower levels (Table 1) . Rat PRL transgenic mice developed marked enlargement of tne prostate. The weight of prostate was examined when the animals were sacrificed at 10-15 months of age. Histological examinations were carried out on all prostates. All prostates from PRL transgenic mice had a higher weight compared to age matched controls (Table 1) . On average, the dorso-lateral lobes from the prostate were 20 times larger (wet weight) than the controls and the ventral parts were 9 times larger (wet weight) (Table 1). The bGH transgenic mice had 1.6-tιmes larger dorso- lateral lobes of the prostate glands (Table 1) and also increased body weight (1.4 times larger than controls, data not shown) . In contrast, the body weight of the prolactin transgenic mice were not increased (data not shown) . The DNA content in the prostate glands were meas- ured in 5 rPRL transgenic animals and 5 controls when sacrificed. The total DNA in the dorso-lateral lobe was increased 4.7 times (155 n 34 eeg DNA/prostate lobe vs. 33 n 5 eeg .DNA/prostate lobe in the controls) and in the ventral lobe 4.2 times (96 n 11 eeg DNA/prostate lobe vs. 23 n 5 eeg DNA/prostate lobe in the controls) .

Histologically, all prostates from PRL transgenic mice showed hyperplasia and glands distended by secretion in contrast to non-transgenic animals. The proportion of stroma cells and connective tissue were increased in the rPRL transgenic animals compared to controls. Occasionally there were parts classified as adenomas and in some areas showing cellular atypia. Focal parts of chronic inflammation were also visible.

The rPRL transgene, the endogenous mPRL gene -and the PRLR were expressed in the prostate gland. Specific mRNA for the rPRL transgene was detected in both in the dorso- lateral part of the prostate and in the ventral lobe (Table 2) . .Two of the lines (LI and L2 ) expressed the transgene at higher levels compared to the third line when measured with the semi-quantitative method RT-PCR. In the normal animals, expression of the mouse PRL gene was detected in all parts of the prostate gland (Table 2). Also PRLR-specific mRNA was detected in the dorso- lateral lobe and the ventral lobe of the prostate (Table 2) . Several different tissues were analyzed for the presence of mRNA corresponding to rPRL using a RT-PCR assay. The primers were selected in a way that the PCR reaction could not amplify cDNA corresponding to expression of the mouse PRL gene. The transgene was expressed in the liver, kidney, pancreas, seminal vesicles, testis, thymus and the prostate gland. Expression of the PRL transgene in different organs were analyzed with RT-PCR as described in the material and methods. Tissue samples were collected from the line LI at 14 months of age. An expected 152 bp fragment was detected in all organs analyzed. As a negative control the PCR reaction was run without any template added. Appropriate lanes from the same gel were selected.

Rat PRL transgenic mice had elevated levels of testosterone and of IGF-I. BGH transgenic mice had elevated levels of IGF-I. The serum levels of testosterone and IGF-I were measured when the animals were sacrificed. PRL transgenic -mice had higher levels of testosterone than controls (Table 1) . The testosterone levels among the rPRL transgenic mice could not be correlated to the prostate weight in neither the dorso-lateral lobe or the ven- tral lobe as can be seen in Figure 2. Figure 2 shows the correlation between the prostate wet weight and the serum testosterone levels in the PRL transgenic mice. r=0.50 for serum testosterone correlated to the weight of the dorso-lateral prostate and r=0.20 for serum testosterone correlated to the weight of the ventral prostate. p>0.05 The IGF-I levels were elevated in the rPRL and the bGH transgenic animals compared to controls (Table 1) .

Conclusion As exemplified above, prolactin transgenic animals may serve as a useful model for studying prostate hyperplasia. In addition to previously described transgenic models using expression of int-2 and large-T the present model represents a hormone dependent hyperplasia and might therefore be closer to the human patophysiology.

Other non-transgenic animal models for prostatic hyperplasia exist, e.g. testosterone induced BPH but our data indicates that prostatic growth in Prl transgenic mice could occur independently of at least serum concen- trations of testosterone. In the human, the influence of PRL on prostate growth is not clear but the present study suggests that the question whether PRL is an important factor for development of prostate hyperplasia in man should be addressed. The use of transgenic animals may be instrumental in order to obtain and/or test e.g. inhibitors of Prl actions.

Table 1 Age when sacrificed, plasma levels of rat prolactin, testosterone and IGF-I and weight of different lobes of the prostate gland in rat-prolactin (PRL) and bovine-growth hormone (bGH) transgenic mice and normal control animals

<Λ

C 09 O

H

m en x m m

3 c r- m ι

Table 1 , cont .

c

03 CtO

m

V)

X m rπ

H

"3 c r- m r

** = p<0 . 01

* = p<0.05 PRL or bGH transgenic compared to control animals

§§ p<0.01 PRL transgenic compared to bGH transgenic

Table 2 Expression of the rat prolactin transgene, the mouse prolactin gene and the prolactin receptor in the prostate measured by RT-PCR as described m materials and methods. Different lobes of the prostate gland were collected at 14 to 15 months of age

na=not analyzed

Example 2 - Cultivation of cells from transσenic animals An expression vector consisting of the rat Prl gene driven by a metallothionens promoter was used in transgenic animals. One of the phenotypic alterations was the occurrence of breast cancer in transgenic animals. Below the applicants describe that mammary glands from rPrl transgenics can be cultivated m vitro either as explants or as established cell lines. It is to be noted that cells modified by transgenic expression behaved differently compared to other mammary gland cells; the cells continued to express Prl and attempts to differentiate breast cancer cells, a process that normally require the action of Prl, did not require Prl because the transgene had taken over this function. Material and methods 2A - Organ culture

Abdominal mammary glands from 9 months old rPRL transgenic mice and control mice at gestation day 14 were dissected out and placed in ice-cold Hanks' balanced salt solution. After gently cutting the glands into small pieces, the explants were transferred into tissue culture dishes. As shown in Table 2A, a subset of cells were exposed to hormonal treatment; dexametasone 1 mM, insulin 5 g/ml and PRL 5 mg/ml.

After 7 days in culture the explants were processed for histological examination.

Table 2A

Results

After culture in presence of insulin and dexametasone, mammary gland explants from rPRL transgenic mice started to differentiate and to produce milk in contrast to normal mammary gland explants which do not differentiate in absence of PRL.

During co-culture between normal and rPRL transgenic mammary explants in the presence of insulin and dexameta- sone the normal explant starts to differentiate as well as the transgenic explants.

Conclusion

The expression of rPRL in the mammary gland from rPRL transgenic animals can substitute for PRL in the culture medium indicating the biological significance of the locally PRL expressed m the mammary gland. Such cells could be used m a procedure to screen for compounds that interfere with the actions of Prl.

2B - Establishment of cellmes from mammary tumors m rPRL transgenic mice

A mammary tumor from a rPRL transgenic female mice was dissected out and gently cut into small pieces. The pieces were either treated in Collagenase 8 mg/ 10 ml for 15 mm in 37 C or stayed untreated before cultured.

Dulbecco' s MEM nut mix F-12 medium was used with 15% FCS, fungizone 500 mg/1, gentamycm sulfate 50 mg/1, L- glutamme 2 mmol/1 and L-ascorbic acid 100 mg/1 n a nu- midified 5% C02 atmosphere at 37°C.

Results

Both cellines established produced rat-PRL detectable by RIA m the cell culture media and such cells are useful to discover new compounds e.g. by screening com- pound libraries, that inhibit or facilitate the action of the transgene.

Example 3 - Generation of transgenic animals that are "humanized" in terms of GH receptor expression A cDNA construction was made that contained the extracellular part of the human GH receptor and the mtracellular part of the rat GH receptor. A transgene was subsequently made that consisted of a metallothionem promoter, a signal sequence from human GH the extracellu- lar part of the hGH receptor fused to the mtracellular rat GH receptor at a common Nco 1 site. The exchange of signal sequence is of significance for its function in transgenic animals.

BRL (buffalo rat liver) cells were transfected witn an expression vector containing the GH receptor cDNA as described above together with a plasmid for neomycm selection. A single cell clone was selected and used for the reporter gene assay. A reporter gene consisting of STAT5 DNA response elements, a TK promoter and a lu- ciferase gene was transfected into the cells (using DOTAB, Boeringer Mannheim) after a 12h incubation, the media was changed and a subset of cells were exposed to GH during an over-night incubation. Luciferase activity was subsequently measured in cell extracts..

The techniques for cell transfection and the design of the GH reporter gene has been described by Wood et al (Wood et al. J. Biol Chem. 129: 9448-9453 (1995). The transgene construct was also used to generate transgenic mice. A 4.2 kbp fragment was cleaved, purified and injected into fertilized CBA/B6 oocytes . Transgenic offspring were identified by polymerase chain reaction (PCR) using primers complementary to the 5^' part of the metallothionem promoter and the 3 'part of the mtracellular domain of the rat GH receptor. 6 mice were found to contain the full length construct and 5 of these transmitted the transgene to offspring. Table 3 shows that this DNA construction activates a GH dependent reporter gene when stably transfected into BRL cells and similar results were obtained in COS cells (not shown) .

Table 3

Function of a GH receptor cDNA consisting of the extracellular part of the human GH receptor and the mtracellular part of the rGH receptor

Conclusion

These results show that non-human cells can be made to respond to GH through the expression of a human GH receptor cDNA variant and that such a gene can be incorpo- rated and inherited in transgenic animals. Alternative ways to achieve expression of human GH receptors is e.g. to use larger receptor constructs that contain the entire hGH receptor gene locus, e.g. on a PI or on a YAC clone (not shown) . The existence of these animals allow the testing of compounds that activate or inhibit the expressed receptor.

Claims

1. A method for the screening and identification of low molecular weight compounds which interact with lacto- genic/somatogenic receptors, characterized by the use of isolated tissues for in vitro cultivation as organ culture, primary cells, immortalized or transformed cells from transgenic non-human animal that over-express hormones/receptors belonging to the somatogenic or lacto- genie family.

2. A method according to claim 1 in which the transgenic non-human animal is used for screening and identifying of substances that influence the phenotypes induced by transgenic over-expression of somatogenic/lactogenic hormones or corresponding receptors

3. A method according to any of claims 1 or 2 in which the utilized tissues derived from transgenic non- human animals have been modified to; (1) withstand in vitro culture conditions and optionally (2) contain a new gene element, a reporter gene, that is characterized by an ability to react to signals activated by the transgene .

4. A method according to claim 3 in which the introduction of the new gene element could be achieved by cel- lular transfection or by cross-breeding of mice; one that express the reporter gene and the other that over-express the lactogenic hormone.

5. A method according to any of claims 1 to 4 in which cells are used to screen for substances that inter- fere with the expressed transgene or pathways activated by the same.

6. A method according to claim 5 in which end point measurement parameters are changes in cell proliferation, cell morphology, gene expression, cell signaling mole- cules, receptor internalization, effector molecules and/or reporter gene activity.

7. A transgenic non-human animal that over-express prolactin which cause phenotypic alterations notably in the prostate and/or mammary gland.

8. A transgenic non-human animal according to claim 7 model which over-expresses human GH receptors.

9. Use of a transgenic non-human animal according to any of claims 7 and 8 in a method according to any of claims 1-6.

10. Use of a transgenic non-human animal according to any of claims 7 and 8 for testing of inhibitory substances that reverse the phenotype induced by transgenic over-expression of prolactin.

11. Use of a transgenic non-human animal according to claim 8 in a method according to claims 1-6 for test- ing of substances that interfere specifically with the human receptor.

RECTlflED SHEET (RULE 91)