SYNDECAN ENHANCER ELEMENT AND ITS USE FOR TARGETING GENE EXPRESSION

Abstract: A syndecan enhancer element, novel proteins that activate the enhancer element, non-human transgenic animals comprising this enhancer element linked to a structural gene, and the use of this enhancer element to regulate the expression of syndecan and other genes are also provided. The enhancer element can also be used to target expression of a gene to wound sites.

Description: Syndecan Enhancer Element and Its Use for Targeting Gene Expression Field of the Invention This invention is in the field of recombinant DNA technology. The invention is directed to transcription regulatory elements associated with the syndecan- 1 gene, such as the FGF-inducible Response Element (FiRE) and to the use of such transcription regulatory elements for controlling gene expression. The FiRE can also be used to target expression of heterologous genes to injured tissue sites in vivo. Background of the Invention Cell surface proteoglycans play an important role in the regulation of cell behavior. Through their covalently bound glycosaminoglycan side chains, such proteoglycans can bind various extracellular effector molecules. One central challenge in proteoglycan biology is to understand the biological consequences which result from the binding of different effector molecules to cell surface proteoglycans. It is important to determine the intracellular responses triggered by effector binding and how these responses lead to altered cellular behavior. One way to investigate these matters is to create biological models which are dependent on the expression of specific proteoglycans. Syndecan- 1 is the best characterized cell surface proteoglycan. It was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan side chains. Syndecan belongs to a family of proteoglycans with conserved plasma membrane and cytoplasmic domains but with dissimilar ectodomains. The conserved structure of syndecans suggests that it could participate in signal transduction through the plasma membrane. International patent application WO 90/12033 discloses the amino acid sequence and corresponding cDNA sequence of the mouse syndecan- 1 molecule. Diagnostic methods for detecting transformed cells by detecting changes in the syndecan expression in transformed cells are described in International Patent Applications WO 92/13274 and WO 93/05167. International Patent Application WO 94/12162 provides methods for altering the level of syndecan within a cell by enhancing syndecan expression by inducing expression of the syndecan gene. This publication also discloses a proximal enhancer region of the syndecan gene comprising 2196 base pairs [SEQ ID NO: 1]. Wound repair is a complex process that requires the ordered expression of several genes. Peptide growth factors seem to play an essential role in the initiation and control of wound healing by regulating the chemotaxis, proliferation and migration of different cells. Growth factors involved in the reepithelialization process include, at least, the epidermal growth factor (EGF) family and the fibroblast growth factors (FGFs). EGFs are produced by several different cell types and act on epithelial cells and fibroblasts by stimulating their migration and cell division, while an FGF- family member, keratinocyte growth factor (KGF or FGF-7), is produced by fibroblasts but acts only on keratinocytes. Binding of EGF is induced in migrating keratinocytes in wounds and EGF has been shown to enhance wound healing. KGF is induced during wound healing, stimulates keratinocyte migration and has also shown to accelerate wound healing. Besides growth factors other extracellular signals, including disruption of cell-cell or cell-matrix contacts and the provisional matrix might contribute to the initiation of migration and reepithelialization. In recent years new insights have evolved on the roles of growth factors and matrix signals on wound repair. See e.g., The molecular and cellular biology of wound repair. Ed. R.A.F Clark. Plenum, New York, 1996. However, the mechanisms triggering gene expression in mammalian cells during these processes are not well understood. Several genes regulated by nuclear AP-1 factors are expressed during wound healing. Fos is rapidly activated on the wound healing edge, jun may also be activated during wounding or wounding induced tumorigenesis. Cancerous cells are also known to be able to activate the AP-1 complex, and c- fos is required for malignant tumor progression. The fact that cell surface proteoglycans can bind both growth factors and matrix components suggests that proteoglycans play a role in regulating, both temporally (timing of expression) and spatially (precise localization), growth promotion by immobilizing effector molecules to the vicinity of cell- matrix interactions. This is supported by the pattern of syndecan- 1 expression during development which follows morphogenetic, rather than histological, patterns and by the observation that syndecan expression is localized to sites of active proliferation. The mechanism behind this restricted expression of syndecan- 1 is, however, so far not known. Gene therapy is based on the concept of aiming the therapeutic effect at the source of a disease, i.e., at correcting the impaired genetic expression leading to altered cell function and thus disease symptoms. Originally gene therapy was contemplated as the therapy of choice for diseases caused by specific mutations, and pioneer trials were aimed at diseases such as cystic fibrosis, muscular dystrophy and ADA-deficiency. There is, however, an increasing amount of evidence that even acquired diseases often have a genetic component, which make them potential targets for gene therapy. To date, the majority of clinical trials for gene therapy are aimed at cancer and there is also a number of trial going on focusing on AIDS. Most of the gene therapy attempts to add a useful gene into a selected cell type to compensate for a missing or impaired gene, or to introduce a new property to the target cell. One of the main challenges in gene therapy lies in the construction of vectors that carry the therapeutic genes to the site of interest in the recipient patient, either by targetted "homing" or by regulatory control of expression. For a recent review of vectors used in different gene therapy approaches, see e.g., Runnebaum, Anticancer rest 17:2887-2890, 1997. Summary of the Invention The present invention is directed to the DNA encoding enhancer elements of the syndecan- 1 gene and to the use of such elements for regulating heterologous gene expression. More specifically the present invention relates to recombinant DNA constructs incorporating an enhancer element of the syndecan- 1 gene. Vectors and host cells which incorporate DNA sequences containing the syndecan enhancer are also encompassed by the invention. The invention also relates to cells into which the constructs have been inserted, whether these cells are of human or animal origin. Methods of expressing a standard or heterologous gene comprising activating the syndecan enhancer element are also provided. In addition, the invention is directed to a method for enhancing syndecan- 1 expression in a host cell by activating the enhancer element of the syndecan- 1 gene and to a method for stimulating cellular proliferation and differentiation, thus promoting tissue regeneration, especially in processes such as wound healing, by enhancing syndecan- 1 expression. More specifically this invention relates to an enhancer element of the syndecan- 1 gene having about 280 nucleotides. In addition, this invention relates to nuclear proteins that bind to the syndecan enhancer element causing enhancer activation, to the binding sites for these nuclear proteins on the syndecan enhancer, and to a complex of these nuclear proteins bound to the syndecan enhancer element. In particular embodiments of this invention, this nuclear protein is FIN-1, AP-1 , or USF. In more specific embodiments, one or more of these proteins are in an oligomeric form, such as a dimer. The invention further relates to a method of targeting expression of a gene product to injured tissue sites, such as cutaneous wounds, using a construct comprising the syndecan enhancer element operably linked to the gene of interest. Non-human transgenic animals comprising the syndecan enhancer operably linked to the gene of interest inserted into this germ line are also provided. Further features, objects and advantages of the present invention will become more fully apparent to one of ordinary skill in the art from a detailed consideration of the following description of the subject invention when taken together with the accompanying drawings. Brief Description of the Figures Figure 1. The assembly of mouse syndecan promoter region and the localization of the enhancer and suppressor elements together with restriction sites for three different enzymes. Figure 2. The nucleic acid sequence of the mouse syndecan enhancer region. Figure 3. A graphical presentation of how the enhancer element is active in growth factor treated 3T3 cells. The vertical axis represents percent expression (expression in untreated cells = 100%). Figure 4. Northern hybridization (NIH3T3) after exposure to FGF-2 (F6-F48). Syn-1 : mouse syndecan mRNA. GAPDH: loading control. Control samples, with no FGF-2 treatment: (C0-C24). Two individual exposures for each time point are depicted. Figure 5. Transfection constructs comprising different fragments of the regulatory region operably linked to the proximal promoter and the CAT gene. CAT activity by transient transfection in 3T3NIH cells with and without FGF-2 treatment. Figure 6. The FGF-2 inducible region from pXb6 further subdivided and assayed for enhancement of CAT expression. Figure 7. 3T3NIH cells exposed to FGF-2 for 4 and 24 hours followed by isolation of nuclei for a run-off experiment, c-jun and nur transcription was used as a positive control, and β-actin as a loading control. Figure 8. Effect of various growth factors on the activation of FiRE in epithelial cells. Panel A: CAT activity assay. Panel B: 125. UdU incorporation assay. Panel C: CAT activity assay (promoter removed). Figure 9A. DNAse I footprinting of the enhancer element. End-labeled pFiRE vector alone (naked) or together with FGF-2 induced (FGF-2) and non- induced (control) nuclear extracts. The products of the A+G sequence reaction of pFiRE are also depicted. Five footprinted motifs are marked with numbered boxes. Figure 9B. The sequence of the pFiRE fragment. The sequence of the footprinted motifs are underlined Figure 10. Gel retardation analysis for each enhancer motif. Nuclear extracts from FGF-2 treated (f or FGF) or untreated (c or control) 3T3 cells incubated with DNA containing motifs 1-5 as indicated. Figure 1 1. Competition test characterizing FiRE binding nuclear proteins. Panel A: motif 5. Panel B: motif 4. Panel C: motif 3. Figure 12. Schematic presentation of the binding of various nuclear factors to the syndecan enhancer element. Figure 13. Enhancer activation for deletion mutants of each motif measured by relative expression of the CAT reporter gene. Figure 14. Results of experiments to determine whether protein synthesis is needed for the induction of FGF-inductible transcription factors. Cells were treated with FGF-2 with our without translation inhibitor cycloheximide. Figure 15. Schematic model of two transgenic constructs containing 2.25 kb of syndecan- 1 basic promoter region (SynPr) in front of β- galactosidase gene, and an additional AP-1 -driven far upstream 280 bp enhancer (FiRE). A detailed structure of nuclear factor binding sites for FiRE is provided in the lower panel. Figure 16. FiRE activation analyzed by the Image Analyzer System. Means and standard deviations of five to eight independent experiments are shown. Figure 17. Effect of intracellular kinase inhibitors on FiRE activity. Means and standard deviations of five to seven independent experiments are shown. Figure 18. Effect of various growth factors on the activation of FiRE in keratinocytes. Definitions In order to provide a clearer and more consistent understanding of the invention the following definitions are provided. Gene targeting as used herein refers in general to approaches whereby gene therapy is targetted to specific cell types or tissue types in the recipient's body. Gene targeting can be achieved e.g., by vector constructs either "homing" to specific recipient cells only or by constructs capable of expression in specific cell types only. Gene therapy is used herein meaning techniques wherein a DNA sequence is provided to a recipient in order to treat, i.e., suppress or attenuate, an existing disease condition, or to provide a prophylactic therapy to individuals having inherited genetic mutations or somatic mutations leading to impaired gene expression. Operably Linked. In the context of an expression vector, "operably linked" refers to a location, juxtaposition, and/or orientation of various elements, e.g., enhancer, promoter, and structural gene, within an expression vector so that the structural gene is expressed in any appropriate host cell. When an enhancer is operably linked to a structural gene, expression of the structural gene is enhanced over the level of expression observed in the absence of the enhancer under conditions that induce enhancer activity. Syndecan Enhancer Element. In the context of this invention, "syndecan enhancer element" is a DNA sequence having significant structural homology to the sequence of Figure 9B and retaining the same functional properties as the sequence of Figure 9B, e.g., enhancing the expression of the syndecan- 1 gene by FGFs. For example, in certain embodiments of this invention, a syndecan enhancer element may retain 95% sequence homology to the sequence in Figure 9B, while retaining enhancer activity. In other embodiments of this invention, a syndecan enhancer element may retain 90% sequence homology to the sequence in Figure 9B, while retaining enhancer activity. Therapeutic gene as used herein means DNA encoding an amino acid sequence corresponding to a functional protein capable of exerting a therapeutic effect on a cell having impaired gene expression. The term "therapeutic gene" also encompasses genes encoding proteins having a regulatory effect on the expression of a function characteristic to cells in certain disease conditions. The following abbreviations are used in this specification: EGF, epidermal growth factor; ERK, extracellular regulated kinase; FGF-2, basic fibroblast growth factor; JNK, jun N-terminal kinase; KGF, keratinocyte growth factor; MAPK, mitogen activated protein kinase; MAPKK, MAPK kinase; PCNA, proliferating cell nuclear antigen; PKC, protein kinase C; PI-3 kinase, phosphatidylinositol 3-kinase; RTK, receptor tyrosine kinase; TGFα, transforming growth factor alpha; X-Gal, 5-bromo-4-cholo-3-indolyl-β-D- Galactopyranoside. The term "E-box" means a DNA sequence that binds basic helix-loop-helix transcription factors, such as c-myc. Detailed Description of the Preferred Embodiments The regulatory elements of a given gene are commonly located upstream from (i.e., 5 prime to) the transcription initiation site. Syndecan- 1, however, has a very peculiar gene structure, in which the first and second exons are separated by a very large intron. This could mean that, in addition to base sequences upstream from the transcription site, syndecan expression may also be susceptible to regulation by base sequences located in the first intron. In international patent application WO 94/12162 the enhancer element was shown to be located approximately 1 1.5 kb upstream from the transcription initiation site. This enhancer element is here further localized to a 280 nucleotide sequence, depicted in Figure 9B (SEQ ID NO.2). This sequence may also be linked in either orientation to recombinant constructs containing a promoter and a structural gene, including heterologous promoters or structural genes, to enhance recombinant expression in a regulated manner similar to that of syndecan- 1 expression in cells that express syndecan- 1. The 280 bp FGF-inducible response element is located 11.6 kb from the translation start site of the syndecan- 1 gene and shows at least about a tenfold activation in FGF-2 treated 3T3 cells. In mesenchymal cells this activation seems to be restricted to FGF-2. No response to serum or other growth factors tested was observed. Interestingly, FiRE also shows a distinctive specificity among the FGF family members, because FGF-1 did not activate or suppress this element. This suggests a separate signaling pathway and function for FGF-1 and -2 in mesenchymal cells. FGF-7 is a poor activator of mesenchymal cells and did not activate the enhancer. However, in cultured keratinocytes FGF-7 was able to activate FiRE. However, serum, PDGF, FGF-1 and surprisingly also FGF-2, failed to activate FiRE in cultured keratinocytes. In another epithelial cell line (NMuMG) FiRE was not inducible by any of the growth factors tested, which was expected, since NMuMG are FGFR negative cells. This indicates that the activation FiRE is cell specific, and in addition, is selective for different members among the FGF family in a cell dependent manner. FiRE binds both FGF inducible and non-inducible nuclear factors bound in an organized array. The FiRE nuclear factors include two AP-1 factors, which are homo- or heterodimers composed of the products from the fos and jun families. Members of the/05 a d jun families include c-Jun, JunB, JunD, c-fos, Fos-B, Fra-1 and Fra-2. Components of AP-\ , fos and jun, as well as other members of the fos and jun families, are rapidly activated by a vast amount of extracellular stimuli. Our results show that FGF treatment results in transcriptionally active fos-jun heterodimers. FiRE is bound by at least two AP-1 complexes, which both seem to be required for FGF induction, since deletion of one AP-1 site dramatically decreases the FGF response. FiRE also binds USF, which is a ubiquitously expressed homodimeric transcription factor. USF is constitutively expressed in 3T3 cells (Miltenberger et αl, Mol. Cell Biol. 75:2527-2535 (1995)). It is not known to be under influence of any growth factor. In FiRE, USF is not activated by FGF but it is involved in the complex with inducible components. Replacement of the E-Box does not, however, strongly reduce the FGF response on FiRE. This might be due to replacement of USF with another constitutively active transcription factor, which would be able to interact with inducible components. Alternatively, protein-protein interactions might hold USF in the FiRE complex although its optional binding target in DNA has been destroyed. However, the integration of the FiRE-reporter gene construct into the chromosome could still result in requirement for USF since these experiments were done with extrachromosomal plasmid DNA in a transient transfection. Besides AP-1 and USF the FiRE element also binds another non- inducible component as well as an FGF-inducible AP-2-like transcription factor. AP-2 is a cell-specific 50 kDa transcription factor expressed, among other tissues, in keratinocytes in high amounts. FIN-1 is a novel FGF- inducible nuclear factor that binds FiRE and can be competed with AP-2 consensus oligonucleotide. This factor is not recognized by AP-2 antibody, but could be a member of a larger AP-2 family. It is known, that AP-2 can form heterodimers without DNA binding and, that AP-2 dimerization is required for binding (Williams and Tijan, Science 257: 1067-1071 (1991 )). Thus, FIN-1 likely binds to FiRE as a dimer. FiRE can be considered a secondary reponse element requiring de novo protein synthesis after initial FGFR activation. This response element can further diversify FGF influence on different cell types. FiRE is the first example of this type of a response element. The binding sites for each of these nuclear proteins on the enhancer element have been mapped, and their sequence has been identified as described in Example V. See also Figure 9. AP-1 binds to motifs 4 and 5; FIN-1 binds motif 3; USF binds motif 2; and p46 binds motif 1. These binding sites constitute 100% of the enhancer activity. These binding sites on the enhancer element for FIN-1 do not contain any previously described consensus sequences for transcription factors. The FLN-1 protein has been isolated and has a molecular weight of 50 kDa as determined by SDS-PAGE. In addition to activating the syndecan promoter, this enhancer element has been shown to activate the SV40 promoter. The effect of the enhancer element on the SV-40 promoter is shown, with activation by the simultaneous administration of FGF-2 and TGF-β. Therefore, FiRE is not syndecan- 1 restricted but can be used with any suitable eukaryotic promoter, e.g., SV 40 and TK promoters. This will allow the use of FiRE in a wide range expression vectors, in viral vectors such as retrovirus and escpecially in adenoviral vectors, that are commonly used in different gene therapy approaches. Several growth factors were tested for the ability to activate the syndecan enhancer in pFiRE transfected 3T3NIH cells. FGF-2 showed clear enhancer activation. However, no significant enhancer activation was shown under these conditions with FGF-2, FGF-7, EGF, PDGF, or TGF-β. Other growth factors could be tested in a similar fashion. 5% fetal calf serum showed a slight stimulation of the enhancer. However, in cells expressing high levels of syndecan- 1, enhancement activation is only slightly evident. FCS, FGF-1 , FGF-2, FGF-7, EGF, PDGF, and TGF-β were also tested for an increase in DNA synthesis by a 125-IdU incorporation assay. All showed a significant increase in DNA synthesis.. This invention also relates to transgenic non-human animals containing the syndecan enhancer operably linked to a promoter and to a structural gene inserted into this germ line. For example, transgenic mice were made containing the syndecan enhancer operably linked to the syndecan- 1 proximal promoter and to β-galactosidase functioning as a reporter gene. Two plasmids were constructed, both containing a 2.25 kb Xhol-Stul restriction fragment comprising the syndecan- 1 basic promoter region ligated to the β-galactosidase gene. On of the plasmids contains an additional 280 bp Pstl-Stul restriction fragment containing the syndecan- 1 enhancement inserted next to the basic promoter. The plasmid constructs are described in detail in the experiments. The plasmids were transferred into pronuclei of fertilized mouse (FVB/N) oocytes by microinjection and the oocytes were transplanted into pseudopregnant mice. Other transgenic animals can be made by appropriately modifying these procedures. For example, rat and bovine transgenic animals can be made. In the transgenic animals containing the syndecan enhancer, reporter gene activity was localized to wounds. Therefore, the enhancer element according to the present invention is useful for the targeting and/or differential expression of the target gene to injured tissue, such as wounds. By linking the enhancer element according to the present invention to a gene of interest in an expression vector suitable for gene therapy the expression of said gene may be targeted to injured tissue. Suitable promoters that can be incorporated into these constructs include the syndecan- 1 promoter, the thymidine kinase promoter, the keratin-6 promoter, and the keratin- 16 promoter. In particular embodiments of this invention, the gene of interest codes for a therapeutic, and the expression of this therapeutic at the tissue injure sites can be accomplished using an expression system containing the syndecan enhancer. Examples of suitable genes of interest include growth factors, such as EGF, KGF, PDGF, TGF and TGFβ, matrix proteins, such as fibronctin, metalloprotease inhibitors and other factors participating in wound healing and tissue repair. FiRE may be considered as a healing-process related gene response element. Initial analysis of the human homologue of FiRE indicates a high similarity with non-human species. The enhancer element of the present invention may represent a tissue regeneration regulating gene element, which is highly conserverd during evolution. Whilst FiRE will be a highly useful tool for growth factor-induced and healing related gene expression studies, it might also have therapeutic applications. This could take place in, for example, poorly healing ulcerative wounds where FiRE-driven therapeutic agents, such as keratinocyte and fibroblast chemoattractants or mitogens, could be introduced into wound margin keratinocytes. The Examples below are for illustrative purposes only and are not intended to limit the scope of the invention. Example I Determination of Mouse Syndecan Promoter and Enhancer Activities. The mouse syndecan- 1 gene has been cloned and characterized up to -10 kbs upstream from the transcription start site. To determine the specific activities of different proximal promoter regions (up to -2 kbs from the start site) and enhancer regions (from -2 to -10 kbs) we have made plasmid constructs in which these regions were cloned into pCAT basic or pCAT promoter vectors, containing the CAT reporter gene. The reporter CAT gene produces the enzyme chloramphenicol acetyltransferase, which transfers the n-butyryl moiety of n-butyryl CoA to chloramphenicol. The n-butyryl chloramphenicol can be separated from native chloramphenicol by extraction with xylene. For the proximal promoter, a deletion series was made (Hindlll, Hindll, Bglll, Stul, Dral, Clal, BamHl and Pstl - Xhol) and the resulting fragments were cloned into the pCAT basic vector. For enhancer areas, three Xbal fragments were cloned into a pCAT promoter vector, where the SV 40 promoter was displaced by the Bglll - Xhol fragment from the syndecan promoter. The plasmid constructs were transiently transfected into eukaryotic cells by calcium phosphate precipitation simultaneously with a β-Galactosidase expressing vector to determine transfection efficiency. After a four hour incubation, cells were treated with 15% glycerol and grown for approximately 48 h in cell culture medium. Cells were then scraped from dishes in TEN- buffer and the cytoplasmic extract was obtained by repeated freezing and thawing. β-Galactosidase activity was obtained in the cytoplasmic extract by adding 0.1 M sodium phosphate, 45 mM mercaptoethanol and 0.2 mg O- nitrophenyl-s-galactopyranoside (ONGP). This was incubated from 2 hours to overnight and the color reaction was measured spectrophotometrically at 420 nm. CAT activity was determined by adding 0.25 M Tris buffer, 25 ng n-butyryl CoA and 0.0626 : Ci of ^C-chloramphenicol to the cytoplasmic extract. Samples were incubated overnight, extracted with xylene and the radioactivity present was measured by scintillation counting. CAT activity was corrected for transfection efficiency as determined by assays of β-galactosidase activity. The cells used for proximal promoter constructs were 3T3 NIH, SI 15 (either hormone-treated or not) and nMuMG cells. For enhancer constructs we used 3T3 NIH cells grown in 2% CMS medium and MCA 3D cells grown in Ham's medium. 3T3 NIH cells were also used to test the effect of growth factors in 2% CMS medium with 10 ng/ml FGF-2 and 2 ng/ml TGFβ- 1. MCA 3D cells were used to test the effect of 10 ng/ml KGF. By measuring the CAT activity present in transfected cells, the effect of inserted syndecan gene regions on gene transcription was determined. Results indicated that both a suppressor element and an enhancer element are upstream of the syndecan transcription initiation site. The suppressor element is located between 250 and 600 base pairs upstream from the transcription initiation site. The enhancer element is located between 8,600 and 9,300 base pairs upstream from the syndecan transcription initiation site. See Figure 1. Example II Activation of Syndecan- 1 Gene Transcription by FGF-2 in NIH3T3 Cells. To determine whether FGF-2 could alone activate the syndecan- 1 gene, 3T3NIH cells were exposed to 10 ng/ml of FGF-2 for 0, 4, 6, 8, 12, 24 and 28 hrs to test the activation of the endogenous syndecan- 1 gene. Following incubation with FGF-2, syndecan mRNA was isolated and quantified. Control incubations without FGF-2 were also performed. Syndecan mRNA was isolated and quantified without FGF-2 treatment following 6, 12, and 24 hr incubations. The quantification of syndecan mRNA production can be seen in Figure 4. GAPDH mRNA was used as a loading control. Based upon these data, syndecan expression is significantly enhanced (several fold) after 4 hrs of incubation with FGF-2. The enhancement appears to be maximal at about 6 hrs of FGF-2 incubation, and decreases thereafter. In the Figure, two individual exposures for each time point are presented. Nuclear run-off experiments revealed that this upregulation was transcriptional. The same cells were exposed to 10 ng/ml of FGF-2 for 4 hours and 24 hours with or without exposure to FGF (Control) followed by the isolation of nuclei for the run-off experiment, c-jun or nur transcription was used as a positive control and β-actin as a loading control. In nuclei isolated 4 hours after FGF exposure the level of transcription of the syndecan- 1 gene was elevated as were the levels for c-jun and nur as positive controls. The transcription of syndecan- 1 was not detectable after 24 hours, in agreement with the results of Northern hybridization. See Figure . This result suggests that the syndecan- 1 gene is a direct target for an FGF-2 induced signal. Moreover, this targeting can be modulated by signals produced by other growth factors, like TGF-β, which alone cannot activate the syndecan- 1 gene. Example III FGF-induced Transcription of the Syndecan-1 Gene is Based on the Activation of a Far Upstream Enhancer. In order to characterize the transcriptional elements responsible for syndecan- 1 expression following FGF-2 exposure, the 5 ' -region of the gene was cloned and sequenced 12 kb upstream from the TATA box. Various recombinant DNA constructs were made containing different fragments of the 11.5 kb syndecan upstream regulatory region together with the 1.1 kb segment containing the proximal promoter of the syndecan-1 gene. Plasmid pXbόCAT contains an Xbal fragment from -9.4 kb to -1 1.6 kb operably linked to the proximal promoter sequence, which in turn is operably linked to the CAT gene. Plasmid pXb4CAT contains an Xbal restriction fragment from -5.0 to -9.4 kb linked to the segment containing the proximal promoter, which is operably linked to the CAT gene. Plasmid pXb2/5 ' CAT contains an Xbal fragment from -2.4 kb to -4.4 kb linked to the segment containing the proximal promoter, which is operably linked to the CAT gene. Plasmid p-2.5 CAT contains a Hindlll-Xhol fragment from -1.3 kb to -2.4 kb linked to the segment containing the proximal promoter, which is operably linked to the CAT gene. Plasmid pSyn Prom CAT contains no enhancer segment, but contains the proximal promoter segment operably linked to the CAT gene. The structure of these plasmids is shown in Figure 5. In each of these constructs the distance between the potential enhancer activity and the Syn Pro segment is conserved; about 2.5 kb separates the fragments containing the potential enhancer from the SynPro segment. Each of these plasmids was transiently transfected into 3T3NIH cells, using the calcium phosphate precipitation method as follows. For transient transfections NIH3T3 cells were plated at equal density on six-well plates (Falcon) two days before transfection. Plasmid DNA was transfected into cells by calcium phosphate method. A β-galactosidase expressing plasmid (pSV-β- galactosidase, Promega) was co-transfected with CAT constructs to monitor transfection efficiencies. Three parallel transfections were used in every assay. Directly after transfection growth factors were added, media change the next day and cells were harvested after two days. CAT activities were measured by liquid scintillation and β-galactosidase activities spectrophotometrically at 420 nm as described by Vihinen et al, J. Biol Chem 265:17261-17269 (1993)). As a control, other samples of the cells transfected with each plasmid were treated with buffer without FGF-2 for the same time periods. Only plasmid pXb6 responded to FGF-2 treatment with an increase in CAT gene expression. See Figure 5. The cells transiently transfected with the other plasmids showed no significant difference in CAT gene expression with FGF-2 treatment and without FGF-2 treatment. These results indicate that enhancer activity is localized to within the region between -9.4 kb to -1 1.6 kb upstream of syndecan transcription initiation site. This region of the syndecan upstream regulatory region was further cleaved into smaller fragments and assayed similarly. See Figure 6. Specifically, plasmid pXSp2 contains a 1.14 kb Xbal-Sphl restriction fragment from -1 1.6 kb to -10.5 kb. Plasmid pXSpl contains a 1.05 kb Sphl-Xbal restriction fragment from -10.5 kb to -9.4 kb. Plasmid pXS2 contains a 0.36 kb Sphl-Earll fragment from -10.5 kb to -10.1 kb. pXB3 comprises a 0.30 kb Earl-Earl restriction fragment segment of the enhancer region from -10.1 kb to -9.8 kb of pXB6. pXXl is a 0.39 kb Earl-Xbal restriction fragment of the enhancer region from -9.8 kb to -9.4 kb of pXbό. pFiRE comprises a 0.28 kb Pstl-Styl restriction fragment of the enhancer region from -10.3 kb to -10.0 of pXb6. pFiRE REV contains the fragment of enhancer region from pXbό inserted in the opposite orientation. For the gene expression analysis the fragments of the syndecan-1 upstream region were subcloned from mouse genomic clones into the Xbal site of the pCATProm vector in which a mouse syndecan-1 promoter region (-1310 to +140) was fused to the CAT reporter gene (Vihinen et al, J. Biol Chem. 271 : 12532-12541 (1996)). The pX-HIIICAT plasmid was prepared by cloning Hindlll-Xhol promoter fragment (-2400 to +140 bp) into the promoterless pCAT basic vector (Promega). For pXSpl and 2 plasmids, Sphl fragments -1 1.4 to -10.5 kb and -10.5 to -9.4 kb, respectively) were deleted from pXbόCAT (-1 1.6 to -9.4 kb). Constructs pXS2, pXB3 and pXXl were generated by ligating S/?M-E rI/blunt (-10.5 to -10.1 kb), Earl/blunt fragment (-10.1 to -9.8 kb) and E rl/blunt-Xέ l (-9.8 to -9.4 kb) from pXSpl into Sphl- Ac T/blunt, Accl/blunt or Xbal-Sphl/blunt sites, respectively, into the pCATProm vector. Blunt ended fragments were made by T4 polymerase (Promega). For FiRΕ and FiRΕ rev a Pstl-Sϊyl/blunt fragment was subcloned into Pstl-EcoRV or Pstl-Smal sites of the pBluescript vector and then transfected to Xba-Sphl sites in pCATProm vector. These constructs were then used to transient transfect NIH3T3 cells as described supra, and CAT activity was measured. The 280 base pair element inserted into pFiRE retained full FGF-2 dependent enhancement of CAT expression. See Figure 6. This enhancement was also maintained when the 0.28 kb fragment was inserted in the opposite orientation (pFiRE REV ). In addition, plasmid pXSpl, which encompasses the sequence of pFiRE, also exhibited a FGF-2 dependent enhancement in CAT expression. These results localize the enhancer element to a 280 bp region from nucleotide -10,280 to nucleotide -10,000 with the first nucleotide at the translation initiation site numbered as +1. Example IV Effect of Various Growth Factors on Enhancer Activation in NIH3T3 Cells. Plasmid pFiRE was transfected into 3T3NIH cells as described. The cells were starved in DMEM medium supplemented with 5% fetal calf serum to about 70% confluence. Fetal calf serum was then replaced with 2% carboxy-methyl-Sephadex eluted fetal calf serum (CMS) 12 hrs before adding the growth factors. Various growth factors in the same medium, 5% fetal calf serum (FCS) supplemented medium or a control containing the medium, without any added growth factors were incubated with the transfected cells for 2 days, and CAT activity was measured. The effect of 10 ng/ml of FGF-2, FGF-1 , FGF-4, FGF-7 (KGF), EGF, IGF1 , TGFα, IFNγ, and PDGF and 2 ng/ml TGF-β were tested. The human recombinant growth factors were purchased from Peprotech (FGF-1 , -2, and -7), Boehringer (FGF-2) or from Calbiochem (PDGF) TGF-β was purchased from Sigma. The effect of the added growth factors and FCS on DNA synthesis was also tested by a 125-IdU incorporation assay. This cell proliferation assay was carried out by incubating cells for 4-6 hrs with 0.25 μCi/ml 5-[ 1 5 I]iodo-2 ' - deoxyuridine (IdU, Amersham), washed several times with PBS and solubilized in 1 M NaOH. Radioactivity was measured using a gamma counter (Wallac). The results of this experiment are reported in Figure 8. The results indicate that all of the tested growth factors significantly increase the level of incorporation of 125-IdU above that of the control, indicating that the growth factors were taken up by the transfected 3T3 NIH cells and were functional . Although all of the growth factors known to act on 3T3 cells stimulated 3T3 proliferation, FGFs were the only growth factors to clearly increase reporter gene activity. FGF-1 and FGF-4 had a lower effect those FGF-2. FGF-7, known to act only on epithelial cells, had no effect. Some FGF-1 preparations, from the different commercial sources, lacked the ability to activate FiRE, while still inducing cell proliferation. Only FGF-2 revealed clear enhancer activation. However, 5% FCS slightly stimulated the enhancer. In addition, 5% FCS only induced a small response despite containing several growth factors. PDGF, IGF and EGF enhanced cell proliferation to a similar extent as FGF-2, but had no effect on enhancer activity. Thus, stimulation of different tyrosine kinase receptors does not lead to activation of FiRE, nor does proliferation alone correlate with activation of FiRE. Consequently, FiRE cannot be regarded as a simple proliferation related target of growth factors. As the original construct contained over 1 kb of syndecan-1 proximal promoter, we wanted to rule out the possible suppressive action of this basal promoter on the function of the other growth factors. Therefore, nearly all of the promoter was deleted, leaving only 98 bp of the proximal promoter (p-271CAT), which included only the putative TATA-box without any upstream regulatory elements. As shown in Figure 8 the removal of the syndecan promoter had no effect on the pattern of growth factor induced FiRE activation, indicating that the growth factor specificity is not regulated by the proximal promoter. Example V The Enhancer Comprises Five DNA Binding Motifs. Plasmid pFiRE was end-labeled using Klenow fragment and α 22 PdCTP. DNAse I footprinting was performed with labeled pFiRE vector alone (naked), together with FGF-induced nuclear extracts (FGF-2), and together with non- induced nuclear extracts (control). To obtain nuclear extracts, NIH3T3 cells were plated on 16 cm dishes and treated with or without FGF-2 for 1 day. Nuclear protein were extracted using a modification described by Lee et al, Gene Anal. Techn. 5:22-31 (1988). Protein concentrations were measured by the Bradford method. For footprinting, pBluescript containing pFiRE was cut with Hindlll, end-labeled with [α 2 P]dCTP using Klenow DNA polymerase (Promega) and digested with Xbal. The labeled and PAGE purified DNA was incubated for 10 minutes at room temperature with about 40 mg of crude nuclear extract, 2 μg of poly-3[I-C] (Boehringer Mannheim) in a reaction buffer (10 mM TrisCl, pH 8; 5 mM MgCl 2 , 1 mM CaCl 2 , 2 mM DTT, 50 mg/ml BSA and 100 mM KC1) containing 0.1 or 1 unit of DNAsel (Boehringer Mannheim). The reaction was stopped after 2 minutes. A chemical G+A sequencing ladder was run along with the digestion products on a 6% sequencing gel. The results are shown in Figure 9. Five footprinted motifs ranging from 14 to 38 bp in length in close proximity to each other and covering a total of 170 bp were revealed and are marked with numbered boxes. Binding of nuclear factors to motifs 3, 4 and 5 appear to be FGF-2 dependent, whereas binding to motifs 1 and 2 appear to be FGF-2 independent. The A+G sequence of pFiRE was run alongside the footprinting ladders. Example VI Sequence of the FGF-2 Inducible Enhancer. The 280 bp fragment of pFiRE containing the syndecan enhancer was sequenced. This sequence is depicted in Figure 9B [SEQ ID NO. 2]. The footprinted motifs observed in Example V are underlined. It was determined that these footprinted motifs together restore 50-70% of the original enhancer activity. The sequence of motif 3, which contains the binding site for FGF-inducible nuclear factor FIN-1 , was compared to consensus binding sites for transcription factors. No significant homology between this motif and the consensus sequences was observed. Example VII The Enhancer is Bound by FGF-2 Inducible and Non- inducible Nuclear Factors. Gel retardation analysis for each enhancer motif (1-6) was performed with double-stranded oligonuceotides. The double-stranded oligonucleotides were end-labeled with [α- 32 P]dATP (ICN Biomedicals) by the polynucleotide kinase (Promega). The sequence of these oligonucleotides is: Motif 1: 5' -dGCTGGCACAC CCACCGTCAC GAGAGCTTCC-3' [SEQ ID NO. 3] Motif 2: 5'-TTGGCACACC TGGGAGGATG-3' [SEQ ID NO. 4] Motif 3: 5'-AGTGGTTCAG GGTGACTCT-3' [SEQ ID NO. 5] Motif 4: 5 ' -AGGAGTGAGC CATGCCACC-3 ■ [SEQ ID NO. 6] Motif 5: 5 ' -CTGGGTCATT GATGACTG-3 ' [SEQ ID NO. 7] In a 12 μl reaction, 5 μg of the nuclear extracts were incubated with labeled oligonucleotide, 2 μg of poly-d[I-C] and 2x reaction buffer, i.e., 30 min Tris, pH 7.5; 100 mM NaCl; 2 mM EDTA; and 10% glycerol for 15 minutes at room temperature. Non-labeled competitor oligonucleotides were used at 100-fold molar excess. The complexes were analyzed by electrophoresis in a 4.5% polyacrylamide gel. For supershifts, 0.1 μg of specific antibody was added to the reaction 15 minutes before the labeled oligonucleotide. Each motif element was treated with nuclear extracts derived from FGF- 2 treated (f or FGF) or untreated (C or control) 3T3 cells. An SPI consensus oligo was used as a control to test the functionality of the nuclear extracts. The results are given in Figure 10. Binding of at least two protein complexes for each motif were observed. The results indicate that enhancer motifs 3, 4 and 5 bind to a factor present in nuclear extracts from FGF-2 treated 3T3 cells. However, binding of enhanced proteins to motifs 1 and 2 were evident in stable FGF-2 stimulated and nonstimulated 3T3 cells. These motifs was further analyzed by competing the binding of nuclear factors with a molar excess of specific (s) and non-specific (ns) oligonucleotides. The non-specific oligonucleotide for motifs 3, 4 and 5 was the motif- 1 sequence, while the non-specific oligonucleotide for motifs 1 and 2 was the motif 3 sequence. The resulting complexes were analyzed by the gel retardation analysis described above. The results are depicted in Figure 10, B:motif 1, C:motif 2, D:motif 3, E:motif 4 and F:motif 5. Specific binding is indicated by an arrow. The result indicated that all motifs shifted at least one specific band in the gel retardation assay. The results also show that motifs 3, 4, and 5 bind at least one specific FGF-2 inducible nuclear protein. Motifs 1 and 2 do not show binding to an FGF-2 inducible factor. From these results it can be concluded that all 5 motifs are in use. Motifs 1 and 2 are occupied constitutively by nuclear proteins, while motifs 3, 4, and 5 are occupied only after FGF exposure of 3T3 cells. Example VIII Deletion Mutants of FiRE. To determine whether all binding sites are required for enhanced activation, deletion mutants for each motif were generated by PCR. For motif 1 , the 3 ' end of FiRE was deleted by generating a PCR product ranging from the 5' end of FiRE to motif 2. For motif 2, the E-Box was replaced by a Kpnl restriction site. For motifs 3 and 4, a central 10 bp sequence was replaced with a 10 bp Spel restriction site. For motif 5, a PCR product from motif 4 to motif 1 was generated. The mutant DNA was ligated into a CAT reporter plasmid as described supra. Cells were treated with FGF-2 and CAT assays were performed as described. The results of this experiment are depicted in Figure 13. The modification of motifs 1 , 3, 4 and 5 resulted in the loss of a significant reduction of FiRE activation. However, the modification of motif 2 has little or no effect on transcriptional activation. This result supports the involvement of 4 of the 5 motifs in the activation of FiRE by FGF. Furthermore, it shows that at least two fos-jun complexes (AP-1) are required, but alone they are not sufficient to activate FGF induced transcription. To determine whether the different inducible components of FiRE is due to direct post-translational modifications or whether they are newly synthesized, simultaneous cycloheximide and FGF treatment and subsequent protein extraction was performed. As shown in Figure 14, the FGF-induced binding of AP- 1 s to motifs 4 and 5 as well as the binding of FIN- 1 to motif 3 was blocked by cycloheximide. This indicates that all inductible proteins involved in FiRE require de novo protein synthesis, and that FiRE represents a secondary response element in FGF initiated signalling. Example IX Characterization of FiRE Binding Nuclear Proteins. The nucleotide sequence of FiRE was compared to known sequences of different transcription factors stored in the Transcription Factor Database (TFD Sites). Motif 2 showed significant homology to a known transcription factor binding consensus site: an E-box. Motif 4 contained an AP-1 -like consensus binding site with one mismatch, and motif 5 has two AP-1-like consensus sites. Motifs 1 and 3 do not contain known consensus sequences for transcription factors. Various commercial consensus oligonucleotides, including AP-1 , AP-2, AP-3, Ets, GATA, SIE, CRE, MEF-1, MEF-2, Max-Myc, SP-1, NFkB, and cEBP were used to test for competition with the nuclear factor- enhanced binding in order to reveal possible protein binding outside the established consensus sequence. The result of this experiment is reported in Figure 1 1. A Max-Myc consensus oligonucleotide was able to abolish binding of nuclear factors to motif 2. (Figure 1 ID). AP-1 consensus oligos were able to compete binding of nuclear factors to motifs 4 and 5. (Figure 1 1A and 1 IB). Binding of nuclear factors to motif 3 were unexpectedly abolished by an AP-2 oligonucleotide even though there is no AP-2 consensus site on motif 3. (Figure 11C). Binding of nuclear factors to motif 1 could not be competed with any of the consensus oligonucleotides tested. These results indicate that motifs 4 and 5 bind on AP-1 complex, motif 3 binds on AP-2 complex, motif 2 binds a basic helix-loop-helix (bHLH) factor such as myc/mad, and motif 1 binds an unknown nuclear factor. In order to further confirm the results obtained from the competition experiments, the effect of adding specific antibodies to the gel retardation reactions was tested. Antibodies to c-fos and c-jun were able to remove specific binding to motifs 4 and 5 and to produce supershifts. Anti-USF or anti-ATF-3 antibodies analyzed at the same time, had no effect. Thus, the nuclear factors that bind to motifs 4 and 5 are AP-1 fos and jun proteins. The effect of an AP-2 on binding to motif 3 was tested. While the AP-2 consensus oligonucleotide was able to abolish binding to motif 3, the AP-2 antibody had no effect (Figure 1 IC). The ability of this antibody to bind antigen was tested with labeled AP-2 oligonucleotide using FGF-2 induced 3T3 cell nuclear extracts. It produced a supershift, showing that it was functional. Thus, the protein that binds motif 3 is an AP-2 related transcription factor, which is distinct from AP-2. This protein has been named FIN-1 for FGF inducible nuclear factor. For motif 2, neither factor Max or Myc antibodies had obliterating or supershifting effects. However, another bHLH protein, USF, is known to be constitutively expressed in 3T3 cells (Millerberger et al, Cell Biol 5(5):2521- 2535 (1995)). USF was shown to occupy motif 2 on USF antibody removed the bond specific for binding to this motif without affecting the binding to other motifs (Figure 11D). Example X Mass Analysis of the Enhancer Binding Nuclear Factors. A gel retardation experiment as described in Example VII was carried out, and the gel containing the oligonucleotides bound to proteins was exposed to UV light to crosslink the protein to the oligonucleotide. Specifically, the gel mobility assays were run as described supra. After the gel was run, it was exposed to 245 nm UV-light (3600J/em^) in a Strategene crosslinker. The gel was exposed for several hours, the specific bands were cut out, eluted overnight at 4EC, precipitated with ethanol, resuspended in Laemmli buffer, denatured at 95EC for 5 minutes, and loaded onto a 10% SDS-PAGE together with a Relabeled molecular weight markers to analyze their molecular weights. The SDS-PAGE gel is shown in Figure 1 1C, with the position of the molecular weight markers shown at the left. Lanes 1-5 correspond to motifs 1-5, respectively. The molecular weights of the nuclear factors were estimated after subtracting the mass of the oligonucleotide from the complex mass as indicated below: Motif MW Oligo +Factor MW Oligo MW Factor 1 66 kDa 20 kDa 46 kDa 3 62 kDa; 90 kDa 12 kDa 50 kDa; 78 kDa This experiment shows a reproducible 46 kDa band for motif 1 and two bands, 78 kDa and 50 kDa, for motif 3. These values have a margin of error of about ±3 kDa. Example XII Targeting a Reporter Gene (lacZ) to Healing Wounds of Mouse Skin Using the FiRE Enhancer. To demonstrate the utility of the FiRE enhancer in targeting a desired gene product to sites of healing wounds, two separate transgenic mouse strains harboring a reporter gene (Lac-Z) ligated to the syndecan- 1 promoter regions were produced: the first with 2.25 kb of the syndecan-1 basic promoter region (SynPr) in front of the β-galactosidase reporter gene, and the second with an additional far upstream 280 bp enhancer element (FiRE) positioned immediately upstream of the SynPr-LacZ gene (Fig. 15) . The SynPr construct carries a Xhol- Sphl fragment in front of a reporter gene in the Lac-Z vector. The FiRE construct is the same, but has the 280 bp far upstream (from -10 kb) FGF inducible element (Pstl- Sty I) next to the SynPr fragment. Constructs were released from vector sequences by Notl digestion, separated on agarose gels, electroeluted and dissolved into the injection buffer. Transgenic mice were generated by microinjecting (C57B1/6 * DBA/2)F1 fertilized eggs using standard methods. Founder animals were identified from tail DNA by PCR analysis using Lac-Z specific oligonucleotides. Subsequently, after the initial experiments the FiRE strain mice were tested by β-galactosidase enzyme expression in a tail wound. Because FiRE was shown to be growth factor dependent in fibroblasts and because several growth factors are known to participate in wound healing, we targeted our investigations to wounded skin, where syndecan-1 expression is also known to be upregulated. The experimental procedures were approved by the ethics committee of the University of Turku. For these wound healing studies from 8 to 12 week old mice of both sexes were anaesthetized by 2,5 % Avertin, the hair was cut and full thickness incisional wounds were made with a scalpel and left unsutured and uncovered. Wounds were equally made to the skin of tails, a piece of the tail was cut after 48 h, fixed in 4% paraformaldehyde for 30 min., washed and stained overnight with X-Gal. Dermal cells did not show Lac-Z activity whereas in all twelve mouse lines that were DNA positive with the FiRE construct, Lac-Z activity was found at the wound sites in contrast to the six DNA positive SynPr mouse lines which illustrated no staining. The intact skin or several other adult organs did not show any consistent staining in either SynPr or FiRE strains. However, during embryonic development both FiRE and SynPr strains showed nonspecific Lac-Z activity, for example, in neural tissue and limbs, demonstrating that the quiescence of the SynPr strain during wound healing was not due to its chromosomal inactivation. Several time points, from 4 hours up to 15 days, were selected to study the timing of FiRE activation. During the early inflammatory phase (from 4 to 18 hours) no Lac-Z activity was seen. However, at 20 to 24 hours post- wounding, β-galactosidase staining, indicating activation of FiRE, appeared at the wound edge. The staining was most abundant from two to three days and started to decline at day 7. To reveal FiRE activation on different stages of wound healing, consecutive sections of a single tail wound were produced. In this model a cross-section from the middle of the incision shows a situation where the keratinocyte migration is just starting, while sections from the ends of the incision present already reepithelialized keratinocyte sheet. Staining was first seen in the stratified epidermal sheet of the keratinocytes nearest to the incision site starting downwards migration. In the more advanced phase FiRE activity was very strong at the leading edge of the migratory keratinocytes and also in the merging epithelium. During subsequent stratification of the epithelial cells FiRE activation was still evident but, at the end of reepithelialization the staining started to decline, as demonstrated by the most distal section of the tail wound. The epidermis further apart from the incision site, as well as the dermal cells and the wound clot, remained negative for beta- galactosidase activity throughout the repair process. During the later phases when dermal integrity was being re-established and the epidermis still maintained a hyperproliferative state, FiRE was not reactivated. In the hair follicle keratinocytes adjacent to the incision site, FiRE was also activated. These cells, as well as the wound edge keratinocytes, are supposed to be starting sources for keratinocyte migration. FiRE is activated in migrating but not in dividing cells and the activation precedes appearance of syndecan-1 protein. The relationship between FiRE activity and cell proliferation was investigated by staining the FiRE wound sections with a proliferating cell nuclear antigen (PCNA) antibody. Tissues were fixed in 4% formalin and dehydrated in ascending concentrations of ethanol and embedded in paraffin. Tail pieces were treated with 4% formic acid for 2-4 days before embedment. Microtome sections (5 μM) were mounted on glass slides treated with poly-L-lysine. Standard techniques were used for hematoxylin- eosin staining. For immunohistochemistry PCNA (Novocastra) and syndecan-1 recognizing 281-2 primary antibodies and subsequent avidin-biotin-peroxidase complex (ABC) techniques (Vectastain, Vector laboratories) were used. During the early time points, e.g. at day 1 , when FiRE activity appears at the wound margin, PCNA staining was found to locate more distally, including the basal keratinocytes, the hair follicle keratinocytes and the glandular epithelium. At a more advanced migratory phase (days 2 and 3) cell proliferation was more marked in the basal layer, yet in totally different cell pool than FiRE activity (not shown). In the fully reepithelialized wound at day 7, where robust cell division was found in the basal keratinocytes and in the stratum spinosum of the thickened epidermis, the β-galactosidase staining was detected only in few cells of the differentiated granular layer. To compare FiRE activation with the appearance of syndecan-1 protein at the cell surface, previously shown to be upregulated in migrating keratinocytes, the wound sections were double stained for X-Gal and for 281- 2, an antibody that specifically recognizes mouse syndecan-1. The epidermal sheet on a 1 day wound was negative or showed low expression of syndecan-1 protein at the sites of FiRE activation. In a totally reepithelialized 10 day wound, FiRE staining was already absent but the hyperproliferating epidermis was still heavily stained by 281-2. This demonstrated that activation of FiRE precedes the appearance of syndecan-1 protein but might not be required to maintain high syndecan-1 expression. Activation of FiRE is inhibited by suramin and genistein. In order to asses the molecular mechanisms inducing FiRE in wounded epithelium, we tested chemicals known to inhibit activation of cell surface receptor tyrosine kinases (RTKs) by growth factors. Suramin is a well characterized heparin analog that blocks the binding of several growth factors to their receptors. It has been shown to have some retarding effects on wound healing without inhibiting reepithelialization. Genistein, on the other hand, is a specific inhibitor of tyrosine kinases, which among other tyrosine kinases blocks the growth factor receptors. The ability of these chemicals to inhibit FiRE was assayed in a model where mice tails were cut into slices following culturing with or without the inhibitors. For these organ culture studies mice were sacrificed and approximately 1 cm^ skin pieces, after hair removal, or approx. 1/2 cm consecutive tail pieces were cut with a scalpel. The skin and tail pieces were maintained in cell culture conditions in DMEM supplemented with 10% FCS or 2% CMS (carboxy- methyl-Sephadex eluted fetal calf serum) in a 24-well plate for one to two days. Growth factors were purchased from PeproTech and tested compounds from CalbioChem or Sigma. SB 203580 was a kind gift from SmithKline Beecham. FiRE activation was evaluated as the appearance of Lac-Z activity on the healing surface of the tail pieces. The activation was evaluated as the proportional area of β-galactosidase staining on the healing surface of cut tails and analyzed by the Image Analyzer System. In each experiment the β- galactosidase activity in the chemical treated wound edges was compared to the non-treated (control) wounds. Both suramin and genistein inhibited FiRE activation in a concentration dependent manner (Fig. 16). 100 μM suramin reduced the staining intensity to approximately 30 % and with 300 μM suramin the activation of FiRE was almost totally blocked. The tyrosine kinase inhibitor genistein was also clearly found to inhibit FiRE at 30 μM, the enzyme activity was reduced to approximately 10 % at 75 μM and was totally abolished at 150 μM. In contrast to growth factor receptor and tyrosine kinase blocking agents, the tyrosine phosphatase inhibitor vanadate had only a marginal effect on FiRE activity at 100 μM. As judged by histological sections none of the chemicals completely prevented keratinocyte migration. These data suggested that activation of FiRE in migrating keratinocytes might be dependent on growth factor induced RTK activation. Simultaneous growth factor and anisomycin treatment activates FiRE in resting skin. Since introducing various growth factors into the media of cultured non-wounded skins did not activate FiRE, we considered the possibility of a requirement for other signaling cascades besides those triggered by growth factors. As the Raf/ MAPKK1/ ERK cascade is one well established pathway known to be induced by growth factors we in addition stimulated the stress activated JNK/SAPK and p38 MAPK signaling pathways by anisomycin and UV irradiation. Pieces of skin were tape-stripped, which is known to induce at least the EGF receptor and treated with a combination of growth factors (a cocktail of EGF, TGFα and KGF) and anisomycin or UV- irradiation in organ culture. FiRE was not activated if anisomycin or growth factors were given separately to the culture medium. However, treatment of the skin simultaneously with the growth factors and anisomycin resulted in the activation of FiRE in the keratinocytes of non-wounded skin. Equivalent results were obtained by treating the skin pieces simultaneously with UV- irradiation (80 J / m ) and growth factors. Okadaic acid, known to activate several intracellular kinases and subsequently fos and jun proteins and AP-1- driven promoters by inhibiting protein phosphatases 1 and 2A, was alone a potent activator of FiRE in resting skin. The skin of SynPr strains was not inducible by any of these treatments. The results suggested that the activation of FiRE depends on two separate signaling pathways: i) of growth factor induced RTKs and ii) a stress induced pathway. Inhibiting either ERK or p38 MAP kinases blocks FiRE activity. To further investigate the signal transduction pathways downstream from the cell surface we used compounds known to specifically inhibit different intracellular kinases. PD 098059, a chemical that inhibits activation of MAP kinases ERK- 1 and -2 by MAPKK 1 at micromolar concentrations was able to reduce activation of FiRE at 5 μM and totally block it at 20 μM, implying involvement of MAP kinases in this activation. A specific inhibitor of stress induced p38/RK MAPK, SB 203580, shown to be selective for p38 MAPK at least up to 100 μM also acted as an inhibitor of FiRE in a concentration dependent manner. The staining was clearly reduced at 10 μM and abolished at 40 μM concentrations. In contrast to these two compounds neither the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, even at 2000 nM concentration, nor the p70 S6 kinase inhibitor rapamycin, at 100 nM, had any effect on the wound induced activation of FiRE. Likewise, the PKC- specific inhibitors, bisindolylmalameide (1.2 μM) and chelerytrin (5 μM), failed to inhibit FiRE activation. As well as their inhibitory effect on wounding induced Lac-Z activity, pretreatment with either PD 098059 or SB 203580 was able to abolish okadaic acid, while wortmannin up to 5 μM had no inhibitory effect. These results, summarized in Figure 17, suggest that though the ERK and p38 pathways might be mandatory for transcriptional activation of AP-1 regulated genes in migrating keratinocytes, the PI-3 kinase or the typical PKCs may not be necessary. Example XIII FiRE is Induced by EGF, TGFa and KGF in Keratinocytes. The ability of FiRE to enhance transcription was tested in the mouse keratinocyte cell line MCA3D (Kuliz-Martin et al, Carcinogenesis 4: 1367-1377 (1983)). MCA3D cells were transfected as described supra for mouse NIH3T3 cells. Following transfection, the cells were treated with fetal calf serum, TGFα, PDGF, EGF, FGF-1 , FGF-2, and KGF as described supra for the NIH3T3 cells. Surprisingly, FGF-2 only slightly activated FiRE although it stimulated cell proliferation. Serum, PDGF, and FGF-1 did not activate FiRE. However, KGF, EGF and TGFα strongly activated FiRE in these cells. See Figure 18. Moreover, syndecan-1 mRNA expression is strongly induced by KGF. This shows that FiRE can be selectively activated by different members of the FGF family in different cell types. The SynPr construct showed no activation with any growth factors when transfected into MCA3D mouse keratinocytes. In another epithelial cell line derived from mouse mammary gland epithelium (NMuMg), which is negative for FGFRs, none of the tested growth factors had an effect on the FiRE construct. This indicates that FiRE is both cell specific and activated through FGFRs. These examples can be extrapolated to other gene products, which have replaced the lacZ-gene to target expression of these genes to wound sites as a therapeutic means. All documents, e.g., scientific articles, cited herein are fully incorporated herein by reference. Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof. SEQUENCE LI STING ( 1 ) GENERAL INFORMATION: ( i) APPLICANT : (A) NAME: OY BIOTIE THERAPIES (B) STREET: TYKISTONKATU 6, BIOCITY (C) CITY: TURKU (E) COUNTRY: FINLAND (F) POSTAL CODE (ZIP) : FIN-20520 (G) TELEPHONE: +358-2-2748900 (H) TELEFAX: +368-2-2748910 (A) NAME: JALKANEN, Mar u (B) STREET: RAUVOLANTIE 79 (C) CITY: TURKU (E) COUNTRY: FINLAND (F) POSTAL CODE (ZIP) : FIN-20760 (A) NAME: JAAKKOLA, Panu (B) STREET: KELLONSOITTAJANK 13 B 20 (C) CITY: TURKU (E) COUNTRY: FINLAND (F) POSTAL CODE (ZIP) : FIN-20500 (A) NAME: VIHINEN, Tapani (B) STREET: KASKENKATU 11 C 54 (C) CITY: TURKU (E) COUNTRY: FINLAND (F) POSTAL CODE (ZIP) : FIN-20700 (ii) TITLE OF INVENTION: Syndecan Enhancer Element and Its Use for Targeting Gene Expression (iii) NUMBER OF SEQUENCES: 7 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 760534 (B) FILING DATE: 02-DEC-1996 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2196 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCTAGAACAC TTATTAAGAG CCAGGCACTG AAAAGTGCAG ACTCCCTCAT TTCATCCTGG 60 CCGTGCTTAC AAGTAGTTTC CATGCTCTGG TAACCCTGTG CAGAGGGCAG CGTGGGAGGC 120 GGGCCGCTTG GTGGACGGTC ATGGGGGCTC TGCATGGGTG GTTGCCCTTG CCTCAGAAGA 180 ACTCCCTAAG TAAGAGCAAG TTAGCCTCCC TAACCCCTGG TGGGTTGTTG CTTCTTTTCT 240 CCTCTTGTTT CTGCCAAGAG AGGGTGGACC AAGAAGACCC CAGCCTACAG AACATGTGAT 300 CCAAATAAAC TTCTTTTTAG TATAAATGTC CTAGCCTGTG ACGTTCTGGT AGACTAGCAC 360 AAGATGGACC AAGACAACTC TCATCGAGAC TCTGAGGAAC GAACTGGCAT CACATGGGAA 420 CAGGAAATGA AGCTTAGAGA GAGGTTCTGT GGCTTGTCCA ACATGGCTGT AGTTTAAATC 480 CAGCTTGCCA CCAAAGCACA CACATTTCAC TGCTGTGCTG GGCCGGGCCT CAGATCCCAG 540 GGGCTCCGGA GCTAGAAGGA CACGTGTATC AGCCATGGCT TCAGTTTATT GCTGTATACT 600 CTGTGCTTCT GGCTCTCATG GAAAAGACAG ACATTGGGGT TCTTATAATC TCTCCCTCTC 660 CCCTCCCCAC ACTCTATCCC CAAAGGAGGC ACCACTTCTG CAGGTAAATG TTATCTTCAA 720 AGCGCTCACA TCGCAACCTT TGCCCACACC ATCTCATTAA AGGAATTGGC AGTGACTTTA 780 AGGTGAAAGA ACTCGGTGGC TACGTGTTAT ATAAATTTGC ATCTGGGTCT CAGAGCTGGA 840 AGGAAGGCAC TCCCATACAT GCAGTCTGTA CATGCAGTCG GATGATGGAC CAACAACACA 900 TTGTGATTTA TGCCCCTGCT GGTGAGCCCA GGAATCCCTG TAGCACTCTC TCTCAGCTCT 960 AGGGCCCTGC TTGTGTATGG AAAACGCTTA GTGTTTTATA GGTATTTTGT CAGAATACTT 1020 TAAGGAACTT GACCAAAGTT ACAGGGAGGT TAGACAGATT GTCATGGTAT ACTCACCTCT 1080 GTCTCTGACC CTCCTAACTG GGACCTCTTT AGTCTCCCTT GAGGCAGGGA GTGCCACATG 1140 CATGTGTCCA GGCACATGTC TCCTGGTTTA CCTCCCAACG CACCTCAAGT CCCCAAGGTA 1200 GGTAGGCACT TGTATTCTGT AATTCAGAGA GGCAAATCAA ACTGTTACAA TGTTTGCCCA 1260 AAGCTCCCCA AGCAAAGTGG CCCTAAGAGT GAGCAAAGAG ACTGCGTGCC TTCACTGCCT 1320 GTGTGAATCC CTGCAGATAG TCTCTCATCT TGGTGCCCTT CCCACAGAGG CTGGGGCGGC 1380 AGGAGGGAGC CTGGACAGCT CAGACACTGG GTCATTGATG ACTGTTGTGT GGGATACCTG 1440 CCGGGGCGCA GGAGTGAGCC ATGCCACCCC AGGAAGTGGT TCAGGGTGAC TCTTCTTGGC 1500 ACACCTGGGA GGATGTAGCT GGTGCTGGCA CACCCACCGT CACGAGAGCT TCCTGTCCAA 1560 ACCTTCAACA AAGGCGGCTT CTTGAGACAG GCTAGACTGA AGTCACCAGC CTTGGGTGGG 1620 GTCCACTATG TAACCTCAGT GCTCAGGAAC CCTTTCCCAT ACTGTCTGGA ACTATACTGT 1680 ATGTAGCTGG GTTTCCACGC ATGTGTGCCT GCACCCAGTC CATCTCATCT TCTATCTCCC 1740 TCCCCTTTCC CGCTTCCCCC CTCCCCACTC TCCATCTCAT CTTCCATCCC CACCTCTTCT 1800 GGTCCCTGCC CTGCTAAACT CAGGGTAGCT GCATTCCGCT GGCCTTCCCC ATGTTCCAGG 1860 CTTCAGTCCC TTCTCTGCAC CTGTCCTTTG TGAAGTGACC AGAGGATTTC TGATCCTGTC 1920 TCTGTCGCTC TGAAGGGTCA GGAGTTCCTC CTGCCTGGAC AAAGCCATCC TGACGCACAT 1980 AAATAAAACA AACATCAAAC TCTATTCAAC CCCCTGGAAC CCGTGTGTGT TACTTACAGG 2040 GCAAAAGAAT GGAGCAGGGG ATGGGTTGTG GGGGGGGGGG GTGGCATCTG GGTTGTCTAC 2100 AGTTGTGCAT TAAGTTGTAA TTAAGATGTG CATTTCTCCA AATAAGGGAA AATTATTCTG 2160 GATTATTTGA GTGAAGCTGA AAGGTGATCA TCTAGA 2196 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 283 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TGCAGATAGT CTCTCATCTT GGTGCCCTTC CCACAGAGGC TGGGGCGGCA GGAGGGAGCC 60 TGGACAGCTC AGACACTGGG TCATTGATGA CTGTTGTGTG GGATACCTGC CGGGGCGCAG 120 GAGTGAGCCA TGCCACCCCA GGAAGTGGTT CAGGGTGACT CTTCTTGGCA CACCTGGGAG 180 GATGTAGCTG GTGCTGGCAC ACCCACCGTC ACGAGAGCTT CCTGTCCAAA CCTTCAACAA 240 AGGCGGCTTC TTGAGACAGG CTAGACTGAA GTCACCAGCC TTG 283 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGGCACACCC ACCGTCACGA GAGCT 25 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGGCACACCT GGGAG 15 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TGGTTCAGGG TGACT 15 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AGGAGTGAGC CATGCCACC 19 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CTGGGTCATT GATGACTGTT GTGTGGGATA CCTGCCGGG 39

Claims: Claims 1 . A recombinant DNA molecule comprising a syndecan enhancer element, wherein said syndecan enhancer element has the sequence depicted in SEQ ID NO. 2. 2. A recombinant DNA molecule comprising a syndecan enhancer element derived from the regulatory region upstream from the syndecan transcription initiation site, wherein: a) said syndecan enhancer element is less than or about 280 nucleotides in length; b) said syndecan enhancer element comprises binding sites for nuclear proteins FLN-1, AP-1 (jun and fos family members) and USF. 3. The recombinant DNA molecule as claimed in claim 2, wherein said syndecan enhancer element comprises each of the sequences depicted in: a) SEQ LD NO. 3; b) SEQ ID NO. 4; c) SEQ ID NO. 5; ) SEQ ID NO. 6; and e) SEQ ID NO. 7. 4. The recombinant DNA molecule as claimed in claim 2 wherein said syndecan enhancer comprises at least one sequence selected from the group consisting of one or more of the following oligonucleotides: a) TGGCACACCCACCGTCACGAGAGCT [SEQ ID NO. 3] ; b) TGGCACACCTGGG AG [SEQ ID NO. 4] ; c) TGGTTCAGGGTGACT [SEQ ID NO. 5] ; d) AGGAGTGAGCCATGCCACC [SEQ ID NO. 6] ; and e) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG [SEQ ID NO. 7] . 5. The recombinant DNA molecule as claimed in claim 2, wherein said syndecan enhancer is FGF-2 inducible. 6. The recombinant DNA molecule as claimed in claim 2, wherein said syndecan enhancer is FGF-7, EGF and/or TGFα inducible. 7. The recombinant DNA molecule as claimed in claim 2, wherein said syndecan enhancer element binds FIN-1. 8. The recombinant DNA molecule as claimed in claim 2, wherein said recombinant DNA molecule further comprises a promoter operably linked to a structural gene. 9. The recombinant DNA molecule as claimed in claim 8, wherein said promoter is not the syndecan promoter. 10. The recombinant DNA molecule as claimed in claim 8, wherein said promoter is the SV40 promoter or the TK promoter. 1 1. A DNA molecule selected from the group consisting of one or more of the following oligonucleotides: a) TGGCACACCCACCGTCACGAGAGCT [SEQ ID NO. 3]; b) TGGCACACCTGGGAG [SEQ ID NO. 4] ; c) TGGTTCAGGGTGACT [SEQ ID NO. 5] ; d) AGGAGTGAGCCATGCCACC [SEQ ID NO. 6]; and e) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG [SEQ ID NO. 7] . 12. An expression vector comprising a syndecan enhancer element derived from the regulatory region upstream from the syndecan transcription initiation site, wherein: a) said syndecan enhancer element is less than or about 280 nucleotides in length; b) said syndecan enhancer element comprises binding sites for nuclear proteins FLN-1, AP-1 (jun and fos family members) and USF. 13. The expression vector as claimed in claim 12, wherein said syndecan enhancer element comprises each of the sequences depicted in: a) SEQ ID NO. 3; b) SEQ ID NO. 4; c) SEQ ID NO. 5; d) SEQ ID NO. 6; and e) SEQ ID NO. 7. 14. The expression vector as claimed in claim 12 wherein said syndecan enhancer comprises at least one sequence selected from the group consisting of one or more of the following oligonucleotides: a) TGGCACACCCACCGTCACGAGAGCT [SEQ ID NO. 3]; b) TGGCACACCTGGG AG [SEQ ID NO. 4] ; c) TGGTTCAGGGTGACT [SEQ ID NO. 5] ; ) AGGAGTGAGCCATGCCACC [SEQ ID NO. 6]; and e) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG [SEQ ID NO. 7]. 15. The expression vector as claimed in claim 12, wherein said syndecan enhancer is FGF-2 inducible. 16. The expression vector as claimed in claim 12, wherein said syndecan syndecan enhancer is FGF-7, EGF and/or TGFα inducible. 17. The expression vector as claimed in claim 12, wherein said syndecan enhancer element binds FL - 1. 18. The expression vector as claimed in claim 12, further comprises a promoter operably linked to a structural gene. 19. The expression vector as claimed in claim 18, wherein said promoter is not the syndecan promoter. 20. The expression vector as claimed in claim 18, wherein said promoter is the SV40 promoter or the TK-promoter. 21. Purified nuclear protein FIN-1 having a molecular weight of about 50 kD. 22. A nuclear protein as claimed in claim 21 , wherein said protein is dimerized. 23. A purified nuclear protein capable of binding to a syndecan enhancer element having the sequence of SEQ ID NO. 2, wherein said nuclear protein binds to motif 1 , and has a molecular weight of about 46 kDa. 24. A method of expressing a structural gene comprising: a) providing a host cell transformed with a recombinant expression vector comprising a syndecan enhancer element as claimed in claim 2 operably linked to a structural gene; and b) inducing an enhanced level of expression of said structural gene by activating said syndecan enhancer element. 25. The method of expressing a structural gene as claimed in claim 24, wherein the syndecan enhancer element is activated by FGF-2. 26. The method of expressing a structural gene as claimed in claim 25, wherein said host cell is a mesenchymal cell. 27. The method of expressing a structural gene as claimed in claim 24, wherein the syndecan enhancer element is activated by FGF-7, EGF and/or TGFα. 28. The method of expressing a structural gene as claimed in claim 27, wherein said host cell is a keratinocyte. 29. The method of expressing a structural gene as claimed in claim 24, wherein the syndecan enhancer element is activated by binding FIN-1 to said enhancer element. 30. A method for differentially expressing a structural gene at a wound site comprising: a) providing a recombinant expression vector comprising a syndecan enhancer element as claimed in claim 2 operably linked to said structural gene; b) introducing said recombinant expression vector into a patient having wound sites; and c) inducing expression of said structural gene. 31. A non-human transgenic animal, wherein the germ line of said transgenic animal comprises a syndecan enhancer operably linked to a non- syndecan structural gene. 32. The non-human transgenic animal as claimed in claim 31 , wherein said structural gene promotes wound healing. 33. The non-human transgenic animal as claimed in claim 31 , wherein said transgenic animal is a mouse. 34. A method of making a recombinant DNA molecule capable of expressing a structural gene, wherein said method comprises: a) providing a syndecan enhancer element less than or about 280 nucleotides in length, wherein said syndecan enhancer element comprises binding sites for nuclear proteins FIN-1, AP-1 and USF; and b) linking said syndecan enhancer element to an oligonucleotide to form a recombinant DNA molecule, wherein said syndecan enhancer element is operably linked to a structural gene in said recombinant DNA molecule.

Inventors: JAAKKOLA PANU, JALKANEN MARKKU, VIHINEN TAPANI, JAAKKOLA, PANU, JALKANEN, MARKKU, VIHINEN, TAPANI
Applicants: BIOTIE THERAPIES LTD OY, JAAKKOLA PANU, JALKANEN MARKKU, VIHINEN TAPANI, JAAKKOLA, PANU, JALKANEN, MARKKU, OY BIOTIE THERAPIES, LTD., VIHINEN, TAPANI
Application year: 1997
Main classification: C12N15
IPC codes: A01K67, A61K48, A61P17, C07K14, C12N15