TRANSGENIC ANIMALS AS URINARY BIOREACTORS FOR THE PRODUCTION OF PROTEIN IN THE URINE, RECOMBINANT DNA CONSTRUCT FOR KIDNEY-SPECIFIC EXPRESSION, AND METHOD OF USING SAME

Abstract: The invention relates to recombinant DNA constructs, a method for producing a recombinant biologically active protein in vivo in the urine of a non-human mammal using a kidney-specific promoter, such as the uromodulin promoter, and the transgenic non-human mammals that serve as urine-based bioreactors for protein production.

Description: TRANSGENIC ANIMALS AS URINARY BIOREACTORS FOR THE PRODUCTION OF PROTEIN IN THE URINE, RECOMBINANT DNA CONSTRUCT FOR KIDNEY-SPECIFIC EXPRESSION, AND METHOD OF USING SAME CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U.S.C. §119 (e) from U.S. provisional application 60/108,195, filed November 13, 1998, and U.S. provisional application 60/142,92 . 5., filed July 9, 1999, the entire contents of which are hereby incorporated by reference. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to transgenic animals as urinary bioreactors for the expression and production of proteins in the urine. The present invention further relates to a recombinant DNA construct for kidney-specific expression of proteins in the urine and to a method for producing such proteins in the urine. Description of the Related Art Significant progress has recently been made in using transgenic animals as bioreactors to produce large quantity and high quality pharmaceuticals. The overall strategy entails the use of tissue-specific promoters to drive the expression of genes encoding medically important molecules. When those molecules are expressed in the target tissue of transgenic animals and secreted into body fluids, they can be harvested, purified and used for treating human diseases. The most notable example is the milk-based bioreactor system, taking advantage of mammary gland-specific gene promoters. U.S. Patent No. 5,476,995 was one of the first patents directed to transgenic female sheep as milk- based bioreactors that expressed the transgene in the mammary gland so as to produce the target protein in its milk. A number of proteins have been produced in milk- based bioreactor systems, such as protein C (U.S. Patent No. 5,589,604), blood coagulation factors (U.S. Patent No. 5,322,775), fibrinogen (U.S. Patent No. 5,639,940), antibodies (U.S. Patent No. 5,625,126) and hemoglobin (U.S. Patent No. 5,602,306) , some of which are now being used in clinical trials. However, even in view of its initial success, a milk-based bioreactor system has several limitations. The first relates to its relatively low degree of cost-effectiveness. For instance, the lactation of transgenic livestock does not occur until an average of one and a half years old. Besides, lactation only occurs in female animals and lasts for a limited period of time. Secondly, purification of target proteins from milk often requires the development of complicated purification schemes (Wilkins et al, 1992) . Thirdly, leakage of biologically active proteins from the mammary gland into the blood stream commonly occurs with the possibility of leading to pathological conditions in transgenic animals. Another potential bioreactor system that can circumvent some of the above-mentioned limitations is a urine- based system where urine is an easily collectable fluid from transgenic livestock animals. This bioreactor system has been recently tested by Kerr and colleagues (1998) , among whom is one of the present inventors, in transgenic mice using a urothelium-specific promoter (uroplakin II promoter) to drive human growth hormone (hGH) expression and production. They found that hGH could indeed be found in the urine of these transgenic mice at a concentration of 0.1 mg/ml, indicating that the urothelium can serve as an alternate bioreactor. The major advantages of this urine-based system over milk-based systems are the ability to harvest the product soon after birth and throughout the life of the animal irrespective of sex or reproductive status and the ease of product purification from urine. In addition, livestock urine is a proven, currently utilized source of pharmaceuticals; it is estimated that urine is being collected from 75,000 pregnant horses annually as a source of estrogenic compounds for postmenopausal hormone replacement therapy (Williams, 1994) . Despite these major advantages, several technical problems still exist with the current urine-based system, the most important being the relatively low yield of urinary hGH (0.1 mg/ml) obtained by Kerr et al (1998), as most of the hGH the urothelium is not known to be a major secretory epithelium and the purification of a minor protein from urine may require sophisticated purification procedures. In addition, low levels of hGH was found to have leaked into the mouse blood stream, possibly being responsible for the infertility observed in the transgenic female mice. Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement. SUMMARY OF THE INVENTION It is an object of the present invention to overcome the above-mentioned deficiencies in the art by providing a urine-based bioreactor system using a kidney- specific promoter for the expression and production of a recombinant biologically active protein. The present invention provides a recombinant DNA molecule containing a kidney-specific promoter operably linked to a heterologous gene, which kidney-specific promoter is capable of expressing the heterologous gene in the kidney of a host animal to produce a recombinant biologically active protein in the urine . The present invention also provides for a method for producing a recombinant biologically active protein in vivo using a urine-based bioreactor system in transgenic animals. Further provided are transgenic animals, all of whose somatic cells and preferably all of whose germ cells contain a recombinant construct or transgene from which a biologically active protein is produced in recoverable amounts in the urine. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a restriction digestion of five phage clones (lanes 1-5) on agarose gel electrophoresis . M represents lanes of molecular weight markers . Figure 2 shows a Southern blot corresponding to the agarose gel shown in Fig. 1 hybridized separately with each of the 5' -end, middle region, and 3 '-end probes. Figure 3 shows an agarose gel electrophoresis of PCR reaction products using the sets of primers for the 5'- end, the middle region, and the 3 '-end of the uromodulin gene. Figures 4A and 4B show agarose gel electrophoresis (Fig. 4A) of EcoRI restriction digests of genomic DNA from various animal species and Southern blot hybridization (Fig. 4B) of the restriction digested genomic DNA with the middle region probe . Figure 5 is a schematic representation of the uromodulin gene structure in the human, bovine and rat genome. The open boxes represent exons with the exon numbering provided, and the thick bars represent the introns, the lengths of which are variable. Figure 6 shows Southern blot hybridization of BAC plasmid clone 1 digested with the restriction enzymes, Pstl (lane 4) , Apal (lane 6) , EcoRI (lane 7) , Sad (lane 8) , and Kpnl (lane 10) and hybridized separately with 5' -end, middle region and 3 '-end probes. Figures 7A-7H show the nucleotide sequence of the mouse uromodulin promoter region (SEQ ID NO:l) which is 9,345 bp upstream of the first mouse uromodulin coding exon. Figure 8 is a schematic presentation of the mouse uromodulin promoter in which the arrow denotes the transcription initiation site, the letters denote restriction sites (A, Apal; P, Pstl; B, BamHI; H, Hindlll; S, Spel), and the short bar denotes the relative size of the DNA. Figure 9 shows the partial cDNA sequence of goat uromodulin gene (SEQ ID NO: 2) . The location of primers AS14 , AS15 and AS17 used for isolation of goat uromodulin genomic DNA is shown in uromoduline. Figure 10A and 10B show the nucleotide sequence of goat uromodulin gene intron 1 (Fig. 10A; SEQ ID NO: 3) and exon 3 (Fig. 10B, SEQ ID NO : 4 ) . The location of primers AS1, AS2, AS3, AS4 and AS5 used in genomic. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the development of a bioreactor system in a transgenic mammal where a recombinant biologically active protein is to be produced and secreted into the urine by the kidney-specific expression of a heterologous gene under the direction of a kidney-specific promoter, such as the uromodulin promoter. This urine-based mammalian bioreactor system, according to the present invention, is obtained by producing a transgenic mammal in which an isolated DNA molecule containing a recombinant construct or "transgene" for kidney- specific expression and production of the biologically active protein of interest is stably introduced. An example of a urine-based bioreactor system where the protein of interest is expressed in urothelial cells, rather than kidney cells, but which serves as guidance to development of a urine-based bioreactor system, is provided by Lin et al (1995) and Kerr et al (1998) . To produce transgenic animals, any method known in the art for introducing a recombinant construct or transgene into an embryo, such as microinjection, cell gun, transfection, liposome fusion, electroporation, and the like, may be used. However, the most widely used method for producing transgenic animals, and the method most preferred according to the present invention, is microinjection, which involves injecting a DNA molecule into the male pronucleus of fertilized eggs (Brinster et al, 1981; Costantini et al, 1981; Harbers et al, 1981; Wagner et al, 1981; Gordon et al, 1976; Stewart et al, 1982; Palmiter et al, 1983; Hogan et al, 1986; U.S. Patent No. 4,870,009; U.S. Patent No. 5,550,316; U.S. Patent No. 4,736,866; U.S. Patent No. 4,873,191). While the above methods for introducing a recombinant construct/transgene into mammals and their germ cells were originally developed in the mouse, they were subsequently adopted for use with larger animals, including livestock species (WO 88/00239, WO 90/05188, WO 92/11757; and Simon et al, 1988) . Microinjection of DNA into the cytoplasm of a zygote can also be used to produce transgenic animals . The present invention for producing a biologically active protein in a urine-based mammalian bioreactor system is not limited to any one species of animal, but provides for any appropriate non-human mammal species. For example, while mouse is a mammal species that is routinely used for producing transgenic animals and, thus, serves as a model system to test the transgene, other non-limiting but preferred examples include farm animals, such as pigs, sheep, goats, horses and cattle, which generate large quantities of urine, may be suitably used. The success rate for producing transgenic animals by microinjection is highest in mice, where approximately 25% of fertilized mouse eggs into which the DNA has been injected, and which have been implanted in a female, will develop into transgenic mice. Although a lower success rate has been achieved with rabbits, pigs, sheep and cattle (Jaenisch, 1988; Hammer et al, 1985 and 1986; Wagner et al, 1984), the production of transgenic livestock is considered by those in the art to be routine and without undue experimentation. Wall et al (1997a), Velander et al (1997), Drohan (1997), Hyttinen et al (1994), Morcol et al (1994) , Lubon et al (1997) , Houdebine (1997) , Wall et al (1997b) , Van Cott et al (1997) , Cameron (1997) , Cameron et al (1994) , Niemann (1998) and Hennighausen (1992) , among others, have reported and discussed the use of livestock as bioreactors or factories for the production of biologically active proteins. The introduction of a DNA containing a transgene sequence at the fertilized oocyte stage ensures that the introduced transgene will be present in all of the germ cells and somatic cells of the transgenic animal. The presence of the introduced transgene in the germ cells of the transgenic "founder" animal, in turn, means that all of the founder animal's offspring will carry the introduced transgene in all of their germ cells and somatic cells. There is no need for incorporating, along with the gene being introduced, any plasmid or viral sequences (Jaenisch, 1988) , although the vector sequence may be useful in some instances. In many cases however, the presence of vector DNA has been found to be undesirable (Hammer et al, 1987; Chaka et al, 1985 and 1986; Kollias et al, 1986; Shani 1986; Townes et al, 1985) . For instance, the transgene construct can be excised from the vector used to amplify the transgene in a microbial host by digestion with appropriate restriction enzymes. The transgene is then recovered by conventional methods, such as electroelution followed by phenol extraction and ethanol precipitation, sucrose density gradient centrifugation, chromatography, HPLC, or combinations thereof. It has been reported in U.S. Patent No. 5,589,604 that high transformation frequencies, on the order of 20% or more, in both mice and pigs were obtained by microinjection with HPLC-purified DNA. In order for the introduced gene sequence to be capable of being specifically expressed in the kidney of the transgenic animal, the gene sequence must be operably linked to a kidney-specific promoter. A DNA molecule is said to be "capable of expressing" a protein if it contains nucleotide sequences which contain cis-acting transcriptional regulatory information, and such sequences are "operably linked" to nucleotide sequences which encode the protein. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. The cis-acting regulatory regions needed for gene expression in general include a promoter region, and such regions will normally include those 5' -non- coding sequences involved with initiation of transcription. A promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. Thus, the gene encoding a protein of interest is operably linked to a kidney-specific promoter to generate a recombinant construct or "transgene" that is then introduced into the fertilized embryo. Also included in the transgene are nucleotide sequences that encode the signal sequences that direct secretion of the expressed biologically active protein of interest into the urine of the transgenic animal. Both endogenous and heterologous signal sequences (either for the host or for the biologically active protein of interest) can be used, although the endogenous signal sequence of the heterologous protein of interest is preferred. Furthermore, other regulatory sequences in addition to the promoter, such as enhancers, splice signals, ribosome binding sites and polyadenylation sites, etc., may be useful in the transgene construct. The preferred promoter in the recombinant construct/ transgene for the kidney-specific expression of a heterologous biologically active protein of interest is the promoter for uromodulin. Uromodulin, also named Tamm-Horsfall protein, is by far the most abundant urinary protein of human and other higher mammals, with an excretion rate of up to 200 mg per day (Hunt et al, 1985; Reinhart et al, 1989) . This -90 kDa glycoprotein has several important features that are relevant to its use in a kidney-expressed urine-based bioreactor system. The protein is synthesized by the epithelial cells of the ascending limb of Henle's loop and the beginning portion of the distal convoluted tubule, delivered exclusively to apical membrane and secreted into the urine (Sikri et al, 1981; Bachmann et al, 1990) . Rindler et al (1990) established that uromodulin is a cell surface protein anchored onto the apical plasma membrane via a glycosylphosphatidyl inositol (GPI) tail, where phospholipase C cleavage in vi tro of the GPI linkage completely releases the molecule into the culture medium. Uromodulin is highly tissue-specific, being expressed only in the kidneys and not in any other epithelial and mesenchymal tissue. Moreover, uromodulin is evolutionarily conserved throughout placental animals. The cDNA sequences reported for rat uromodulin (Fukuoka et al, 1992) and human uromodulin (Hession et al, 1987; Pennica et al, 1987) were found to be 91% and 77% identical with the mouse uromodulin cDNA sequence, respectively (Prasadan et al, 1995) . Prasadan and colleagues (1995) also reported that an alignment of uromodulin amino acid sequences from mouse, rat and human showed 91% similarity and 86% identity between mouse and rat, and 79% similarity and 70% identity between mouse and man. As discussed in the example presented herein, the present inventors have isolated and sequenced a 9,345 base pair region including about 7 Kb upstream of the coding region of the mouse uromodulin gene, which region contains the mouse uromodulin promoter. This DNA promoter region, or a fragment thereof which retains the tissue specific promoter activity thereof, can be used for construction of a transgene with a biologically active protein of interest, i.e., human growth hormone (hGF) . While knowledge of the nucleotide sequence of the mouse uromodulin promoter would facilitate the construction of a transgene which is capable of kidney- specific expression of a biologically active protein of interest, such sequence information is not necessary because it is well within the skill of the art to isolate a functional promoter sequence given a uromodulin genomic clone with the upstream promoter region. There is a wealth of scientific literature directed to the isolation and identification of a promoter for a given gene, with the Kahari et al (1990) article on the delineation of functional promoter and regulatory cis-elements being just one representative citation. Clones containing the goat uromodulin gene promoter have also been obtained as disclosed herein in Example 2. Other uromodulin gene promoters can be isolated using the genomic walking procedure described for the isolation of the mouse and goat uromodulin gene promoters in the Examples herein. Although there is an abundance of evidence suggesting that many important regulatory elements are located 5' to the mRNA cap site (e.g., McKnight et al, 1982; Payvar et al , 1983; Renkowitz et al, 1984; Karin et al , 1984), it also appears that important regulatory elements, particularly those mediating tissue-speci ic expression, may reside within the structural gene or even 3' to it (Charnay et al, 1984; Gillies et al, 1983; Reecy et al, 1998; James- Pederson et al, 1995; Sternberg et al, 1988; Belecky-Adams et al, 1993) . Thus, alternate constructs can also be made in which the intron sequences of the uromodulin gene and, if necessary, the 3 ' -untranslated sequences are present in the event that such sequences are needed. The approach to alternate constructs is outlined in the example provided herein and would be well recognized by those of skill in the art. The 3 ' -untranslated sequences of the uromodulin gene can be added downstream of the coding sequence for the biologically active protein of interest. Uromodulin has already been reported to be evolutionarily conserved, being detectable immunologically in all placental mammals (Kumar et al, 1990) . The laboratory of the present inventors has shown by Southern blot hybridization that the uromodulin gene is present as a single copy in many mammals, including all important livestock, such as cattle, sheep, goat, horse and pig. Not only do the uromodulin cDNAs from human, mouse and rat share a high level of identity (on the order of 80% or more) , but even the high mannose glycosylation of uromodulin is highly conserved among different species of mammals. This strongly suggests that the promoter sequences of uromodulin are also likely to be conserved among mammals . Moreover, as evidenced by the numerous examples in the scientific literature of promoters that are interchangeable among species, the uromodulin promoter from one mammal species is believed to be functional in another species. Accordingly, the mouse uromodulin promoter identified herein can be used directly in transgenic livestock to drive kidney-specific expression of the biologically active protein of interest in a urine-based bioreactor system. Alternatively, the uromodulin promoter used in the transgenic livestock to drive kidney-specific expression of the biologically active protein can be its own endogenous uromodulin promoter or an interchangeable uromodulin promoter from another species of livestock. The bovine and rat uromodulin promoter regions have already been identified in Yu et al (1994) , the entire contents of which are hereby incorporated herein by reference. Specifically, Fig. 5 of Yu et al (1994) shows the nucleotide sequence of the bovine and rat uromodulin promoter regions. These promoter regions, or a fragment thereof with kidney-specific promoting activity, can be used to drive the kidney-specific expression of a heterologous gene in those respective species. Furthermore, in view of the conservation of these regions, it is expected that the bovine and the rat uromodulin promoters will also be active to promote the kidney-specific expression of a heterologous gene in any other mammal, such as goat, horse or sheep, which might be used as a urinary bioreactor. If it is determined that the regions of the approximately 600 base pairs upstream of the transcription start site in the bovine and rat sequences of Fig. 5 of Yu et al (1994) do not contain the complete kidney-specific uromodulin promoter sequence for these species, additional nucleotides upstream of the disclosed sequences can readily be obtained and sequenced using the specific sequences as a probe of bovine and rat genomic libraries, or using the technique of genomic walking as described in the examples herein, without the use of undue experimentation. Uromodulin promoters from other mammalian species can be isolated using the same approaches outlined in the examples provided herein, or using the same approach used in Yu et al (1994) , or by hybridization or PCR amplification of genomic libraries or genomic DNAs using probes or primers from the genomic clones of the mouse, rat or cow uromodulin gene. If the need to use a uromodulin promoter from another livestock animal species arises, then information generated from the mouse uromodulin promoter or from the bovine and rat uromodulin promoter region of Yu et al (1994) can be used to facilitate this process. For instance, as the sequence of the mouse uromodulin promoter has now been determined and is reported herein, and the bovine, rat and human promoter regions have been previously reported, oligonucleotide primers based on these sequences can be designed for PCR reactions. Long-range PCR can be performed to directly isolate uromodulin promoters from a pool of genomic DNAs extracted from various livestock animal species. DNA fragments containing the uromodulin promoter from livestock animal species can also be identified by hybridization of genomic libraries of corresponding species with mouse, bovine, rat or human uromodulin promoter probes under hybridization conditions similar to or the same as that used for the Southern blots (Zoo-blots of genomic DNA from various species) as disclosed in Example 1 provided herein. As will be appreciated by those in the art, the uromodulin promoter or any other kidney-specific promoter used in the transgene for directing kidney-specific expression of the biologically active protein of interest can include relatively minor modifications, such as point mutations, small deletions or chemical modifications that do not substantially lower the strength of the promoter or its tissue-specificity. In addition, the identification of additional promoters active in directing gene expression in the kidney can be routinely performed using the suppression subtraction hybridization library technique. Using this technique, which eliminates the cDNAs that are shared by multiple tissues (Diatchenko et al, 1996) , a library highly enriched in kidney- specific cDNAs can be generated. Total RNAs are isolated from stomach, intestine, colon, liver and brain, and Northern blot analysis of these RNAs using an actin cDNA as a probe is used to demonstrate the intactness of the actin mRNA in all of these preparations. Kidney cDNAs are then used as the "tester", and the cDNAs of all the other non- kidney tissues, referred to as the "drivers", are subtracted from the kidney cDNAs . Using the subtraction library technique, the laboratory of the present inventors had earlier probed the cDNAs of the non-subtracted and the subtracted libraries with actin cDNA or uroplakin lb cDNA, and the results indicated that the original (non-subtracted) bovine bladder cDNA preparation contained abundant actin mRNA and relatively little uroplakin lb mRNA. In contrast, the subtracted library contained almost no detectable actin mRNA (at least 50 fold reduction) but greatly increased uroplakin lb mRNA (>10 to 15 fold enrichment) . Multiple cDNA clones have been isolated from the subtraction library and used to probe the mRNAs of various bovine tissues. For example, a uroplakin lb probe confirmed its bladder specificity. The laboratory of the present inventors has already been successful in obtaining three unidentified cDNAs in which the tissue distribution pattern showed bladder specificity. Sequencing data indicate that these three bladder-specific clones are novel genes not described previously. In the same manner, kidney-specific genes can be isolated, and any gene that is involved in the structure and function of the excretory tract of the kidney, including proximal, distal tubules, Henle's loop, collecting duct system can be applied in this system to isolate its promoter for use in expressing and producing a biologically active protein in a urine-based kidney bioreactor. Although the suppression subtraction hybridization library technique is the preferred procedure for obtaining tissue-specific genes, kidney- specific genes can also be identified through other well-known methods, including biochemical methods, protein chemistry, monoclonal antibody production, two-dimensional gel electrophoresis, cDNA library screening, expression library screening, differential display, phage display, etc. As used herein, "biologically active protein" refers to a protein capable of causing some effect within an animal and preferably not within the animal having the transgene. Examples of such proteins include, but are not limited to, adipokinin, adrenocorticotropin, blood clotting factors, chorionic gonadotropin, corticoliberin, corticotropin, cystic fibrosis transmembrane conductance regulators, erythropoietin, folliberin, fcllitropin, glucagon gonadoliberin, gonadotropin, human growth hormone, hypophysiotropic hormone, insulin, lipotropin, luteinizing hormone-releasing hormone, luteotropin, melanotropin, parathormone , parotin, prolactin, prolactoliberin, prolactostatin, somatoliberin, somatotropin, thyrotropin, tissue-type plasminogen activator, vasopressin, antibodies, peptides, and antigens (for use in vaccines) . It will be appreciated by those of skill in the art that the above list is not exhaustive. In addition, new genes for biologically active proteins that will function in the context of the present invention are continually being identified. Proteins which degrade or detoxify organic material may also be produced by means of the present invention. Such proteins may be those discussed in WO 99/28463, the entire contents of which is hereby incorporated by reference. The biologically active protein produced in the urine-based bioreactor system according to the present invention can be isolated from the urine of these transgenic animals. Accordingly, the present invention provides a means for isolating large amounts of biologically active proteins from the urine of transgenic animals which can be used for a variety of different purposes. Furthermore, the biologically active protein can be readily recovered and purified from the urine as would be well within the skill of those in the art. While the production of transgenic animals by the introduction of the transgene into germ line cells is most preferred, it is also contemplated that the transgenic animals, which serve as a urine-based bioreactor system, can be produced by vectors that are useful for transforming the kidney into a bioreactor capable of producing a biologically active protein in the urine for isolation. The transformed cells may be germ line or somatic cells. In an alternative embodiment to introduction into germ line cells, the vector according to the present invention includes a system which is well received by the cells lining the excretory tract of the kidney, including proximal, distal tubules, Henle's loop and collecting duct system. An example of a useful vector system is the Myogenic Vector System (Vector Therapeutics Inc., Houston, TX) . In this embodiment, the heterologous gene of the biologically active protein linked to a viral promoter construct capable of directing kidney-specific expression and carried in the vector is introduced into the kidney of an animal in vivo. Introduction of the vector can be carried out by a number of different methods routine to those of skill in the art. Vectors of the present invention can also be incorporated into liposomes and introduced into the animal in that form. The transgene is absorbed into one or more epithelial cells capable of expressing and secreting the biologically active protein into the urine collecting in the bladder. It may be preferred for some biologically active proteins to also engineer a signaling sequence into the vector to ensure that the protein is secreted from the apical surface into the lumen. Use of signaling sequences, such as the GPI linkage in anchoring proteins to a selected surface is well known in the art . The biologically active protein is then voided from the lumen where it can be collected and separated from other components in the urine. Another alternative embodiment for generating a transgenic animal as a kidney-based bioreactor is through the use of targeted homologous recombination, where one copy of the endogenous uromodulin gene is disrupted by insertion of a heterologous gene encoding a biologically active molecule of interest, which heterologous gene is flanked by sequences complementary to the endogenous uromodulin gene. These flanking complementary sequences which direct homologous recombination to an endogenous uromodulin gene are at least 25 base pairs in length, preferably at least 150 base pairs. This technique for generating transgenic animals and cells by homologous recombination is disclosed in WO 90/11354 and U.S. Patent 5,272,071, the entire contents of which are hereby incorporated by reference. Accordingly, if it is desired for the kidney to express and secrete a selected biologically active polypeptide into the urine, then a short sequence on either side of the start codon of the uromodulin coding sequence in a given species can be used as flanking sequences to create a construct that can be inserted at the specific location in the genome of the host animal species which is between the endogenous uromodulin gene promoter and the endogenous uromodulin gene coding sequence. In this way, the expression of the biologically active polypeptide of interest will be driven by the endogenous uromodulin promoter in the transgenic animal. The bovine genomic uromodulin sequence has already been reported (Yu et al . , 1994), and the mouse genomic uromodulin sequence as well as the clone containing the goat genomic uromodulin gene sequence surrounding the start codon are disclosed herein. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting of the present invention. EXAMPLE 1; ISOLATION OF MOUSE UROMODULIN GENE PROMOTER Generation of Uromodulin cDNA Probes Three probes corresponding to the 5' -end, the middle region and the 3 ' -end of the full-length uromodulin cDNA (Prasadan et al, 1995) were generated using the reverse transcription-polymerase chain reaction (RT-PCR) method, with three pairs of oligonucleotide primers chemically synthesized based on the published uromodulin cDNA sequence. The set of primers for the 5' -end are 5 ' -TGGACCAGTCCTGTCCTGGTTCAG-3 ' (SEQ ID NO: 5; sense), and 5 ' -GGGTGTTCACACAGCTGCTGTTGG-3 ' (SEQ ID NO: 6; antisense) . The set of primers for the middle region are 5 ' -AGGGCTTTACAGGGGATGGTTG-3 ' (SEQ ID NO: 7) and 5'- GATTGCACTCAGGGGGCTCTGT-3' (SEQ ID NO: 8) The set of primers for the 3 '-end are 5 ' -GGAACTTCATAGATCAGACCCGTG-3 ' (SEQ ID NO: 9) and 5 ' -TGCCACATTCCTTCAGGAGACAGG-3 ' (SEQ ID NO: 10). These three pairs of oligonucleotide primers were used to amplify uromodulin cDNA fragments using, as a template, a pool of cDNAs reversed transcribed from mouse kidney RNAs . PCR conditions included the first cycle of 94 °C for 1 min, 55 °C for 1 min, and 72 'C for 2 min; 35 cycles of 95 °C for 2 min, 55°C for 1 min, and 72 °C for 2 min; and the last cycle of 94 °C for 2 min, 55 °C for 1 min, and 72 'C for 8 min. Agarose gel electrophoresis revealed a 400 bp, a 440 bp and a second 400 bp PCR product for the three sets of primer amplifications, 5' -end, middle region, and 3 '-end, respectively. These PCR products were purified by extraction and chromatography using a QIAEX II method (QIAGEN, Valencia, CA) . Screening of Mouse Kidney cDNA Library A mixture of the above three uromodulin cDNA probes were 32 P-labeled and used to screen a BALB/c mouse kidney cDNA library (Clontech, Palo Alto, CA) . A total of 2 x 10 s phage clones from the cDNA library were plated, lifted onto nylon membrane and hybridized with the mixture of probes at 42 * C for 16 hours in a solution containing 50% Formamide, 5X SSPE, 5X Denhardt's solution, 0.1% SDS and 100 mg/ml denatured salmon sperm DNA. After hybridization, the nylon filters were washed at 65 °C for 1 hour in IX SSC and 0.1% SDS, and autoradiographed. Five phage clones were identified from the primary screening, and they were plaque-purified and subjected to the secondary screening using the same conditions as the primary screening. Purified phage clones were amplified by plate lysate and analyzed by EcoRI restriction digestion and agarose gel electrophoresis . On agarose gel, the five clones are of different sizes, ranging from 0.2 kb to 2.7 kb (Fig. 1) . A 2.7 kb clone hybridized with all three probes indicating that this band likely represented the full-length mouse uromodulin cDNA clone (Fig. 2) . This 2.7 kb band was excised from the bacteriophage with EcoRI restriction enzyme, gel-purified, subcloned into the same site of pBluescript KS " (Stratagene, LaJolla, CA) , and sequenced. The sequence matched precisely with the published mouse (uromodulin cDNA sequence of Prasadan et al, 1995) , further establishing the authenticity of this as mouse uromodulin. Isolation of Mouse Uromodulin Gene For the isolation of the mouse uromodulin gene, a commercial genomic screening service (Genomic System, St. Louis, MO) was used. Briefly, two pairs of PCR primers located in exon 3 (exon information derived from human uromodulin gene, Pennica et al, 1987) were designed and pretested by the present inventors. These primers were then used by Genomic System to mass -screen by PCR pooled genomic (BAC) plasmid clones of the MAC ES Mouse II library which harbors 129/SVJ mouse genomic DNAs. The first pair of primers, sense 5'- AGGGCTTTACAGGGGATGGTTG-3 ' (SEQ ID NO: 11), and antisense 5'- GATTGCACTCAGGGGGCTCTGT-3 ' (SEQ ID NO:12) , was used for the initial screen which yielded two uromodulin clones, each about 60-70 kb in length. These clones were confirmed independently by using a second set of nested primers, sense 5'- GCCTCAGGGCCCGGATGGAAAG-3 ' (SEQ ID NO: 13) and antisense 5'- GCAGCAGTGGTCGCTCCAGTGT-3 ' (SEQ ID NO: 14) . In addition, PCR reactions using the three pairs of primers located at the 5'- end, the middle region and the 3 '-end (SEQ ID NOs:5-10) showed that these two clones contained all the coding sequence information, indicating that it contained the entire uromodulin gene (Fig. 3) . Identification of the Uromodulin Gene in Multiple Animal Species An analysis of the conservation of the uromodulin gene sequence in other animal species is shown in Figs. 4A and 4B . The genomic DNA of human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast were digested with EcoRI restriction enzyme and hybridized with the uromodulin middle region probe described above, using the same Southern blot hybridization conditions used above for screening the mouse kidney cDNA library. The results of the Southern blot hybridization shown in Fig. 4B show that the uromodulin gene is conserved in mammals and is present as a single copy in human, monkey, rat, mouse, dog, cow and rabbit. Pennica et al (1987) and Yu et al (1994) reported that the gene structure (exons and introns) of human, bovine and rat uromodulin are highly conserved (Fig. 5) . Identification of Gene Fragments Containing the Uromodulin Gene Promoter Southern blotting was performed to identify DNA fragments containing the uromodulin promoter sequence. This approach is based on the differential reactivity of DNA restriction fragments of BAC clone 1 DNA with three different uromodulin probes located in the 5' -end, middle region, and 3 '-end of the uromodulin cDNA. Thus, BAC plasmid clone 1 was digested with the restriction enzymes Notl, BamHI, Hindlll, Pstl, EcoRI, Apal, Ncol, Sad, Xhol and Kpnl . After agarose gel electrophoresis, DNA fragments were transferred onto nylon membrane, UV-crosslinked and hybridized with the 5'- end, middle region, and 3 '-end cDNA probes. A 6.9 kb Pstl DNA fragment (Fig. 6, lane 4), an 8.3 kb Apal DNA fragment (Fig. 6, lane 6), and an 8.5 kb Sad DNA fragment (Fig. 6, lane 8) reacted with only the 5' -end probe, but not with middle region probe or the 3 '-end probe. This strongly indicates that these three DNA fragments contain portions of the 5 '-end of the uromodulin coding sequence and, more importantly, a large fragment of the 5 '-upstream region of the uromodulin gene. In contrast, a 9 kb Kpnl fragment reacted with all three probes (Fig. 6, lane 10), indicating that this fragment contains all the coding sequences for uromodulin. Finally, a 10 kb EcoRI fragment reacted only with the 3' -probe (Fig. 6, lane 7), indicating that this fragment contains the 3 '-end of the coding region and the non-coding region. The identification of DNA fragments containing the entire uromodulin gene, particularly the 5'- upstream sequence facilitates the cloning of the uromodulin gene promoter . Sequencing of Uromodulin Promoter The 8.3 kb Apal DNA fragment was used for further promoter analysis. A genomic walking method was employed to sequence the entire uromodulin promoter from both 5'- and 3'- ends by sequentially walking the sequence and synthesizing the new primers based on newly obtained sequences . Sequences were determined by the dideoxynucleotide chain termination method of Sanger et al (1977) on an automatic DNA sequencer. Listed below are sense- and anti-sense primers used for the sequencing purposes . Sense Primers SI: 5' -TGTCCTATGTGACTCCAGCT-3' (SEQ ID NO: 15) S2 5' -TCTCCTCAGCTCTCCTGGTC-3' (SEQ ID NO:16) S3 5' -TCCTGCCACCACCATGACCA-3' (SEQ ID NO:17) S4 5 ' -AAGCACCGGTGTGCTTGTAT- 3 ' (SEQ ID NO : 18 ) S5 5 ' -ATGGGGCTGCTGAGACTAAG-3 ' (SEQ ID NO : 19 ) Anti- sense Primers AS1 5 ' -AAGTCAGACTGTGTTAGGAT- 3 ' (SEQ ID NO : 20 ) AS2 5' -ATTGACTGAGCAGGAAGCAT-3' (SEQ ID NO: 21) AS3 5' -ATTTTATAACCTCCCTCTAG-3' (SEQ ID NO:22) AS4 5' -ATGCATTCCAGTCTCAGTGC-3' (SEQ ID NO: 23) AS5 5 ' -TGGGGAGAGGACAAAGCCTTG- 3 ' (SEQ ID NO : 24 ) AS6 5' -TGACGTGCCAACTCCACTGA-3' (SEQ ID NO:25) AS7 5 ' -AGGACCTGTAGGGTAAGAAA- 3 ' (SEQ ID NO : 26 ) AS8 5' -TCTGGCTGTGGGCTCTATAT-3' (SEQ ID NO:27) Analysis of the Uromodulin Promoter The 9,345 bp nucleotide sequence of the promoter region and the genomic coding region including exon 3 of the mouse uromodulin gene is shown in Fig. 7. These results (1) establish the authenticity of the isolated uromodulin clone, (2) indicate that a 7 kb uromodulin promoter has been obtained which is more than adequate to be used in the urine- based transgenic bioreactor system. This mouse promoter can be used in other mammalian species, such as farm animals, to drive the kidney-specific expression of any heterologous gene. Subcloning of Uromodulin Promoter Having identified the uromodulin promoter region, this region can be subcloned for further amplification, and for constructing transgenes . Since the clone containing the uromodulin promoter region is at least 70 kb in size, restriction digestion of each of this clone gives rise to multiple bands. Although the relative sizes of uromodulin promoter-containing bands can be determined by Southern blotting using the 5' -end probe, this does not allow for pinpointing a specific band for subcloning, as most bands are not well-resolved. To circumvent this problem, a dot-blot approach by gel-purifying each individual band in the close vicinity of the area where Southern blot hybridization revealed a positive band will be taken. DNA in each band will be eluted using a QIAEX column (QIAGEN) , and then blotted onto nylon membrane, UV-crosslinked and hybridized with a uromodulin 5 '-probe. The bands reacting with the probe will then be subjected to subcloning. The plasmid pBluescript (Stratagene, LaJolla, CA) , which was used as the cloning vector, is to be restriction- digested using Pstl, Apal and Sad, respectively, phosphatase-treated, and the linearized pBluescript cloning vectors will be mixed with the correspondingly digested inserts, ligation buffer, T4 DNA ligase, and incubated at 16 "C for 16 hours. Half of this ligation mixture will be used to transform CaCl 2 -prepared competent JM109 bacterial cells and then screened using small-scale plasmid preparations, which are carried out using mini-prep columns (Promega) and then restriction-digested to release the inserts. Through these procedures the DNA fragments containing mouse uromodulin promoter are to be subcloned. Detailed Restriction Mapping of Uromodulin Promoter Restriction mapping of the 5 '-flanking sequence of uromodulin, an important step for determining the restriction fragments for constructing transgenes has been performed. Although the detailed restriction map is not shown here, such a restriction map can be generated quite readily using any of the numerous publicly or commercially available DNA analysis software programs . EXAMPLE 2 : ISOLATION OF GOAT UROMODULIN GENE PROMOTER Isolation of Goat Uromodulin cDNA The goat uromodulin cDNA was isolated using reverse transcriptase/polymerase chain reaction (RT-PCR) approach (Wu, et al . , 1993) . Briefly, a sense and an antisense primer were synthesized based on the mouse uromodulin gene sequence that was isolated in the laboratory of the present inventors. The sequences of these two primers are: 5' -GACTGAGTACTGGCGCAGCACAG-3' (SEQ ID NO: 28) and 5' -GATTGCACTCAGGGGGCTCTGT-3' (SEQ ID NO:29). Total RNA was isolated from goat kidneys using the guanidine isothiocyanate method, reverse-transcribed using AMV reverse transcriptase, and the second strand of cDNA was synthesized using DNA polymerase I. PCR amplification was performed using total kidney cDNAs as templates and the two mouse uromodulin as primers, in the presence of NTP, Taq polymerase, and PCR buffer. The PCR reaction was performed for 35 cycles of denaturation at 94°C, annealing at 55°C and extension at 72°C and the resulting PCR products were resolved by agarose gel. The products having the predicted size were subcloned into the TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced. RT-PCR of goat kidney-derived mRNAs, using the pair of primers derived from mouse uromodulin, yielded a single, approximately 300 bp product upon agarose gel electrophoresis. The PCR product was subcloned and sequenced. A Blast search of Genbank of the PCR product sequence (SEQ ID NO: 2; Fig.9) showed that the top four hits were uromodulin sequences from several species. Thus, the sequence of the PCR product shared a 96% identity (287 bp/297 bp) with bovine uromodulin, 90% identity (218/241) with human uromodulin, a 78% identity (239/304) with rat uromodulin, and an 80% identity in a shorter stretch (125/156) with mouse uromodulin. The high degree of sequence identity of the PCR product with known uromodulin sequences firmly established that the product is a partial goat uromodulin cDNA. Isolation of Goat Uromodulin Genomic DNA By Genomic Walking, Cloning and Sequencing A genomic walking approach was employed to isolate the goat uromodulin gene using specific sequence information obtained from goat uromodulin cDNA. Genomic DNA was isolated from goat kidneys and used as templates for PCR-based genomic walking (Clontech, Palo Alto, CA) . The genomic DNA was digested using five restriction enzymes (Dral, Seal, EcoRV, PvuII, Stul), each of which created a blunt end in the genomic DNAs. The ends were ligated with adaptors. PCR was then performed using the ligated DNA library as templates, and two independent anti-sense primers synthesized based on the newly obtained uromodulin cDNA sequence as well as a sense primer located on the adaptor. The sequences for the two anti-sense primers are 5 ' -GTACCAGCCGCCCAGACTGACATCACAG-3 ' (SEQ ID NO:30; primer AS14), and 5'- CAGGTTGTACACGTAGTAGCCGCCGGCA-3' (SEQ ID NO: 31; primer AS17) . The PCR was performed for 1 cycle of denaturation at 99°C for 5 sec, annealing and extension at 68°C for 4 min., followed by 7 cycles of denaturation at 94 °C for 2 sec, annealing and extension at 68°C for 4 min., followed by 32 cycles of denaturation at 94 °C for 2 sec, annealing and extension at 63°C for 4 min., and followed by 1 cycle at 63°C for 4 min. After the first round of PCR, the products were used as templates and subjected to a second round of PCR amplification using two new, nesting sense and anti-sense primers. The specific products were subcloned into the TA cloning vector and the identity of the goat uromodulin gene was confirmed by DNA sequencing of both ends of the product. Based on the newly identified goat uromodulin cDNA, the two above anti-sense primers were designed for genomic walking using goat genomic DNA to identify DNA sequences that are located in the upstream region. After the first and second rounds of PCR and nesting PCR amplifications, a 1.5 kb, single PCR product was obtained. Subcloning and sequencing of this product revealed that its 3 '-end shares 94% identity (494/522) with bovine uromodulin cDNA sequence, thus confirming that the PCR product is a portion of the goat uromodulin gene. The 5' -sequence did not share any significant homology with any of the known uromodulin cDNA sequences and therefore most likely represents intron sequences. Based on the gene structure of mouse uromodulin and the relative length (1.5 kb) of the PCR product, this 5'- sequence is most likely located in intron 1. The nucleotide sequences of intron 1 (SEQ ID NO: 3) and exon 3 (SEQ ID NO: 4) of the goat uromodulin gene are shown in Figs. 10A and 10B, respectively. Isolation of Goat Uromodulin Promoter by Secondary Genomic Walking For the isolation of goat uromodulin promoter, the 5 '-end of the genomic clone that was isolated from the first round of genomic walking was used to design new antisense "walking primers" located in intron 1. The five primers are: 5 ' -AAGATTTACCAGCCCGGGCCGTCGACC-3 ' (SEQ ID NO : 32 ; AS1) 5 ' -AATAAAGTGCCAGGGCAGGGGGGCTTA-3 ' (SEQ ID NO : 33 ; AS2 ) 5' -CTTGTGTGGTTGAGTGTGTTCTTGACC-3' (SEQ ID NO: 34; AS3 ) 5 ' -TGTGAAAGGGGATGTCTTTGGGTACCA-3 ' (SEQ ID NO : 35 ; AS4 ) 5 ' -ACAGCAATGTGCAACCCAATGGAAGGG-3 ' (SEQ ID NO : 36 ; AS5) . Fresh goat genomic DNA as template was digested by the five blunt-ending restriction enzymes (see above) and subjected to PCR walking using the se five anti-sense primers and the aforementioned conditions. PCR and nesting PCR yielded a highly specific, 1.0 kb product in three independent primer combinations. The fact that this product is highly specific and reproducible in different reactions, that all primers used were located in intron 1, and that exon 1 in mouse uromodulin gene is less than 25 bp, is highly indicative of its authenticity as a goat uromodulin gene fragment containing a significant portion of the promoter region. Subcloning and DNA sequencing of the subcloned fragments are under way to establish the identity of the goat uromodulin promoter and its detailed structural features. EXAMPLE 3: CONSTRUCTION OF KIDNEY-BASED BIOREACTOR SYSTEM Construction of Chimeric Genes To test the tissue-specificity of the uromodulin gene promoter and its utility in a kidney-based bioreactor system, a chimeric gene containing the uromodulin promoter and a gene encoding a pharmaceutically- important protein is to be constructed. For this purpose, the human growth hormone gene (hGH) , whose expression has been recently assessed in a uroplakin II -based, bladder bioreactor system (Kerr et al, 1998) will be tested first. A potential limitation has been recognized with the bladder bioreactor system in that it produced relatively low amounts of hGH. Such a potential limitation may possibly be associated with the less than optimal secretory activity of the urothelium. Since uromodulin is normally synthesized in the ascending limb of Henle's loop and the distal tubules where active secretion takes place, the present inventors expect that there will be an active secretion of synthesized hGH into the urine of mice, resulting in high protein yield. The presence of this uromodulin/hGH gene in transgenic mice will allow a comparison of the efficiency between the kidney-based and the bladder-based reactor systems. The uromodulin/hGH chimeric gene will be constructed using a pBluescript cloning vector in two steps. The first step is to clone the restriction-mapped uromodulin 5' -flanking region (above) into pBluescript, and the second is to clone the hGH gene downstream of the uromodulin 5'- flanking region so that the transcription of hGH is under the exclusive control of the uromodulin promoter. Efforts will be made to select restriction sites that would permit efficient cohesive ligation. If necessary, however, blunt- end ligations could be performed. The proper uromodulin/hGH orientation will be verified by restriction mapping, PCR and sequencing using a sense primer at the 3 '-end of the uromodulin promoter and an antisense primer located inside the hGH coding region. The uromodulin/hGH fusion gene will then be excised en bloc using two restrictions enzymes at the 5'- and 3 '-ends of the fusion gene and gel-purified for use in generating mice transgenic for this uromodulin/hGH fusion. Generation of Transgenic Mice Mouse strain C57BL/6XDB2, that has been well adopted by a NYU Transgenic Mouse Facility, will be used for transgenic mouse production. All transgenic techniques, including embryo manipulation, fusion gene microinjection, implanting of embryos into pseudopregnant recipients, will be based on standard procedures, such as Hogan et al (1986), where the transgene is microinjected into the pronuclei of fertilized eggs, which are then implanted into pseudopregnant mice. The offspring are then analyzed for transgene incorporation by Southern blotting of DNA isolated from the tails of mice. Two probes are to be used, one corresponding to the mouse uromodulin promoter, and the other corresponding to hGH. The mouse uromodulin probe will reveal an endogenous uromodulin gene, which will allow calculation of the copy number of the transgene by comparing their relative intensity. Other Considerations for Achieving Kidney-Specific and High- Level Expression of Transgenes Although in a great majority of cases the 5'- flanking region is sufficient to convey the tissue- specificity and high-level expression of a tissue-specific gene, it has been reported that in some instances intron, or even the 3 ' -untranslated sequences, contribute to promoter activity. For example, intron I sequences were found to be important for high-level and tissue-specific expression of an alpha-skeletal actin gene, a beta-globin gene and a peripherin gene (Reecy et al, 1998; James-Pederson et al, 1995; Belecky-Adams et al, 1993) . In view of these examples of introns or 3 ' -untranslated sequences contributing to promoter activity, the constructs to be made will include intron I sequences of the uromodulin gene and, when necessary, 3 ' -untranslated sequences. In the former case, a fragment will be isolated that spans the 5' -flanking region, the first exon and the first intron, followed by the hGH gene. The translation initiation codon of the uromodulin gene could also be mutated to avoid translation of truncated uromodulin, and other regions of the uromodulin gene could also be used to ensure the tissue-specific and high-level expression of the uromodulin transgene. Expression of hGH in Mouse Kidney The expression of hGH in transgenic mouse kidney is to be assessed at both the mRNA and protein levels. First, RT-PCR is to be performed to determine the expression of mRNA using primers specific for hGH. Total RNAs will be extracted from transgenic mouse kidneys and from control tissues, including rat liver, skin, intestine, stomach, brain, skeletal muscle, thymus, thyroid gland, bladder, lungs, heart, pancreas, spleen, prostate, seminal vesicles, uterus and ovaries. The total RNAs are to be reverse-transcribed, PCR amplified and analyzed by agarose gel electrophoresis. The results will reveal whether hGH is expressed in kidney- dependent fashion. To determine whether hGH was synthesized in the ascending limb of Henle's loop and the distal tubules of the kidney, immunofluorescent staining of the kidney using anti-hGH antibody will be performed. Frozen kidney sections are to be stained using an indirect immunofluorescent method (Wu et al, 1993) . Furthermore, the amount of hGH in the urine of transgenic mice will be determined according to Kerr et al (1998) . Having now fully described this invention, it will be appreciated that by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation . While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference. Reference to known method steps, conventional method steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. 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Claims: WHAT IS CLAIMED IS : 1. An isolated DNA molecule, comprising a kidney- specific promoter operably linked to a heterologous gene encoding a biologically active protein, wherein said kidney- specific promoter is capable of expressing said heterologous gene in vivo in the kidneys to produce a recombinant biologically active protein in the urine. 2. The isolated DNA molecule according to claim 1, wherein said kidney-specific promoter is a uromodulin promoter. 3. The isolated DNA according to claim 2, wherein said uromodulin promoter is a mouse uromodulin promoter. 4. The isolated DNA according to claim 1, further comprising a secretion signal sequence operably linked to said heterologous gene . 5. A self-replicable vector, comprising the DNA molecule of claim 1. 6. A host cell transformed with the self- replicable vector of claim 5. 7. A method for producing a recombinant biologically active protein, comprising the steps of: introducing the isolated DNA molecule according to claim 1 into a fertilized embryo of a non-human mammal to generate a transgenic non-human mammal which expresses the heterologous gene and which produces the heterologous protein secreted into the urine of the transgenic non-human mammal; collecting urine from the transgenic non-human mammal ; and recovering the produced heterologous protein as a recombinant biologically active protein. 8. The method according to claim 7, wherein said introducing step comprises injecting the isolated DNA molecule into a pronucleus of a fertilized embryo. 9. The method according to claim 7, wherein the isolated DNA comprises a uromodulin promoter operably linked to a heterologous gene . 10. The method according to claim 9, wherein the uromodulin promoter is a mouse, bovine or rat uromodulin promoter . 11. A transgenic non-human mammal all of whose germ cells and somatic cells contain a recombinant construct corresponding to the DNA molecule according to claim 1, said DNA molecule having been introduced into said mammal , or an ancestor of said mammal, at an embryonic stage, and wherein said mammal produces recoverable amounts of a recombinant biologically active protein in its urine. 12. The isolated DNA according to claim 3, wherein said mouse uromodulin promoter is the sequence of Fig. 7, or a fragment thereof with kidney-specific promoting activity. 13. The isolated DNA accordinig to claim 2, wherein said uromodulin promoter is the bovine or rat uromodulin promoter. 14. The method according to claim 7, wherein said non-human mammal is a goat, cow, sheep, pig or horse. 15. A transgenic non-human mammal according to claim 11 which is a transgenic goat, cow, sheep, pig or horse .

Inventors: SUN TUNG-TIEN, WU XUE-RU, SUN, TUNG-TIEN, WU, XUE-RU
Applicants: UNIV NEW YORK, NEW YORK UNIVERSITY
Application year: 1999
Main classification: C12P21
IPC codes: C07K14, C12N15