KR100458792B1 - Expression of a heterologous polypeptide in renal tissue of transgenic non-human mammals using promoter for tamm-horsfall uromodulin protein - Google Patents

Abstract

The present invention relates to a transgenic mammal other than human, and more particularly, a DNA sequence encoding a uromodulin promoter and one specific heterologous protein of a bovine or mouse. A non-human transgenic mammal containing a genome comprising a DNA construct constructed in an operable state. In addition, the present invention relates to a vector comprising a uromodulin promoter construct bound to a non-homologous gene encoding a selected molecule having biological activity, and more particularly, the uromodulin promoter construct is transfected. A vector comprising a Euromodulin promoter construct configured to direct expression of said non-homologous gene in a kidney cell-specific manner in a transgenic mouse. .

Description

Expression of a heterologous polypeptide in renal tissue of transgenic non-human mammals using promoter for tamm- horsfall uromodulin protein}

The present invention relates to a transgenic mammal other than human, and more particularly, a DNA sequence encoding a uromodulin promoter and one specific heterologous protein of a bovine or mouse. A non-human transgenic mammal containing a genome comprising a DNA construct constructed in an operable state. In addition, the present invention relates to a vector comprising a uromodulin promoter construct bound to a non-homologous gene encoding a selected molecule having biological activity, and more particularly, the uromodulin promoter construct is transfected. A vector comprising a Euromodulin promoter construct configured to direct expression of said non-homologous gene in a kidney cell-specific manner in a transgenic mouse. .

In an effort to obtain large quantities of proteins that are useful biologically, therapeutically and industrially, but are produced in small amounts or in difficult-to-purify forms, several methods have been tried, particularly those that utilize microorganisms such as bacteria and yeast. Recombinant technology has enabled the production of proteins. However, some proteins produced from bacterial expression systems, including yeast, baculovirus, etc., require a maturation step that requires chemical denaturation, such as chemical denaturation and glycosylation of specific amino acids after they are produced. Done. Lack of proper glycosylation of specific amino acids, proteins produced from these amino acids usually do not have sufficient biological activity, so these proteins are produced in eukaryotic cells capable of posttranslational modification. It is desirable to do this, but culturing tissue cells in large quantities involves a number of technical and economic difficulties.

Another method of producing such a protein is to synthesize it in vivo using transgenic animals, and it is desirable that such a method not only can produce a large amount of protein but also be easily obtained. Recently, there has been a movement of commercialization using animals as biological reactors for the production of proteins.

Milk has already established itself as a major productive mode, and the expression of proteins present in the human body in cattle, pigs, sheep and goats has already been used commercially (Drohan, Thrombosis and Hemostasis, 1997, 78, 543-547). ). The lactating animals add a charm to this method because they supply abundant milk, but this method also has some drawbacks as follows. First, purification is required to obtain the desired protein from proteins present in large quantities in milk, such as caseins, lactoferrins and lactoglobulins (paleyanda et al., Nature biotech, 1997, 15, 971-975). Second, since milk contains a large amount of calcium, it has the disadvantage of insolubilizing certain proteins. Third, this method requires females of pregnant or postpartum animals and therefore a lot of time for protein production.

Recently, many patent documents have introduced a method for producing a specific protein from mammalian urine or semen. For example, US Pat. No. 6001646 discloses the use of a Europlakin II promoter to drive expression of a desired protein from the bladder of a mouse, while US Pat. No. 6201167 describes a recombinant protein in genital tract tissue. Disclosed is the use of a promoter having specificity to the gonad or accessory glands for production and secretion.

Urine contains a relatively high osmolality of 50 to 1000 mosmol / kg, a pH of 4 to 5, high concentrations of urea and ammonium, especially due to its high osmolality and urea It can also be used as a chaotropic agent, but urine contains little protein when compared to milk or semen. The glomeruli and proximal tubules of the kidneys are designed to prevent protein loss. Renal specific promoters, which drive limited protein expression in the lower tract of the kidney, act after the glomeruli and proximal tubule sections, which are the upper urinary pathways of the kidney, so that the proteins produced thereby remove the upper urinary tract. It is able to pass through the lower urinary tract without affecting it and discharge in solution without contaminating the host's endogenous proteins. Compared with other organs, the molecular mechanism of tissue-specific gene expression in the kidney is still unknown. Tam-Horsfall (THP; also known as' Euromodulin ') protein is the most abundant in all mammals' urine and is the thick ascending limb of the kidney's loop of Henle. ) Is expressed exclusively. Euromodulin, a glycoprotein, has been studied for the past 50 years, but the physiological role of euromodulin remains an open challenge. The genomic sequences of cow and rat uromodulin have already been cloned (Yu et al, Gene Expr. 1994, 63-75), but so far the promoters of bovine and rat uromodulin It has not been found to direct the expression of renal cell specificity in vitro and externally of transgenic animals. To see if the promoter of uromodulin highly expresses the genes of interest in the kidney and can excrete such recombinant protein into the urine of transgenic animals, we cloned 5.4 kb of the promoter portion of bovine uromodulin. One selected bovine uromodulin sequence containing key elements essential for the activity of the promoter was tested to direct the expression of a reporter gene specific for the kidney of the transgenic mouse. The 3.9 kb portion of the 5 'proximal region of the bovine euromodulin gene was found to contain regulatory elements that direct the expression of foreign genes that are specific for the kidney and efficient in transgenic mice. In order to achieve the object of the present invention, the present inventors also cloned the 2.3 kb region of the 5 'proximal region of the mouse's uromodulin gene, and then the promoter of the uromodulin of the mouse was ex vivo. It was tested for activity, and the results demonstrated that, as in the case of cows, the uromodulin gene promoter in mice also directs renal cell-specific expression of the reporter gene.

The present invention provides a method for recovering from urine by expressing in a kidney of a transgenic animal and excreting after expression a protein having a biological activity that is targeted to and is driven by the 5 'proximal region of the euromodulin gene. Its purpose is to. The present inventors provided sequences of 5.4 kb and 2.3 kb portions of the upstream region of the uromodulin genes of bovine and mouse, respectively, and the present invention can operate the desired recombinant protein on DNA sequences in more detail. To a transgenic construct comprising a renal-specific uromodulin protein promoter coupled to one another. The transforming construct may include a DNA sequence encoding a signal peptide between a DNA sequence encoding a desired recombinant protein and a 5 'upstream region of the euromodulin gene, and thus the desired recombinant protein. The protein is secreted into the urinary tract.

It is another object of the present invention to provide transgenic animals other than humans using such vectors. The promoter comprises about 3.9 kb BamHI-PstI 5 ′ adjacent site of bovine uromodulin gene (+47 to −3878) and about 1.1 kb DraI-HindIII 5 ′ adjacent site of uromodulin gene in mouse.

1 shows the composition of bovine uromodulin genomic DNA and the nucleotide sequence of the 5 ′ flanking region. Base positions are numbered in the left and right directions relative to the putative transcription initiation site indicated by an underlined '+1'. The portion indicated by the arrow represents the 5 'portion of the bovine euromodulin cDNA sequence based on the already published bovine euromodulin cDNA sequence (Genbank accession number S75958). Part of the first intron sequence (subscript) is included.

Figure 2 is a model of the construct of the transgene. The BamH1-Pst1 fragment (3.9 kb) in the 5 'contiguous region of the bovine uromodulin cDNA sequence comprising the sequence of exon 1 and some of the sequences of intron 1 is a bovine growth hormone polyadenylation (bGHpA). Is linked to the LacZ gene encoding beta galactosidase of E. coli. This chimeric gene was cut in the vector, purified by gel and then microinjected into the egg of the mouse. Restriction enzyme sites are abbreviated as follows: BamHI (B), EcoRI (E), PstI (P) and ApaI (A).

3a and 3b show the results of Southern blot using the tail of genomic DNA of the mouse. Figure 3a shows a restriction map of the transgene construct, a 3.7 kb EcoRI fragment of the transgene using a 600 bp HincII fragment in the beta galactosidase coding gene of E. coli as a probe. Was probed. Figure 3b shows the presence of a transgene on the chromosome, taking about 5 μg of the genomic DNA from the tail of four founder mice with EcoRI and probed with DNA probes of the 600 bp HincII fragments described above, respectively. . The number displayed on the top of the gel represents the number of each founder mouse. Lane 1: DNA molecular weight size marker λ / HindIII, lane 2: negative control mouse (NC). Complete digestion of the integrated transgene with EcoRI results in an ecoRI fragment of about 3.7 kb that contains a 635 bp bovine uromodulin promoter moiety and a 3.0 kb lacZ cDNA.

4A and 4B show the expression of bovine uromodulin-lacZ transplant gene using reverse transcription PCR (RT-PCR) in the tissues of transgenic mice. Figure 4a shows the structure of the transgene construct showing the location of the reverse transcriptase. As primers of the reverse transcription PCR, clacZF1 (5'-GCTTACGGCGGTGATTTTGG) and clacZB1 (5'-AATGTCGTTATCCAGCGGTGC) were used to obtain fragments of 621 bp in size. 4B shows renal RNA (K) and liver RNA (L) of transgenic lines F1-16, F1-20, F1-23, F1-32, F0-52 and negative control mice (NC) Reverse transcription PCR results are shown. 10 ng of the transformed construct plasmid DNA was used as a positive control (PC).

5 shows the renal specific expression of bovine uromodulin-lacZ transplant gene using reverse transcription PCR (RT-PCR) in transgenic mice. In reverse transcription PCR, clacZF1 and clacZB1 were used as primers. Reverse transcription PCR was performed using RNA from tissues of line 16 mice and each lane was brain (B), heart (H), kidney (K), liver (Li), lung (Lu), prostate (Pro) and spleen. And pancreas (Sp / Pan). The lower panel shows the results of simultaneous electrophoresis of the reverse transcription PCR product of rig / S15, a house keeping gene, to demonstrate that the RNA sample used for amplification with the primer specific for lacZ mRNA is normal. have.

Figures 6a and 6b show the construction of the uromodulin genomic DNA of the mouse and the nucleotide sequence of the 5 'flanking region. Figure 6a is a model of the uromodulin gene structure of the mouse, restriction enzyme sites are shown as NheI (N), DraI (D), EcoRI (E), SpeI (S) and HindIII (H). Figure 6b shows the configuration of the uromodulin gene of the mouse and the nucleotide sequence of the 5 'adjacent region. Base positions are numbered in the left and right directions relative to the putative transcription initiation site indicated by an underlined '+1'. The portion indicated by the arrow represents the first base sequence that matches the 5 'end of the mouse's euromodulin cDNA sequence based on the already published mouse euromodulin cDNA sequence (Genbank accession number L33406). Part of the first intron sequence (subscript) is included.

7A and 7B show the structure of the Euromodulin reporter constructs in mice and their renal cell specific promoter activity. 7A shows the structure of the uromodulin promoter-luciferase chimeric constructs of the mouse. Fragments at the 5 ′ neighbor of the progressively smaller mouse uromodulin gene were linked to the firefly luciferase cDNA. The first number represents the first base of the promoter sequence. That is, '-1274' indicates a position corresponding to '-1274' based on the putative transcription initiation site. Filled boxes represent 30 bp of untranslated exon 1 and FIG. 7B shows the flow path of mice commonly analyzed in kidney cells (ST-1 cells) and mesenchymal cells (NIH 3T3). The activity of the renal cell specific promoter in the 5 'upstream region of the modulin gene is shown. Constructs were transfected into ST-1 and NIH 3T3 cells, all constructs were transfected with pRL-CMV vector to compensate for the efficiency of transfection and these cells were treated for 24 hours. Recovery was made after elapsed. Each column shows the efficiency of transfection by the activity of Renilla luciferase; The percentage of firefly luciferase activity for each construct after normalization with Renilla luciferase activity is based on the pGL3 basic plasmid. Each of the ST-1 cells (black histogram) and NIH 3T3 (white histogram) represents the mean ± standard deviation of three independent experiments performed in triplicate.

The present invention relates to a transgenic mammal other than a human comprising a genome comprising a DNA construct consisting of an euromodulin promoter and a DNA sequence encoding any heterologous protein. BamHI-PstI (-3878 to +47) of about 3.9 kb developed upwards of intron 1 defined as the 5286 base of the bovine uromodulin gene DNA sequence shown in FIG. 1 to which the uromodulin promoter is attached A fragment, wherein the promoter comprises a nucleic acid selected from the sequence of FIG. 1 attached thereto.

The present invention also provides a vector comprising a promoter construct linked to a nonhomologous gene encoding a protein having a selected biological activity, wherein the promoter construct is disclosed in FIG. 1 to direct expression of a gene homologous to the kidney. Another feature is a vector which is a bovine uromodulin promoter having a sequence.

Referring to the present invention in more detail as follows.

Although studies of renal specific gene expression help to understand the transcriptional regulation of renal cell differentiation and function, the molecular mechanisms of renal tissue specific gene expression are very unknown. This is especially true when compared to other institutions. In recent years, many cDNAs that have been expressed exclusively or almost exclusively in the kidney have been cloned, among them sodium phosphate cotransporter (NPT2), Na-glucose cotransporter (SGLT2), aquaporin- 2 (aquaporin-2 (AQP2)), chloride channels (ClC-K1, ClC-K2, ClC-5), Na-Cl cotransporter (NCCT), sodium-potassium-chlorine Na-K-Cl cotransporter (NKCC2), hydrogen ion peptide cotransporter (H ⁺ -peptide cotransporter (PEPT2)), vacuola hydrogen ion ATPase V-ATPase B1 subunit (vacuolar H ⁺ -ATPase ( There are many membrane transporters such as V-ATPase) B1 subunit) and organic solute transporters (OAT1, RST, OCT2). Examples of cDNAs with other kidney specificities include erythropoietin, tam-holspal protein, gamma glutamyl transpeptidase (GGT, type 2), vitamin D ₃ 1-α hydroxide Hydroxylase, V2 vasopressin receptor and kidney-specific cadherin (Ksp-cadherin). In addition, GGT (Sepulveda et al., 1997), AQP2 (Nelson et al., 1998), ClC-K1 chloride channel (Uchida et al., 1998), NPT2 (Hilfiker et al., 1998), NKCC2 (Igarashi et al., 1996) and Promoters such as Ksp-cadherin (Whyte et al., 1999) have been cloned and have been demonstrated to direct the expression of renal cell specificity in vitro and ex vivo in transgenic mice.

Previous studies have shown that euromodulin, the most abundant protein in mammalian urine, can bind to fimbriated Type 1 E. coli, thus guessing the role of euromodulin in urinary defense (Parkinnen). Et al., 1988; Reinhart et al., 1990). The lacZ reporter gene is configured to be regulated by the 3.9 kb 5 'region of the bovine euromodulin gene to define a euromodulin promoter element for renal specific expression and to show that the nonhomologous gene is targeted to the kidney. A transgenic mouse comprising the chimeric gene was constructed (FIG. 2). The DNA construct was injected into the fertilized egg of the mouse to produce a transgenic mouse. Analysis of the Southern blot using mouse tail DNA revealed that the transplanted gene was integrated into the genome in 15 of 102 mice (FIG. 3), and some of them reported the reporter genes in a Mendelelli manner. Passed to offspring. The tail DNA of the mouse was digested with various restriction enzymes (HindIII, EcoRV, and KpnI) and Southern blot analysis showed that in all four mouse lines examined, the transplanted genes were tandem repeats and independent sites. Showed that it has been integrated. We used reverse transcription PCR to show the expression of lacZ gene in F0 and F1 transgenic mouse tissues. Total RNA obtained after removing DNA from kidneys and livers of transgenic mice F0, F1 and control mice was examined. Reverse transcription PCR is used to identify the expression of the transgene in the presence of lacZ transcripts (FIG. 4), in which the kidneys of F1-16, F1-20, and F1-23 mice in the lines F0-39 were found to have lacZ transcripts. Turned out to have. Total RNA from kidney, spleen, liver, pancreas, lung, brain, skeletal muscle and heart of F1-16 mice was examined and strong signals were detected in the kidneys (FIG. 5). The above data suggest that a promoter that is active in directing expression in the kidneys of animals, such as the 3.9 kb 5 ′ neighboring sequence of the bovine uromodulin gene, drives the expression of the heterologous reporter gene in the kidneys. Shows. Lack of expression in non-renal tissues indicates a high degree of kidney specificity, in which the regulatory elements of the promoter portion of the bovine uromodulin gene are located at the promoter site and the tissue specificity and differentiation- It is shown that very tight control is performed with respect to dependency-dependence. This tight regulation ensures that the host transgenic animal can be admitted because it can accurately deliver to the target production site, express transduced genes with high efficiency, and minimize the toxic effects of aberrant expression. It is considered a very desirable property for a promoter to produce in protein. The vector of the present invention is responsible for converting the euromodulin promoter into a bioreactor to drive kidney specific expression of exogenous proteins and to purify the biologically active molecules present in the urine. Showed. Here, a "biologically active molecule" refers to a molecule capable of producing a certain effect in an animal and does not necessarily mean an animal containing a transplanted gene, and may include antibodies, growth factors, interleukins, Stimulating factors, kinases, coagulation factors, alpha-antitrypsin, hirudin, erythropoietin, urokinase ), Protein C and coagulation factor VIII.

In order to achieve the above object, the inventors of the present invention cloned the 2.3 kb 5 'adjacent region of the uromodulin gene of the mouse and shows the nucleotide sequence of the 2.3 kb promoter. Comparing the sequences of each species showed a high degree of homology. The nucleotide sequence of the 5 'upstream region of the uromodulin promoter in the mouse was the base sequence of the 625 bp uromodulin promoter in the rat and 88% homology and the base of the uromodulin promoter of 626 bp in the bovine. It was found to have 62% homology with the sequence (Yu et al., Gene Exp., 1994, 4, 63-75). Among the promoters of mice, rats and bovines, there was the greatest homology between the transcriptional initiation region and the exon 1 contiguous region. In addition, it can be seen that the transcription of the gene needs to be closely performed in a similar manner. Using cells transiently transfected into the euromodulin promoter of the mouse through a series of fusions with the firefly luciferase cDNA, the inventors have included the entire exon 1 and a portion of intron 1. Only the reporter construct was found to direct renal cell specific expression of the reporter gene in vitro (FIG. 7B). This fact indicates that the untranslated exons 1 and intron 1 sequences of mouse uromodulin play a very important role in the promoter activity of mouse uromodulin. It is interesting to note that there is a high degree of homology between each species with respect to untranslated exon 1 of euromodulin, which indicates that the specific transcription factor exon 1 of the euromodulin gene is essential for the promoter activity of euromodulin. a major consensus binding site for the factor. Lack of expression in non-renal mesenchymal tissue (NIH 3T3) indicates a high level of renal cell specificity, with regulatory elements in the uromodulin promoter portion of the mouse used for genes downstream of the promoter. It shows a very tight adjustment. This very tight regulation of the promoter indicates that the promoter is affected by several transcription factors and clearly shows that very complex transcriptional control is being performed.

The present invention relates to DNA sequences and a vector required for performing the following process, in particular, the DNA sequences include at least one non-homologous gene encoding a protein of interest, wherein the protein The DNA size of the uromodulin promoters of bovine and mouse and the uromodulin promoter of the bovine and mouse used were determined to be at least 3.9 kb and 1.1 kb in length from the intron 1, exon 1 and 3 'ends, respectively. Located.

The invention also relates to a transgenic animal used to carry out the process according to the invention and to a transformed cell comprising the construct according to the invention.

In one embodiment according to the present invention, the present inventors have provided a transgenic mammal in which a foreign DNA sequence is stably integrated into the genome of the transgenic mammal, wherein the foreign DNA sequence is bound to a DNA sequence encoding a polypeptide. It includes a promoter.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Identification of Bovine Euromodulin Gene

Bovine primary fibroblasts were extracted from bovine genomic DNA. PCR was performed using DNA and Taq polymerases as described in the prior art to obtain a portion of the promoter in the euromodulin gene of the small genome (Yu et al., Gene Exp. 1994, 4, 63-75). Forward primer 5'-GGTCCAATGATGTCTGAATTGC-3 'spanning 25-46 of the bovine genomic promoter sequence disclosed in Genbank accession number S75961 published in the prior art and 623-602 of the genome sequence A reverse primer 5'-GCAGATAGCCTTACCTGAAACC-3 'was used.

The conditions of the PCR used were as follows: Initial denaturation was performed for 25 seconds at 95 ° C. for 90 seconds, then at 95 ° C. for 90 seconds, at 54.6 ° C. for 30 seconds, at 72 ° C. for 45 seconds, and finally at 72 ° C. Elongation was performed at < RTI ID = 0.0 > PCR products amplified using these two primers were cloned into pCR2.1 vectors and sequenced. The comparison of sequences was performed using Blast and MacVector. Inserts were cut using EcoRI and used as searchers to screen bovine genomic libraries. The bovine genome library (Stratagene, La Jolla, Calif.) Used standard plaque hybridization and positive cloning performed secondary and tertiary screens. Clonings that were positive in order to find cloning containing the 5 ′ adjacent site were screened by PCR using the aforementioned primers. A 10 kb fragment containing the 5 'region of the bovine uromodulin gene was subcloned into the XhoI position of the pBluescript II SK (Stratagene) plasmid vector for restriction map and sequencing. The 5.4 kb contiguous region of the bovine uromodulin gene is shown in FIG. 1.

Example 2: Expression of Chimeric Gene (THP-lacZ) in Transgenic Mice

Transplant Gene Construct

For the purpose of testing this concept, the inventors have regulated the β-galactosidase (β-gal) lacZ cDNA of E. coli to a 3.9 kb fragment (BamHI-PstI fragment) at the 5 'neighbor of the bovine uromodulin gene. A chimeric construct (pBSbTHP38lacZ) was constructed. In order to stabilize the mRNA, bovine growth hormone polyadenylation (bGHpA) sequence was added. BamHI-Pst1 of Bovine Euromodulin Gene (3.9 kb Fragment of Sequence Listing) The fragment was inserted at the polylinker position (between BamHI-PstI) of the pBS SK vector to obtain plasmid pBSbTHP38. Plasmid pBSbTHP38lacZ was obtained by inserting lacZ cDNA and bGHpA between PstI and ApaI in the polylinker of pBSbTHP38 (FIG. 2). The construct was separated from the vector using BamHI and ApaI.

Construction of Transgenic Mice

Transgenic mice were obtained through conventional microinjection. 1-2-pl containing about 500 genes were injected into male pronuleus (Jackson Laboratory) of male embryos. The BamHI-ApaI fragment of the pBSbTHP38lacZ vector containing the transforming construct was injected. The embryos were transferred to hormone-prepared female oviducts. Genomic DNA (5 μg) extracted after tail biopsies of mice was cut with EcoRI and Southern blotted to reveal the presence of the transplanted gene in the offspring of the mouse. The DNA fragments were separated by electrophoresis on 0.8% agarose gel and then transferred to nylon membrane. Blot hybridization was performed by random priming of 0.6 kb Hinc II fragments isolated from pBSTHP38lacZ with radioactive ³² P-dCTP. Hybridization was carried out for 16 hours in a solution containing 100 μg / ml of semen DNA of 6 × SSC, 25 mM phosphate buffer (pH 7.2), 5X Denhardt's, 0.5% SDS, EDTA (pH 8.0) and denatured salmon at 65 ° C. Was performed. The blot was washed twice with 2X SSC and 0.1% SDS for 15 minutes at room temperature, then washed twice for 30 minutes with 0.1X SSC and 0.1% SDS at 45 ° C and left at -80 ° C for 16 hours. As a result, about 13% of the progeny of the mice were found to be transgenic animals.

Example 3: LacZ Expression in Transgenic Mice

Total RNA extracted from liver, spleen, pancreas, brain, kidney, lung, heart and skeletal muscle were isolated using Trizol reagent (Invitrogen Inc.). Reverse transcription was performed using an Ultra HF RT-PCR system kit (Stratagene). 1 μg of total RNA was treated with DNase and briefly mixed with oligo (dT) primer and dNTP mixture. After incubating the RNA / primer mixture at 65 ° C. for 5 minutes, 0.5 μl of Stratascript reverse transcriptase was added to the RNA / primer mixture and reacted at 42 ° C. for 1 hour. The cDNA thus synthesized was performed using Taq polymerase and the following primers according to standard PCR protocols: clacZF1, 5 '(5'-GCTTACGGCGGTGATTTTGG) and clacZB1, 3' (5'-AATGTCGTTATCCAGCGGTGC). Fragments amplified by PCR were electrophoresed on 1% agarose gel. Oligo primers specific for rig / S15 (small ribosomal subunit protein), a type of housekeeping gene that is expressed constitutively to demonstrate that the RNA used in reverse transcription PCR reactions is intact (forward: Amplification with 5'-TTCCGCAATTCACCTACC; reverse: 5'-CGGGCCGGCCATGCTTTACG) was also performed in parallel.

Example 4 Cloning of the 5 'Adjacent Site of the Mouse Euromodulin Gene Promoter

A genome walker kit (Clontech, Palo Alto, Calif.) Was used according to the manufacturer's instructions for cloning the promoter region of the mouse uromodulin gene. GSP1 (5′-GCTCCAGACAGTCTTACCTGAACCCAG) was used as the uromodulin specific mouse antisense primer for the first PCR reaction. GSP2 (5'-TCTTACCTGAACCCAGGAC AGGACTG) was used as an antisense primer for nested PCR. The primary PCR reaction was carried out under the following conditions: After 1 cycle of denaturation for 15 seconds at 95 ° C., 7 cycles were performed for 2 seconds at 94 ° C. and 4 minutes at 70 ° C., and 2 seconds at 94 ° C. 36 cycles were performed during and at 65 ° C. for 4 minutes, and a final extension was performed at 68 ° C. for 10 minutes. The first PCR product was diluted 1:50 and 1 μl was used for the second amplification reaction. Cycling parameters in the secondary PCR are the same as those used during the primary PCR except that the primary and secondary steps consisted of 5 and 32 cycles, respectively. This technique was used to identify a single major PCR product in the Dral library, one of the mouse libraries provided in the genome walker kit. The PCR product was gel-purified, cloned into pCR2.1 TOPO vector (Invitrogen) and sequenced bidirectionally (ABI Prism Automated Fluorescence Sequencer). This approach was used to clone and sequence about 1.1 kb (-1037 to +35 bp) mouse uromodulin promoter. Additional 5 'sequence upstream of the mouse uromodulin gene was obtained in a similar manner using two gene-specific primers, GSP3 and GSP4. The latter was based on a sequence of 1072-bps obtained primarily (GSP 3; 5-CCCAGAGGAGCCCAGAATGG and GSP4; 5-GCACGCGTCCCAGAATGGGCAGGACCAC, respectively). This approach was used to clone and sequence an additional 1.0 kb of the mouse uromodulin promoter. The 3 ′ sequence downstream of the mouse uromodulin gene was obtained in a similar manner using two gene-specific primers, GSP5 and GSP6. A 2.0 kb of said mouse uromodulin gene was obtained comprising partial sequences of intron 1, exon 2, and intron 2. The sequence of about 2.3 kb (+ -116 bp at -2090) 5 ′ flanking of the mouse euromodulin gene is shown in FIG. 6.

Example 5 Luciferase Reporter Construct Generation and Transfection Analysis

Certain sites in the cloned 5 'sequence were deleted by careful use of available restriction enzyme sites and subcloning into compatible enzyme sites. In some cases, PCR was used to generate contiguous restriction enzyme sites for cloning purposes. Plasmids used in the transfection experiments were purified using EndoFree Plasmid Isolation Kit (Qiagen) and purity was confirmed using A ₂₆₀ / A ₂₈₀ and agarose gel electrophoresis. For all constructs, the orientation of the inserts was confirmed by direct sequencing. ST-1 cells received from Dr. Adam Sun were maintained in DMEM fed with 10% FBS. These cells exhibit phenotypic properties of the medullary thick ascending limb of Henlesloop (MTAL) in the body, including the expression of uromodulin (Kone et al., Am. J. Physiol). 1995, 269: F718-F729). NIH 3T3 fibroblasts were obtained from the American Type Culture Collection and maintained in DMEM fed 10% fetal bovine serum. The day before transfection, cells were placed in 6-well tissue culture dishes at a density such that 70-80% confluence can be reached upon transfection. Transfection was performed according to the protocol provided by the manufacturer using LipofectAMINE reagent (Invitrogen). Each transfection was performed using 1 μg luciferase reporter construct DNA and 30 ng of internal control plasmid pRL-CMV (Promega). After 4 hours after transfection, the transfection culture was removed, 2 ml of complete culture (including serum and antibiotics) was added and the plate was placed back into the incubator. After 24 hours after transfection, the culture was removed and wells were washed with phosphate-bufer saline to remove detached cells and residual growth culture. Thereafter, 400 μl of 1 × passive lysis culture provided in the Dual-Luciferase Reporter Assay System (Promega) was added per well. Cells were dispersed by scraping with a disposable plastic cell lifter. The samples were transferred to a 1.5 ml microcentrifuge tube, followed by one cycle of freezing (-80 ° C.) / Thawing (room temperature) to complete cell lysis, then 3 at 12,000 × g. Centrifuged in cold microcentrifuge for minutes. Supernatant was used to analyze luciferase activity. Firefly and Renilla luciferase activity was measured sequentially using a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. Luciferase activity was measured using a Turner model 20 Luminometer. The activity of the Renilla luciferase expressed from the CMV promoter was used as an internal control to monitor transfection efficiency. Firefly luciferase activity was normalized based on Renilla luciferase activity in each well.

As described above, according to the present invention, it is possible to induce renal cell-specific expression of transgenic animals of non-homologous genes using a uromodulin gene promoter of a bovine or mouse. It has also been used to enable the mass production of proteins that are useful biologically, therapeutically and industrially but whose natural output is small or not easy to purify.

<110> IN2GEN CO., LTD. <120> Expression of a hetero; ogous polypeptide in renal tissue of transgenic non-human mammals using promoter for tamm-horsfall uromodulin protein <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 5333 <212> DNA <213 > Bos taurus <400> 1 gatccaggtt ggatgacttt atttcaagga cacatgctat tagaaaggca gagttgagaa 60 acacataaaa ttaataaatt tatattaaaa aaaaaaaaag gacaacgtct cacacagctc 120 ctaggctgct tccctaggtc cactattctt gagtgcttaa ccacatgtcc tgattggctc 180 ctgtgccctg gcattgttct acaatttcca ttccctccta attctgtcca tgcctcagta 240 agctgaaagg ggtcatctgt attagttagg accctttcag ttgcaaatgt cagaaactat 300 agcactgttt ggcttaagca gcaatgggaa tttatcccct ataaccagga gaacatcaaa 360 ggtgggtgga ttctgaagct canagtcatt ttctgtctcc ccatctctgt tttcctctgt 420 ttgcttcatt cccaagcagg cacttcccca cggaggggca gtgactgctc cacagctctc 480 agctaacatc ttctcagctt gcaaagaaag tattattaat aataccggt ctttagacat gtctagtagcat gccaggcct ggggcagcag cacctctgag 600 cttagagcag tgaaaggctg cttggcaata caatggcaat tactgcataa ttcttgcctc 660 tatttatttt tcctcctcca accctttctc cctccctctc ttcttttctt aaatagctac 720 tctctgttga gcaaagtgaa gagacagaaa agaaggtatt agcggaggaa aaggaggaac 780 attttatgaa gaaagagcta ccagaacaga atttgcctga gctggagaat gtgaccaagc 840 tcgaagggcc aactgagcag aaagaaaagg ctgaggagga gaaggtagct gagccaaagg 900 aacctccaac acangcagag aaaccaagaa tgaaggaaga cctgtaaggc tctccaaagc 960 cagnaagaga gggtgcccat tttccagatc ctggcatcat tttctgagtt tatcccactc 1020 caaataaaat tccatataat tgtggtaggt gggtggaggc tggataataa aagcccctga 1080 atgtctttgg ccccgtgtta ctcattacct tttttctgct ggtgttgctt tagaagagct 1140 ctttggcctc gaattcagat agggctgcaa gggaagccat aatagtaaca tttaatgagt 1200 gtttacaaca tactgggcat tcgcctagtt ctcacagcct cactgagagc taggtttcat 1260 catccccatt ttacagatga ggaaactgag gcaaagagag gttgagcaac caggcaggat 1320 cacaaggcta gaaagccctt gaaatatgat tctgggcaat ctcactccac aggtggtgca 1380 cacatcacca tcaccactac cacactggga tccccagaga gggtggctga cgctttcaaa 1440 gaggtggact gatctttttt tttgcttcat tttagcacgg taagaaagag atttgaccca 1500 aaatctctcc ctagtctttc actcaaaatg acccattctc gtaagcaacc atatgctagg 1560 ctaacgctca agtcccttct ttccttttat taccataaaa gtaactccac caaaatacat 1620 gactcctcct acttccttcc atgtgactcc atccactatt ccccatctcc aaccaccagt 1680 ctcagccaca gaagtcttct aattggtccc tctactgcgg ctctgtacct ccaagccccc 1740 aacacataca tgcacacaat ccgtattccc taaagctgtt aaagtgatct ttttaaaagc 1800 cgtttcatga gagctcacgg tactaccctg tttttctagt ttctagtggc tacctgccat 1860 tcatggtttc ctcttcaaag aactctgacc atttgtttcc atcctcactt gtcctgcttc 1920 ctctgactct aattcttcct gcatctctct cataagcacc actgtaattg cactgggcct 1980 gcccagataa cctcatctca agacgctgat cttaattata cccacaaagt cccttttgcc 2040 atgtaaggta acattcacac ggagaaggaa atggcaaccc actccagtgt tcttgcctgg 2100 agagtcccag ggaccgggga gcctggtggg ctgcccgtct atggggtcga acagtcagac 2160 acgactgaag tgacttagca gcagcagcag cagcatattt atgctgctgg tttgtgacat 2220 tcacttaatt cgggtataat agaccttctc agtaaaagga gggcatacta ttttagaaac 2280 taaggtctac acaaaccacc tgggcctctc cacagccttc ctattatgta acgcaaaggc 2340 tgattgtact taatcaggtc tcaatagcct gtgccccagt ctctcctttc ccaaacccgg 2400 gcctgtccac atccagacta agcagggaac agcctgcggc cctggaactc accggaacca 2460 gttgccctgg caggagcagg tgttagtatt caagacacga ttccaagctc acctgcttca 2520 aagccatgtg gctgccagtt gaaggagggg tccccggggg cttctaggaa atcacgtgga 2580 actaaatcac atggaacaga gctccttact ggccatgagc cagaaaatgg gtgtgctact 2640 tgcctgtccc ccagatgcaa ctggggcctt aggaagtggg ggctcatatt ggtgaataca 2700 gtatgggtag gtggggcagg tgatgactct ctgttggggg gtcattagag catacaggga 2760 gagaaaaacc ttttacagaa ggatactgtc tcaatgaaag gatgggatgt ggaagatgag 2820 tccagaggca ggtagatgga gaagatgtgg gaggcatggc ttaatgactc caatgaagtt 2880 gccatgtcgt cattaagtgt gtggggctg g tgggggcagg gaagggtgcg aagaacaaag 2940 agtggaaaaa tagccaatta gagaaatata ccacaagatg atctagctgt gttgaatgaa 3000 ggtggtaatt actgttctaa aatacagatt ttattataat tcaagtctga caaaggcaac 3060 agaacaggag ataactgtca tcaaaagata gtttattata ctctcagatc ctaagagaag 3120 gnaatatatc atgccatgaa ggngggcata cagaaaagtg ttagggttgg tcggaaagcc 3180 agagaaagca aggaagaatc tgactatacc ntgaaaatgt aggctttatt gtggttcccn 3240 caggaaggga cagaagaggc aaggtaagca ggtttaggat tggctagttg gaataatgtc 3300 agtaggctct gggacttgga aattgtccct agttatctgg tacctggtcc tgagtgtgag 3360 cctgacagag gagagcttgg gtgatgggct ctagactggc tgttttgcat gtgaaaggca 3420 tgctctcagg aatatgatat tcttagagat agcaaggtgt atctccaatg ctacctatct 3480 ccaaggcata gttcaccctc agtggggcag tccctccaga atcaccaaag tttcaagatg 3540 taaaagcatc aaacacaaaa acttgaataa tgtggttggt tattagactt aagaatagtc 3600 tcagcccaaa ctccctattt tcctacccaa gatctctgcc cagacaactt caggagctac 3660 ctggagctcc atctttaatc ctttaaag ac acccagactt aggttttgac agagcctcag 3720 aaggaaatgg caacccaggg acgggggagc ctggtgggtt gccgtctctg gggttgcaca 3780 gagtcggaca caactgaagc gacttagcag cagcagcagc agcatgtcac caaccagaaa 3840 tgacactcac cacctaggat tgagaaaaaa aatattaaga acttttattt tcttctgaag 3900 ttatagcaaa gaaagggaaa aaaacattct tacggggggt ggggaggaaa tgggcaaagg 3960 atacaaacag ttcagaaaag aataaatagt aagcaaatga aaagataact tcctttttca 4020 ttaaagaact gcaaaaggaa ataacgatga gatatttctc acttttccac aaagatgaaa 4080 gttaatgccc agggtggctg agtactgtgc tgggattgta aactaattgt tatagatctc 4140 tctggggtgc tgtttgggaa gaaatatcgc tgaaaactgt gctacctctt ttcctatgaa 4200 attcccctga ggaggtgagt gagcctctgc tgatagtcac ccgaccacta ggccagacag 4260 aaggagaaag ccctcaaaga ggcaatgctg tggatcactg tcatactttc ctgctcaagc 4320 ctgagttcac acggtacctg atttttctca atatggcatt gccattaatg tggaattagg 4380 tcaggagacc taaggctgaa ccaagccctg tcattctcta ccccatgatt gcacatcacc 4440 aaagcagcat cggcagtgac ttccaca gtt ggtaccattg ctatatgcct taacttgcat 4500 catcttcttt aatggccata acaattctag gacacgggta ttctcgtttt atagatgatg 4560 aaaattacct ctggaaggaa agaaataagc ccactggcac acaaaaaacg ctgaccagga 4620 ttcagataga ctgactccaa agtcagtctg ttcatttaca aaattatcta cttctcaagg 4680 accttccttc atgggaattc aaatttcttg attcacagag catctggtcc aatgatgtct 4740 gaattgcctt ctgtctctga ccttcaggca ttctcagctc ctttcctgct cacatcggga 4800 ccccagggaa gctggttgaa cccatgagna tggaacttgc tttgnaactg agtggccaca 4860 agtatacatc ccagtggggc cagtgagcac cccttttctc ctggagcagc ctgggcttcc 4920 agattttggg cctctgctgg gctccacttt gtgcttttca atgaccaaga aaagcccagg 4980 cacttggaat tttttaccca gttaatttct aactaaagaa cctctcgttg ccaaaagata 5040 taaaacagag cccttgtaac tctgggcaca actgtgaccc ccagtgtcaa tcatttgggg 5100 tctctaccta ttagggaaaa gaacaacaac cacctcacag cctagaaaag gaaaacactg 5160 tgtcaaaagg gaaaaatatt ccacccccat taaaataatt aagaaacaga accagaggat 5220 cattggagga aagactgcca gtgggg gaca gatgtatata tatagatatg atagtcacct 5280 acttgtaaaa ggattaattc tacctttctg gtttcaggta aggctatctg cag 5333 <210> 2 <211> 2206 <212> DNA <213> Mus musculus <400> 2 tggggaggaa tgggtgggag aagcaaattt aagactggtt gatttcttga atttcagtgg 60 gcttgggaaa tagcagctat atattcagtt tcctcgttcc tggctggctt cctggggtga 120 tcagagcaga gtatagtagc cctgtgtggc agtcacacca agcagacagg agatagggca 180 tggctctggt gtggctggta gacataggaa aggatccttg tagcaagatg tttgccatct 240 ccagagacta gacagcccag gaaagtttgt cctcccagga ccagccagca ctgagactgg 300 aatgcatcaa atccagagac cagaaagcac ggtgctagca cttangaaga gacactagcc 360 caaaagtctc cttgctcctg cctaaagctt tgccaattct tgcaaacctt gaaaaattag 420 catctttaaa ttcagaaggg ataccagaag agaacttaca tgggaccttt gtaaaaaaag 480 catagggcat cagtaactaa agntaccaag ataacaatca agtggngagt gaacaaanga 540 catgggcatg tttttttttg ttaagtgtgc gcacgggggg cacacanaca acaggggcac 600 acacacacac aggcatgcac acatgcacca cacacaaaac tggcaaaagt gaataaaaag 660 atatttctca ctttggcaaa gtggatgaaa agttgatcaa aatgaaagtt atactcagaa 720 ctattttgta ctagagggag gttataaatt attgttattg ttatattcta ttttactgtt 780 tgtggcagcc taagttggtc ttgaactcac tatgaagcta gcaatgacct tgagcttctg 840 atccttatat ctacactctc aagtgcccag attataagtg tgcaccacta tactcagttt 900 atgctgtgct aaggactaag cccaattata caaacacaca cacatatata cacacataca 960 cacacacaca cgtatatata tgtatatata tacatacata cacacacaca cacacatata 1020 tgtaaaattt gggaagatat agcaatcttc tttaaagtac atgctacttt ggtccaaaac 1080 tttcactttt aggaagttag gaaggaagag acagaataag agatgtccca agaaagtcag 1140 tgtggttgtc ttagttatgc ttcctgctca gtcaatgttt cagatttttc tcagcacaat 1200 gacatctatt ctatcaagtt tttgataact ctttacatgg gactgggtgt ggcttgtggc 1260 tctagctatt tctatttgtg actgcctatc agcaaagcat ccacttcaga ctttgactca 1320 aacatcacca agtattccca cttgcattgt ctctgttaac cagcatcact gttcacaggg 1380 cagggcatca catctcacaa agggaaaggg aaagggaaga gttaaattcc ctgggatact 1440 agtcacggtg gactcaggca aacagcctct tcaattgtaa gaagattccc tagtccaaag 1500 gaccctctac tggtttggac tccagtcttg tctgacagag gtccagttca ggagtgtcca 1560 gatggtctga taacctgatg ccattctcag agactctttc ctgtctggaa tctagtgagg 1620 aggacttatc tggtgaagct gtcctttaga acaggggtgt gttccagtct tcaaagcaaa 1680 cattcctttt atcctaacac agtctgactt cagatatact gtctttttcc tggctccttg 1740 ggcttaggtc taccttgtcc ttgcccaggt ccaagaaaag gcccagaacc ttggcactgt 1800 tttgccagtt aatgtctaac tgaggaatgt cttgctgcca aaaggtgaaa acagagacct 1860 tgtatttcca ggcacaggtg tgaccccaat gtcaatcatt ttgtgtctaa ctcccagggg 1920 aaaaactaac aacaacagac tcatggcttg gaaaaggtga attctatgcc aaaagggaag 1980 gaaagttcta cccccacaga aacaatctca gagggcagaa gcagagaata atctgaggga 2040 gagggccagc caagggcagg caagtatata ttgatcacag gcacttactt gtgaatggac 2100 cagtcctgtc ctgggttcag gtaaggctgt atgaaactgt cacccccata tccacttctc 2160 ctctatctaa tcccattata tttcaggga g gttgtggtag aagctt 2206

Claims (9)

In a non-human transgenic mammal containing a genome comprising a DNA construct composed of an uromodulin promoter and a DNA sequence encoding any heterologous protein, The uromodulin promoter is a bovine uromodulin promoter represented by the sequence shown in FIG. 1, and the promoter is a transgenic mouse characterized in that the recombinant vector pBSbTHP38lacZ was inserted into the vector pBS SK. 2. The BamHI-PstI of 3.9 kb of claim 1 wherein said uromodulin promoter is developed in an upward direction of intron 1 defined as the 5286 base of the bovine uromodulin gene DNA sequence of FIG. 1. 47) transgenic mouse, characterized in that it is a fragment. delete delete delete In a vector comprising a promoter construct linked to a nonhomologous gene encoding a protein having a selected biological activity, Wherein said promoter construct is a bovine uromodulin promoter represented by the sequence disclosed in FIG. 1 that directs the expression of a gene that is homologous to the kidney. delete In a DNA construct for the production of a non-human transgenic mammal consisting of an uromodulin promoter and a DNA sequence encoding any non-homologous protein, The euromodulin promoter is a DNA construct for the production of a transgenic mammal other than human, characterized in that the bovine euromodulin promoter represented by the sequence shown in FIG. 9. The BamHI-PstI of 3.9 kb according to claim 8, wherein the uromodulin promoter is developed in an upward direction of intron 1 defined as the 5286 base of the bovine uromodulin gene DNA sequence shown in FIG. 47) DNA construct, characterized in that the fragment.