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WO1994023570A1 - Nouveaux elements regulateurs destines a l'expression genique - Google Patents

Nouveaux elements regulateurs destines a l'expression genique Download PDF

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WO1994023570A1
WO1994023570A1 PCT/US1994/004141 US9404141W WO9423570A1 WO 1994023570 A1 WO1994023570 A1 WO 1994023570A1 US 9404141 W US9404141 W US 9404141W WO 9423570 A1 WO9423570 A1 WO 9423570A1
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gene
sequence
regulatory
seq
expression
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Sylvia Lee-Huang
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New York University NYU
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New York University NYU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12N9/1033—Chloramphenicol O-acetyltransferase (2.3.1.28)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/30—Vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region

Definitions

  • the present invention in the field of molecular biology relates to nucleic acid molecules encoding a number of transcriptional regulatory elements which are found in human genomic DNA both 5' and 3' from a DNA sequence encoding human erythropoietin (hEPO) as well as nucleic acid molecules representing these 5' and/or 3' regulatory sequences.
  • These regulatory sequences may be operably linked to protein coding sequences.
  • the regulatory sequences are useful in the construction of recombinant molecules wherein the expression of a coding sequence of interest is subject to control by the various regulatory factors which interact with the above regulatory elements. Such regulatory sequences may be used to modulate production of a gene product.
  • EPO Erythropoietin
  • EPO synthesis is regulated by tissue oxygen availability. Under conditions of hypoxic or anemic stress, EPO production increases and serum concentrations may reach several hundred fold above normal ((Krantz et al . , supra ; Eschbach, J.W. et al . , Kidney Int . 28 : 1- 5 (1985); Bondurant, M.C. et al., Mol .
  • EPO E.D. et al., J.
  • the transgenic material included sequences containing 0.4 kb, 6 kb, 14 kb, and 16.5 kb of DNA 5' to the EPO coding region. This analysis suggested that the region between 0.4 kb and 6 kb upstream from the EPO coding region was critical to tissue- specific expression, since transgenes that lacked this region were inappropriately expressed in a variety of tissues. This region also confers hypoxic inducibility to hepatic EPO production. Despite the foregoing, remarkably little is known about the molecular mechanisms involved in tissue-specific production, developmental regulation and inducible expression of EPO as well as other physiologically important proteins.
  • Recombinant hEPO is known in the art ((Lin, F-K. et al. , Proc. Natl . Acad. Sci . USA 82 7580-7584 (1985); Lin, U.S. Patent 4,703,008; Jacobs, K. et al . , Nature 313:806-810 (1985); Fritsch et al . , PCT Publication O8603520, 19 June 1986); Egrie, J.C. et al . , In: HUMAN CYTOKINES, Aggarwal et al . , eds. , Blackwell Scientific Publications, 1992, pp.
  • the present inventor has reported the isolation of a hEPO cDNA clone (Lee-Huang, S., Proc. Natl . Acad. Sci . USA 81:2708- 2712 (1984) ) which was used to isolate a novel genomic EPO clone containing both extended 5' and 3' flanking regions with a multitude of regulatory elements, which are the basis for the present invention described below, and which have never been found in EPO genomic clones reported to date.
  • Gene therapy involves introduction of a "foreign" gene into a cell and ultimately, into a live animal.
  • Several general strategies for gene therapy have been studied and have been reviewed extensively (Yang, N-S., Cri t . Rev. Biotechnol . 12:335- 356 (1992); Anderson, .F., Science 255:808-813 (1992); Miller, A.S., Nature 357:455-460 (1992); Crystal, R.G. , Amer. J. Med. 92 (suppl 6A) :44S-52S (1992); Zwiebel, J.A. et al. , Ann . N. Y. Acad. Sci . 518:394-404 (1991); McLachlin, J.R.
  • One approach comprises gene transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue.
  • a second strategy involves direct transfer of a functionally active "foreign" gene into mammalian somatic tissue or organ in vivo.
  • Examples of successful transfer of genes include: (a) direct injection of plasmid D ⁇ A into mouse muscle tissues, which led to expression of marker genes for an indefinite period of time (Wolff, J.A. et al . , Science 247:1465 (1990) ; Acsadi, G. et al .
  • retroviral vectors are effective for in vivo and in si tu infection of blood vessel tissues;
  • portal vein injection and direct injection of retrovirus preparations into liver effect gene transfer and expression in vivo Horzaglou, M. et al . , J. Biol . Chem . 255:17285 (1990); Koleko, M. et al. , Human Gene Therapy 2:27 (1991); Ferry, N. et al . , Proc . Natl . Acad. Sci .
  • Retroviral-mediated human gene therapy utilizing amphotrophic, replication-deficient retrovirus systems have been used to introduce a functional adenosine deaminase gene into lymphocytes of deficient patients and treatment of patients with such genetically modified cells.
  • the gene NPT-II and the gene for tumor necrosis factor have been introduced into tumor infiltrating lymphocytes which were then infused into tumor- patients.
  • Retrovirus-mediated gene transfer is performed ex vivo, followed by implantation of the transformed cells into the recipient animal. Retrovirus-mediated gene delivery generally requires target cell proliferation for successful gene transfer (Miller, D.G. et al . , Mol . Cell . Biol .
  • Herpes simplex virus is neuron-specific and persists in infected cells for prolonged periods, allowing it to serve as a useful vector for transfer of foreign genes into the nervous system (Ahmad et al. , supra) .
  • the present inventor obtained from a human leukocyte genomic library, and characterized and sequenced, a 9.3 kb human genomic clone a portion of which encoded hEPO.
  • the genomic clone extends in both directions beyond the boundaries of previously reported EPO-encoding DNA.
  • the genomic DNA disclosed herein does contain classic canonical TATA boxes and a CAAT box.
  • the newly described 5' and 3' flanking regions contain a large number of important sites which are linked to physiologic regulation of EPO expression and which may be utilized in recombinant DNA constructs to produce novel controllable genetic sequences any desired coding sequence of interest which can be brought under the desired regulatory control.
  • regulatory sites identified in the novel DNA sequence 5'and 3' from the coding sequence include: cytokine-responsive consensus sequences, tissue-specific nitrogen regulatory/oxygen- sensing elements and metal responsive elements. Also present are cAMP-responsive elements (CRE) , glucocorticoid-responsive elements (GRE) , and binding sites for transcription factors, including API, CRE, GRE, NF- ⁇ B, Spl and many others.
  • CRE cAMP-responsive elements
  • GRE glucocorticoid-responsive elements
  • binding sites for transcription factors including API, CRE, GRE, NF- ⁇ B, Spl and many others.
  • nucleic acid molecule of the invention may consist essentially of regulatory sequences natively associated with the human erythropoietin gene, said molecule being substantially free of protein-coding nucleotide sequences with which the regulatory sequences are natively associated.
  • the invention is directed to a purified and isolated nucleic acid molecule which comprises at least one regulatory region or element present in the 5' or 3' regulatory regions native associated with the human erythropoietin gene.
  • the 5' extension is encompassed by SEQ ID NO:2 and the 3' extension by SEQ ID NO:3. Both of these extensions are found in a 9.3 kb genomic clone designated hEpSLH, deposited with ATCC (Accession Number 69281) .
  • the regulatory elements may be operably linked to coding sequences for desired proteins and used to regulate the expression of the coding sequence.
  • the present invention includes a nucleic acid molecule in isolated and purified form comprising a first nucleotide sequence consisting of one or more regulatory regions contained in SEQ ID NO:2 or SEQ ID NO:3.
  • the first nucleotide sequence may consist of SEQ ID NO:2 or SEQ ID NO:3.
  • nucleic acid molecule which further comprises a second nucleotide sequence encoding a desired protein in operable linkage with control sequences capable of effecting its expression and in operable linkage with at least one the regulatory region.
  • the above nucleic acid molecule is preferably an expression vehicle such as a plasmid or viral vector.
  • the present invention is directed to a eukaryotic host cell, such as a hemopoetic cell or a liver cell, and preferably a mammalian cell, transfected with the above nucleic acid molecule.
  • a eukaryotic host cell such as a hemopoetic cell or a liver cell, and preferably a mammalian cell, transfected with the above nucleic acid molecule.
  • the present invention also provides a nucleic acid molecule encoding a gene product of interest, for example, hemopoietic growth factors such as colony stimulating factors, GM-CSF, G- CSF, lymphokines, stem cell growth factor and platelet-derived growth factors, under the transcriptional control of regulatory sequence natively associated with the human erythropoietin gene, preferably SEQ ID NO:2 or SEQ ID NO:3.
  • hemopoietic growth factors such as colony stimulating factors, GM-CSF, G- CSF, lymphokines, stem cell growth factor and platelet-derived growth factors
  • the present invention is also directed to a process for preparing a protein, preferably a human protein, or a functional derivative thereof, in a eukaryotic cell or host, the process comprising culturing the cells described above.
  • the present invention is directed to a transgenic non-human mammal essentially all of whose germ cells and somatic cells contain a nucleic acid molecule or DNA molecule as described above.
  • the nucleic acid or DNA molecule has been introduced into the mammal or an ancestor of the mammal at an embryonic stage.
  • a chimeric non-human mammal at least some of whose cells contain a nucleic acid molecule as described above which preferably encodes human erythropoietin.
  • Figure 1 shows gel patterns of Southern hybridization and restriction endonuclease analysis of human leukocyte DNA and the hEpSLH clone. Electrophoresis was carried out on 1% agarose gel in Tris-phosphate buffer, transferred to nitrocellulose filter and hybridized with nick translated 32 P-labeled hEPO cDNA at 65°C overnight. The filter was then washed at 65°C with 0.15M
  • Panel 1A Lane 1, Southern hybridization of human leukocyte DNA BamHI digest with nick translated 3 P-labeled hEPO cDNA; Lane 2, Southern hybridization of hEpSLH genomic clone BamHI fragment; Lane 3, molecular weight marker, 32 P-labeled fragments of phage ⁇ Hind III digest.
  • Panel IB shows results of Southern hybridization of hEpSLH digested with: lane 1, Sad; lane 2, Hind III partial digest; lane BamHI; lane 4, SacI partial digest; lane 5, Hindi; lane 6, BstEII; lane 7, BamHI; lane 8, unlabeled phage ⁇ Hind III digest, molecular marker.
  • Panel 1C shows restriction endonuclease digests of hEpSLH as in Panel IB.
  • Figure 2 is a schematic representation of the genomic structure and restriction enzyme map of the 9.3 kb hEpSLH genomic clone and its comparison with the 3.6 kb genomic clone reported by others (Lin et al . , supra; Jacobs et a . , supra; Egrie et al . , supra) .
  • Figure 3 is a schematic representation of the classical canonical CAAT Box, TATA Boxes and potential transcriptional regulatory elements and their positions in the 5' -flanking region of hEpSLH.
  • Figure 4A, 4B, 4C, 4D, 4E, 4F and 4G shows the nucleotide sequence of the entire 9.3 kb genomic clone hEpSLH [SEQ ID NO:l] . Restriction enzyme sites, CAAT box and TATA boxes are noted.
  • Figure 5A, 5B and 5C shows the nucleotide sequence (SEQ ID NO:2) of the extended 5' -flanking region (BamHI to Hindlll) of hEpSLH. This region, consisting of the first 3892 bases of SEQ ID NO:l, was not found in the 3.6 kb EPO genomic clone from fetal liver reported by others (Lin et al . , supra; Jacobs et al . , supra ; Egrie et al . , supra) .
  • the CAAT box and TATA boxes are underlined.
  • Figure 6A and 6B shows the nucleotide sequence (SEQ ID NO:3) of the extended 3' -flanking region (to the BamHI site), consisting of the last 1777 bases of SEQ ID N0:1.
  • the oxygen- sensing and tissue-specific regulatory elements are underlined.
  • the extended 5' and 3' flanking sequences are unique new sequences having no significant homology with any DNA sequences in the GenBank database.
  • Figure 7 provides a schematic representation of the locations of oxygen sensing (N/O) , tissue specific, and other selected transcriptional regulatory elements in the 3' flanking region of hEpSLH.
  • the lower portion of the figure shows a schematic map of the locations of 14 potential stem-loops in this region.
  • the present inventor has cloned a human genomic DNA fragment which includes not only a protein coding sequence (for hEPO) but also large 5' and 3' regulatory regions containing a number of regulatory elements not heretofore known to be associated with the hEPO gene or to occur together in a single large cluster.
  • the compositions and methods of the present invention thus useful for understanding and treating diseases associated with insufficeint production or overproduction of the protein which they regulate.
  • Methods and compositions are provided for the controlled expression of a gene in a eukaryotic cell or host, in particular, a mammalian gene in a mammalian host, most - y — preferably, a human gene in a mammalian host.
  • the coding sequence and the 5' and 3 ' regulatory sequences are useful in a host cell culture similar or identical to the human host in which the gene is naturally expressed.
  • the present invention provides a substantially purified nucleic acid molecule which corresponds to a human genomic DNA sequence (SEQ ID NO:l) .
  • This nucleic acid molecule includes a coding sequence, a 5' flanking region containing multiple regulatory elements and a 3' flanking region containing multiple regulatory elements. The sequence is shown in Figure 4A, 4B,
  • nucleic acid molecule of the present invention may include a coding sequence, with either the 5' or the 3' flanking region.
  • the present invention provides a substantially purified nucleic acid molecule (SEQ ID NO:2) which corresponds to the non-coding 5' portion of SEQ ID NO:l, and which includes all the 5' regulatory elements contained therein.
  • the nucleic acid molecule of the present invention consists of a portion of SEQ ID NO:2 containing a combination or subcombination of regulatory elements.
  • the present invention provides a substantially purified nucleic acid molecule (SEQ ID NO:3) which corresponds to the non-coding 3' flanking region of SEQ ID N0:1, and which includes all the regulatory elements contained therein, or a subcombination thereof.
  • SEQ ID NO:3 substantially purified nucleic acid molecule
  • the nucleic acid molecules of the present invention may be used to produce recombinant DNA molecules, vectors, plasmids, and the like for expression of any eukaryotic protein.
  • Such recombinant DNA molecules may contain the coding sequence of interest and the full array, or a subcombination of, the regulatory elements contained in the 5' (SEQ ID NO:2) and/or 3' (SEQ ID NO:3) flanking regions natively associated with the hEPO coding sequence in human genomic DNA.
  • the nucleic acid molecules containing the regulatory sequences according to the present invention include sequences responsible for both transcriptional and translational control of the regulated gene. This may include the promoter region (RNA polymerase recognition and binding sites) , and possibly other control sequences which have been modified in such a way that they allow constitutive expression of downstream coding sequences.
  • the regulatory region may also include other portions of the translated and untranslated regions of a gene which participate in transcriptional and/or translational regulation, including 3' untranslated sequences.
  • regulatory region or “regulatory element” refers to any nucleotide sequence the presence of which influences the expression of a coding sequence contained in the same nucleic acid molecule.
  • the ability of the regulatory element or regulatory region to exert such control may in turn be influenced by additional factors such as interleukins or transcription control proteins.
  • An entire protein coding sequence, including the structural region as well as the 5' and 3' flanking regions may conveniently be employed; a second gene of interest may be inserted within the structural region to produce a fused protein upon translation which contains either a fragment of, or the entire, first protein and a fragment of, or the entire, product of the second gene.
  • a gene (or protein coding sequence) of interest may be inserted in operable linkage with the 5' regulatory region or with the 3' regulatory region or with both these regions, as described herein.
  • DNA constructs including selection markers, enhancers, promoters, and the like, which result in enhanced production of a protein in a eukaryotic host.
  • Such constructs and methods for their production and use are well-known in the art. See, for example Kaufman, U.S. 5,079,159, U.S. 4,740,461; Stunnenberg et al . , U.S. 5, 017,487; DeV Amsterdam, U.S. 4,963,481; Etcheverry et al . , 4,940,661; Tibbetts et al. , 4,775,630; Smith et al. , 4,745,051; Skoultchi, PCT Publications WO91/06666 and WO91/06667) ;
  • Figure 5A, 5B, 5C shows the nucleotide sequence (SEQ ID NO:2) of the extended 5' flanking region from the BamHI site to the HindiII site of the genomic clone hEpSLH (see Example, below) .
  • This flanking region comprises 3892 bp and contains the CAAT and TATA boxes and at least 321 potential transcriptional regulatory elements. Table I, below, lists several of these elements and their positions.
  • a schematic diagram is presented in Figure 3.
  • Figure 6A, 6B shows the nucleotide sequence (SEQ ID NO:3) of the extended 3' flanking region from the 5'-most PST I site 3'of the end of the coding sequence to a BamHI site.
  • This flanking region comprises 1777 bp exhibiting many stem-loop structures. It also contains TATA boxes in forward or reverse orientation, and at least about 184 potential transcriptional regulatory elements. Table II, below, lists several of these elements and their positions.
  • TFIID TFIID
  • metal responsive elements include GR/PR-MMTV
  • glucocorticoid responsive elements including GR/PR-MMTV
  • NF- B NF- B
  • API AP2
  • Spl Spl
  • lymphokine responsive consensus sequences and many others.
  • This region also contains two nitrogen regulatory/oxygen sensing sequences at positions 1246 and 1278.
  • hEpSLH clone In the hEpSLH clone, and in embodiments of the nucleic acid molecule of the present invention, are included three TATA boxes with the canonical sequence TATAAA (located at nucleotides 1954, 2006, and 2304 of hEpSLH) . In addition, three potential weak TATA boxes of the sequence TACAAA are found at positions 1754, 1778, and 3554.
  • TATAAA canonical sequence located at nucleotides 1954, 2006, and 2304 of hEpSLH
  • TACAAA three potential weak TATA boxes of the sequence TACAAA are found at positions 1754, 1778, and 3554.
  • IL-6-responsive elements are grouped into two types of consensus sequences (Heinrich, P.C. et al . , Biochem. J. 265:621-636 (1990); Hattori, M. et al . , Proc . Natl . Acad. Sci . USA 87:2364-2368 (1990) ; Majello, B. et al . , EMBO J. 9:457-465 (1990) ) :
  • a decanucleotide GRGRTTYCAY [SEQ ID NO:11] (wherein R is purine base and Y is a pyrimidine) ; and (2) a heptanucleotide, TGR 4 A.
  • the decanucleotide sequence GRGRTTYCAY (SEQ ID NO:11) occurs in the 5' flanking regions of genes encoding the hematopoietic growth factors GM-CSF, IL-3 and IL-2. These elements are also known as “lymphokine consensus elements” and are involved in the coordinate regulation of these lymphokines by each other and by themselves ( (Heinrich et al . , supra) .
  • heptanucleotide sequences are found in genes whose expression is regulated by IL-6, including acute phase hepatic proteins, C-reactive protein, haptoglobin and hemopexin (Gauldie, J. et al . , Proc . Natl . Acad. Sci . USA 84:7251-7255
  • NF-IL6 A nuclear factor, NF-IL6, binds to these elements and mediates IL-6 induction (Poli, V. et al., Proc . Natl . Acad . Sci . 86:8202-8206 (1989)).
  • the promoter region of hEpSLH (and SEQ ID NO:l and 2) contains both of the above sequence motifs, located at both the proximal and distal regions of the promoter.
  • the proximally located element confers tissue specificity
  • the distally located element confers cytokine responsiveness.
  • the nucleic acid molecules of the present invention may contain two copies of the decanucleotide lymphokine consensus sequence, one located at position 1271 (GGGGTTTCAC; SEQ ID NO: ) and other at 1871 (GAGGTTTCAC; SEQ ID NO:5) in hEpSLH. These consensus sequences are located several hundred bases upstream from canonical TATA boxes, but are separated from the main protein coding sequence by a significant distance (see Table IV, below) .
  • the nucleic acid molecule of the present invention preferably contains seven copies of the heptanucleotide IL-6 responsive element.
  • TGGGAGA clustered upstream at positions 304 (TGGGAGA) , 319 (TGGGGAA) , 432 (TGGGGGA) , 442 (TGGGGGA) , 508 (TGAGGGA) , and 601 (TGGGAGA) , and one is located further downstream at position 1837 (TGAGAGA) .
  • IL-6 REs multiple copies of these IL-6 REs at specific distal and proximal regions of the EPO gene play an important role in tissue-specific and inducible coordinated expression of EPO. This is consistent with the observation that IL-6 upregulates hypoxia-induced EPO production in Hep3B cells (Faquin et al . , supra) .
  • the 5' regulatory region of the present invention may be combined with an extrachro osomal replication system for a predetermined host to provide an expression vector for that host.
  • Such vectors will include DNA sequences having one or more restriction sites for insertion of a gene or genes 3' to the regulatory region to provide the regulated transcription and translation of the inserted gene or genes.
  • the vector may also include markers for selection in host cells and other DNA regions of interest.
  • the expression control region can be joined to a desired structural gene and the resulting DNA construct introduced directly into a host cell. Methods of such direct transfer include injection of DNA into the nucleus (Cappechi, M, Cell 22:479-488 (1980)) or co- transformation by the calcium phosphate precipitation (Wigler et al .
  • the expression control region of the present invention may be combined with a terminator for complete transcriptional control of an inserted coding sequence.
  • the terminator may be derived from the gene itself, or the inserted structural gene may carry its own or any other suitable terminator sequence.
  • Extrachromosomal replication systems may be used, for example, autonomously replicating sequences as described by Struhl et al . , Proc . Natl . Acad. Sci . USA 73:1471-1475 (1979)) and the 2 ⁇ plasmid for replication in yeast.
  • Integrating mammalian replication systems may be also be used, for example those derived from papovaviruses such as SV40 and bovine papilloma virus, adenoviruses, avian retroviruses or mammalian retroviruses.
  • papovaviruses such as SV40 and bovine papilloma virus, adenoviruses, avian retroviruses or mammalian retroviruses.
  • a DNA molecule encoding a protein of interest, or a functional derivative thereof may be recombined with vector DNA in accordance with conventional techniques, including blunt- ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, ligation with appropriate ligases, or the synthesis of fragments by the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain signals for transcriptional and translational initiation, and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the signals for transcriptional and translational initiation and the DNA sequence sought to be expressed are connected in a way which permits gene expression. The precise nature of the signals required for gene expression may vary from organism to organism. Intact native proteins can be made in an appropriate host cell by providing the appropriate regulatory sequences or elements, including a promoter.
  • promoter region contains a promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5' -non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the preferred control region is the 5' flanking sequence of described herein, most preferably, SEQ ID NO:2.
  • the non-coding region 3' to the protein coding sequence may be obtained by standard methods.
  • This region may be retained for its multiple regulatory elements as well as its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences (such as a regulatory sequence and a protein-encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation,
  • a regulatory sequence is operably linked to a DNA coding sequence if the promoter is capable of effecting transcription of that coding sequence.
  • transcriptional and translational signals recognized by an appropriate host cell are necessary.
  • the nucleic acid molecule of the present invention may be used for gene therapy of disorders associated with a defective or inappropriately regulated gene.
  • another gene of interest placed under control of the regulatory sequences of the present invention can be used for therapy of an animal in which the gene of interest is dysfunctional.
  • Such an approach is useful for a number of growth factor genes, including genes encoding a variety of hemopoietic growth factors, including GM- CSF, G-CSF, CSF-1, stem cell growth factor, and the like, as well other cytokines such as IL-2 and IL-6.
  • the regulatory sequences may also be used to control stress responses via proteins such as heat shock proteins.
  • the tissue-specific oxygen regulatory sequences present in the nucleic acid molecules of this invention are particularly important for regulating stress responses associated with varying oxygen levels.
  • tissue specific regulatory elements such as those described herein that are in the 5'and 3' regulatory regions, fucntioning alone or in combination, can be used for example, to target the expression of heterologous genes not normally expressed in kidney or liver to kidney or liver cells.
  • the preferred regulatory sequence for liver specific expression includes, but is not limited to, the Bglll - BstEII region of the 5' regulatory region of the present invention (see Figure 2) .
  • the preferred regulatory sequence for kidney specific expression includes, but is not limited to, the BamHI-Hindlll region ( Figure 2) .
  • pancreatic peptides are normally produced in the a , ⁇ , ⁇ , and PP cells of the pancreatic islets of Langerhans; the expression of these proteins are often affected in patients with pancreatic diseases, including diabetes.
  • the promotor-enhancer control elements disclosed herein can be used to target the expression of these pancreatic proteins to either kidney or liver cells by producing DNA constructs comprising the regulatory regions described herein operably linked to the heterologous gene of interest.
  • the expression of a defective gene product can be restored by preparing a construct comprising DNA encoding the functional gene product in operable linkage with liver-specific or kidney-specific regulatory sequences of the human genomic DNA.
  • Retrovirus-mediated gene transfer is performed ex vivo, followed by implantation of the transformed cells into the recipient animal.
  • a DNA molecule according to the present invention for example a coding sequence together with the 5' and/or 3' regulatory sequences described herein (SEQ ID NO:2 and SEQ ID NO:3) , can be used to transform human cells such as bone marrow cells, lymphocytes, fibroblasts or the like from a deficient patient and infused into the patient to provide a source of the missing protein.
  • a selected gene encoding a protein for which a patient is defective can be recombined with the present regulatory sequences, such as SEQ ID NO:2 or SEQ ID NO:
  • the gene therapy approach can be utilized in a site- specific manner to deliver the retroviral vector to the tissue or organ of choice.
  • a catheter delivery system can be used (Nabel, E.G. et al . , Science 244:1342 (1989) ) .
  • Such methods using either a retroviral vector or a liposome vector, is particularly useful to deliver the gene to a blood vessel wall.
  • vascular endothelial cells are exposed to blood circulation, this approach is useful for releasing the protein product of the therapeutic transgene into the bloodstream for systemic delivery.
  • the usual requirement for cell proliferation to achieve gene transfer in a retrovirus-mediated system may be bypassed by using a recombinant adenovirus vector (Horowitz, M.S., In: VIROLOGY, Fields, B.N. et al . , eds, Raven Press, New York, 1990, p. 1679; Berkner, K.L.
  • adenovirus vectors for human gene therapy include the fact that recombination is rare, no human malignancies are known to be associated with such viruses, the adenovirus genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovirus is a safe human vaccine organisms.
  • Adeno- associated virus is also useful for human gene therapy (Samulski, R.J. et al . , EMBO J. 10:3941 (1991) according to the present invention.
  • Herpes simplex virus is neuron-specific and persists in infected cells for prolonged periods, allowing it to serve as a useful vector for transfer of DNA constructs according to the present invention into the nervous system.
  • Gene transfer can also be achieved using "carrier mediated gene transfer" (Wu, C.H. et al., J. Biol. Chem. 264:16985 (1989) ; WU, G.Y. et al . , J. Biol. Chem. 263:14621 (1988) ; Soriano, P. et al., Proc. Natl. Acad. Sci. USA 80:7128 (1983) ; Wang, C-Y. et a . , Proc. Natl. Acad. Sci. USA 84:7851 (1982) ; Wilson, J.M. et al. , J. Biol. Chem. 267:963 (1992)).
  • Preferred carriers are targeted liposomes (Nicolau, C. et al., Proc. Natl. Acad. Sci. USA 80:1068 (1983); Soriano et a . , supra) such as immunoliposomes, which can incorporate acylated monoclonal antibodies into the lipid bilayer (Wang et al., supra), or polycations such as asialoglycoprotein/polylysine (Wu et al. , 1989, supra) .
  • the liver is targeted by producing a conjugate between a liver-recognizing compound and a DNA binding compound.
  • an asialoglycoprotein is produced by isolating orosomucoid from human serum, disialylating it to form asialoorosomucoid.
  • the asialoorosomucoid is conjugated to polylysine which binds DNA without damaging it.
  • This asialoglycoprotein/polylysine conjugate is complexed with plasmid DNA according to the present invention for transfer.
  • the present invention is also directed to a transgenic non- human eukaryotic animal (preferably a rodent, such as a mouse) whose germ cells and somatic cells contain genomic DNA according to the present invention, or which contains the regulatory sequences of the 5' flanking sequence (preferably SEQ ID NO:2) , or the 3' flanking sequence (preferably SEQ ID NO:3) introduced into the animal, or an ancestor of the animal, at an embryonic stage, preferably the one-cell, or fertilized oocyte, stage, and generally not later than about the 8-cell stage.
  • the activated sequence as the term is used herein, means a gene or DNA sequence, which, when incorporated into the genome of the animal, is expressed or functions in the animal.
  • One method of introducing such a gene to achieve chromosomal incorporation and expression is to transfect the embryo with the DNA as it occurs naturally, and select transgenic animals in which the DNA has integrated into the chromosome at a locus which results in expression.
  • Other methods for ensuring expression involve modifying the coding sequence or the regulatory or control sequences of the DNA being inserted prior to introduction into the embryo.
  • One such method is to transfect the embryo with a vector containing an already modified gene.
  • Other methods utilize a gene the transcription of ' which is under the control of the 5' regulatory sequence as disclosed herein, or to use a gene activated by one or more base pair substitutions, deletions, or additions, as is well-known in the art.
  • fertilized eggs are collected by flushing oviducts of mated females.
  • the fertilized eggs contain two pronuclei, one from the sperm and the other from the egg, that form the nucleus of the one-celled embryo.
  • Target DNA sequences in about 2 picoliters, containing about 500 to 600 copies, are introduced into one of the two pronuclei by microinjection.
  • the injected eggs are then transferred into the oviduct of a foster mother (made pseudopregnant by mating with vasectomized males) .
  • a significant proportion of the microinjected fertilized eggs implant in the uterus of the foster mother and develop to full term animals.
  • mice about three weeks after birth, the offspring are tested for the presence of the transgene by Southern blotting or PCR analysis of DNA isolated from a small piece of tissue obtained from the tail.
  • transgene is present in all of the germ cells and somatic cells of the transgenic animal and has the potential to be expressed in all such cells.
  • the presence of the transgene in the germ cells of the transgenic "founder" animal in turn means that all its progeny will carry the transgene in all of their germ cells and somatic cells.
  • Introduction of the transgene at a later embryonic stage in a founder animal may result in limited presence of the transgene in some somatic cell lineages of the founder; however, all the progeny of this founder animal that inherit the transgene conventionally, from the founder's germ cells, will carry the transgene in all of their germ cells and somatic cells.
  • Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the transgenic DNA sequence of the present invention, produced, for example, when fewer than all of the cells of the morula are transfected in the process of producing the transgenic mammal, are also intended to be within the scope of the present invention.
  • Transgenic animals will also serve as an animal model enabling testing of treatment and diagnostic methods for any of a number of human diseases of genetic origin associated with malfunction of one or more of the 5' or 3' regulatory elements which natively flank the hEPO gene.
  • Diseases with abnormal EPO production include, but are not limited to polycythemia, heart failure, high cholesterol and other microvascular disorders.
  • Transgenic animals according to the present invention can also be used as a source of cells for cell culture.
  • Transgenic animals may also be used as bioreactors for the production of large quantities of a protein. Production of a physiologically functional hormone which requires glycosylation, for example, can only be achieved in certain mammalian cell cultures where this hormone is folded and processed properly.
  • the protein is produced and secreted into the milk of a transgenic sheep or cow.
  • the regulatory sequences of genes encoding (8-lactoglobulin, casein, and whey acidic proteins have been cloned and used to control the production of heterologous genes in transgenic animals. Expression of these proteins is regulated by promoters specific for the mammary tissue.
  • a vector is produced comprising a coding sequence (with or without portions of the flanking regulatory sequences) under the control, for example, of a ⁇ -lactoglobulin promoter. This vector is then introduced to sheep or cow ova by microinjection.
  • tPA tissue plasminogen activator
  • Overabundance of a protein may have pathologic consequencs.
  • an overabundance of EPO is associated with polycythemias of chronic obstructive pulmonary disease, cyanotic heart disease, sleep apnea, high affinity hemoglobinopathy, smoking, localized renal hypoxia, primary erythrocytosis, - 2.6 - ectopic EPO secretion by tumors, androgen therapy, Cushing' s disease, Bartter's syndrome and post-renal transplantation.
  • mice deficient in the regulatory sequences described herein, as well in a protein coding sequence which they regulate will be valuable in studying the roles of interactions between regulatory elements and their regulated proteins.
  • they allow the analysis of the effect of specific regulatory elements in an intact animal as opposed to isolated tissues.
  • the absence of the regulatory elements may induce the organism to express compensatory, possibly alternative or redundant, mechanisms. The relative importance of these other mechanisms can thus be studied in the intact animal.
  • targeted disruption of coding or regulatory sequences provides a valuable means to study the role of various parts of an entire gene in the development of the animal and in the pathogenesis of human disease including development defects and cancer.
  • Step 1 Construction of the targeting vector.
  • Step 2 Incorporation of the targeting vector into embryonic stem (ES) cells and selection of targeted cells
  • Step 3 Generation of chimeric mice expressing ES cell clones
  • Step 4 Breeding of chimeric mice to obtain homozygous protein-deficient mice
  • the design and construction of the target vector begins with selection of a region of a coding sequence or regulatory sequence, as described herein, which is to be replaced by a selectable marker, preferably the neomycin-resistance gene (Ne ⁇ *) .
  • a selectable marker preferably the neomycin-resistance gene (Ne ⁇ *) .
  • the first exon of the EPO coding sequence, or a sequence between the BamHI and Hindlll in the 5'-flanking regulatory region of hEPO genomic D ⁇ A may be selected as the sequence to be replaced.
  • the Neo* gene confers resistance to the antibiotic G418.
  • a second selectable marker such as a herpes simplex virus thymidine kinase (HSV-tJc) gene is positioned at the 3' end of the targeting vector.
  • HSV-tk gene confers susceptibility ⁇ to the drug gancyclovir.
  • the basis for target vector integration into the cellular genome and selection of cells undergoing homologous recombination is as follows. A key part of the coding or regulatory sequence is interrupted by the presence of the Ne ⁇ * gene. Specific homologous recombination then occurs between two sites flanking the Neo gene and their homologous site in the cellular D ⁇ A. As the HSV- tk gene is outside these regions, this gene is not incorporated into the cellular D ⁇ A. Thus, cells in which homologous recombination occurs (the desired cells) will not express this thymidine kinase and will therefore not be sensitive to gancyclovir.
  • the genomic D ⁇ A is broken and the ends join the ends of the linearized vector.
  • the resulting cells express the viral thymidine kinase and are therefore susceptible to gancyclovir.
  • the desired cells are selected by exerting (a) positive selection, for example, with the G418 antibiotic, to obtain those cells with the Ne ⁇ * gene, and (b) negative selection with gancyclovir to exclude cells that contain the HSV-tJ gene.
  • ES cells obtained from a mouse strain with an agouti coat color (see below) are grown in complete medium, preferably Dulbecco's MEM (D-MEM) containing 20% fetal calf serum (FCS) , over a feeder layer of irradiated fibroblasts from day 13 mouse embryos.
  • D-MEM Dulbecco's MEM
  • FCS fetal calf serum
  • the ES cells remain undifferentiated and pluripotent for embryo development.
  • Electroporation is preferably conducted by a Gene Pulser apparatus.
  • ES cells are grown to confluence, collected by trypsin treatment and resuspended in PBS at about 10 7 cells/ml.
  • the cells are incubated with linearized target vector DNA at a concentration of about 150 ⁇ g/ml for electroporation.
  • the cells are fed with medium containing G418 (preferably 150 ⁇ g/ml) 24 hours later, to initiate positive selection for cells containing the NecP gene.
  • Gancyclovir preferably at a concentration of 2 ⁇ M is added 48 hours later for negative selection.
  • Double resistant colonies are selected, plated onto 96-well plates and expanded on 24-well plates.
  • Genomic DNA is isolated from transfectants by standard methods of proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. Southern blot analysis is used to confirm targeted disruption of the protein coding sequence or regulatory sequence. DNA of the Neo gene is also useful as a probe to verify successful homologous recombination.
  • Blastocysts are obtained from B6 females 3 days after mating with a stud male B6 mouse. Donor mouse uteri are dissected and flushed with D-MEM containing 10% FCS and 20 mM HEPES buffer (0.5 ml). Blastocysts are individually collected using a capillary pipette and placed in microdrop cultures at 37°C.
  • the coat color of the B6 blastocyst donor mice is black.
  • the mice which donated the ES cells are derived are preferably agouti. This enables easy identification of chimeric offspring mice by the appearance of dominant agouti patches on the black background. Use of such embryonic cells is disclosed, for example by Zijlstra, M. et al . , Nature 342:435-438 (1989))
  • Cells that have undergone homologous recombination and selection, as described above, are microinjected into blastocysts using a microinjection apparatus, which consists of a inverted microscope, micromanipulators, and a cooling stage.
  • a microinjection apparatus which consists of a inverted microscope, micromanipulators, and a cooling stage.
  • ES cells are injected into the blastocoele of each embryo (blastocyst) using a needle with a bevelled and sharpened tip of approximately 20 ⁇ m diameter.
  • the blastocysts are allowed to recover in medium at 37°C and then implanted into uteri of foster mothers.
  • Preferred foster mothers are (C57BL/6 x DBA/2)Fj (BDF1) mice which are pseudopregnant resulting from mating with vasectomized male mice two days earlier. Six to eight blastocysts are surgically introduced into each uterus using methods well-known in the art. These foster mothers generally carry the embryos to term and give birth 17 days later.
  • the litters obtained above will include chimeric mice expressing genes of the blastocyst as well as genes incorporated via the target vector. These chimeric offspring can be identified by coat color.
  • the ES cells are derived from black-agouti mice with dominant agouti coat color, whereas the host blastocysts are from B6 mice with non-agouti black coat color. Chimeric mice are therefore identified by the presence of agouti coat patches (derived from the ES cells) , on a background of black coat color from the host mice. The overall degree of chimerism in all tissue is reflected by the degree of chimerism in the coat color.
  • mice heterozygous for the disrupted protein coding sequence or regulatory sequences are also chimeric, offspring of these heterozygous mice with B6 mice will be entirely agouti in coat color, and will be heterozygous for the disrupted gene or sequences. Intercrossing of heterozygous animals will generate homozygous mice which have a homozygous defect in the gene, either in the coding or regulatory sequences. These are the "gene knockout" mice, as intended herein.
  • tissue specific regulatory sequences present in human genomic DNA can also be used to target cells of the liver or kidney lineage for killing.
  • tumor cells or infected cells can be targeted for death, as has been done using antibodies specific for the tumor cell or the infected cell to deliver a toxic agent to the diseased cell.
  • the preferred regulatory sequence for liver or kidney specific expression includes the 5' and 3' regulatory regions of the present invention (see Figure 2) .
  • the regulatory sequences can be used to target expression of a "toxic" peptide of bacterial, plant or animal origin (Frankel, A.E. et al . , Ann . Rev. Med . 37:125-142 (1986)) to a liver or kidney cell.
  • any of a number of toxic proteins of bacterial origin such as Pseudomonas exotoxin A or Diphtheria toxin (Endo, Y. et al . , J. Biol . Chem. 262:5908-5912 (1987)) are known.
  • Examples of well-known toxic proteins, primarily single chain ribosomal inhibitory proteins, of plant origin, include ricin or abrin A chain, Trichosanthin (Gu, Z. et al . , Acta Chemica Sinica 43:943-945 (1984)) and the non-toxic anti-HIV protein, TAP 29, derived therefrom (Lee-Huang, S. et al. Proc. Natl . Acad.
  • PAP pokeweed anti-viral proteins
  • dianthins stripe, F. et al., Biochem. J. 195:399-405 (1981)
  • gelonin stripe, F. et al . , J. Biol . Chem . 255:6947-53 (1980)
  • GAP 31, DAP 32 and DAP 30 Lee-Huang, S. et al . FEBS Lett .
  • An animal-derived toxic protein is tumor necrosis factor- ⁇ . Such toxic proteins may act as antiviral agents or an antitumor agents.
  • the regulatory sequences of the present invention may also be used to target an antisense oligonucleotide (Hambor, J.E. et al . , J. Exp . Med. 168:1237-1245 (1988); Holt, J.T. et a . , Proc . Nat 'l . Acad. Sci . 83:4794-4798 (1986); Izant, J.G. et al. , Cell 36:1007-1015 (1984); Izant, J. G., et al . , Science 229:345-352 (1985) and De Benedetti, A. et al . , Proc . Natl . Acad. Sci .
  • the antisense oligonucleotides may range from 6 to 50 nucleotides, and may be as large as 100 or 200 nucleotides.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • tissue specific regulatory sequences for targeted expression and for killing of specific cells are known in the art, including expression of (1) SV40 large T antigen in pancreatic ⁇ cells by insulin regulatory sequence (Hanahan, D., Nature 325:115-122 (1985) , (2) diphtheria toxin A chain in pancreas by elastase I regulatory sequence (Palmiter, R.D. et al . , Cell , 50:435-443 (1987) , and (3) ricin or diphtheria toxin A chain in lens cells by the tissue specific regulatory sequence of crystallin genes (Borrelli, E.R. et al . , Proc . Natl . Acad. Sci . USA 85:7572-7576 (1988) ) .
  • agents capable of interacting with hEPO regulatory sequences and affecting expression of a protein coding sequence can be identified by virtue of their ability to interact with one or more regulatory sequence in the 5' and 3' flanking regions described herein (SEQ ID NO:2 and SEQ ID NO:3) .
  • Such interactions may be measured as the ability of the agent to bind to the DNA, or to influence the binding of a regulatory agent to the DNA sequence.
  • binding assays are well-known in the art (see, for example, Sambrook et al. , supra) .
  • a regulatory DNA sequence according to the present invention can be linked to a reporter gene or sequence (or its complement) having a length of between about 5 and 1000 nucleotides, more preferably, 10 to 100 nucleotides.
  • the reporter sequence will have a physical characteristic such as size, suitability to cleavage by restriction endonuclease, sequence, etc., which will permit or facilitate its identification or detection by means well known in the art.
  • the reporter sequence can contain modified bases which may be used to facilitate its detection.
  • the presence of the reporter sequence will be detected through the use of an antibody or an antibody fragment, capable of specific binding to either the reporter sequence, the complement of the reporter sequence or a double-stranded nucleic acid molecule composed of the reporter sequence complement and the reporter sequence.
  • reporter gene sequences include the chloramphenicol acetyltransferase (CAT) gene, ⁇ -galactosidase and human growth hormone. These systems are described in Sambrook et al . , supra .
  • cells transfected with a construct comprising part or all of the 5' (SEQ ID NO:2) or 3' (SEQ ID NO:3) regulatory flanking sequences disclosed herein, and capable of producing a heterologous protein can be tested directly for the effect of an agent on the protein production.
  • transgenic animals expressing human gene in conjunction with part or all of the 5' or 3' regulatory sequences as described herein can be administered the agent and the effect on protein production, its stimulation by a stimulatory factor, or its inhibition by an inhibitory factor, can be ascertained using routine methods, as disclosed herein.
  • Use of such systems to test interactions between the agent being tested and the regulatory sequences will allow detection of agents which can modulate, either stimulate or inhibit, production of a heterologous, preferably human, protein.
  • the cDNA was used to screen a human genomic library cloned in ⁇ gtll. Hybridizing phage were plaque purified, and phage DNA was prepared using established methods (Sambrook, J. et al . , Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)) . A 9.3 kb genomic clone was identified and selected for further analysis. This clone was designated hEpSLH
  • the BamHI insert of hEpSLH was subcloned into pUC19 for large scale preparation.
  • the clone was characterized by restriction analysis and Southern blotting (Southern, E., J " . Mol . Biol . 98:503-517 (1975)) using EPO cDNA as a probe.
  • the sequence of the genomic DNA was determined by the dideoxy method (Sanger, F. et al., Proc . Natl . Acad. Sci . USA 74:5463-5467 (1977)) using ml3mpl8 and ml3mpl9 subclones.
  • the nucleotide sequence of hEpSLH was analyzed using the
  • Transient expression of hEPO was determined by a well- established bioassay, the in vivo exhypoxic polycythemic mouse bioassay (Cotes, P.M. et al. , Nature 192:1065-1067 (1961)) with modifications as described by Lee-Huang, S., Blood 56:620-624 (1980) .
  • FIG. 1 shows the results of restriction and Southern blot analysis of human genomic DNA and of the hEpSLH clone using EPO cDNA as a probe.
  • panel A BamHI digestion of human leukocyte DNA revealed a single 9.3 kb band which hybridized with hEPO cDNA (Panel A, lane 1) .
  • This hybridizing band was the same as that of the BamHI fragment of hEpSLH (Panel A, lane 2) , indicating that hEpSLH consisted of the same single EPO gene known to exist in fetal liver.
  • Lanes 3 and 7 in both figures are the 9.3 kb BamHI fragment, the starting material of the restriction digestion.
  • Figure 1, panel C, lanes 1 and 4 are near complete and partial digests, respectively, of the 9.3kb clone digested with Sad.
  • hEpSLH has a unique Sad site such that complete digestions with this enzyme results in two fragments.
  • a 6.9 kb BamHI-SacI fragment contains the 5' -flanking region and the entire coding region of the clone, it was thus detected by the EPO probe ( Figure 1, panels B and C, lanes 1 and 4) .
  • the other 2.3 kb Sa -BamHI fragment consists mainly of the extended 3'-flanking region. This fragment was not recognized by the probe and thus appears only in lanes 1 and 4 of Figure 1, panel C but not in the corresponding lanes of Figure 1, panel B.
  • hEpSLH has a unique Hindlll site.
  • Hindlll Figure 1, panel C, lane 2
  • Hindlll Figure 1, panel C, lane 2
  • the other fragment of 5.4 kb contains the reported 3.6 kb portion of the hEPO gene and a 1.8 kb fragment of the extended 3' region. Only the 9.3 kb and the 5.4 kb were detected by Southern blot hybridization ( Figure 1, panel B, lane 2) whereas the 3.9 kb band was not detected by the probe and thus absent in lane 2 of Figure IB. These results indicate that the hEpSLH clone extends beyond the fetal liver EPO clones by 3.9 kb at the 5' end, and 1.8 kb at the 3' end. These results were also confirmed by sequence data. Lanes 5 and 6 of Figure 1, panel C, show Hin i and BstEII digests, respectively, of hEpSLH respectively. Lane 8 shows an unlabelled size marker, the Hindlll fragments of ⁇ DNA.
  • the complete nucleotide sequence of the hEpSLH clone was determined.
  • the coding region consisted of five exons and four introns, with intron/exon boundaries flanked by consensus donor and acceptor splice sites. This organization matches that of the fetal liver EPO clones. Over 99% nucleic acid sequence identity was also found in that portion of the 5' flanking region where the hEpSLH clone overlaps with other known clones.
  • Biologically active hEPO was produced in COS-7 cells that had been transiently transfected with an expression vector pSVL, containing either the genomic hEpSLH 9.3 kb BamHI-BamHI or the 5.4 kb Hindlll-BamHI fragments of the present invention under the control of the SV40 late promoter. EPO production was measured by the exhypoxic polycythemic mouse bioassay as described above.
  • the 9.3 kb fragment yielded about 12-19 units hEPO/ml, whereas the 5.4 kb fragment yielded or 7-11 hEPO units/ml.
  • vectors containing these two fragments yielded only 3-5 units hEPO/ml or ⁇ 1 unit hEPO/ml, respectively.
  • Figure 5A, 5B, 5C shows the nucleotide sequence of the extended 5' flanking region from the BamHI site to the Hind III site of hEpSLH (SEQ ID NO:2) . A total of 3892 bp were found in this region. In addition to the CAAT and TATA boxes, at least 321 potential transcriptional regulatory elements were identified in this region. Table I, above, lists some of these elements and their positions. A schematic diagram is presented in Figure 3.
  • TATA BOXES AND CAAT BOX Most eukaryotic promoters for RNA polymerase II contain the sequence TATAA ("TATA box") located about 25 to 30 bp upstream from the transcription initiation site and the sequence CCAAT (CAAT box) located 60 to 80 bp further upstream (McKnight, S.L. et al . , Science 217:316-324 (1982)) .
  • the TATA box is recognized by TFIID, and the TFIID-promotor complex directs the assembly of an initiation complex with RNA polymerase II and other initiation factors (McKnight, S.L. et al . , Cell 46:795-805 (1987)) .
  • the rate of transcription is specified by the interaction of DNA sequence-specific binding proteins and their recognition sites at the TATA box, at upstream activating sequences, and at initiator elements at the start site itself.
  • the hEPO genomic clones disclosed prior to the present invention did not contain classic TATA or CAAT boxes (Lin et al . , supra; Jacobs et al . , supra; Egrie et al . , supra) , leading to the suggestion that the EPO gene may be an example of a "housekeeping gene” that also lack such elements.
  • the EPO gene may be an example of a "housekeeping gene” that also lack such elements.
  • McKnight, supra housekeeping gene expression is highly tissue-specific, is developmentally regulated, and occurs in response to specific stimuli such as hypoxia or cytokines.
  • the hEpSLH disclosed herein contains three TATA boxes with the canonical sequence TATAAA located at nucleotides 1954, 2006, and 2304. In addition, three potential weak TATA boxes, having the sequence TACAAA, were found at positions 1754, 1778, and 3554.
  • the identification of multiple promoters with both canonical and noncanonical TATA sequences in hEpSLH is consistent with, and indeed, provides an explanation for, unexplained observations of multiple transcription initiation sites.
  • Transgenic mice that contain various fragments of the hEPO gene utilize multiple RNA transcription initiation sites detected by RNAse protection assays (Semenza et al. , 1989, 1990, 1991, supra) .
  • RNAse protection assays Semenza et al. , 1989, 1990, 1991, supra
  • fragments that corresponded to protection up to the 5' end of the probe were noted in several reports. These fragments were derived from transcription upstream from the 5' limit of the probe, but were unexplainable.
  • Hypoxic human hepatoma cells similarly utilize multiple transcription start sites, proving that this is not an artifact associated with expression of a transgene in mice. The present inventor concluded that the occurrence of these transcripts results from the canonical TATA boxes described herein.
  • the genomic organization described here is similar to that of the genes encoding GM-CSF and the IL-3, where transcription from a site close to the majority of the coding sequences is detected using RNA probes from this region, but alternative transcripts that arise far upstream, some beyond 10 kb, are also present (Stanley, E. et al . , EMBO J. 4:2569-2573 (1985)). These longer transcripts are associated with a cytokine consensus sequence, and may be related to coordinate expression of multiple cytokines.
  • a CAAT box was identified at position 875 of the hEpSLH clone, with the sequence AGCCACT.
  • This latter sequence represents a high affinity CP2 binding site that is also present in other human genes such as fibrinogen and H-2 b (Chodosh, L.A. et al . , Cell 18:11-24 (1988)). No sites that bind to CP1 or NF1 (CTF) were found. This CAAT box is located far upstream from the TATA boxes.
  • Interleukin-6 plays a central role in host defense mechanisms by regulating immune responses, hematopoietic events and acute phase reactions (Kishimoto, T., Blood 74:1-10 (1990)).
  • IL-6 also enhances hypoxia-induced EPO production in Hep 3B cells (Faquin et al . , supra) .
  • IL-6-responsive elements can be grouped into two types of consensus sequences (Heinrich, P.C. et a . , Biochem. J.
  • the promoter region of hEpSLH was found to contain both of the above sequence motifs. Interestingly, both are also found in the IL-6 REs of C-reactive protein. These elements are located at both the proximal and distal regions of the promoter and are involved in the coordinated regulation of gene expression. The proximally located element confers tissue specificity, and the distally located element confers cytokine responsiveness. — -5 y -
  • the decanucleotide sequence SEQ ID NO:11 has been found in the 5' flanking regions of genes encoding the hematopoietic growth factors GM-CSF, IL-3 and IL-2. These elements are also known as "lymphokine consensus elements" and are involved in the coordinate regulation of these lymphokines by each other and by themselves ( (Heinrich et al . , supra) . EPO genomic DNA contains two copies of this lymphokine consensus sequence one located at position 1271 (GGGGTTTCAC; SEQ ID NO:4) and other at 1871 (GAGGTTTCAC; SEQ ID NO:5) in hEpSLH.
  • these consensus sequences are located within several hundred bases upstream from canonical TATA boxes, but are separated from the main protein coding sequence by a significant distance (see Table IV) . These consensus sequences are thought to be related to basal and induced expression under coordinate control.
  • heptanucleotide sequences are found in genes whose expression is regulated by IL-6, including acute phase hepatic proteins, C-reactive protein, haptoglobin and hemopexin (Gauldie, J. et al. , Proc . Natl . Acad. Sci . USA 84:7251-7255 (1987)).
  • a nuclear factor, NF-IL6 binds to these elements and mediates IL-6 induction (Poli, V. et al . , Proc. Natl . Acad. Sci . 86:8202-8206 (1989)). Seven copies of the IL-6 RE were identified in hEpSLH.
  • TGGGAGA Six of these elements are clustered upstream at positions 304 (TGGGAGA) , 319 (TGGGGAA) , 432 (TGGGGGA) , 442 (TGGGGGA) , 508 (TGAGGGA) , and 601 (TGGGAGA) , and one is located further downstream at position 1837 (TGAGAGA) .
  • the presence of multiple copies of these IL-6 REs at specific distal and proximal regions of the EPO gene may play an important role in tissue-specific and inducible, coordinated expression of EPO, as demonstrated by the upregulation of hypoxia-induced EPO production in Hep3B cells by IL-6 (Faquin et al . , supra) .
  • Table IV Table IV
  • a Lymphokine-encoding gene and species of origin b Consensus recognition sequence, 5' to 3' end. See Table I. c Nucleotide position of consensus sequence relative to TATA boxes of lymphokine-encoding gene. For hEPO, locations relative to each of the 3 TATA boxes are shown d Distance between consensus sequence and start codon ATG in lymphokine-encoding gene.
  • ACTIVATOR PROTEIN BINDING SITES API. AP2 , AND AP4 Two API binding sites were found at positions 2271 (TGACTTCT) and 2909 (CTGACTAA) of hEpSLH, with the latter being a palindromic site. Three AP2 sites at nucleotides 896
  • TCCCCCTCCC SEQ ID NO:6
  • 2621 TCCCCCACCC, SEQ ID NO:7
  • 2785 TCGCCCAGGC, SEQ ID NO:8
  • TCAGCTGCGG single AP4 site at 3040
  • API transcription factor is a heterodimer of the jun and fos proto-oncogene products, which dimerize by a leucine zipper mechanism (Landschulz, W.H. et al. , Science 240:1759-1764 (1988)) .
  • API mediates responsiveness to phorbol esters, but may be involved in basal expression as well (Curran, T. et al . , Cell 55:395-397 (1988)) .
  • AP2 is a tissue-specific factor, present, for example, in HeLa cells but not in hepatocytes (Masayoshi, I.
  • AP4 is involved in tissue-specific and developmental regulation (Mermod, N. et al . , Nature 332:557-561 (1988)) , for example, in pituitary cell type-specific expression of prolactin and growth hormone (Nelson, C. et al . , Nature 322:557-562 (1986)) .
  • Phorbol ester, cAMP, and steroid hormones all stimulate the production of EPO (Rodgers, G.M. et al . , Am. J. Med . 58:31-38 (1975)) .
  • these multiple copies of different types of the AP binding sites may play a coordinated role in the regulation of EPO production.
  • CYCLIC AMP (cAMP) RESPONSIVE ELEMENT cAMP is involved in the expression of numerous eukaryotic genes.
  • the cAMP-responsive element (CRE) enhancer sequence, ACGTCA, is responsible for the induction of many genes in response to increased intracellular cAMP concentrations
  • cAMP causes phosphorylation of the transcription factors that bind to the CRE, leading to transcriptional activation.
  • a protein kinase which activated physiologically by diacylglycerols (DAGs) and by the tumor promoter phorbol myristyl acetate (PMA; also known as tetradecanoyl phorbol acetate or TPA) also regulates the expression of certain genes. This again involves the phosphorylation of transcriptional factor API.
  • the present inventor has identified a CRE sequence, ACGTCA, in the promoter region of hEpSLH at position 1438.
  • GRE STEROID HORMONE GLUCOCORTICOID-METAL RESPONSIVE ELEMENTS
  • GREs Glucocorticoid responsive elements
  • GREs are generally palindromic, often consisting of an inverted repeat sequence separated by a three nucleotide gap, for example, the sequence AGAACA NNN TGTTCT [SEQ ID NO:12] .
  • GREs also include elements responsive to androgens, mineralocorticoids (GR-MT) and progestins (von der Ahe, D. et al . , Nature 313:706-711 (1985)) .
  • steroid hormones are mediated by a family steroid receptors which are specific DNA binding proteins consisting of an N-terminus of variable length, followed by about 60 amino acid residues containing two zinc fingers, and a C-terminus that contains the hormone-binding domain (Yamamoto, K.R. Annu. Rev. Genet . 29:209-252 (1985)) .
  • Free steroid receptor proteins are found complexed with heat shock proteins (hsp) in the cytosol .
  • hsp heat shock proteins
  • a steroid hormone binds to the receptor causing dissociation from the hsp and migration of hormone-receptor complex to the nucleus. There, the complex recognizes and binds to its specific responsive sequence via its zinc fingers. Binding of these hormone- receptor complexes to DNA can either stimulate or inhibit transcription.
  • glucocorticoid-metal responsive element GR- MT
  • TGTCCT glucocorticoid-metal responsive element
  • NF-KB a ubiquitous protein, is a member of the rel-related family of transcription factors (Lenardo, M.J. et al . , Cell
  • NF- ⁇ B-I ⁇ B complex dissociates and the NF-KB subunits dimerize and translocate to the nucleus where they recognize and bind to specific decanucleotide binding sites and activate transcription.
  • NF- ⁇ B binding is known to participate in regulating expression of a number of cytokines, including IL-1, IL-2, IL-6, IFN/3 and TNF- ⁇ .
  • NF- ⁇ B is also involved in the cytokine- mediated expression of liver proteins during inflammatory responses, and may also be exploited by viruses in the expression of viral genes.
  • Two NF-KB binding sites were identified in hEpSLH at nucleotides 1271 (GGGGTTTCAC, SEQ ID NO:10) and 3199 (GGGAATCTC) .
  • the 1271 site overlaps with the lymphokine consensus element, similar to overlaps found in such elements in the IL-2 and IL-6 genes.
  • Spl is a ubiquitous transcription factor that selectively binds to GC-rich regions (GC boxes) with the sequence of GGGCGG or its reversed orientations (Jones, K.A. et al . , Nature 327:179-182 (1985)) .
  • GC-rich polynucleotide elements have been identified in multiple copies in promoter regions of several viruses including SV40, the herpes viruses, and HIV, as well as in many cellular genes.
  • Spl is involved in maintaining basal transcription level, and the core sequence which Spl recognizes has been noted in GC-rich regions of many enhancers, such as the metal responsive element (MRE) , and hsp.
  • MRE metal responsive element
  • the Spl protein contains three zinc fingers at its C-terminus.
  • the hEPO gene is shown herein to contain three Spl binding sites; two are located in the promoter region of hEpSLH at nucleotides 2390 (CCCCGCCC) and 1969 (GGCGGG) , and one is located further downstream, 3' to the Hindlll site.
  • the human genome contains a single copy of the EPO gene, according to Southern blot analysis.
  • the genomic organization and nucleotide sequence of the hEpSLH clone of the present invention indicate that this clone includes the DNA of known fetal liver EPO clones.
  • hEpSLH differs from the known isolated and characterized hEPO clones in that it extends beyond the boundaries of the previously reported hEPO genes by 3.9 kb in the 5' direction, and 1.8 kb in the 3' direction.
  • the extended 5' and 3' flanking sequence contain many transcriptional regulatory elements, described above.
  • the hEPO gene is shown to indeed contain classic canonical TATA boxes and a CAAT box.
  • the newly described 5' flanking region of the gene also contains consensus cytokine sequences, tissue-specific and metal responsive elements, and binding sites for transcription factors, including API, CRE, GRE, NF- ⁇ B, and Spl, and heat shock protein hsp70 recognition sites. These regulatory elements have not been found in the EPO genomic clones thus far described (Lin et al . , supra; Jacobs et al . , supra; Egrie et al . , supra) .
  • nucleotide sequence of the extended 5' flanking region from the BamHI site to the Hindlll site of hEpSLH (SEQ ID NO:2) is a new and unreported sequence.
  • a computer-aided homology search of the entire 3892 bp in the 5' flanking region and the entire 1777 bp in the 3' flanking region against the sequences in the GenBank database did not reveal significant homology with any published DNA sequence.
  • the 3' flanking region of the EPO gene has also been implicated in oxygen sensing.
  • Transient transfection studies showed that a 70 base sequence located 120 bases downstream from the poly-A addition signal conferred oxygen-regulated expression on a variety of heterologous promoters, although only imparting a 20-fold increase in mRNA levels (Semenza, G.L. et al . , Proc . Natl . Acad. Sci . USA 88:5680-5684 (1991); Pugh, C.W. et al . , Proc . Natl . Acad. Sci . USA 88:10553-10557 (1991)).
  • FIG. 7 The genomic structure of the extended 3' flanking region of hEpSLH is shown schematically in Figure 7.
  • Figure 6A, 6B shows the complete nucleotide sequence (SEQ ID NO:3) of the extended 3' flanking region. It extends from the 5-'most Pst 1 site of the reported clones to the 3' BamHI site. This region consists of 1,777 bp.
  • a computer-aided homology search of the entire sequence against the nucleotide sequence data in the GenBank database did not reveal significant homology with any published nucleotide sequence. Thus, the sequence described herein is new and unique.
  • Table IV presents a selected list of the regulatory elements and their nucleotide positions in the extended 3' flanking region.
  • tissue specific regulatory elements and cytokine responsive element many other potential transcriptional regulatory elements were also identified in this region.
  • a schematic map of these regulatory elements is shown in Figure 7.
  • B Stem-Loops
  • the extended 3' flanking region contains many inverted repeats that allow the formation of stem-loop structure.
  • a total of 14 possible stem-loops with maximum loop size of 20 nucleotides were identified.
  • Figure 7 provides a schematic map of these stem-loops and their nucleotide positions.
  • DNA looping is an important regulatory feature of gene expression, and stem loop formation is involved in the regulation of transcription of many genes. The effect of enhancers is often mediated by loop formation. Formation of a stem-loop facilitates the interaction between DNA binding proteins and their cis-acting elements, and is therefore expected to be important in the regulation of hEPO. Furthermore, stem-loop formation may also increase the stability of mRNA.
  • EPO mRNA levels in mouse liver and kidney increase several hundred fold in response to anemia or hypoxia (Bondurant et al . , supra ; Schuster et al . , supra) .
  • An oxygen sensing enhancer element is located in the 3' flanking region of the EPO gene (Semenza et al . , 1991, supra; Beck et al . , Goldberg et al . , supra; Imagawa et al . , supra; Pugh et al . , supra; Maxwell, P.H. et al . , Proc . Natl . Acad. Sci . USA 90:2423- 2427 (1993) .
  • hypoxia-regulated expression of the mouse EPO gene is provided by a DNA fragment located 120 nucleotides 3' to the polyadenylation site. About 70 bp in this fragment are necessary and sufficient for the enhancer activity.
  • This enhancer element was originally reported to be cell type- specific and active only in hepatoma cells but not in CHO and MEL cells (Pugh et al . , supra) , although this observation appears to have been an artifact of the low cell density used.
  • transient transfection of an ⁇ l-globin reporter gene coupled to the hypoxia-enhancer region of the mouse EPO gene revealed than an oxygen sensing system similar to that of EPO is commonly present in mammalian cells (Maxwell et al . , supra) .
  • This enhancer is active in other cell types including human fetal lung fibroblasts, skin fibroblasts, monocyte/macrophages, monkey renal fibroblasts, pig renal epithelium, Chinese hamster - 47 - ovary cells and mouse renal adenocarcinoma cells. This suggests a common mechanism of oxygen-regulated gene expression in biological systems.
  • hypoxia enhancer element in hEPO The status of a hypoxia enhancer element in hEPO is less clear.
  • a hypoxic-enhancer-like activity was observed in a 255 bp fragment at the 3' flanking region (Imagawa et al . , supra) .
  • This fragment bore no homology with the hypoxia-regulatory element of mouse EPO and demonstrated no enhancer-like activity when linked to an c-1-globin reporter gene (Pugh et al . , supra) .
  • the present inventors have found that the extended 3' flanking region of human genomic EPO DNA contains two copies of nitrogen-regulatory/oxygen- sensing consensus sequences, namely, 5'-TTTTGCA and 5'-CCCTGCA at positions 1278 and 1246, respectively.
  • sequences 5'-TTTGCA and its homologues are common nitrogen-regulatory/oxygen-sensing sites on all of the bacterial nif genes, except nifH, responsive to both the ntrC (nitrogen assimilation) and nif A gene products (nitrogen fixation) .
  • 5'-CCCTGCA is found only at the regulatory site of the nifH (nitrogenase) gene of the enteric bacterium Klebsiella pneumoniae, and is responsive only to the nif A gene product (Ow, D.W. et al., Proc . Natl . Acad. Sci . USA 80:2524-2528 (1983)) .
  • Nitrogenase the nitrogen fixation enzyme, fixes atmospheric N 2 to ammonia reductively. This reaction is under stringent hypoxia regulation and is inhibited by oxygen. Oxygen also represses the expression of the nitrogenase gene via the nifL oxygen-sensing regulation (Fay, P. Microbiol . Rev. 56:340-373 (1992)) . Thus, the identification of multiple copies of the nitrogen-regulatory/oxygen-sensing consensus sequences in the hEPO gene is very important .
  • 5'-TTTTGCA is responsive to both of the nitrogen fixation and nitrogen assimilation regulators
  • 5'-CCCTGCA is responsive only to nitrogen fixation regulation
  • the ntr and nif genes are evolutionarily related and conserved between species (Fay, supra) . This conservation from nitrogen fixation genes in bacterial cells to the human EPO gene underscores the likelihood that a common mechanism exists these regulatory functions among very different cell types. D. Tissue Specific Regulatory Elements
  • tissue specific regulatory elements are found in the extended 3' flanking region of hEPO as disclosed herein. These are shown in Table IV, above, and include: (1) the binding sites for A-activator (AABS) , 5-GTGGTGCAA at position 409;
  • CAAT/enhancer binding protein C/EGP
  • 5'TGGTGCAAT 5'- TTTTGCAAT at positions 410 and 1278;
  • D site of albumin promoter (DBP), 5'-TGATTTTGT at position 345;
  • Hepatocyte nuclear factor (HNF) 5'-TATTTTGT at position 339, 5' -TGTTTGT at positions 351, 355 and 359, as wells as its complementary sequence, 5'ACAAACA at positions 1, 1140 and 1144.
  • AABS is a liver-specific transcriptional regulatory element with the consensus sequence 5' -GTGNNGYAA. It is found in the A2 vitellogenin gene of Xenopus laevis, as well as in liver- specific, IL6-responsive, acute phase genes of human including the hemopexin, haptoglobin, and C-reactive protein genes (Kaling, M. et al., Mol . Cell . Biol . 12:93-101 (1991)) . Both AABS and IL-6 responsive element interact with at least three transcriptional factors, C/EBP and LFB/HNF1. C/EBP is also known as the CCAAT/enhancer binding protein and is found in fully differentiated liver cells, fat and lung tissue.
  • LFB/HNF1 was originally identified as a liver-specific transcriptional factor which recognizes the HPl element of liver specific genes, but was found to be present in the intestine and kidney as well. It is noteworthy that the C/EBP binding site, 5'TTTTGCAAT at position 1278 overlaps with the nitrogen-regulatory/oxygen-sensing consensus sequence, 5'TTTTGCA. The identification of this array of tissue-specific and oxygen-sensing regulatory elements in the 3' flanking regulatory region of hEPO DNA provides an abundant resource for further examination of the control and regulation of EPO expression in the human.
  • a Pst linker is added at the 3' end of chloramphenicol acetyltransferase (CAT) coding sequence by PCR amplification of a 792 base pair Hindlll fragment containing the CAT gene coding sequence (CAT GenBlock, available from Pharmacia Biotech) .
  • the 9.3 kb hEpSLH fragment (SEQ ID NO:l) cloned into the BamHI site of pSVL, is cleaved with Hindlll/PstI, and the PCR-amplified Hindlll-PstI CAT fragment is inserted by ligation.
  • the resulting pSVL construct containing CAT coding sequence flanked by 5' (SEQ ID NO:2) and 3' (SEQ ID NO:3) regulatory regions, is used to transfect COS-7 cells (ATCC#1651) by the calcium phosphate method (Wigler M. et al., Cell 22:223- 232 (1977) ) .
  • Transient expression of CAT enzyme is determined using standard methods as described in Sambrook et al . , supra (sections 16.59 through 16.65) .
  • CAT enzymatic activity is expressed, indicating that the 5' and 3' sequences effect expression of CAT when they are operably linked to the CAT coding sequence.
  • GGGATCTCAC TATGTTGGCC AGGCTGGTCT CAAACTCCTG GGCTCAAGAA ATCCTCCTGG 840
  • CTCAGGCTCC CAAAATGTTG GGATTACAGG TGTGAGCCAC TACACTTGGG CCAAATCCCC 900
  • TTGTAAAGTA GGGGTTTCAC CATGTTGCCC AGGCTGGTCA AGCCAACTCC TGGGCTCAAG 1320
  • CTGTAATCCC AGCACTTTGG GAGGCTGAGG TGGGTGGATC ATCTGAGGTC AGGTGTTTGA 8460
  • GGGATCTCAC TATGTTGGCC AGGCTGGTCT CAAACTCCTG GGCTCAAGAA ATCCTCCTGG 840
  • CTCAGGCTCC CAAAATGTTG GGATTACAGG TGTGAGCCAC TACACTTGGG CCAAATCCCC 900
  • TTGTAAAGTA GGGGTTTCAC CATGTTGCCC AGGCTGGTCA AGCCAACTCC TGGGCTCAAG 1320
  • CTCCTCCCTA AGCAGCACCC TGGCCTCATC TTCCACCTCT CTCTTCTCCA TCTTCCCTTC 1680
  • CTAAACTTAA TAATAAATGA TGTACATATG GTGCATTGTT GACACCACGG GACCAGAAGC 2460 GGTGACCCCC CTGGACCAGC TTTCACTATC TTGTGTGTGT CTATTATTTC TCAACCTGCT 2520
  • GCTCGGGGAC AAGGGCCACT CTAGGTGGTC CATTTATATA TTTGTGATTT TGTTTGTTTG 360
  • CTACAGTGCC CTCTTCAGGT CTTACTAGGA ACTGCTAGAG AAACCTACAA GCGATAGATC 1500

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Abstract

Nouvelles molécules d'acide nucléique contenant un certain nombre d'éléments régulateurs transcriptionnels qui se trouvent dans les régions 5' et 3' par rapport à la séquence codante d'érythropoiétine (EPO) dans l'ADN génomique humain. Ces séquences de régulation peuvent être utilisées pour la production de molécules d'ADN recombinées, l'expression d'une séquence codante d'ADN à étudier étant soumise au contrôle des différents facteurs régulateurs qui interagissent avec les éléments régulateurs décrits ci-dessus. Des procédés permettant d'obtenir différents produits géniques sousmis au contrôle des séquences d'ADN de régulation décrites ci-dessus sont également décrits, ainsi que des animaux transgéniques dans lesquels ces séquences d'ADN ont été indroduites.
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND CELLULAR BIOLOGY, Volume 10, No. 3, issued March 1990, G.L. SEMENZA et al., "Human Erythropoietin Gene Expression in Transgenic Mice: Multiple Transcription Initiation Sites and cis-Acting Regulatory Elements", pages 930-938. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 9, No. 11, issued November 1989, J.A. BOKAR et al., "Expression of the Glycoprotein Hormone Alpha-Subunit Gene in the Placenta Requires a Functional Cyclic AMP Response Element, where as a Different cis-Acting Element Mediates Pituitary-Specific Expression", pages 5113-5122. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Volume 81, issued May 1984, S. LEE-HUANG, "Cloning and Expression of Human Erythropoietin cDNA in Escherichia Coli", pages 2708-2712. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Volume 88, issued October 1991, G.L. SEMENZA et al., "Cell-Type-Specific and Hypoxia-Inducible Expression of the Human Erythropoietin Gene in Transgenic Mice", pages 8725-8729. *

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