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WO1993010135A1 - Characterization of estrogen responsive mouse lactoferrin promoter - Google Patents

Characterization of estrogen responsive mouse lactoferrin promoter Download PDF

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Publication number
WO1993010135A1
WO1993010135A1 PCT/US1992/009459 US9209459W WO9310135A1 WO 1993010135 A1 WO1993010135 A1 WO 1993010135A1 US 9209459 W US9209459 W US 9209459W WO 9310135 A1 WO9310135 A1 WO 9310135A1
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sequence
promoter
construct
lactoferrin
gene
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PCT/US1992/009459
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French (fr)
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Christina T. Teng
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The United States Of America As Represented By The Secretary Department Of Health And Human Services
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Publication of WO1993010135A1 publication Critical patent/WO1993010135A1/en

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    • 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
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • 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
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to identification and characterization of the promoter region of the gene which encodes mouse lactoferrin. In another aspect, it relates to a method of targeting a particular nucleotide to a specific tissue and controlling its timing and level of expression by attaching the mouse lactoferrin promoter to a particular nucleotide and using the promoter and nucleotide to obtain a transgenic animal or to perform gene therapy in humans.
  • Lactoferrin is an iron-binding glycoprotein originally found in milk (Masson, P.L., and Heremans, J.F. (1971) Comp. Biochem. Physiol. 39B, 119) .
  • This protein, together with transferrin and melanomas antigen p97 belongs to a gene family which arose from ancient intragenic duplication 300 to 500 million years ago (Bowman, B.H., Yang, F. , and Adrian, G.S. (1988) Advance in Genetics 25, 1- 38; Park, I., Schaeffer, I.E., Sidole, A.A. , Baralle, F.E., Cohen, G.N. , and Zakin, M.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3149-3153).
  • Lactoferrin is expressed at high levels in neutrophil (Masson, P.L., Heremans, J.F., and Shonne, E.J. , (1969) Exp. Med. 130, 643-656) , in lactating mammary gland (Masson et al.
  • lactoferrin gene activity is a complex process. Its expression in mouse uterus is controlled by estrogen (Pentecost et al. (1987)), while prolactin plays an important role in regulating its expression in mammary gland (Green et al. (1978)). Lactoferrin is not expressed in the brain but is constitutively expressed in neutrophil as it matures (Teng et al. (1989) , Panella et al. (1991)). In view of such a complicated regulation of the lactoferrin gene in various tissues in a variety of ways, the 5- lactoferrin promoter region has been isolated and analyzed in order to understand more about the molecular mechanisms involved.
  • mouse lactoferrin gene 5*-flanking regions The functional structure of the mouse lactoferrin gene 5*-flanking regions has been isolated and characterized. It has been shown that the mouse lactoferrin promoter region contains various putative regulatory elements found in both housekeeping and inducible genes. Regions of negative and positive regulation of the basic lactoferrin promoter activity have been identified with transient transfection into various cell lines of CAT reporter plasmids. Plasmid containing the estrogen responsive element consensus sequence of the lactoferrin gene responds to estrogen stimulation in the presence of estrogen receptors. SUMMARY OF THE INVENTION
  • the present invention relates to a DNA segment having a nucleotide sequence as shown in FIG. 2 (b) and allelic variations thereof.
  • the present invention further relates to the DNA segment having a nucleotide sequence as shown in FIG. 2 (b) wherein the segment is a promoter region for a gene encoding mouse lactoferrin protein.
  • FIG. 1 Primer extension to determine the 5 1 end of the lactoferrin mRNA.
  • RNA isolated from DES-treated mouse uteri, was used as the template for primer extension. 5* end-labeled oligonucleotide, corresponding to the antisense sequence of the first exon was hybridized to 20 ⁇ g of total RNA as described in Experimental Procedures. The reaction products were analyzed on 12% polyacrylamide/urea sequencing gel. One major band at position -32, two minor bands at positions -24 and -23 upstream from the first ATG site, were detected (arrow) .
  • FIG. 2 Nucleotide sequence of the 5' end of mouse lactoferrin gene.
  • A Diagrammatic representation of the sequencing strategy and partial restriction map of ⁇ J14.
  • a 3053 bp of the lactoferrin gene containing the first exon, part of the first intron and 2661 bp 5*-flanking sequence were restriction-digested and subcloned into either bacteria phage M13mpl8/19 or pGem 32 plasmid as described in Experimental Procedures.
  • Solid arrows indicate the regions were sequenced on a single strand from M13mpl8/19 and the dashed arrow indicates the regions were sequenced on double-stranded plasmid with synthetic oligonucleotide as the primer.
  • H Hind III; E, Eco RI; P, Pst I.
  • FIG. 3 DNase I footprinting of mouse lactoferrin promoter region.
  • a 298 bp Hinc II/Xba I DNA fragment (- 589 to -291) was 3 » end-labeled at position -291 (sense strand) and incubated with a different amount (10 or 20 ⁇ g) of DES-treated mouse uterine nuclear extract (ut) or liver nuclear extract (L) , and then treated with DNase I. Control sample treated with DNase I but without added protein (0) . DNA "G+A" and "G” sequencing reaction on the same probe were included as markers. Major protected region designated as I. The number alongside refers to the nucleotide position relative to the transcription initiation site.
  • FIG. 4 Structure and activity of lactoferrin promoter-CAT chimeric gene.
  • lactoferrin 5'- flanking sequences inserted in front of the CAT reporter gene.
  • the chimeric constructs were transiently transfected into RL95-2 and C6 cells as described in the Examples. All DNA fragments were inserted at 5• to 3' orientation relative to the transcription initiation site; the nucleotide boundaries were indicated (left panel) .
  • the 100% CAT activity determined from 1.7 mL14-CAT in RL95-2 cells corresponds to 50% chloramphenicol acetylation/40 ⁇ l cell extract /2 hrs.
  • the 1.7 mL14-CAT construct was constitutively expressed in RL95-2 cells and the promoter was most active among all constructs tested in these cells.
  • Relative CAT activity in relationship to the 1.7 mL14-CAT in RL95-2 cells are shown next to each column (right panel) .
  • the plasmid pCAT-Basic and pSV-CAT were also used as negative and positive controls in these experiments. Each bar represents an average of six determinations.
  • CAT activity was normalized with the co-transfected ⁇ -galactosidase activity. D TATA box; O CAAT boxes.
  • FIG. 5 Estrogen responsiveness of the lactoferrin promoter-CAT chimeric gene transfected into RI-95-2 cells.
  • A Schematic illustration of the chimeric gene 0.6 mL14-CAT which contains 568 bp of the lactoferrin 5'-flanking region was inserted in front of the CAT reporter gene.
  • the region contains the consensus sequence of ERE and COUP is indicated.
  • TATA box, two CAAT boxes, reversed AP 2 site and Sp 1 site are also indicated.
  • RL95-2 cells containing low level of estrogen receptor (Way, D.L., Grosso, D.S., Davis, J.R., Surwit, E.A. , and Christian CD. (1983) In Vitro 19, 147-158) were used either directly (lane 1 and 2) or were co-transfected with HEO (lane 3 and 4) , the estrogen receptor expression vector (Green, S., Walter, P., Kumar, v., Krust, A., Borneert, J.M., Argos, P., and Chambon, P. (1986) Nature 320, 134-139), to test its estrogen responsiveness.
  • the estrogen receptor expression vector Green, S., Walter, P., Kumar, v., Krust, A., Borneert, J.M., Argos, P., and Chambon, P. (1986) Nature 320, 134-139
  • the cells were incubated in the presence (lane 2 and 4) or absence (lane 1 and 3) of 10 ⁇ 8 M DES as described in the Examples. Cell extract from these cultures were assayed for CAT activity with ⁇ -galactosidase normalization. The results were visualized by using thin layer chromatography, followed by autoradiography.
  • the present invention relates to a promoter region for a gene encoding mouse lactoferrin protein.
  • This promoter region contains: a TATA like sequence, two CAAT boxes, three GC boxes including one within the first intron, an AP2 site, seven PU boxes, an AC rich region, A Bl sequence and an estrogen responsive element (ERE) consensus sequence overlapping with a COUP binding element.
  • the present invention further relates to an isolated DNA segment having a promoter sequence from -2661 to +1 as shown in FIG. 2 (b) or variations or segments thereof having promoter activity.
  • the present invention additionally relates to portions of the above isolated DNA segment which serve as promoter regions for a gene encoding mouse lactoferrin protein.
  • the present invention also relates to allelic variations or variations achieved through mutations, insertions, deletions, or substitutions, all of which are functionally equivalent to a promoter region of the isolated DNA segment.
  • allelic variations achieved through mutations, insertions, deletions, or substitutions, all of which are functionally equivalent to a promoter region of the isolated DNA segment.
  • the present invention further relates to a DNA construct having a promoter sequence which is operably linked to a heterologous structural gene in a manner sufficient to modulate the expression of the structural gene.
  • this DNA construct can further be linked to a plasmid or a vector.
  • a plasmid or a vector One skilled in the art would understand the methods used to achieve such a construct which can be linked to a plasmid or vector.
  • the present invention also relates to such transfected host cells constructed using methods well-known in the art.
  • host cells can include but are not limited to human endometrium carcinoma RL95 2 cell or C6 rat glioma cell.
  • the present invention relates to a method of carrying a particular nucleotide sequence to a specific tissue in an animal by operably linking at least a portion of the isolated DNA segment of the present invention having a promoter sequence to a particular nucleotide sequence.
  • This combination of DNA promoter sequence and nucleotide sequence can be administered to an animal.
  • One skilled in the art would understand the methods that would be used to combine the two segments and to administer the combination to an animal.
  • the present invention also relates to a method of treating a condition in a patient characterized by a genetic abnormality. . This can be accomplished by administering to the patient a sufficient amount of a nucleotide sequence which is operably linked to a portion of the promoter DNA sequence.
  • a nucleotide sequence which is operably linked to a portion of the promoter DNA sequence.
  • One skilled in the art would understand the relevant technology and methods of administering a sufficient dosage to achieve the desired clinical results.
  • the present invention further relates to an animal cell which contains the construct having the promoter sequence operably linked to a heterologous structural gene so that the heterologous structural gene is modulated by the promoter sequence which does not naturally modulate the gene.
  • the animal cell which contains the construct is preferably an embryo cell.
  • the present invention further relates to an animal whose genetic material contains the above construct, otherwise known as a transgenic animal. Such an animal is preferably a mammal and in more preferably a mouse.
  • the above described cell containing the construct can be obtained by the steps of obtaining a recently fertilized egg from an animal, injecting the combination of promoter sequence and particular nucleotide sequence into the fertilized egg, placing the fertilized egg which has been injected with promoter and nucleotide into the uterus of the animal from which the egg was obtained.
  • One skilled in the art would be familiar with the technology necessary to obtain such a cell.
  • the DNA segment promoter can be used to carry oncogenes, drug resistant genes, growth factors, etc. to various target organs in transgenic animal studies. Under controlled regulation, those genes will be expressed in specific tissues.
  • the 5* genomic regulatory region of the mouse lacto errin gene can regulate DNA in a tissue specific manner, i.e., it can be in an "on” mode in breast tissue and in an "off” mode in skin. It also can be hormonally regulated, i.e., "on” in mid-cycle menstrual cycle, "off” at menses. This regulation ability can be used in several ways. Genes targeted for transgenic mice and genes to be used in therapy in human disease conditions (gene therapy) can be used with the lactoferrin promoter.
  • genes are epidermal growth factor (EGF) cDNA, P53 tumor suppressant cDNA, and granulocyte macrophage-colony stimulating factor (GM-CSF) cDNA.
  • EGF epidermal growth factor
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • a genomic library of mouse 129/J liver DNA clone was screened in ⁇ Dash (Stratagene) .
  • the hybridization probe was a nick-translated EcoRl fragment of the full length nucleotide cDNA encoding mouse lactoferrin (Pentecost et al. (1987)), and end-labeled oligonucleotides specific to the 5' regions of the same cDNA.
  • a 14 kbp genomic ⁇ phage clone ( ⁇ J14) containing the 7.6 kbp 5'-flanking and 6.5 kbp of lactoferrin gene sequences was isolated and plaque-purified (Maniatis, T., Fritsch, E.F., and Sarubrook, Jr.
  • mL14p9E contained 2.6 kbp of 5*- flanking sequences and the first eight exons of the lactoferrin gene, while mL14p5E contained 5'- flanking sequences only.
  • a 3.0 kbp EcoRl/Hinc II fragment from mL14p9E containing the first exon, part of the first intron and 5'-flanking sequences was further restriction-digested and subcloned into either bacteria phage M13mpl8/19 or pGem 3Z plasmid.
  • a synthetic oligonucleotide corresponding to the antisense sequence of the first exon (nucleotide +52 to +71) of the mouse lactoferrin cDNA was 5' end-labeled with 32 P (1 x 10 8 cpm/ ⁇ g) and used in a primer extension experiment.
  • Total RNA from the diethylstilbestrol (DES)-stimulated uteri of 21 day-old immature mice were isolated by the guanidine isothiocyanate/cesium chloride method according to Chirgwin et al. (Chirgwin, J.M. , Przybyla, A.E., McDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5304).
  • the 32 P-labeled oligonucleotide was hybridized to 20 ⁇ g of total RNA, then the primer was extended using reverse transcriptase as described by Wong et al. (Wong, G. , Itakura, T., Kawajiri, K. , Skow, L., and Negishi, M. (1989) J. Biol. Chem. 264, 2920-2927).
  • a 1 kbp Hinc II genomic fragment containing exon 1 served as template for dideoxynucleotide sequencing using the same oligonucleotide primer.
  • the sequencing reaction products were run parallel with the primer- extended fragments on a 12% polyacrylamide/urea sequencing gel.
  • Immature (17-day-old) female CD-I mice (Charles River, Wilmington, MA) received subcutaneous injections of DES (20 ⁇ g/kg body weight/day) in corn oil at 0, 24 and 48 hr. The animals were killed 72 hr.
  • tissuemizer in buffer A containing protease inhibitor (10 mM Hepes pH 7.6, 10% glycerol, 0.25 M sucrose, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 M PMSF, 0.15 M sper ine and 0.5 mM spermidine, l ⁇ g/ml each of leupeptin, aprotinin and chymostatin) .
  • protease inhibitor 10 mM Hepes pH 7.6, 10% glycerol, 0.25 M sucrose, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 M PMSF, 0.15 M sper ine and 0.5 mM spermidine, l ⁇ g/ml each of leupeptin, aprotinin and chymostatin
  • Nuclei were collected by centrifugation at 2,500 xg, washed twice and resuspended in buffer B (10 mM Hepes, pH 7.6, 100 mM KCl, 10% glycerol, 3 mM MgC12, 0.1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 1 ⁇ g/ml each of leupeptin, aprotinin and chymostatin) .
  • High salt solution (buffer B-2 M KCl) was added drop-by-drop to the crude nuclear suspension with continuous gentle shaking until a final KCl concentration of 0.4 M was achieved.
  • the nuclei were then homogenized with a Dounce homogenizer (type B) to release the DNA and protein.
  • the viscous lysate was centrifuged at 200,000 xg for 45 min at 4°C to pellet the DNA.
  • Nuclear proteins precipitated by ammonium sulfate (0.33 g/ml) collected by centrifugation at 50,000 xg for 50 min and resuspended in buffer C (25 mM
  • DNase I protection of the 5*-flanking sequence with nuclear protein extract was performed according to the instruction specified by the manufacturer (Hotfoot DNase I footprinting kit, Stratagene, La Jolla, CA) .
  • DNA fragments, 270 bp Xba I/Sma I (-291 to -21) , 298 bp Hinc II/Xba I (- 589 to -291) and 33 bp Sph I/Hinc II (-589 to -291) and 333 bp Sph I/Hinc II (-922 to -589) were labeled with reverse transcriptase and ⁇ 32 P dCTP before being used in footprints.
  • the specific activity of the G- 50 spin column purified DNA fragments were approximately 0.5-2 x 10 8 cpm/ ⁇ g. After the reaction, the DNA was separated on 8% denaturing polyacryla ide gels. The chemical sequencing reactions (Maniatis et al. (1982)), G+A and G on the same DNA fragments used in footprinting were included as a marker.
  • Plasmid mL14p9E was cleaved with EcoRl/Sma I (from position -2644 to - 21) and Kpn I/Sma I (from position -1739 to 21) to generate a 2623 bp and 1718 bp fragment, respectively. Blunt ends were created by incubating the fragments with either Klenow polymerase or T4
  • the resultant plasmids were designated as 2.6 mL14- CAT and 1.7 mL14-CAT respectively.
  • the 1.3 mL14- CAT was constructed by ligating the 1300 bp Pst I fragment (-1534 to -234) from mL14p9E into Pst I site of the pCAT.
  • the 0.6 mL14-CAT was constructed by inserting the 568 bp Hinc II/Sma I fragment (589 to -21) (also taken from mL14p9E) into blunt ended Sal I site of the pCAT.
  • the mL14p9El was cleaved, which contains the 2623 bp Eco Rl/Sma I genomic DNA fragment (-2644 to -21) in pGem 3Z, with Sph I/Sal I.
  • the 196 bp restriction-fragment (-922 to -21 and 15 bp of the pGem 3Z linker sequence) was then inserted into the Sph I/Sal I site of the pCAT.
  • the 0.3 mL14-CAT and 0.2 mL14-CAT were constructed by cutting the 0.9 mL14-CAT with Xba I (from position -291 to -21 and 18 bp of linker sequences) alone or together with Pst I (from position -234 to -21 and 18 bp of linker sequences) to generate a 288 bp fragment and a 231 bp fragment before being cloned into the Xba I or Xba I/Pst I site of another pCAT, respectively.
  • the coordinates of 5* and 3' boundaries, relative to the transcription initiation site, were verified by detailed restriction mapping and nucleotide sequencing. All supercoiled plasmid DNA used for transfection experiments were subjected to CsCl gradient, twice purified and agarose gel examined.
  • Human endometrium carcinoma RL95-2 cells (ATCC # CRL 1617) and C6 rat glioma cells (ATCC # CCL 107) were grown in 1:1 Dulbecco's minimal essential medium : F12 supplemented with 10% fetal bovine serum (GIBCO) and 100 ⁇ /ml of penicillin/streptomycin under 95% air and 5% C0 2 - Cells were transfected at 50% confluency using the DNA calcium phosphate method according to the instruction of the CellPhect transfection kit (Pharmacia) .
  • Dulbecco's minimal essential medium F12 supplemented with 10% fetal bovine serum (GIBCO) and 100 ⁇ /ml of penicillin/streptomycin under 95% air and 5% C0 2 - Cells were transfected at 50% confluency using the DNA calcium phosphate method according to the instruction of the CellPhect transfection kit (Pharmacia) .
  • RL95-2 cells were co-transfected with HEO (I ⁇ g/well) expression plasmid (Green et al. (1986) ) ; incubations were carried out for another 24 h in the presence or absence of 10 M estradiol (Sigma) before harvest. All experiments were repeated at least three times (duplicated dish per experiment) to assume reproducibility. Relative CAT activity after normalization for ⁇ -galactosidase activity was reported and values were expressed as the mean + S.E.
  • the transcription initiation site was determined by primer extension analysis.
  • the major primer extension product was at 32 bp with two minor bands at 23 and 24 bp nucleotide upstream from the ATG start codon (FIG. 1) .
  • FOG. 1 ATG start codon
  • the promoter region that was found contained a noncanonical TATA box (ATAAA) at position -28 and two CAAT boxes at positions -69 and -98.
  • ATAAA noncanonical TATA box
  • the sequence from the transcription initiation site to the TATA box and from the TATA box to the first CAAT box had a G+C content of 78% and 75% respectively.
  • the G and C-rich residues in a region upstream from the start of transcription are commonly found in housekeeping genes (Guerin, S.L., Pothier, F. , Robidoux, S., Gosselin, P., and Parker, M.G. (1990) J. Biol. Chem. 265, 22035-22043) .
  • GC boxes There were three GC boxes whose sequences could serve as binding sites for the transcription factor Spl (Dynan, W.S., and Tijian, R. (1983) Cell 35, 79-87). One of the sites was located in the first intron (position +105) . The two other sites were located within the 5'-flanking region (at positions -530 and -908), with one in close proximity to a sequence resembling the binding site for transcription factor AP-2 (Mitchell, P.J., Wang, C, and Tijian, R. (1987) Cell 50, 847-861) . The AP-2 binding sequence at position -509 to -516 differed from the consensus sequence by one nucleotide and the orientation was reversed.
  • Positions -329 to -341 contained a sequence highly homologous to the consensus sequence of the estrogen responsive element (ERE) (Klein- Hitpass, L. , Ryffel, G.U., Heitlinger, E. , and Cato, A.C.B., (1988) Nucleic Acids Res. 16, 647-663).
  • ERE estrogen responsive element
  • a COUP transcription factor binding sequence (Sagami, I. Tsai, S.Y., Wang, L.H., Tsai, M.J., and O'Malley, B.W. (1986) Mol. Cell Biol. 6, 4259-4267) was found at position -337 to -349 and overlapped the ERE.
  • the putative estrogen responsive element differed by only one nucleotide from the consensus ERE sequence. Furthermore, seven PU.l/Spi.l DNA binding sequences (PU box) (Pettersson, M. , and Schaffner, W. (1987) Gene Dev. 1, 962-972) were found in the 5'-flanking region. One of the sequences at position -646 was present in reverse orientation; two others were found at positions -154 and -614 at reverse complement. Five of the PU boxes were clustered within the 80 bp region (-614 to -694) . A 63 bp stretch of alternating purine:pyrimidine, with the potential to form Z-DNA (Hamada, H.
  • mL14-CAT-reporter plasmids were constructed which contained various lengths of 5' lactoferrin promoter sequences in both 5' to 3' and 3' to 5* orientation with respect to the transcription initiation site. These chimeric constructs were transfected into RL95-2 and C6 cells by the DNA-calcium phosphate precipitation method.
  • the highest CAT activity in RL95-2 cells transfected with 1.7 mL14-CAT was expressed as 100% (50% conversion of 14 C-chloramphenicol to the acetylated form) .
  • All mL14-CAT reporter constructs in a 3• to 5' orientation had very low CAT activity relative to 1.7 mL14-CAT in 5' to 3' orientation (data not shown).
  • the mL14-CAT constructs were expressed in human endometrium carcinoma cells at varying levels, depending on the length of lactoferrin 5'-flanking sequences.
  • the 0.2 mL14- CAT which contained a noncanonical TATA box, two CAAT boxes and a PU box efficiently expressed the CAT activity in both RL95-2 and C6 cells. Deletion of this region resulted in a complete loss of CAT activity in both cell types (FIG. 4, 1.3 mL14-CAT) .
  • Basic lactoferrin promoter activity found in the RL95-2 cells was regulated by multiple cis-acting regulatory elements 5' upstream of the gene. At nucleotide positions -2644 to -1739 and - 589 to -291, two negative regulatory elements were found.
  • the negative element located between position -589 and -291 seems to be particularly strong because this same region also contained several transcription factor binding sites known to cause a positive effect on promoter activities (Klein-Hitpass. t al. (1988); Sagami et al. (1986)).
  • a positive regulatory element was located between nucleotide positions -1739 and -922; deletion of this region resulted in a three-fold decrease of CAT activity. Tissue-specific expression was observed by lactoferrin gene sequence extended beyond position -922. There was, however, very little CAT activity detected in C6 cells transfected with 2.6 mL14-CAT or 1.7 mL14-CAT chimeric reporter plasmid, while full activity was found in RL95-2 cells.
  • Reporter plasmid 0.6 mL14-CAT, containing the mouse lactoferrin gene promoter and the estrogen response element region (-21 to -587) was tested for its estrogen responsiveness in transient transfection experiments. There was very little hormone response in RL95-2 cells in the absence of exogenous estrogen receptors, although a low level of estrogen receptor has been found in these cells (Way, D.L., Grosso, D.S., Davis, J.R. , Surwit, E.A., and Christian CD. (1983) In Vitro 19, 147-158). A 2.5-fold stimulation was observed when 1 ⁇ g of HEO plasmids were also introduced (FIG. 5) .
  • reporter plasmid 0.3mL14-CAT lacking the estrogen response element (-21 to -291) , did not respond to estrogen stimulation (data not shown) .
  • the conventional method comprises removing a superovulated fertilized mouse egg from a mouse uterus; microinjecting a male pronucleus of a fertilized mouse egg with an amount of a nucleotide sequence attached to at least a portion of mouse lactoferrin promoter; replacing said microinjected egg into said mouse uterus.
  • the 5' genomic regulatory region of mouse lactoferrin contains various pertinent regulatory elements. The most interesting one is the estrogen responsive module. Under the influence of estrogen, the regulatory region becomes a strong promoter only in the uterus. Therefore, this particular promoter can be used to carry oncogenes, drug-resistant genes, growth factors and tumor-suppressor genes to the uterus in transgenic animal studies.
  • those genes will then be regulated by estrogen.
  • those genes can also be targeted to the mammary gland and bone marrow by the lactoferrin gene promoter-enhancer sequence and regulated under different control.
  • the 2.7 kbp 5' flanking sequence of the lactoferrin gene is being used for this transgenic animal study.
  • the lactoferrin 5' sequence is linked to the cDNA or gene of interest and a SV40 3' region with poly(A) addition site or equivalent poly (A) addition site from another source. Plasmids from commercial sources such as pUC, pGEM and Bluescript are used to construct the chimeric plasmid for transgenic animal study.
  • lactoferrin 5'- flanking sequence uses for the lactoferrin 5'- flanking sequence as follows: (1) The 9 kbp lactoferrin fragment in mL14p9E or the entire 14 kbp lactoferrin insert in J14 is introduced into embryonic stem (ES) cells to produce mutant mouse that lacks the lactoferrin.
  • ES embryonic stem
  • Lactoferrin 5 '-flanking sequence is used in selective ablation of a specific cell type.
  • Cell ablation is achieved by introducing genes encoding cytotoxins, such as the catalytic subunits of DT or ricin, driven by the lactoferrin promoter/enhancer sequence.
  • An alternative strategy is conditional ablation, using the HSV-tk gene under the control of lactoferrin promoter/enhancer sequence in which cells are rendered susceptible to drugs such as gancyclovir.
  • this DNA fragment (- 586 to -21) was under negative regulation in RL 95- 2 cells and the repression appeared to be released by estrogen.
  • the footprinting data indicated at least four different protein binding sites within this region. Whether interaction between protein and DNA plays any roles in regulating the lactoferrin promoter activity requires a detailed deletion and mutational analysis together with band- shift and methylation interference study. The surrounding sequences and protein to protein interactions may have also been differently involved in different cell types, as has been found in many genes (Grosschedl et al. (1985) ; Edlund et al. (1985)) .
  • a very strong positive regulatory element was present between -1739 and -922, and deletion of this region resulted in a drastic decrease of lactoferrin promoter activity.
  • a distinctively different pattern of lactoferrin promoter activity was obtained from the same chimeric reporter genes transfected into C6 cells. DNA sequence, extended beyond -922, completely silenced the lactoferrin promoter in C6 cells. Numerous studies of cell hybrids have indicated that somatic cells produce negative regulators, "extinguishers", that prevent the expression of genes foreign to their own differentiation (Killary et al. (1984); Petit et al. (1986)). It is likely that the negative effect observed in C6 cells transfected with the 2.6 mL14- CAT construct, was mediated through the recognition of negative regulatory elements by trans-acting protein that was present in the C6 cells and absent in the RL 95-2 cells.
  • DNA sequence up to -291 was sufficient for the maximum expression of lactoferrin promoter in C6 cells, while 1739 bp was necessary for RL 95-2 cells.
  • the basic lactoferrin promoter activity resided within the -234 to -21 region; deletions of this DNA sequence completely abolished its function as indicated by 1.3 mL14-CAT construct.
  • a noncanonical TATA element at -32 to -28, two CAAT elements at -72 to -69 and -101 to -98 were present in this region, in addition to a GC rich sequence between -98 to +1.
  • lactoferrin promoter The presence within the lactoferrin promoter of individual features characteristic of both housekeeping and inducible promoters could contribute to the differential regulation of lactoferrin gene in a variety of cell types. Identification of multiple regulatory elements of the lactoferrin gene is the initial step toward understanding its differential control of expression. Deletion, mutation and nuclear protein interaction with those regulatory elements are currently under investigation.

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Abstract

The promoter region of the mouse lactoferrin gene was cloned and sequenced. Several clones containing lactoferrin gene fragments were isolated from a mouse (129/J) genomic library including clone μJ14 which contains 7.5 kbp 5'-flanking sequence. Sequence analysis of the region flanking the transcription initiation site revealed the following: a TATA like sequence, two CAAT boxes, three GC boxes including one within the first intron, an AP2 site, seven PU boxes, an AC rich region, a B1 sequence and an estrogen responsive element (ERE) consensus sequence overlapping with a COUP binding element. Footprinting analysis demonstrated that several regions, including the putative ERE region, in the 5'-flanking sequence were protected from DNase I digestion. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter-plasmid to study functional activity. The mouse lactoferrin gene promoter was active in human endometrium carcinoma RL 95-2 cells and in rat glioma C6 cells.

Description

CHARACTERIZATION OF ESTROGEN RESPONSIVE MOUSE LACTOFERRIN PROMOTER
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to identification and characterization of the promoter region of the gene which encodes mouse lactoferrin. In another aspect, it relates to a method of targeting a particular nucleotide to a specific tissue and controlling its timing and level of expression by attaching the mouse lactoferrin promoter to a particular nucleotide and using the promoter and nucleotide to obtain a transgenic animal or to perform gene therapy in humans.
Background Information
Lactoferrin is an iron-binding glycoprotein originally found in milk (Masson, P.L., and Heremans, J.F. (1971) Comp. Biochem. Physiol. 39B, 119) . This protein, together with transferrin and melanomas antigen p97 belongs to a gene family which arose from ancient intragenic duplication 300 to 500 million years ago (Bowman, B.H., Yang, F. , and Adrian, G.S. (1988) Advance in Genetics 25, 1- 38; Park, I., Schaeffer, I.E., Sidole, A.A. , Baralle, F.E., Cohen, G.N. , and Zakin, M.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3149-3153).
Some of the proteins from this gene family have been shown to play important roles in cell growth and differentiation (Barnes, D., and Sato, G. (1980) Anal. Biochem. 102, 255-270; Broxmeyer, H.E., Williams, D.E., Hangoc, G. , Cooper, S., Gentile, P., Shen, R.N., Ralph, P., Gillis, S., Bicknell, D.C. (1987) Blood Cells 13, 31-48). Extracellular lactoferrin has been observed to have both antibacterial and antiviral activity (Arnold, R.R. , Cole, M.F., and McGhee, J.R. , (1976) Science, 197, 263-265; Lu, L., Hangoc, A., Oliff, L.T., and Chen, R.N., (1987) Cancer Res. 47, 4184-4190). The wide variety of biological functions highlights the importance of the regulation of these genes in individual tissue and cell types. Lactoferrin is expressed at high levels in neutrophil (Masson, P.L., Heremans, J.F., and Shonne, E.J. , (1969) Exp. Med. 130, 643-656) , in lactating mammary gland (Masson et al. (1971); Green, M.R., and Pastewka, J.V., (1978) Endocrinology 103, 1510-1513), and in uterus (Pentecost, B.T., and Teng, C.T., (1987) -T. Biol. Chem. 262, 10134-10139; Teng, CT., Pentecost, B.T., Chen, Y.H., Newbold, R.R. , Eddy, E.M. , and McLachlan, J.-A., (1989) Endocrinology 124, 992-999) under different types of control. Conversely, it is expressed constitutively at low levels in some other tissues and is present in many biological fluids
(Teng et al. (1989); Masson, P.L. , Heremans, J.F., and Divec, (1966) Clin. Chim. Acta 14, 735-739). Although many studies have been done on the distribution of the protein, relatively little is understood about the molecular mechanisms that regulate its expression. It would be particularly important to determine which mechanisms control the differential expression of the lactoferrin gene in various tissues. In mammalian cells, the transcriptional regulation of a particular gene is a complex process. It involves interaction between multiple cis-acting regulatory elements and their cognated protein factors (Grosschedl, R. , and Baltimore, D., (1985) Cell 41, 885-897; Edlund, T., Walker, M.D. , Barr, P.J. , and Rutter, W.J. (1985) Science 230, 912-916) . In order to understand the mechanisms of expression of the lactoferrin gene in various cell types, it is necessary to study the structural and functional organization of the regulatory regions of the lactoferrin gene.
Previously, the cloning of the lactoferrin cDNA from estrogen-stimulated mouse uterus has been reported (Pentecost et al. (1987)). Furthermore, the gene has been mapped to both mouse chromosome 9 and human chromosome 3 band 3q21-q23 (Teng, CT. , Pentecost, B.T., Marshall, A., Solomon, A., Bowman, B.H., Lalley, P.A., and Naylor, S.L., (1987) Somatic Cell and Molecular Genetics 13, 689-693; McCombs, J.L. , Teng, CT. , Pentecost, B.T., Magnuson, V.L. , Moore, CM., and McGill, J.R. , (1988) Cytogenet. Cell Genet. 47, 16-17). Subsequently, human lactoferrin cDNA has also been cloned from both mammary gland and bone marrow (Rado, T.A., Wei, X., and Benz, E.J., Jr., (1987) Blood 70, 989-993;
Powell, M. . , and Ogden, J.E., (1990) Nucleic Acids Res. 18, 4013; Rey, M.W., Woloshuk, S.L., deBoer, H.A. , Pieper, F.R. , (1990) Nucleic Acids Res. 18, 5288; Panella, T.J., Liu, Y. , Huang, A.T. , and Teng, CT., (1991) Cancer Res. 51, 3037-3043). The deduced primary structure of mouse lactoferrin exhibits internal homologies between N-terminal and C-terminal domain similar to the human lactoferrin protein (Metz-Boutigue, M.H., Jolles, J. , Mazurier, J. , Schoentgen, F. , Legrand, D. , Spik, G. ,
Montreuil, J. , and Jolles, P., (1984) Eur. J. Biochem. 145, 659-676; Anderson, B.F., Baker, H.M. , Dodson, E.J., Norris, G.E., Ru ball, S.V., Waters, J.M. , and Baker, E.N. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1769-1773). The amino acid sequences of mouse lactoferrin shared 70% homology to human lactoferrin and 56% to human transferrin (Metz- Boutigue et al. (1984), Yang, F., Lum, J.B. , McGill, J.R. , Moore, CM., Na lor, S.L., vanBragght, P.H. , Baldwin, W.D., and Bowman, B.H. , (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2752-2756).
Cell-type specific regulation of lactoferrin gene activity is a complex process. Its expression in mouse uterus is controlled by estrogen (Pentecost et al. (1987)), while prolactin plays an important role in regulating its expression in mammary gland (Green et al. (1978)). Lactoferrin is not expressed in the brain but is constitutively expressed in neutrophil as it matures (Teng et al. (1989) , Panella et al. (1991)). In view of such a complicated regulation of the lactoferrin gene in various tissues in a variety of ways, the 5- lactoferrin promoter region has been isolated and analyzed in order to understand more about the molecular mechanisms involved.
The functional structure of the mouse lactoferrin gene 5*-flanking regions has been isolated and characterized. It has been shown that the mouse lactoferrin promoter region contains various putative regulatory elements found in both housekeeping and inducible genes. Regions of negative and positive regulation of the basic lactoferrin promoter activity have been identified with transient transfection into various cell lines of CAT reporter plasmids. Plasmid containing the estrogen responsive element consensus sequence of the lactoferrin gene responds to estrogen stimulation in the presence of estrogen receptors. SUMMARY OF THE INVENTION
It is an object of the present invention to isolate and characterize the promoter region for a gene encoding mouse lactoferrin. It is another object of the present invention to use the promoter region for mouse lactoferrin gene in combination with a recombinant gene to be used either to make transgenic animals or in gene therapy in humans, the promoter region serving to target the recombinant gene to a specific tissue and/or to place it under hormonal control. The present invention relates to a DNA segment having a nucleotide sequence as shown in FIG. 2 (b) and allelic variations thereof. The present invention further relates to the DNA segment having a nucleotide sequence as shown in FIG. 2 (b) wherein the segment is a promoter region for a gene encoding mouse lactoferrin protein. Various other objects and advantages of the present invention will become apparent from the following figures and description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Primer extension to determine the 51 end of the lactoferrin mRNA.
Total RNA, isolated from DES-treated mouse uteri, was used as the template for primer extension. 5* end-labeled oligonucleotide, corresponding to the antisense sequence of the first exon was hybridized to 20 μg of total RNA as described in Experimental Procedures. The reaction products were analyzed on 12% polyacrylamide/urea sequencing gel. One major band at position -32, two minor bands at positions -24 and -23 upstream from the first ATG site, were detected (arrow) .
FIG. 2. Nucleotide sequence of the 5' end of mouse lactoferrin gene.
A. Diagrammatic representation of the sequencing strategy and partial restriction map of λ J14. A 3053 bp of the lactoferrin gene containing the first exon, part of the first intron and 2661 bp 5*-flanking sequence were restriction-digested and subcloned into either bacteria phage M13mpl8/19 or pGem 32 plasmid as described in Experimental Procedures. Solid arrows indicate the regions were sequenced on a single strand from M13mpl8/19 and the dashed arrow indicates the regions were sequenced on double-stranded plasmid with synthetic oligonucleotide as the primer. H, Hind III; E, Eco RI; P, Pst I.
B. Nucleotide sequences and the putative regulatory elements. Amino acids encoded by the first exon are indicated below the appropriate codons. The first exon, noncanonical TATA box and the two CAAT boxes are indicated by a box. Putative regulatory elements are indicated by either overlining or underlining the sequences. Mouse speci ic Bl sequence is indicated with a double under line. Numbers alongside refer to nucleotide position relative to the major transcription start site.
FIG. 3 DNase I footprinting of mouse lactoferrin promoter region.
A. A 298 bp Hinc II/Xba I DNA fragment (- 589 to -291) was 3» end-labeled at position -291 (sense strand) and incubated with a different amount (10 or 20 μg) of DES-treated mouse uterine nuclear extract (ut) or liver nuclear extract (L) , and then treated with DNase I. Control sample treated with DNase I but without added protein (0) . DNA "G+A" and "G" sequencing reaction on the same probe were included as markers. Major protected region designated as I. The number alongside refers to the nucleotide position relative to the transcription initiation site.
B. Identical experiment as described in A. Three regions were protected from DNase I treatment. They were designated as II, III and IV. C A 333 bp of Sph I/Hinc II DNA fragment (-922 to -589) was 3' end-labeled at position -922 (antisense strand) . DNase I footprinting with DES- treated uterine nuclear extract were examined. One protected region was observed (V) .
FIG. 4. Structure and activity of lactoferrin promoter-CAT chimeric gene.
Various lengths of lactoferrin 5'- flanking sequences inserted in front of the CAT reporter gene. The chimeric constructs were transiently transfected into RL95-2 and C6 cells as described in the Examples. All DNA fragments were inserted at 5• to 3' orientation relative to the transcription initiation site; the nucleotide boundaries were indicated (left panel) . The 100% CAT activity determined from 1.7 mL14-CAT in RL95-2 cells corresponds to 50% chloramphenicol acetylation/40 μl cell extract /2 hrs. The 1.7 mL14-CAT construct was constitutively expressed in RL95-2 cells and the promoter was most active among all constructs tested in these cells. Relative CAT activity in relationship to the 1.7 mL14-CAT in RL95-2 cells are shown next to each column (right panel) . The plasmid pCAT-Basic and pSV-CAT were also used as negative and positive controls in these experiments. Each bar represents an average of six determinations. CAT activity was normalized with the co-transfected β-galactosidase activity. D TATA box; O CAAT boxes.
FIG. 5 Estrogen responsiveness of the lactoferrin promoter-CAT chimeric gene transfected into RI-95-2 cells.
A. Schematic illustration of the chimeric gene 0.6 mL14-CAT which contains 568 bp of the lactoferrin 5'-flanking region was inserted in front of the CAT reporter gene. The region contains the consensus sequence of ERE and COUP is indicated. TATA box, two CAAT boxes, reversed AP 2 site and Sp 1 site are also indicated.
B. Representative CAT activities resulting from transient transfection of 0.6 mL14- CAT into RL95-2 cells. RL95-2 cells containing low level of estrogen receptor (Way, D.L., Grosso, D.S., Davis, J.R., Surwit, E.A. , and Christian CD. (1983) In Vitro 19, 147-158) were used either directly (lane 1 and 2) or were co-transfected with HEO (lane 3 and 4) , the estrogen receptor expression vector (Green, S., Walter, P., Kumar, v., Krust, A., Borneert, J.M., Argos, P., and Chambon, P. (1986) Nature 320, 134-139), to test its estrogen responsiveness. The cells were incubated in the presence (lane 2 and 4) or absence (lane 1 and 3) of 10~8 M DES as described in the Examples. Cell extract from these cultures were assayed for CAT activity with β-galactosidase normalization. The results were visualized by using thin layer chromatography, followed by autoradiography.
C. Graphical presentation of CAT activity in cell lysate of experiments described in B. The relative CAT activities in relationship to the constitutively expressed 1.7 mL14-CAT in RL95-2 cells were presented. Each bar represents the mean + S.E. of at least six determination (***=p<0.01) .
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a promoter region for a gene encoding mouse lactoferrin protein. This promoter region contains: a TATA like sequence, two CAAT boxes, three GC boxes including one within the first intron, an AP2 site, seven PU boxes, an AC rich region, A Bl sequence and an estrogen responsive element (ERE) consensus sequence overlapping with a COUP binding element. The present invention further relates to an isolated DNA segment having a promoter sequence from -2661 to +1 as shown in FIG. 2 (b) or variations or segments thereof having promoter activity.
The present invention additionally relates to portions of the above isolated DNA segment which serve as promoter regions for a gene encoding mouse lactoferrin protein. The present invention also relates to allelic variations or variations achieved through mutations, insertions, deletions, or substitutions, all of which are functionally equivalent to a promoter region of the isolated DNA segment. One skilled in the art would understand what would be included within the terms "allelic variations", "mutations", "insertions", "deletions", or substitutions". and would know how to make such variations of the DNA segment.
The present invention further relates to a DNA construct having a promoter sequence which is operably linked to a heterologous structural gene in a manner sufficient to modulate the expression of the structural gene.
Additionally, this DNA construct can further be linked to a plasmid or a vector. One skilled in the art would understand the methods used to achieve such a construct which can be linked to a plasmid or vector.
The present invention also relates to such transfected host cells constructed using methods well-known in the art. Examples of such host cells can include but are not limited to human endometrium carcinoma RL95 2 cell or C6 rat glioma cell.
In addition, the present invention relates to a method of carrying a particular nucleotide sequence to a specific tissue in an animal by operably linking at least a portion of the isolated DNA segment of the present invention having a promoter sequence to a particular nucleotide sequence. This combination of DNA promoter sequence and nucleotide sequence can be administered to an animal. One skilled in the art would understand the methods that would be used to combine the two segments and to administer the combination to an animal.
The present invention also relates to a method of treating a condition in a patient characterized by a genetic abnormality. . This can be accomplished by administering to the patient a sufficient amount of a nucleotide sequence which is operably linked to a portion of the promoter DNA sequence. One skilled in the art would understand the relevant technology and methods of administering a sufficient dosage to achieve the desired clinical results.
The present invention further relates to an animal cell which contains the construct having the promoter sequence operably linked to a heterologous structural gene so that the heterologous structural gene is modulated by the promoter sequence which does not naturally modulate the gene. The animal cell which contains the construct is preferably an embryo cell. Additionally, the present invention further relates to an animal whose genetic material contains the above construct, otherwise known as a transgenic animal. Such an animal is preferably a mammal and in more preferably a mouse.
The above described cell containing the construct can be obtained by the steps of obtaining a recently fertilized egg from an animal, injecting the combination of promoter sequence and particular nucleotide sequence into the fertilized egg, placing the fertilized egg which has been injected with promoter and nucleotide into the uterus of the animal from which the egg was obtained. One skilled in the art would be familiar with the technology necessary to obtain such a cell.
For example, the DNA segment promoter can be used to carry oncogenes, drug resistant genes, growth factors, etc. to various target organs in transgenic animal studies. Under controlled regulation, those genes will be expressed in specific tissues. The 5* genomic regulatory region of the mouse lacto errin gene can regulate DNA in a tissue specific manner, i.e., it can be in an "on" mode in breast tissue and in an "off" mode in skin. It also can be hormonally regulated, i.e., "on" in mid-cycle menstrual cycle, "off" at menses. This regulation ability can be used in several ways. Genes targeted for transgenic mice and genes to be used in therapy in human disease conditions (gene therapy) can be used with the lactoferrin promoter. Examples of such genes are epidermal growth factor (EGF) cDNA, P53 tumor suppressant cDNA, and granulocyte macrophage-colony stimulating factor (GM-CSF) cDNA. Such genes, when linked to the lactoferrin promoter may be regulated in a tissue specific or hormonal pattern.
The following non-limiting examples are provided to further describe the present invention.
EXAMPLES
Example 1
Genomic cloning and sequencing of the mouse lactoferrin 5'-flanking region.
A genomic library of mouse 129/J liver DNA clone was screened in λ Dash (Stratagene) . The hybridization probe was a nick-translated EcoRl fragment of the full length nucleotide cDNA encoding mouse lactoferrin (Pentecost et al. (1987)), and end-labeled oligonucleotides specific to the 5' regions of the same cDNA. A 14 kbp genomic λ phage clone (λ J14) containing the 7.6 kbp 5'-flanking and 6.5 kbp of lactoferrin gene sequences was isolated and plaque-purified (Maniatis, T., Fritsch, E.F., and Sarubrook, Jr. (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. Molecular Cloning: A laboratory manual) . DNA insert from λ J14 clone cut by EcoRl at a single location, resulted in 9 kbp and 5 kbp fragments. These fragments were subcloned in pGe 3Z and designated as mL14p9E and mL14p5E accordingly. Further analysis of the subcloned plasmids by restriction mapping. Southern blotting (Maniatis et al. (1982)) and hybridization to specific oligonucleotides, revealed that mL14p9E contained 2.6 kbp of 5*- flanking sequences and the first eight exons of the lactoferrin gene, while mL14p5E contained 5'- flanking sequences only. A 3.0 kbp EcoRl/Hinc II fragment from mL14p9E containing the first exon, part of the first intron and 5'-flanking sequences was further restriction-digested and subcloned into either bacteria phage M13mpl8/19 or pGem 3Z plasmid. Restriction DNA fragments subcloned into pGem 3Z were designated as mL14p9E 0-14 series. Both double-stranded sequencing and single-stranded sequencing were performed by dideoxynucleotide chain termination using cloned T7 DNA polymerase (Sequenase, United States Biochemical Corp.) (Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467). Oligonucleotide primers used for sequencing were synthesized on a Beckman System One Plus automated DNA synthesizer. The entire 3.0 kbp EcoRl/Hinc II fragment of the 5' end of the gene was sequenced at least two times on both strands. Example 2
Primer extension analysis.
A synthetic oligonucleotide corresponding to the antisense sequence of the first exon (nucleotide +52 to +71) of the mouse lactoferrin cDNA was 5' end-labeled with 32P (1 x 108 cpm/μg) and used in a primer extension experiment. Total RNA from the diethylstilbestrol (DES)-stimulated uteri of 21 day-old immature mice were isolated by the guanidine isothiocyanate/cesium chloride method according to Chirgwin et al. (Chirgwin, J.M. , Przybyla, A.E., McDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5304). The 32P-labeled oligonucleotide was hybridized to 20 μg of total RNA, then the primer was extended using reverse transcriptase as described by Wong et al. (Wong, G. , Itakura, T., Kawajiri, K. , Skow, L., and Negishi, M. (1989) J. Biol. Chem. 264, 2920-2927). A 1 kbp Hinc II genomic fragment containing exon 1 served as template for dideoxynucleotide sequencing using the same oligonucleotide primer. The sequencing reaction products were run parallel with the primer- extended fragments on a 12% polyacrylamide/urea sequencing gel.
Example 3
Preparation of nuclear protein extract.
Immature (17-day-old) female CD-I mice (Charles River, Wilmington, MA) received subcutaneous injections of DES (20 μg/kg body weight/day) in corn oil at 0, 24 and 48 hr. The animals were killed 72 hr. post-injection and immediately afterward their uteri were removed and homogenized (Tissuemizer) in buffer A containing protease inhibitor (10 mM Hepes pH 7.6, 10% glycerol, 0.25 M sucrose, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 M PMSF, 0.15 M sper ine and 0.5 mM spermidine, l μg/ml each of leupeptin, aprotinin and chymostatin) . Nuclei were collected by centrifugation at 2,500 xg, washed twice and resuspended in buffer B (10 mM Hepes, pH 7.6, 100 mM KCl, 10% glycerol, 3 mM MgC12, 0.1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 1 μg/ml each of leupeptin, aprotinin and chymostatin) . High salt solution (buffer B-2 M KCl) was added drop-by-drop to the crude nuclear suspension with continuous gentle shaking until a final KCl concentration of 0.4 M was achieved. The nuclei were then homogenized with a Dounce homogenizer (type B) to release the DNA and protein. The viscous lysate was centrifuged at 200,000 xg for 45 min at 4°C to pellet the DNA. Nuclear proteins precipitated by ammonium sulfate (0.33 g/ml) , collected by centrifugation at 50,000 xg for 50 min and resuspended in buffer C (25 mM
Hepes, pH 7.6, 10% glycerol, 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 μg/ml each of leupeptin, aprotinin and chymostatin) . The residue ammonium sulfate and insoluble material were removed by dialysis against buffer C and by centrifugation, respectively. Small aliquots of nuclear extract were quickly frozen and stored in liquid nitrogen after its protein concentration had been determined (BioRad Protein Assay, Richmond, CA) .
Example 4
Footprint analysis.
DNase I protection of the 5*-flanking sequence with nuclear protein extract was performed according to the instruction specified by the manufacturer (Hotfoot DNase I footprinting kit, Stratagene, La Jolla, CA) . DNA fragments, 270 bp Xba I/Sma I (-291 to -21) , 298 bp Hinc II/Xba I (- 589 to -291) and 33 bp Sph I/Hinc II (-589 to -291) and 333 bp Sph I/Hinc II (-922 to -589) were labeled with reverse transcriptase and α32P dCTP before being used in footprints. The specific activity of the G- 50 spin column purified DNA fragments were approximately 0.5-2 x 108 cpm/μg. After the reaction, the DNA was separated on 8% denaturing polyacryla ide gels. The chemical sequencing reactions (Maniatis et al. (1982)), G+A and G on the same DNA fragments used in footprinting were included as a marker.
Example 5
Construction of plasmids.
Various lengths of the 5'-flanking sequence from the lactoferrin gene were cloned into the polylinker region of the pCAT-Basic plasmid (Promega, Biotec, Madison, WI) . Plasmid mL14p9E was cleaved with EcoRl/Sma I (from position -2644 to - 21) and Kpn I/Sma I (from position -1739 to 21) to generate a 2623 bp and 1718 bp fragment, respectively. Blunt ends were created by incubating the fragments with either Klenow polymerase or T4
DNA polymerase and dNTP's, and then were ligated to a similar blunt-ended Hind III site of the pCAT-B. The resultant plasmids were designated as 2.6 mL14- CAT and 1.7 mL14-CAT respectively. The 1.3 mL14- CAT was constructed by ligating the 1300 bp Pst I fragment (-1534 to -234) from mL14p9E into Pst I site of the pCAT. The 0.6 mL14-CAT was constructed by inserting the 568 bp Hinc II/Sma I fragment (589 to -21) (also taken from mL14p9E) into blunt ended Sal I site of the pCAT. To construct the 0.9 mL14- CAT, the mL14p9El was cleaved, which contains the 2623 bp Eco Rl/Sma I genomic DNA fragment (-2644 to -21) in pGem 3Z, with Sph I/Sal I. The 196 bp restriction-fragment (-922 to -21 and 15 bp of the pGem 3Z linker sequence) was then inserted into the Sph I/Sal I site of the pCAT. The 0.3 mL14-CAT and 0.2 mL14-CAT were constructed by cutting the 0.9 mL14-CAT with Xba I (from position -291 to -21 and 18 bp of linker sequences) alone or together with Pst I (from position -234 to -21 and 18 bp of linker sequences) to generate a 288 bp fragment and a 231 bp fragment before being cloned into the Xba I or Xba I/Pst I site of another pCAT, respectively. The coordinates of 5* and 3' boundaries, relative to the transcription initiation site, were verified by detailed restriction mapping and nucleotide sequencing. All supercoiled plasmid DNA used for transfection experiments were subjected to CsCl gradient, twice purified and agarose gel examined.
Example 6
Cell transfection and CAT assay.
Human endometrium carcinoma RL95-2 cells (ATCC # CRL 1617) and C6 rat glioma cells (ATCC # CCL 107) were grown in 1:1 Dulbecco's minimal essential medium : F12 supplemented with 10% fetal bovine serum (GIBCO) and 100 μ/ml of penicillin/streptomycin under 95% air and 5% C02- Cells were transfected at 50% confluency using the DNA calcium phosphate method according to the instruction of the CellPhect transfection kit (Pharmacia) . In each experiment 2xl05 cells were transfected with 1.5xl0-12 M of the plasmid and 0.25 μg of the β-galactosidase reference plasmid pCH 110 (Pharmacia) as an internal standard for transfection efficiency. Cell extracts were prepared 24-48 h later; the CAT enzyme activity was performed with the whole cell extract in 150 μl reaction containing 14C-chloramphenicol (40-50 Ci/mmol) (DuPont, NEN) as described (Maniatis et al. (1982)). The reaction products were analyzed with an ascending thin layer chromatography followed by x-ray autoradiograph and by liquid scintillation counting. To test the estrogen responsiveness of the lactoferrin gene 5* flanking sequences, RL95-2 cells were co-transfected with HEO (Iμg/well) expression plasmid (Green et al. (1986) ) ; incubations were carried out for another 24 h in the presence or absence of 10 M estradiol (Sigma) before harvest. All experiments were repeated at least three times (duplicated dish per experiment) to assume reproducibility. Relative CAT activity after normalization for β-galactosidase activity was reported and values were expressed as the mean + S.E.
Example 7
Determination of transcription initiation site and nucleotide seguence of the 5'-flanking region.
The transcription initiation site was determined by primer extension analysis. A 20-mer oligonucleotide, complementary to bases in the first exon, was used as primer. The major primer extension product was at 32 bp with two minor bands at 23 and 24 bp nucleotide upstream from the ATG start codon (FIG. 1) . To identify possible regulatory sequence elements in the 5 *-flanking region of the lactoferrin gene, a 2661 bp 5 '-flanking region and a 393 bp region of the gene including the first exon and part of the first intron were sequenced. The sequencing strategy is presented in FIG. 2A and the nucleotide sequence is presented in FIG. 2B. The promoter region that was found contained a noncanonical TATA box (ATAAA) at position -28 and two CAAT boxes at positions -69 and -98. The sequence from the transcription initiation site to the TATA box and from the TATA box to the first CAAT box had a G+C content of 78% and 75% respectively. The G and C-rich residues in a region upstream from the start of transcription are commonly found in housekeeping genes (Guerin, S.L., Pothier, F. , Robidoux, S., Gosselin, P., and Parker, M.G. (1990) J. Biol. Chem. 265, 22035-22043) . There were three GC boxes whose sequences could serve as binding sites for the transcription factor Spl (Dynan, W.S., and Tijian, R. (1983) Cell 35, 79-87). One of the sites was located in the first intron (position +105) . The two other sites were located within the 5'-flanking region (at positions -530 and -908), with one in close proximity to a sequence resembling the binding site for transcription factor AP-2 (Mitchell, P.J., Wang, C, and Tijian, R. (1987) Cell 50, 847-861) . The AP-2 binding sequence at position -509 to -516 differed from the consensus sequence by one nucleotide and the orientation was reversed. Positions -329 to -341 contained a sequence highly homologous to the consensus sequence of the estrogen responsive element (ERE) (Klein- Hitpass, L. , Ryffel, G.U., Heitlinger, E. , and Cato, A.C.B., (1988) Nucleic Acids Res. 16, 647-663). A COUP transcription factor binding sequence (Sagami, I. Tsai, S.Y., Wang, L.H., Tsai, M.J., and O'Malley, B.W. (1986) Mol. Cell Biol. 6, 4259-4267) was found at position -337 to -349 and overlapped the ERE. The putative estrogen responsive element differed by only one nucleotide from the consensus ERE sequence. Furthermore, seven PU.l/Spi.l DNA binding sequences (PU box) (Pettersson, M. , and Schaffner, W. (1987) Gene Dev. 1, 962-972) were found in the 5'-flanking region. One of the sequences at position -646 was present in reverse orientation; two others were found at positions -154 and -614 at reverse complement. Five of the PU boxes were clustered within the 80 bp region (-614 to -694) . A 63 bp stretch of alternating purine:pyrimidine, with the potential to form Z-DNA (Hamada, H. , Petrino, M.G., and Kakunaga, T. (1982) Proc. Natl Acad. Sci. U.S.A. 79, 6465-6469), was found at position -740 to -803. A purine-rich region found at position -637 to -694 had 95% AG content. Bl sequence (Krayev, A.S.,
Kramerov, D.A., Skryabin, K.G., Ryskov, A.P., Bayev, A.A., and Georgiev, G.P. (1980) Nucleic Acids Res. 8, 1201-1215) , a mouse repetitive eHP LaserJet IID (Additional)HPLAIIAD.PRS nuclear protein which interacted with the 5'-flanking region of the lactoferrin gene, nuclear protein extract was prepared from DES treated animals. Specific DNA- protein interactions were examined by the footprinting DNase I protection method. A DNA fragment from -291 to -21 was not protected by the uterine nuclear extract (data not shown) , while a DNA fragment from -589 to -291 showed several protected regions (FIG. 3A and B) . One region corresponding to nucleotide -353 to -327 which houses both putative estrogen responsive element and COUP sequence was protected by uterine nuclear extract but not by hepatic nuclear extract (FIG. 3A) . Three protected regions (region II, III and IV) further upstream were found. Region II contained a PU box while regions III and IV had no described regulatory consensus sequence (FIG. 3B) . DNA from -922 to -589 has one protected region at nucleotide position -845 to -824, which was a 22 bp stretch of alternating purine:pyrimidine (FIG. 3C) .
Example 9
Detection of lactoferrin promoter activity in RL 95- 2 and C6 cells.
To test lactoferrin promoter activity, a series of mL14-CAT-reporter plasmids were constructed which contained various lengths of 5' lactoferrin promoter sequences in both 5' to 3' and 3' to 5* orientation with respect to the transcription initiation site. These chimeric constructs were transfected into RL95-2 and C6 cells by the DNA-calcium phosphate precipitation method.
The highest CAT activity in RL95-2 cells transfected with 1.7 mL14-CAT was expressed as 100% (50% conversion of 14C-chloramphenicol to the acetylated form) . All mL14-CAT reporter constructs in a 3• to 5' orientation had very low CAT activity relative to 1.7 mL14-CAT in 5' to 3' orientation (data not shown). As shown in FIG. 4, the mL14-CAT constructs were expressed in human endometrium carcinoma cells at varying levels, depending on the length of lactoferrin 5'-flanking sequences. The 0.2 mL14- CAT, which contained a noncanonical TATA box, two CAAT boxes and a PU box efficiently expressed the CAT activity in both RL95-2 and C6 cells. Deletion of this region resulted in a complete loss of CAT activity in both cell types (FIG. 4, 1.3 mL14-CAT) . Basic lactoferrin promoter activity found in the RL95-2 cells, however, was regulated by multiple cis-acting regulatory elements 5' upstream of the gene. At nucleotide positions -2644 to -1739 and - 589 to -291, two negative regulatory elements were found. The negative element located between position -589 and -291 seems to be particularly strong because this same region also contained several transcription factor binding sites known to cause a positive effect on promoter activities (Klein-Hitpass. t al. (1988); Sagami et al. (1986)). A positive regulatory element was located between nucleotide positions -1739 and -922; deletion of this region resulted in a three-fold decrease of CAT activity. Tissue-specific expression was observed by lactoferrin gene sequence extended beyond position -922. There was, however, very little CAT activity detected in C6 cells transfected with 2.6 mL14-CAT or 1.7 mL14-CAT chimeric reporter plasmid, while full activity was found in RL95-2 cells. This observation provided evidence for the existence of cis-acting negative regulatory elements in the lactoferrin gene which could be recognized by "tissue-specific extinguishers" (Killary, A.M. , and Fournier, R.E.K. (1984) Cell 38, 523-534; Petit, C, Levillies, J., Ott, M.O., and Weiss M.C (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2561-2565 ,27) in nonexpressing tissue. In the C6 cell, further deletion of the lactoferrin promoter sequence to position -589 resulted in the progressive increase of CAT activity to the basal level found in RL95-2 cells. No other positive or negative regulatory element was found in C6 cells. Together, these results demonstrated the complicated transcriptional regulation of the lactoferrin gene in a variety of cell types.
Example 10
Identification of estrogen responsive element in the 5'-flanking region of the mouse lactoferrin gene.
Reporter plasmid, 0.6 mL14-CAT, containing the mouse lactoferrin gene promoter and the estrogen response element region (-21 to -587) was tested for its estrogen responsiveness in transient transfection experiments. There was very little hormone response in RL95-2 cells in the absence of exogenous estrogen receptors, although a low level of estrogen receptor has been found in these cells (Way, D.L., Grosso, D.S., Davis, J.R. , Surwit, E.A., and Christian CD. (1983) In Vitro 19, 147-158). A 2.5-fold stimulation was observed when 1 μg of HEO plasmids were also introduced (FIG. 5) . Therefore, this chimeric reporter with the lactoferrin estrogen response element did respond to estrogenic hormone in the presence of its receptor. In contrast, reporter plasmid 0.3mL14-CAT, lacking the estrogen response element (-21 to -291) , did not respond to estrogen stimulation (data not shown) .
Example 11 Developing transgenic mouse models with the lactoferrin promoter
To generate a transgenic mouse model that is useful in medicine and biology, it is necessary to target the gene to a specific tissue and to control the timing and level of expression (Hogan, B. , Constantini, F., and Lacy, E. , (1986) Manipulating the Mouse Embryo, A Laboratory Manual, pages 153-173; Mullins, L. and Mullins J. , (1991) Current Opinion in Cell Biology 3:192-198; Cory, S. and Adams, J. , (1988) Ann. Rev. Immunol. 6:25-48; Iwamoto, T., Takahashi, M., Ito, M., Hamatani, K. , Ohbayashi, M. , Wajjwalku, W. , Isobe, K. , Nakashima, I. (1991) EMBO 10: 3167-3175; Hanahan, D. (1988) Ann. Rev. Genet. 22:479-519).
To obtain a transgenic mouse, the conventional method is used which comprises removing a superovulated fertilized mouse egg from a mouse uterus; microinjecting a male pronucleus of a fertilized mouse egg with an amount of a nucleotide sequence attached to at least a portion of mouse lactoferrin promoter; replacing said microinjected egg into said mouse uterus. The 5' genomic regulatory region of mouse lactoferrin contains various pertinent regulatory elements. The most interesting one is the estrogen responsive module. Under the influence of estrogen, the regulatory region becomes a strong promoter only in the uterus. Therefore, this particular promoter can be used to carry oncogenes, drug-resistant genes, growth factors and tumor-suppressor genes to the uterus in transgenic animal studies. The expression of those genes will then be regulated by estrogen. Similarly, those genes can also be targeted to the mammary gland and bone marrow by the lactoferrin gene promoter-enhancer sequence and regulated under different control. The 2.7 kbp 5' flanking sequence of the lactoferrin gene is being used for this transgenic animal study. The lactoferrin 5' sequence is linked to the cDNA or gene of interest and a SV40 3' region with poly(A) addition site or equivalent poly (A) addition site from another source. Plasmids from commercial sources such as pUC, pGEM and Bluescript are used to construct the chimeric plasmid for transgenic animal study.
Other uses for the lactoferrin 5'- flanking sequence are as follows: (1) The 9 kbp lactoferrin fragment in mL14p9E or the entire 14 kbp lactoferrin insert in J14 is introduced into embryonic stem (ES) cells to produce mutant mouse that lacks the lactoferrin.
(2) Lactoferrin 5 '-flanking sequence is used in selective ablation of a specific cell type. Cell ablation is achieved by introducing genes encoding cytotoxins, such as the catalytic subunits of DT or ricin, driven by the lactoferrin promoter/enhancer sequence. An alternative strategy is conditional ablation, using the HSV-tk gene under the control of lactoferrin promoter/enhancer sequence in which cells are rendered susceptible to drugs such as gancyclovir.
EXAMPLE 12 • Analysis of Sequence Data
From the sequence data, it was found that a number of interesting consensus regulatory elements resided within 1000 bp of the 5'-flanking region. Transient transfection of the chimeric gene (0.6 mL14-CAT) containing a putative estrogen response element (position -589 to -21) , rendered estrogen responsiveness in human endometrium cells in the presence of estrogen receptors. The region containing the imperfect ERE and COUP (position - 353 and -327) (Sagami et al. (1986); Killary et al. (1984)) was protected by DES-treated mouse uterine nuclear extract from DNase I digestion, but not by liver nuclear extract from the same animal. These results demonstrated the presence of a functional estrogen responsive module in the lactoferrin gene which is responsible for the estrogen regulation in the uterus. Deletion of the DNA sequence between - 589 and -291 resulted in a doubling of lactoferrin promoter activity and a simultaneous loss of the estrogen responsiveness. Therefore, despite the presence of several characterized transcription factor binding elements. (SP l, AP 2, PU.l/Spi.l, ERE and COUP) residing in this region, there could be an additional silencer present. It was interesting to find that the same construct (0.6 mL14-CAT) had higher promoter activity in C6 cells than in RL 95- 2 cells. It is possible that this DNA fragment (- 586 to -21) was under negative regulation in RL 95- 2 cells and the repression appeared to be released by estrogen. The footprinting data indicated at least four different protein binding sites within this region. Whether interaction between protein and DNA plays any roles in regulating the lactoferrin promoter activity requires a detailed deletion and mutational analysis together with band- shift and methylation interference study. The surrounding sequences and protein to protein interactions may have also been differently involved in different cell types, as has been found in many genes (Grosschedl et al. (1985) ; Edlund et al. (1985)) .
A very strong positive regulatory element was present between -1739 and -922, and deletion of this region resulted in a drastic decrease of lactoferrin promoter activity. A distinctively different pattern of lactoferrin promoter activity was obtained from the same chimeric reporter genes transfected into C6 cells. DNA sequence, extended beyond -922, completely silenced the lactoferrin promoter in C6 cells. Numerous studies of cell hybrids have indicated that somatic cells produce negative regulators, "extinguishers", that prevent the expression of genes foreign to their own differentiation (Killary et al. (1984); Petit et al. (1986)). It is likely that the negative effect observed in C6 cells transfected with the 2.6 mL14- CAT construct, was mediated through the recognition of negative regulatory elements by trans-acting protein that was present in the C6 cells and absent in the RL 95-2 cells.
DNA sequence up to -291 was sufficient for the maximum expression of lactoferrin promoter in C6 cells, while 1739 bp was necessary for RL 95-2 cells. The basic lactoferrin promoter activity, however, resided within the -234 to -21 region; deletions of this DNA sequence completely abolished its function as indicated by 1.3 mL14-CAT construct. A noncanonical TATA element at -32 to -28, two CAAT elements at -72 to -69 and -101 to -98 were present in this region, in addition to a GC rich sequence between -98 to +1. The presence within the lactoferrin promoter of individual features characteristic of both housekeeping and inducible promoters could contribute to the differential regulation of lactoferrin gene in a variety of cell types. Identification of multiple regulatory elements of the lactoferrin gene is the initial step toward understanding its differential control of expression. Deletion, mutation and nuclear protein interaction with those regulatory elements are currently under investigation.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. All publications cited in this application are specifically incorporated by reference herein.

Claims

WHAT IS CLAIMED IS:
1. An isolated DNA segment having a promoter sequence from -2661 to +1 as shown in FIG. 2 (b) or variations thereof having promoter activity.
2. The DNA segment according to claim 1 wherein at least a portion of the segment is a promoter region for a gene encoding mouse lactoferrin protein.
3. The DNA segment of Claim 1 wherein said variations are allelic variation or variations achieved through mutations, insertions, deletions or substitutions.
4. A DNA construct having a promoter sequence as set forth in claim 1, operably linked to a heterologous structural gene in a manner sufficient to modulate expression of said structural gene.
5. A construct according to claim 4 wherein said construct is further linked to a vector.
6. A construct according to claim 4 wherein said construct is further linked to a plasmid.
7. A host cell transfected with the construct of claim 4.
8. The host cell of claim 7 wherein said host cell is a human endometrium carcinoma RL95 2 cell or C6 rat glioma cell.
9. A method of carrying a nucleotide sequence to a specific tissue comprising the steps of i) operably linking at least a portion of said DNA segment of claim 1 to said nucleotide sequence; ii) administering said operably linked
DNA segment and nucleotide sequence of step (i) to an animal.
10. A method of treating a condition in a patient characterized by a genetic abnormality, comprising the step of administering to said patient an effective amount of the construct of claim 4.
11. An animal cell containing a DNA construct of claim 4 wherein said heterologous structural gene is modulated by said promoter sequence which does not naturally modulate said gene.
12. An animal cell according to claim 11 which is an embryo cell.
13. An animal whose genetic material contains the construct of claim 4.
14. An animal derived from the embryo cell of claim 12 which is a mammal.
15. An animal according to claim 14 which is a mouse.
PCT/US1992/009459 1991-11-12 1992-11-12 Characterization of estrogen responsive mouse lactoferrin promoter WO1993010135A1 (en)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO1996006938A1 (en) * 1994-08-26 1996-03-07 Hoechst Aktiengesellschaft Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle
WO1996006940A1 (en) * 1994-08-26 1996-03-07 Hoechst Aktiengesellschaft Genetic therapy of tumours with an endothelium cell-specific active substance which is dependent on the cell cycle

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Title
ADVANCES IN GENETICS, Volume 24, issued 1987, G. SCANGOS et al., "Gene Transfer into Mice", pages 285-322. *
PHARMACEUTICAL THERAPEUTICS, Volume 29, issued 1985, M.J. CLINE, "Perspective for Gene Therapy: Inserting New Genetic Information into Mammalian Cells by Physical Techniques and Vectors", 69-92. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 262, No. 21, issued 25 July 1987, B.T. PENTECOST et al., "Lactotransferrin is the Major Estrogen Inducible Protein of Mouse Uterine Secretions", pages 10134-19139. *
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WO1996006938A1 (en) * 1994-08-26 1996-03-07 Hoechst Aktiengesellschaft Genetic therapy of vascular diseases with a cell-specific active substance which is dependent on the cell cycle
WO1996006940A1 (en) * 1994-08-26 1996-03-07 Hoechst Aktiengesellschaft Genetic therapy of tumours with an endothelium cell-specific active substance which is dependent on the cell cycle
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