WO1987005935A1 - Procedes et compositions d'expression de produits genetiques eucaryotiques competitifs - Google Patents

Procedes et compositions d'expression de produits genetiques eucaryotiques competitifs Download PDF

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WO1987005935A1
WO1987005935A1 PCT/US1987/000805 US8700805W WO8705935A1 WO 1987005935 A1 WO1987005935 A1 WO 1987005935A1 US 8700805 W US8700805 W US 8700805W WO 8705935 A1 WO8705935 A1 WO 8705935A1
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gene
heat
shock
expression
control region
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Peter Bromley
Richard Voellmy
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Battelle Memorial Institute At Columbus, Ohio Gene
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention is a continuation-in-part of U.S. Serial Number 626,588 filed July 3, 1984, which was a continuation of Serial Number 568,176 filed January 5, 1984, which was a continuation-in-part of application serial number 504,593 filed June 17, 1983 which was a continuation-in-part of application serial number 464,232 filed February 7, 1983.
  • the industrial production of proteins coded by specific genes involves selecting and isolating gene sequences from viral, eukaryotic and other RNA or DNA, splicing of the sequences in the form of DNA into DNA vectors to provide recombinant DNA, and introducing said recombinant DNA into host cells capable of expressing these genes.
  • the vectors may impart to the transformed host cells a phenotypic trait used for isolation and cloning purposes.
  • the gene products are isolated from cell cultures by usual techniques.
  • Non-proteolytic post-translational modifications of certain proteins may not occur correctly, if at all, in bacteria or cell types significantly different from the cell type in which the gene product of interest is normally produced in the organism.
  • the correct form of these modifications may prove critical in the synthesis of fully competent gene products by molecular cloning techniques.
  • the gene product is a glycoprotein, such as the haemagglutinin of influenza virus
  • the precise nature of glycosylation may influence the efficiency of antibodies raised against this synthetic protein to protect human beings or animals against influenza virus infections.
  • glycosylation, acetylation or phosphorylation or other modifications are required to stabilize, activate, or mediate intracellular transport or excretion of a protein, the precision of these modifications may be critical to the utility of these genetically engineered protein products in practice.
  • DNA can be introduced by co-transformation into suitable mutant cells which are deficient in the production of a particular enzyme, such a thymidine kinase or hypoxanthine phosphoribosyl transferase. Then by culturing the cells in a selective medium deficient in the enzyme product, transformed cells may be selected for by their ability to grow, i.e., their ability to produce the enzyme.
  • a particular enzyme such as thymidine kinase or hypoxanthine phosphoribosyl transferase.
  • cells can be co-transfected in a positive sense to add a gene that will transduce cells to become selectively resistant to a drug or selective medium such methotrexate, neomycin, or others.
  • a drug or selective medium such methotrexate, neomycin, or others.
  • the host expression unit system employed consists of an expression vector derived from one cell type or organism that is capable of synthesizing the said fully competent gene products, and that this expression vector directs the synthesis of said gene products in the same or similar cell type or organism as host.
  • DNA constructions comprising control elements derived from heat-shock genes associated with genes of interest, which permit the expression of the gene of interest in both procaryotic and eucaryotic cells.
  • control elements derived from heat-shock genes associated with genes of interest, which permit the expression of the gene of interest in both procaryotic and eucaryotic cells.
  • a homologous combination of expression unit and host cell for expression is used and the nature of this homologous system is that of the same or similar cell type or organism that normally produces the said gene product.
  • Fig. 1a is a schematic representation of a partial restriction map of part of plasmid 132E3 containing two 70 kdal heat-shock protein (hsp) genes (heavy lines) and their orientation (arrows).
  • the lower part of Fig.1a represents the relevant partial restriction map of plasmid 51 for comparison purposes.
  • Fig. 1b represents on an enlarged scale a more detailed restriction map of part of plasmid 51 with additional restriction sites, including the two characteristic Sau3A sites.
  • Fig. 2 represents the complete DNA sequence of one strand of the DNA preceding the 70 kdal hsp gene in plasmid 51 except for the white sequences on a black background, which are present in another 70 kdal gene sequence, as determined by the procedure of [Maxam and Gilbert (1977), PNAS 74: 560], and in addition some important restriction sequences as well as the site of the start of transcription and translation (arrows).
  • Figs. 3a-3d represent a diagrammatic representation of the procedure for the construction of plasmid pRV15.
  • Fig. 3a shows a restriction map of plasmid 51 which after digestion with Sau3A gives rise to an approximately 650 bp fragment referred to henceforth as the 650 bp fragment, one end of which is indicated in detail in Fig. 3c (left part).
  • Fig. 3b is a restriction map of plasmid pMC1403 which, after restriction with BamHI, leaves one end with the structure indicated in detail in Fig. 3c (right part).
  • Fig. 3c represents details of the sequence of the ends produced by the restriction digests of plasmids
  • Fig. 3d represents the structure of plasmid pRVl5 showing the linkage of the Sau3A fragment of plasmid
  • Fig. 4a is a photograph showing characterization of the structure of plasmid pRVl5 indicated in Fig. 3d by the colony hybridization assay of Grunstein and Hogness (1975) PNAS 72 : 3961. This was performed using radioactive probes prepared by nick translation [Maniatis et al., (1975) PNAS 72: 1184] of either a 2 kbp XbaI fragment excised from plasmid 132E3 ( see Fig . 1a ) or a label led 650 bp Sau3A fragment from plasmid 51 ( Fig . 4b ) . A number of transformants produced using the construction scheme of Fig. 3 were tested as were some colonies of pMCl403 as a control; these colonies are indicated by arrows in Fig. 4a.
  • Fig. 4b represents a similar colony hybridizaiton assay but using the Sau3A 650 kbp fragment from plasmid 51 as a probe. Control colonies of pMC1403 are indicated by arrows.
  • Fig. 5 represents the characterization, by restriction analysis, of the structure for plasmid pRV15 indicated in Fig . 3d . Fragment sized in bps are indicated . In particular:
  • Fig . 5a is a photograph showing the migration of various DNA fragments under electrophoresis.
  • DNA was prepared from mini-plasmid preparations [Davis et al., (1980), Methods in Enzymoloy, Grossmann and MoIdave, eds. 65: 404-414] for plasmid pMC1403, pRV15 and two similar isolates, pRV25 and pRV5. These plasmids were digested with restriction enzymes as indicated below and the fragments of DNA produced were electrophoresed on 0.85% agarose gels at 150 v for 4 hours after which bands were visualized using ethidium bromide staining and U.V. photography. Identification of the lanes is as follows: Lane 1 PMC1403 - Sau3A/XhoI
  • Fig. 5B is similar to Fig. 5a for a series of digests and analyses performed identically.
  • the lanes are as follows:
  • Fig. 5c is similar to Figs. 5a and 5b and concerns restriction digests and analyses performed for fragments separated on gels of 5% polyacrylamid. Electrophoresis was at 40 v for 14 hours. Lane 1 51 - Sau3A/XbaI
  • Figs. 6a-6b refer to an analysis of the orientation of the Drosophila 70 kdal hsp gene Sau3A fragment in plasmid pRV15.
  • Fig. 6a is a diagrammatic representation of the sizes of predicted restriction fragments that would result if the desired orientation of Drosophila elements and the E. coli ⁇ -galactosidase gene is indeed the case in plasmid pRV15.
  • Fig. 6b is a photograph of an acrylamide gel (5%) analysis of the results of restriction of plasmid pRV15 and pBR322 to test the prediction presented in the diagram of Fig. 6a. Analyses were performed as described for the cases represented by Fig. 5c.
  • Figs. 7a-7e which show plasmids or fragments of plasmids after digestion with restriction enzymes, represent schematically the construction of plasmid 520, which consists of incorporating the SamI - SalI fragment of pRVl5 containing the lac operon fragment and the Drososphila 650 bp control element into plasmid pSVod using the BamHI - SalI restriction site.
  • plasmid 520 which consists of incorporating the SamI - SalI fragment of pRVl5 containing the lac operon fragment and the Drososphila 650 bp control element into plasmid pSVod using the BamHI - SalI restriction site.
  • Fig. 7a represents pRV15
  • Fig. 7c represents pSVod
  • Fig. 7b represents a SmaI - SalI fragment of pRV15
  • Fig. 7d represents pSVod after excision of a BamHI - SalI fragment
  • Fig. 7e represents p520.
  • Fig. 8a is a restriction map for plasmid vector 520 indicating the position of selected restriction sites in the structure of the plasmid.
  • Fig. 8b is a photograph of an agarose gel analysis of restriction enzyme digestions of plasmid 520 performed as described in the case of pRVl5 (see Fig. 5a.) .
  • Fig. 8c is a photograph also referring to the characterization of plasmid 520. It shows agarose gel analysis of various restriction digest of plasmids pSVod, pRV15 and 520-like plasmids, i.e., plasmids 639, 520, 519, X, Y, Z. Lane 1 639 - SalI/EcoRI
  • Fig.8d is a photograph showing further restriction digests of plasmid 520 by comparison with digests of plasmids pSVod and pRV15.
  • Figs. 9a-9c refer to the construction of plasmid PR81.
  • Figs. 9a-9c refer to the construction of plasmid PR81.
  • Fig. 9a represents the Mount Sinai A/PR 8/ms/-4.76 plasmid from which a fragment (ref. no. 20) was excised with BamHI and HindIII;
  • Fig. 9b represents pSVod less a HindIII - SalI fragment;
  • Fig. 9c represents plasmid PR81, i.e., the ligation product of the BamHI - HindIII fragment 20 with a SalI - HindIII digest.
  • Figs. 9d and 9e are photographs of a Grunstein colony hybridization analysis of PR81-like plasmids, performed as described for pRV15 (see Fig. 4 ).
  • the nick--translated probe used was the BamHI/HindIII fragment for A/PR 8/ms/4.76.
  • Fig. 9f is a photograph of a restriction analysis of PR81-like plasmids shown by colony hybridization analysis in Figs. 9d and 9e.
  • Figs. 10a-10e refer to the construction scheme of plasmidPR84. This plasmid was constructed as shown in the figure and consists of substituting the haemagglutinin (HA) gene 20 present in plasmid PR81 for the ⁇ -galactosidase gene in plasmid 520.
  • Fig. 10a represents plasmid 520
  • Fig.10c represents plasmid PR81
  • Fig. 10b represents an NcoI - PvuII fragment excised from 520
  • Fig. 10d represents PR81 after digestion with HindIII and NcoI
  • Fig. 10e represents schematically PR84.
  • Fig. 10f is a more detailed representation of
  • PR84 with one strand of base-pairs including the sequence originating from PR 8/ms; only the crucial joining region between the Drosophila HS control element and the HA start sequence is detailed.
  • Fig. 11 refers to checking the results from the construction of PR84.
  • Fig. 11a is a photograph referring to the restriction digestion of plasmids PR81, PR 8/ms/34, pSVod and 520.
  • a nick-translated radioactive probe (approximately 10 8 cpm/ ⁇ g) of the plasmid 51 Sau 3A/Xho I Drosophila control element fragment was used in these assays.
  • Fig. 11c refers to a 5% polyacrylamide gel analysis of the XhoI digestion products of PR84-like plasmids selected from the positive colonies identified in Fig. 11b.
  • Fig. 11d refers to further restriction analysis of plasmids giving the XhoI digestion pattern shown in Lanes 2, 4, 6 and 8 of Fig. 11c. Analysis was performed as described for Fig. 11c.
  • Fig. 12a refers to a 5% polyaerylamide gel analysis of some candidate 629 series plasmids. Plasmid
  • Fig. 12b refers to another 5% polyacrylamide gel analysis of all eight candidate 629 series plasmids.
  • Fig. 12c shows a Grunstein colony hybridization assay using the nick-translated Xho-BglII fragment from plasmid p622b.
  • Fig. 12d shows a Grunstein colony hybridization assay using the nick-translated HindIII/BamHI.HA gene fragment from plasmid A/PR 8/mx/34.
  • Fig. 13 relates to Example 3 herein and depicts heat-shock hybrid gene constructs. Symbols used: D.- melanogaster hsp70 gene promoter regions 1 E. coli ⁇ -galactosidase-coding sequence 2 D. melanogaster hsp70 gene 3'trailer sequence 3 thymidine kinase gene RNA leader sequence 4 chicken lysozyme gene 5 ; SV40 origin of replication 6 ; SV40 gene 3' trailer sequences 7
  • Hsp70 gene sequences are underlined. The transcriptional orientation of the hybrid genes is indicated by arrows.
  • Fig. 14 relates to Example 3 herein and depicts electrophoretic analysis of hybrid gene products synthesized by heat-treated (H) or untreated (C) Xenopus oocytes.
  • H heat-treated
  • C untreated
  • A Examination of cytoplasmic extracts prepared from oocytes that had been injected with pR84 (Lanes 1,2,8 and 9), p17-lys (Lanes 3,4,10 and 11), p522-lys (Lanes 5,6,12 and 13) or p17hGH dhfr DNA (Lanes 14 and 15). Electrophoresis was carried out following immunoprecipitation with the antisera indicated in the Figure.
  • B Electrophoresis was carried out following immunoprecipitation with the antisera indicated in the Figure.
  • Fig. 15 relates to Example 3 herein and depicts hybrid gene products made by heat-treated (H) or untreated (C) transfected COS 1 (Lanes 1 to 13) or CHO (Lanes 14 to 16) cells.
  • Cells had been transfected with p522 (Lanes 1,2,3,4,9 and 10), p522-lys (Lanes 5 and 6), p17-lys (Lanes 7,8,11 and 14) or p17hGH dhfr DNA (Lanes 12,13,15 and 16).
  • Cytoplasmic extracts from p522-containing cells were analysed by electrophoresis following immunoprecipitation with either anti-bromelain or anti- -galactosidase antisera (Lanes 1 to 4). Lanes 5 to 16 show immunoprecipitated proteins from media samples.
  • Methods and compositions are provided for the controlled expression of a gene in a eukaryotic host.
  • the present invention is particularly useful for expressing mammalian genes in mammalian hosts, typically in a host cell culture similar or identical to the host in which the gene is naturally expressed. In this way, post-transcriptional and post-translational modifications of the gene products will be achieved, thus assuring the competence of such resulting gene products.
  • DNA sequences which comprise an inducible expression control region derived from a eukaryotic heat-shock protein (hsp) gene are employed.
  • control regions are inducible for example by elevated temperatures and include sequences responsible for both transcriptional and translational control of the hsp gene activity, e.g., the promoter region (RNA polymerase recognition and binding sites), and possibly also heat-shock protein gene control sequences which have been modified in such a way that they allow the constitutive expression of downstream coding sequences.
  • the control region may also include other portions of the translated and untranslated regions of the hsp gene which participate in transcriptional and/or translational regulation including 3' untranslated sequences.
  • sequences derived from the 3' untranslated region flanking the structural gene i.e., the noncoding portion of the gene of interest may be included.
  • hsp gene including the structural region as well as the 5' and 3' flanking regions may be employed.
  • the gene of interest may be inserted internal to the structural region to produce a fused protein upon translation.
  • the protein of interest may then be recovered by conventional means.
  • the term heat-shock protein (hsp) gene will be employed in the specification and the claims to denote the entire gene locus responsible for the inducible expression of heat-shock proteins, specifically including the 5' regulatory sequences upstream of the structural re- gion, the structural reigon which encodes the mRNA transcript (including untranslated leader sequences) and the 3' flanking region downstream of the structural gene.
  • Heat-shock protein genes may be obtained from most higher eukaryotic organisms. Particular heat-shock proteins have been identified in fruit flies (Drosophila), mice, frogs, monkeys, and man. Heat-shock genes suitable as a source for the control regions of the present invention may be obtained from any of these or other eukaryotic organisms. Although heat-shock genes drived from one species can be expressed in other species and even in procaryotes, it is generally desirable that in order to optimize the expression advantages of heat-shock control elements at both the transcriptional and the translational levels, these elements should be derived from the same or similar organism or cell type that will be used for expression of the gene of commercial interest.
  • constructions including genes under control of Drosophila heat-shock elements can be most advantageously expressed in Drosophila cells in culture.
  • an inducible expression control region obtained from a heat- shock protein gene which encodes for a 70 kdal heat-shock protein found in Drosophila was used to express E. coli ⁇ -galactosidase gene in E. coli, COS I Monkey cells, and in Xenopus occytes. The same region was used to control the expression of the influenza haemagglutinin gene in COS I cells.
  • the inducible expression control region of the present invention may be combined with an extrachromosomal replication system for a predetermine host to provide an expression vector for that host.
  • Such vectors will include DNA sequences having restriction site(s) for insertion of gene(s) 3' to said control regions to provide the regulated transcription and translation of the inserted genes.
  • the vector can also include markers for selection in bacteria and in eukaryotic host cells, a prokaryotic replication system allowing cloning of the vector in a prokaryotic host, and other DNA regions of interest.
  • the expression control region can be joined to a desired structural gene and the resulting DNA constructs introduced directly into the host cells.
  • Methods for such direct transfer include injection of the DNA into nuclei [Cappechi (1980) cell 22 : 479-488] and co-transformation by calcium phosphate precipitation [Wigler et al. (1979) Cell 16: 777-785] or DEAE dextran [McCutchan, J.H. and Pagano J.S. (1968) J. Nat. Cancer Inst. 41: 351-357].
  • the DNA constructs of the present invention may include differing portions of the hsp gene.
  • the constructs will include at least the 5' regulatory region which carries the promoter, regulatory sequences such as operators, activators, cap signals, signals enhancing ribosomal binding, and other sequences as well as additional DNA leader sequences responsible for transcriptional and translational control.
  • the DNA constructs may also include part of the protein coding sequence of the hsp gene, resulting in the production of fused proteins when the foreign gene to be expressed is inserted downstream of the hsp sequences. If a fused protein is not desired, it will be necessary to remove the hsp coding sequences from the isolated hsp DNA by usual methods such as restriction by enzymes and exonuclease digestion.
  • a fused protein including the amino-terminal amino acid sequences of the heat-shock protein is provided.
  • the expression control region of the present invention will usually be combined with a terminator for complete transcriptional control of the inserted structural gene.
  • the terminator can be derived from the heat-shock gene itself, although the inserted structural gene may carry its own or any other suitable terminator sequence.
  • Extrachromosomal replication systems may also be used. Suitable replication systems include autonomously replicating sequences as described by Struhl et a l . 9(1979) PNAS 73 : 1471-1475 and the 2 ⁇ m plasmid for replication in yeast. Mammalian replication systems could be derived from papovaviruses, such as simian virus 40 and bovine papilloma virus; adenoviruses; avian retroviruses, such as avian sarcoma virus; and mammalian retroviruses such as Moloney leukemia virus.
  • papovaviruses such as simian virus 40 and bovine papilloma virus
  • avian retroviruses such as avian sarcoma virus
  • mammalian retroviruses such as Moloney leukemia virus.
  • prokaryotic replication system In addition to the optional eukaryotic replication system, it is advantageous to provide a prokaryotic replication system to allow for cloning of the vector in a bacterial host. This allows large quantities of the vector to be grown in well characterized bacterial systems prior to transforming a eukaryotic host.
  • Suitable prokaryotic replication systems are well known and include plasmids such as pBR322, pRK290, ColE1, and bacteriophages, e.g. ⁇ dv.
  • the prokaryotic replication systems will necessarily include an origin of replication recognizable by a prokaryotic host, and will usually include one or more markers for the selection of transformants in the prokaryotic host. Such markers include biocide resistance, toxin resistance, and the like. Alternatively, complementation allowing the growth of an auxotrophic host in a selective medium may be employed. Such techniques are well known in the art and need not be described further.
  • markers employed will be different for selection in prokaryotic and eukaryotic hosts.
  • Various dominantly acting markers are useful in selecting for transformed mammalian cell lines. They usually comprise a specific gene whose expression confers a new drug-resistant phenotype to the mammalian cells in an appropriate selective medium.
  • Specific markers include the bacterial xanthine-guanine phosphoribosyl transferase gene which can be selected in medium containing mycophenolic acid and xanthine [Mulligan et al.
  • transformants with vectors carrying a mouse cDNA fragment coding for dihydrofolate reductase may be selected for using medium containing aminopterin [Subramani et al. (1981) Mol. Cell Biol. 1: 854-861]; and a bacterial plasmid gene specifying an amino-glycoside phosphotransferase that inactivates the anti-bacterial action of neomycin-kanamycin derivatives may be selected for using medium containing G418 a neomycin derivative toxic for most mammalian cell lines [Colbere-Garapin et al. (1981) J. Mol. Biol. 150: 1-14].
  • the number of copies of gene expression units introduced into a host cell may vary and that by proximal combination of one of the amplifiable genes such as the mouse dihydrofolate reductase gene the number of copies of integrated gene expression units may likewise be amplified by induction of the amplification of the associated amplifiable gene and the proximal DNA.
  • proximal combination of one of the amplifiable genes such as the mouse dihydrofolate reductase gene
  • the number of copies of integrated gene expression units may likewise be amplified by induction of the amplification of the associated amplifiable gene and the proximal DNA.
  • dihydrofolate reductase cDNA and the E. coli XGPRT (xanthine-guanine phorphoribosyl transferase) gene in Chinese ovary cells [Rungold, G. et al. (1981). J. Mol. and Appl. Genetics, 1, 165-175].
  • both the expression control region of the heat-shock protein gene and the eukaryotic replication system are inserted into a suitable prokaryotic plasmid.
  • the manner and order of the insertion are not critical, and it is necessary only that the resulting vector retains viable replication systems for prokaryotic and when necessary eukaryotic hosts.
  • the DNA constructs containing Drosophila heat-shock gene control segments functionally linked to a structural gene of interest can be used to synthesize products of said gene in Drosophila cells or cultured cells closely related to Drosophila. Since the structural gene is under the transcription and if necessary translation control of the heat-shock control element, its expression can be enhanced by for example increasing the ambient temperature of the cells for instance in the range 37-42°C. By such induction a large fraction of the newly made mRNA will be derived from said structural gene.
  • the structural gene is a human gene encoding a protein requiring complex processing
  • the expression vector preferably comprises, in addition to said structural gene, replication elements and marker genes, a heat-shock control element derived from a human heat-shock gene. This construct will be introduced advantageously into cultured human cells by procedures described above to produce products of said human gene. As necessary the choice of recipient human cells will be made on the basis of their competence in correctly expressing the fully processed gene product.
  • a wide variety of structural genes may be introduced into the subject vectors to permit the production of various gene products including polypeptides, such as enzymes, proteins, hormones, novel protein structures, and the like.
  • Plasmid pRV15 which contains the E. coli ⁇ -galactosidase gene under the control of a Drosophila heat-shock control element.
  • Plasmid 132E3 contains two comlete genes for the 70 kdal heat-shock protein, and the aforementioned isolated sub-clone, plasmid 51 [Karch et al.
  • Fig. 1a is a representation of a portion of plasmid 132E3 indicating the two genes encoding 70 kdal heat-shock proteins (thick line segments) and some of the identified restriction sites.
  • the lower part of Fig. 1a represents a portion of p51 containing the BglII - BamHI segment containing the aforesaid 650 bp fragment, bounded by the Sau3A cleavage sites.
  • Fig.1b is a more detailed representation of the portion of interest contained in p51 with additional restriction sites indicated, and also showing the position of the 650 bp fragment between two Sau3A sites.
  • Fig. 2 provides the DNA sequence data for one strand contained in the aforementioned 650 base pair fragment. The limits of this sequence are indicated by the position of the Sau3A restriction sites (GATC). Also, XbaI, XhoI and PstI recognition sites for restriction are indicated, and the position of the transcription and translation start sequence are indicated by arrows la and lb, respectively.
  • the 650 bp fragment was functionally inserted into plasmid pMC1403 in a position to control the expression of test genes in this plasmid. Plasmid pMC1403 [Casadaban et.al. (1980) J. Bacteriol.
  • plasmid pBR322 (Bolivar et.al. (1977) Gene 2 : 95-113] containing the entire E.coli lac operon with the exception of the sequences coding for the first seven amino acids of ⁇ -galactosidase and all sequences 5' to the ⁇ -galactosidase protein coding region (promoter, ribosomal binding site, translation initiation codon). Consequently lac strains of E.coli such as MC 1061 [Casadaban et.al. (1980) J. Mol. Biol 138: 179-207] carrying plasmid pMC1403 do not produce ⁇ -galactosidase.
  • a polylinker (EcoRI, SmaI, BamHI) at the 5' end of the lac sequences in pMC1403 permits the introduction of foreign DNA sequences upstream from the ⁇ -galactos idase coding region. Insertion of a segment containing a functional promoter and the RNA leader sequences will result in the production of ⁇ -galactosidase activity. It should be noted that the amino-terminal end of ⁇ -galactosidase is not essential for its enzymatic activity [Muller-Hill B. and Kania, J. (1974) Nature 249: 561-563].
  • Plasmid p51 is digested with Sau3A to excise the aforesaid 650 bp fragment represented on the left of Fig. 3c by numeral 2; this fragment was purified from polyacrylamide gels (see Fig. 5c) by electroelution.
  • This fragment was inserted into the BamHI site of pMC1403 in either orientation.
  • the ligation mixture was then used to transform the lac strain of E.coli MC 1061.
  • Transformants were plated on media containing ampicillin and Xgal (Xgal is 5-bromo-4-chloro-3-indolyl-N-D-galactoside) which is a substrate for ⁇ - galactosidase, and which after cleavage by the enzyme produces an identifiable colored product (see Miller, J. Experiments in Molecular Genetics, pp. 47-55, Cold Spring Harbor, N.Y. 1972).
  • Plasmid pMC1403 did not produce ⁇ -galactosidase activity in this assay while plasmid pRV15 and a large number of other transformants, prepared in the aforesaid manner, were found to produce substantial amounts of ⁇ -galactosidase as determined by the color change produced on Xgal plates.
  • ⁇ -galactosidase-coding plasmid constructions such as pRV15 were analyzed further by the colony hybridization assay of Grunstein [Grunstein and Hogness (1975) PNAS 72 : 3961-3966] using either radioactively labelled 650 bp Sau3A fragments from p51 (see Fig.
  • Plasmid pMC1403 contains unique restriction sites for EcoRI and SalI, but no XhoI or XbaI sites.
  • the Sau3A 650 bp fr&gment from plasmid p51 however contains unique sites for restriction by XhoI and XbaI but has no sites for restriction enzymes such as EcoRI and SalI.
  • recombinant plasmids such as pRV15 should contain unique sites for all four aforementioned enzymes (see Fig. 3 ).
  • Fig. 5a shows a photograph of DNA fragments produced by the aforementioned restriction enzymes after electrophoresis on agarose gels, and confirms the structure of plasmid pRV15 shown in Fig. 3.
  • pRV15 contains the 650 bp Sau3A fragment of plasmid p51 inserted into the BamHI site of pMC1403, then it should be possible to recover the 650 bp Sau3A fragment by digestion of recombinant DNAs such as pRV15 with the restriction enzyme Sau3A. That this is the case is shown in Fig. 5c lane 7 by the arrows.
  • the identity of the 650 bp excised fragment is confirmed by its restriction by XbaI or by XhoI. See Fig. 5c lanes 5 and 6.
  • ⁇ -galactosidase protein produced in E.coli under the control of a Drosophila heat-shock control element has also been clearly demonstrated by immune precipitation of protein extracts of E.coli containing plasmid pRV15 after radioactive labelling a newly synthesized protein with 35 S-methionine.
  • a polypeptide of moleculnr weight approximately 120,000 daltons was precipitated from such protein extracts by immune sera directed against authentic E.coli ⁇ -galactosidase.
  • a Sau3A digest of pBR322 was electrophoresed in parallel and served to identify the 650 bp 51 Sau3A promoter fragment (Fig. 5c). DNA fragments were visualized with ethidium bromide (EtBr). The region containing the promoter fragment, indicated by the arrows in Fig.5c, was cut out of the gel and the fragment was electroeluted into a dialysis bag. Electroelution was carried out for 5 hours at 200v in 1 x TBE buffer. The eluate was collected in a 15 ml siliconized Corex tube and ethanol-precipatated overnight at -20°C.
  • EtBr ethidium bromide
  • the precipitate was collected by centrifugation (Sorvall, 30 min., 10,000 rpm, SS34 rotor), dried in a lyophilizer and resuspended in 200 jal of TE buffer (10 mM Tris. HCl, pH 7.5, 1 mM Na 2 EDTA) .
  • the DNA was then extracted twice with TE-saturated phenol and twice with ether.
  • the solution was then passed through a Sephadex G75 mini-column (in a Pasteur pipet). DNA in the column eluate was ethanol-precipitated twice, dried and resuspended in 20 ⁇ l of TE buffer .
  • plasmid pMC1403 was digested with 4 units of BamHI for 30 min. at 37°C.
  • the digested DNA was extracted 3 times with phenol and then 3 times with ether.
  • the DNA was further purified by 2 subsequent ethanol precipitations.
  • the pelleted DNA was dried and then resuspended in 20 ⁇ l of TE buffer.
  • the 2 kbp 70 kdal heat-shock protein gene fragment was eluted electrophoretically from the gel and was purified as described above.
  • the latter fragment and the p51 Sau3A fragment were then 32 p-labelled by nick translation to a specific radioactivity of 2x10 8 cpm/ ⁇ g.
  • the two probes were then denatured and hybridized to two nitrocellulose filters containing DNA of the 80 selected transformants and of pMC1403 (Figs. 4a, b). Both probes strongly hybridized to DNAs of all 80 recombinants, suggesting that all recombinants contained the 650 bp promoter fragment. Clones 5, 15, 25, 35, 45, and 55 were selected for further studies.
  • Plasmid pRV15 (50 - 100 ug) was digested with an excess of SalI and EcoRI. The two resulting restriction fragments were then separated on 0.85% agarose gels. The 7kb fragment containing the hybrid gene but no vector sequences was purified by electroelution and gel filtration on Sephadex ® G75. This fragment was then incubated with an excess of T4 DNA ligase in a total volume of 25 ⁇ l as described above, to permit circle formation. The ligated fragments were injected into oocytes as described in Voellmy and Rungger (1982) PNAS 79 : 1776-1780.
  • RNA prepared from these oocytes or from oocytes that did not contain foreign DNA was hybridized to Southern blots of Sal I/Eco RI digests of plasmid PMC1403. Oocytes that did not contain the hybrid gene did not produce measurable amounts of RNA complementary to the ⁇ -galactosidase gene. Both heat-treated and untreated oocytes containing the pRV15 fragment were found to have made ⁇ -galactosidase RNA in approximately similar quantities.
  • RNA polymerase B i.e. II
  • Plasmid PR84 which Contains a Human Influenza Virus Haemagglutinin Gene under the Control of a Drosophila Heat-shock Control Element
  • Plasmid 520 was constructed from plasmid pRV15 and plasmid pSVod [Mellon et.al. (1981) Cell 27: 279-288] which is capable of replicating either in procaryotic cells or in certain eukaryotic cells. Plasmid 520 allows rapid analysis of transcriptional control since it includes the SV40 virus origin of replication and is able to replicate efficiently in SV40 mutant-transformed, transformation antigen positive COS cells [Gluzman (1981) Cell 23: 175-182].
  • Plasmid pRV15 (Fig. 7a, representation in linear form) was digested with SmaI, subsequently digested with SalI and the resulting 7 kbp fragment (Fig. 7c) containing the 650 bp Drosophila control element 2 and the lac gene fragment 3 originating from pMC1403 present in pRV15 was isolated by electrophoretic elution from a preparative agarose gel. Plasmid pSVod (Fig. 7c) was digested with Bam-HI, the cohesive ends were filled in with DNA polymerase Klenow fragment and the DNA finally digested with SalI. In Fig. 7 , Plasmid pSVod (Fig. 7c) was digested with Bam-HI, the cohesive ends were filled in with DNA polymerase Klenow fragment and the DNA finally digested with SalI. In Fig.
  • numeral 10 indicates the site of the deletion of the lkb pBR322 sequence which has been claimed to be inhibitory for replication in eukaryotic cells.
  • the truncated pSVod fragment (Fig. 7d ) so formed was ligated to the 7 kbp fragment isolated from pRV15, and the ligation mixture was used to transform E.coli MC1061.
  • the structure of plasmid 520 is shown in Fig. 7e in which a more detailed representation of the 650 bp segment also appears with the XbaI, XhoI and PstI restriction sites.
  • Plasmid 520 is shown in circularized form in Fig. 8a where the segments a, y and z represent genes of the lac operon. Numeral 11 designates the fragment containing the SV40 origin. Transformants were plated out on Xgal-Ampicillin plates as described previously, and blue transformants suspected of containing plasmid 520 were isolated. From the structure shown in Fig. 8a, plasmid 520 should provide the following size fragments when digested with the named restriction enzyme combinations.
  • the 520 plasmids indeed produced fragments of the correct sizes when digested as evidenced in the gel analyses of Figs. 8b, c and d.
  • Other fragments produced by various combinations of enzyme digestions serve as controls for the interpretion of the digestions noted above.
  • Plasmid p81 places a gene which encodes for a eukaryotic protein in the correct reading frame with the 650 bp heat-shock control element of p520.
  • plasmid A/PR 8/ms/34 Mount Sinai Clone No. 4.76 containing a human influenza haemagglutinin gene (1775 bp, numeral 20) inserted into the PvuII site of vector PAT 153/Pvu II/8 was obtained from Dr. G. Brownlee (University of Oxford, Department of Pathology, U.K.).
  • the plasmid containing the haemagglutinin (HA) gene insert 20, see Fig.
  • plasmid PR81 was digested with BamHI, and the ends were filled in with DNA polymerase Klenow fragment. Following digestion with Hind-III, the haemagglutinin gene fragment was purified on agarose gels.
  • the insert 20 contains a complete HA protein coding sequence, an RNA leader segment, and 40 bp of 3' nontranslated sequence.
  • the HA gene-containing fragment was inserted into plasmid vector pSVod to produce plasmid PR81 as follows: pSVod DNA was digested with SalI, ends were filled in as described above, and the DNA was further digested with HindIII (see structure in Fig. 9b) .
  • the HA fragment 20 and the SalI/ HindIII digest of pSVod were ligated together using T4 DNA ligase in the presence of BamHI and SalI to give PR81 (see Fig. 9c), and the ligation mixture was used to transform E.coli C 600.
  • Transformants were analyzed for the presence of PR81-like plasmids (for predicted structure, see Fig. 9c) as follows: Grunstein colony hybridization was performed in a manner similar to that described earlier in connection with pRV15 (see Fig. 4). A nick translated probe of the HA gene (BamHI/HindIII fragment from pA/PR 8/34/ms/4.76 kb) was prepared and hybridized to the PR81-like transformants (see Figs. 9d and 9e). Figs. 9d and 9e show the autoradiograms of eight colonies. It can be seen from Figs. 9d and 9e that this DNA probe hybridizes to most of the transformants. Negative controls are provided by the pSVod containing colonies indicated in the figure by arrows.
  • PR81-like plasmids were further characterized by restriction analysis, as shown in Fig.9f.
  • the structure indicated for PR81 in Fig. 9c is verified by the formation of DNA fragments of the following sizes after digestion with the restriction enzymes indicated below:
  • Fig. 9f The results provided in Fig. 9f indicate the presence of the above fragments.
  • the construction scheme for plasmid PR84 is presented in Figs. 10a to 10e. Briefly, plasmid PR84 places the HA gene under the control of expression control region of the Drosophila heat-shock gene described heretofore. The hybrid gene itself is placed under the replication control of plasmid pSVod. Digestion of plasmid 520 (Fig. 10a) with PvuII and NcoI results in the formation of two fragments of about 1 kbp in length, one of which (Fig.
  • 10b contains part of the SV40 origin of replication sequence and part of the 650 bp heat-shock control element, including 400 bp of 5'-nontranscribed sequence and 60 bp of the RNA leader sequence.
  • the two 1 kbp fragments were isolated by electrophoresis on preparative gels of 1.2% low melting agarose.
  • Plasmid PR81 (Fig. 10c) was digested with HindIII, the ends were filled up with DNA polymerase
  • NcoI which has a unique cutting site in this plasmid situated within the SV40 origin of replication sequence (see Fig.10d).
  • the resulting PR81 fragments were ligated with the 1 kbp fragments isolated from plasmid 520 and the ligation mixture was used to transform E.coli C 600.
  • the resulting transformants include plasmids such as PR84 whose predicted structure is shown in Fig. 10e.
  • PR84 comprises from the 5' end, successively, a 250 bp origin of replication segment of SV40 origin, a 346 bp sequence from pBR322, a 400 bp Drosophila 5' nontranscribed sequence [p51; p132E3, see Karch et.al. (1981) J. Mol. Biol.
  • RNA leader fragment (base pairs numbered 1 to 65), a linker segment of 8 base-pairs, a haemagglutinin (HA) RNA leader segment (base pairs numbered 1-32), a HA signal peptide coding segment (base pairs numbered 33-83) and a HA protein coding segment (bp 84 onwards).
  • HA haemagglutinin
  • PR81 1NcoI site, 1 HindIII site, the NcoI site in the SV 40 origin of replication sequence.
  • PR 8/ms/34 1 NcoI site, no site in HA gene. 520: 1 Ncol site, several PvuII sites in the lac operon a pair of 1 kbp NcoI/PvuII fragments, one of them containing the origin - hsp 70 promoter fragment.
  • the expected pattern of digestion products formed by XhoI digestion as predicted from the structure of PR84 presented in Fig. 10 are: one XhoI fragment of 405 bps and one very large fragment.
  • Fig. 11c shows the results of this analysis and the plasmid clones in lanes 2, 4, 6 and 8 have the expected pattern as compared to the HinfI standard digest pattern of pSVod. These plasmids were selected for further analysis where they were digested either with XhoI/NcoI or with XbaI/NcoI. The results obtained are shown in Fig. lid. The expected sizes of fragments produced from the map shown in Fig.
  • Fig. 11d shows the validity of these predictions for all of the plasmids analyzed.
  • DNA from plasmid PR84 was used to transfect eukaryotic cells .
  • suitable cel ls for bringing about such experiments COS cells [Gluzman (1981) Cell 23: 175- 182] were selected because of their usefulness for the rapid expression analysis of gene constructions [Gething and Sambrook (1981) Nature 293: 620-625].
  • plasmid pRV15 Fifty ⁇ g of plasmid pRV15 were digested first with 50 units of SmaI for 2 hours at 37°C and subsequently with 50 units of SalI for 2 hours at 37°C (buffers as recommended by New England Biolabs). The digest was then electrophoresed on a 0.9% agarose gel. The region containing the 7 kb lac fragment was cut out of the gel, and the DNA was recovered by electroelution (200 v. 6 hours). DNA in the eluate was collected by ethanol precipitation. The fragment was then dried and resuspended in 200 ⁇ l of TE buffer, phenol- and ether-extracted twice, and then passed through a small Sephadex ® G75 column as described before. The DNA in the column eluate was collected by ethanol precipitation.
  • plasmid pSVod Ten jug of plasmid pSVod were digested with 10 units of BamHI for 2 hours at 37°C.
  • the digested DNA was purified by 3 phenol-extractions, 3 ether-extractions and 2 ethanol precipitations.
  • the dried DNA was then resuspended in TE buffer and incubated with several units of DNA polymerase Klenow fragment in the presence of 0.5 mM deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP) at 6 - 7°C for one hour.
  • dATP deoxyadenosine triphosphate
  • dCTP deoxycytidine triphosphate
  • dGTP deoxyguanosine triphosphate
  • dTTP deoxythymidine triphosphate
  • the DNA was purified again by repeated phenol- and ether-extraction and ethanol precipitation.
  • the purified DNA was then digested with 10 units of SalI for 2 hours at 37°C.
  • the digestion products were purified again as described above.
  • the 7 kbp pRV15 SmaI/SalI fragment and the above described pSVod fragments were then incubated overnight (in a molar ratio of 10:1) with an excess of T4 DNA ligase at 14°C.
  • This ligation mixture was used to transform E.coli MC 1061. Smal and BamHI were included in the ligation mixture.
  • Transformants were plated on LB plates containing 10 ⁇ g/ml ampicillin and 40. ⁇ g/ml Xgal. Blue transformants were isolated. DNA from such recombinants was prepared as described by Schedl et.al. (1978) Cell 14 : 921-929. Recombinants were identified by restriction analyses.
  • plasmid A/PR 8/34 Clone No. 4.76 obtained from Prof. G. Brownlee, University of Oxford, U.K. were digested with 30 units of HindIII and BamHI for 2 hours at 37oC. The digest was electrophoresed on a 0.85% agarose gel. The HindIII-BamHI haemagglutinin gene fragment was purified by electroelution as described above. Aliquots of this DNA were nick translated to serve as probes for identifying PR81 and similar recombinants by Grunstein colony hybridization (specific radioactivity of probes about 10 8 cpm/ ⁇ g).
  • A/PR 8/34/4.76 were digested with 50 units of BamHI for 2 hours at 37°C.
  • the DNA was phenol-extracted three times, ether-extracted and ethanol-precipitated.
  • the dried DNA was then resuspended in 25 ul of TE buffer and incubated with several units of DNA polymerase Klenow fragment and 0.3 mM of all four deoxynucleotide triphosphates in a total volume of 100 ⁇ l for 90 minutes at 6-7°C.
  • the DNA was then purified by phenoland ether-extractions as above. Following ethanol precipitation, the DNA was dried and then resuspended in 25 ⁇ l of TE buffer. It was digested subsequently with 54 units of HindIII for one hour at 37°C. The digested DNA was repurified by phenol-extraction.
  • A/PR 8/34 digest (1.5 ⁇ g) and a pSVod digest (2 ⁇ g ) were incubated overnight at 14°C with an excess of T4 DNA l igase in a total volume of 30 ⁇ l .
  • Several units of BamHI and SalI were included in the ligation mixture.
  • the ligated DNA was purified by phenol extraction as described above. It was then digested with 6 units of BamHI for one hour at 37oC, followed by incubation with 12 units of SalI for one hour at 37°C. Aliquots of this reaction mixture were used to transform CaCI 2 -treated E.coli C 600. Transformants were isolated on LB plates containing 10 ⁇ g/m l ampicillin.
  • A/PR 8/34 3.3 kbp, 1.7 - 1.8 kbp pSVod 2.7 kbp, 0.6 - 0.7 kbp
  • PR81 is one of these five clones.
  • PR81 DNA was prepared as described by the method of Schedl et.al. (1978) Cell 14: 921-929 and was analyzed further by restriction enzyme digestion.
  • Plasmid PR84 p520 DNA (75 ⁇ g ) was digested with 25 units of PvuII for one hour at 37°C and subsequently with 20 units of NcoI for 2 hours at 37°C.
  • the digest was electrophoresed on a 1.2% low melting agarose gel containing 0.05 ⁇ g/ml EtBr.
  • the region including the two 1 kbp NcoI/PvuII fragments (one of them is a hsp 70 gene promoter fragment) was cut out of the gel.
  • the agarose piece and 10 volumes of TE buffer were heated to 65°C for 10 minutes, and the DNA was extracted subsequently with an equal volume of phenol. After one additional phenol/chloroform and 3 ether extractions, the DNA was precipitated two times with ethanol.
  • PR81 DNA (10 ⁇ g ) was digested with 15 units of HindIII for one hour at 37°C. Following purification by phenol extraction the DNA was incubated with several units of Klenow fragment and 0.5 mM of all four deoxyribonucleotide triphosphates for one hour at 6-7°C. After repurification, the DNA was digested with 10 units of NcoI for 90 minutes at 37°C and was repurified by phenol extraction.
  • the medium was removed from cell cultures at room temperature and the cultures were washed twice with 5 ml of phosphate-buffered saline (PBS). Subsequently one ml of the following preparations per dish were added. 1. 1 ml of Dulbecco's MEM with no serum, but containing DEAE Dextran (500 ⁇ g) and chloroquine (200 ⁇ g ).
  • Cell cultures were labelled by addition of 1 ml of this same low methionine medium per dish but in the presence of 35 S-Methionine (50 ⁇ Ci/ml, 968 Ci/mmol). Cultures were incubated for one hour at either 37°C or 42°C in the case of the heat-shock samples. After this period of labelling, the 35 S-Methionine medium was removed, the cultures washed three times with ice-cold PBS, and cells were scraped off in 1 ml of NET buffer (0.1 M NaCl, 0.01 M Tris-HCI pH 7.8, 0.001 M EDTA, 0.5% NP 40).
  • 35 S-Methionine 50 ⁇ Ci/ml, 968 Ci/mmol
  • cytoplasmic extracts were kept on ice, 5 ⁇ l of specific antibodies were added and the solutions were mixed gently at 4°C for two hours.
  • the antibodies used were the following: Normal rabbit antiserum, antiserum raised against purified E.coli ⁇ -galactosidase, and antiserum raised against PR8 influenza virus obtained from Dr. J. Shekel, MRC Laboratories, Mill Hill, London, U.K.
  • Anti HA antisera was preadsorbed for 30 minutes in NET buffer using cytoplasmic extracts of unlabelled COS cells prior to use for immune precipitation of labelled samples.
  • Antigen-antibody complexes were isolated by the addition of 50 ⁇ l of a 50% suspension of protein A-Sepharose ® (Pharmacia), continuing the shaking at 4°C for one hour, and recovering the bound complexes by centrifugation at 15,000 x g for one minute.
  • the complexes on protein A-Sepharose ® were washed and centrifuged four times using 1 ml of NET and three times using 1 ml of RIPA buffer (0.15 M NaCI, 0.05 M Tris-HCl pH 7.5, 0.02 M EDTA, 1 M Urea, 1% Triton x 100, 1% sodium deoxycholate).
  • Sepharose pellets were boiled for 3 minutes in 100 ⁇ l of sample buffer [Laemmli (1970) Nature 227: 680-685]. After centrifugation at 15,000 x g for 1 minute, the samples were subjected to electrophoresis on 10% polyacrylamide gels as indicated by Laemmli (1970) supra.
  • COS cells transfected with PR 84 were subjected to a period of heat-shock of five hours at 42°C and were subsequently labelled with 35 S-Methionine as described above but at 37°C for 14 hours. Subsequent immunoprecipitations with anti HA antisera indicated a larger quantity of HA product than in non heat-treated samples labelled under similar conditions.
  • expression vectors are provided which yield efficient transformation of eukaryotic hosts and high levels of expression within the host.
  • the transcriptional/translational control region of a 70 kdal heat-shock protein has been isolated and joined to a replication system derived from a simian virus.
  • a human influenza virus haemagglutinin gene was inserted into the resulting vector under the control of the said control region, and the resulting plasmid used to transfect a mammalian cell culture and apparently glycosylated haemagglutinin was produced.
  • a further Drosophila control element/HA gene construction was made to extend the utility of plasmid constructions such as PR84.
  • the HA gene being a viral gene is rather specialized as a model for any eucaryotic gene with its 3' end signals, and its RNA leader sequence, hence the PR84 construction contains both an HA RNA leader and a Drosophila RNA leader.
  • plasmid p629 was constructed. This plasmid contained in addition to the Drosophila heat-shock promoter the entire heat-shock RNA leader region, but was attached to a HA gene lacking the first 40 amino acid codons.
  • a plasmid p622b (a gift and personal communi cation of R. Voellmy) was employed; this plasmid contains a 450 bp XhoI/BglII fragment carrying the Drosophila hsp 70 promotor and RNA leader sequence (represented by the XhoI/Sau3A fragment of plasmid pRV15 described earlier).
  • p622b is a derivative of the COS cell vector pSVod. The sequence around the BglII site is given below:
  • Both DNAs were again phenol-extracted and purified as above and again dissolved in 20 ⁇ l of TE buffer. They were then digested individually with 15 units of BamHI (total volume 100 ul; 37°C for 2 hours). Following phenol extraction the DNAs were collected by EtOH precipitation and dissolved in 15 ⁇ l of TE buffer. Different sets of ligations containing 1 ⁇ l of 622b digest and 1-5 ⁇ l of PR 8/ms/34 digest in a total volume of 25 ⁇ l were carried out (enzyme: 1-2 ⁇ l, T4 DNA ligase. New England Biolabs) incubated overnight at 14°.
  • the ligase was inactivated at 65°C for 10 min and the samples were then digested with several units of ClaI and SalI (successively, using the conditions suggested by New England Biolabs) for 2 hrs at 37°C in volumes of 50 ⁇ l. The DNA mixtures were then used to transform E.coli MC1061.
  • probes used were: a) the 450 bp Xho-BglII fragment from p622b and b) the 1775 bp HindIII/BamHI gene fragment from PR8.4.76. Fragments had been separated in low melting agarose gels and were subsequently eluted from these gels. They were then labelled by nick translation (10 7 -10 8 cpm/ug; 10 6 cpm/filter). Eight (8) colonies that hybridized to both probes were selected, (see Figs. 12c and d) and DNA was prepared from them using the mini-DNA preparation procedure described previously.
  • the DNAs were digested with XhoI or with BglII/EcoRI and the digests were analyzed on 5% polyacrylamide gels.
  • XhoI was expected to yield two fragments with sizes of 450bp and of about 5 kb.
  • the BglII/RI digest should give fragments of 1130 bps, 1100 bps and of about 2.6 kb.
  • Three of the eight clones tested had the correct restriction patterns (see Figs. 12a and b).
  • Promoters of 70 kd heat-shock protein (hsp70) genes of human and Drosophila origin have been used to express a number of different genes in a heat-inducable fashion.
  • hsp70 heat-shock protein
  • Hybrid genes have been introduced into widely divergent cell types, in which they have been observed to direct the synthesis of encoded proteins in a fully heat-inducible manner.
  • the more prominent ones are those derived from various metallothionein genes and those from certain retroviruses, in particular mouse mammary tumor virus (MMTV).
  • MMTV mouse mammary tumor virus
  • the metallothionein gene promoters of both mouse and human origin have been described (17,18), but both the degree of inducibility at the protein level (1.3 to 2.5 fold using a Bovine Papilloma virus vector, ref. 33) as well as the practicality of induction in large cell cultures remain problematic.
  • the MMTV promoter inducible by glucocorticoid hormones (5), has been used to express, for example, the thymidine kinase gene in mouse cells (15), but this system shares the inconvenience of hormone induction in mass culture and adds the unknown risk of using a tumor virus sequence; in addition a limited host range of applications is available for this system.
  • the versatility of the heat shock promoter system may permit the expression of a given gene construct in a large number of different cells and may, therefore, obviate a search for a homologous promoter system for every specific expression problems.
  • Plasmid R81 plasmid A/PR 8/ms/34 (Mount Sinai) clone 4.76 which contains, in between a HindIII (5' end of the inserted gene) and a BamHI site, an influenza virus haemagglutinin gene, including short segments of 5' and 3 ' nontranslated sequences was obtained from G . Brownlee. This DNA was digested with BamHI, and cohesive ends were filled in with DNA polymerase fragment A. The DNA was digested further with HindIII, and the haemagglutinin gene fragment was isolated. Vector pSVOd (28) was cut with SalI, and following incubation with DNA polymerase fragment A, was digested further with HindIII, and then ligated to the above haemagglutinin gene fragment to give plasmid R81.
  • a 650 bp Sau3A fragment was isolated from plasmid 51 (16). This fragment which included about 400 bp of promoter sequence of a D.melanogaster hsp70 gene, a complete RNA leader region and the first seven hsp70 codons was inserted into the unique BamHI site at the 5' end of the lac operon segment in plasmid MC1403 (6) to give plasmid RV15. Plasmid RV15 DNA was digested with SamI and SalI, and a 7 kb long fragment, including the above- mentioned 650 bp hsp70 gene segment and the lac operon segment, was isolated.
  • Plasmid SVOd DNA was digested with BamHI, and the cohesive ends were filled in by DNA polymerase fragment A. The DNA was then digested with SalI and ligated to the purified 7 kb hybrid gene fragment to give plasmid 520.
  • Plasmid R84 p520 was digested with Ncol and Pvull, and a fragment of about 1 kb was isolated which included part of the pSVOd SV40 origin of replication segment, 400 bp of D.melanogaster hsp70 gene promoter and 65 bp of hsp70 gene RNA leader sequences. Plasmid R81 was digested with HindIII , ends filled as above , and the DNA was then further digested with Ncol, which cuts within the SV 40 origin of replication sequence, and then ligated to the isolated p520 fragment. Plasmid 522-lys: p522 DNA was digested with
  • Plasmid 17-lys DNA of plasmid 17 which contains a fragment of a human hsp70 gene (see map in ref .42) was digested with BgllI and Sad and then treated with DNA polymerase fragment A. A 1.2 kb long fragment containing a functional heat shock promoter was isolated. This fragment was ligated to a 2.8 kb long pSP65 (29) HindI-II/PvuII fragment that had been blunt-ended as above. The resulting construct p17-65 was then digested with EcoRI and HindIII, and the larger of the two restriction fragments (4 kb) was isolated.
  • Plasmid 522-lys was digested with EcoRl and partially with Hind/III and a 1.5 kb fragment including the lysozyme gene and pSVOd vector sequences (containing the origin of replication region) was isolated and ligated to the above p17-65 fragment.
  • Plasmid pl7hGH dhfr pl7 DNA was digested with EcoRI, blunt ended, and digested further with HindIII.
  • a 3.25 kb fragment containing 3.15 kb of 5' nontranscribed sequence and about 110 bp of RNA leader sequence of a human hsp70 gene was isolated and ligated to a 2.8 kb Pvull/- HindIII fragment from pSP65.
  • DNA of the resulting construct was digested with Bglll and partially with BamHl, and then religated to give p17Jo (the latter step eliminated all but a small region of the 5' nontranscribed sequence of the hsp70 gene).
  • a human growth hormone cDNA gene (obtained from Celltech Ltd., Slough, G.B.) was subcloned into the unique BamHl site of pSP65 to give phGH.
  • This plasmid was then digested with HindIII and Sacl (both sites are from the polylinker), and the hGH fragment was isolated and inserted in between the same sites of p17Jo to give p17hGH.
  • Plasmid p17hGH6 was derived by digesting p17hGH with NcoI and XbaI followed by treatment with DNA polymerase fragment A and religation (this step deletes 6 bp between the hGH and hsp gene, sequences). Finally, p17hGH6 was digested with EcoRI and
  • COS 1 African Green Monkey kidney, human Wish, and 293 cells were grown in Dulbecco's modified Eagle's medium (Gibco), Chinese hamster ovary (CHO) cells in Ham's nutrient mixture F12 (KC Biologicals) and Drosophila melanogaster S3 cells in Schneider's Drosophila medium (KC Biologicals). All cells were grown in the presence of 10% calf serum. CaCl 2 - or DEAE-dextran-based procedures (12, see also ref. 1) were used to transfect the different cell types used in this study (10 - 20 g DNA per 10 cm dish). Prior to expression analysis, transfected COS 1 cells were incubated for two days at 37°C to permit amplification of the introduced plasmids.
  • Cell extracts were prepared by repeated pipeting in buffer containing 0.5% NP40. Concentrations of hybrid gene products were measured by a classical ELISA procedure (43) employing rabbit and mouse anti-E.coli ⁇ -galactosidase antibodies, and peroxidase-conjugated rabbit anti-mouse Ig antibodies, the latter from DAKO Co.
  • the normal medium was removed from COS 1 or CHO cell cultures and replaced by 2 ml/10 cm dish of methionine-free medium containing 200 Ci opf 35 S-methionine. Following overnight incubation at 37°C, the cells were harvested and lysed. Immunoprecipitation and electrophoresis of the precipitated gene products were carried out according to standard procedures (14,22). Heat-treated or untreated Xenopus oocytes were labeled by overnight incubation at 21°C in 200 1 OR2 buffer per 10 oocytes ( 41 ) containing 100 Ci of 35 S-methionine . Rabbi t anti-haemagglutinin antiserum was provided by M. Thibon (Pasteur Institute, Paris), and rabbit anti-human pituitary growth hormone serum was obtained from DAKO Co.
  • Fig. 1. presents the DNA constructs with the selected genes under the expression control of heat shock promoter elements derived from D.melanogaster and human cells.
  • heat shock promoter elements derived from D.melanogaster and human cells.
  • restriction sites occuring early in the hsp70-coding sequences have been exploited to make hybrid genes that encode fusion proteins.
  • Sufficient coding sequence is retained in the hybrid genes to ensure enzymatic activity of ⁇ -galactosidase.
  • the D.melanogaster heat-shock gene fragment is fused at amino acid 7 of its coding sequence to the truncated 3-galactosidase gene to give plasmid 522 (23), and the human gene fragment at amino acid 124 of its respective coding sequence, yielding plasmid 173 (42).
  • a human influenza virus haemagglutinin gene was placed under D.melanogaster heat-shock control in pR84.
  • a chicken lysozyme gene has been incorporated into constructs using either D.melanogaster (p522-lys) or human (pl7-lys) heat-shock gene promoters and a human growth hormone gene into a construct with a human heat-shock gene promoter (p17hGH dhfr).
  • the vector sequences present in p522, pR84, p522-lys, p17-lys and p17hGH dhfr include a Simian Virus 40 (SV40) origin of replication fragment, permitting amplification of the plasmid constructs in COS monkey cells (11,28).
  • SV40 Simian Virus 40
  • DNA constructs to be tested for heat-shock expression control were microinjected into Xenopus oocyte nuclei. Ten min. after injection, oocytes were subjected to a heat shock of 90 min. at 36°C, followed by incubation at 21°C for 12 hrs. in the presence, in the case of the immunoprecipitation experiments described below, of 35 S-labelled methionine. Measurements of gene expression under heat--shock control of the ⁇ -galactosidase constructs have been reported before (1,23,30,42). In those experiments, levels of ⁇ -galactosidase had been quantified by means of a standard colorimetric activity assay.
  • Heat treatment of hybrid gene-containing oocytes resulted in about a 30-fold increase in the level of ⁇ -galactosidase produced.
  • constructs containing a D.melanogaster hsp70 gene promoter we have been able to demonstrate (30) that the accumulation of ⁇ -galactosidase in heat-shocked oocytes is dependent on a functional heat shock gene promoter and cannot be explained merely as a consequence of preferential translation during (27,31,-39), or following, heat shock of hybrid gene products that may have arisen from read-through transcription.
  • the upstream promoter element required for heat-regulated transcription in Xenopus as well as in COS monkey cells lies 48 to 62 bp upstream from the start of transcription site of the hsp70 gene (32,34).
  • Our own studies with hsp70 - ⁇ -galactosidase hybrid genes with promoter segments of different lengths have indicated that 67 bp but not 50 bp of promoter sequence are sufficient for high level heat-regulated synthesis of ⁇ -galactosidase in Xenopus oocytes (30; Ananthan and Voellmy, unpublished results).
  • the efficient, heat-induced synthesis of the protein products of hsp hybrid genes such as the one in p522 is under the control of the heat-shock transcription signal.
  • a gene lacking this signal is essentially inactive at heat shock and control temperature. Identical results were obtained from analogous experiments with COS monkey cells (1).
  • the ⁇ -galactosidase hybrid genes encode hsp70 - ⁇ -galactosidase fusion proteins.
  • the RNA leader regions, and the sequences around and including the translation initiation sites are from heat-shock genes.
  • Such fusion proteins will not be acceptable gene products for most practical applications; the synthesis of the authentic product of a gene of interest will be required. For this reason all constructs with eucaryotic protein genes had been prepared by joining heat-shock gene fragments, and protein-coding genes to be expressed, within their RNA leader regions.
  • Fig. 2 Expression experiments with such constructs are shown in Fig. 2.
  • the presence of labelled protein products was demonstrated by immunoprecipitation of Xenopus oocyte extracts or suspension media, using either anti-haemagglutinin, anti-chicken lysozyme or anti-human growth hormone sera. Immunoprecipitated proteins were analyzed by electrophoresis on SDS-polyacrylamide gels. Extracts from hybrid gene-containing oocytes are analyzed in Fig. 2!a: lysozyme antibodies precipitate a 14.3 kd polypeptide which coelectrophoreses with a chicken lysozyme standard.
  • Both p17-lys and p522-lys only give rise to significant synthesis of lysozyme protein after a heat shock (lanes 4 and 6); plasmid-injected oocytes that have not sustained a heat shock produce little or no lysozyme (lanes 3 and 5).
  • Injection of pR84 and immunoprecipitation with anti-haemagglutinin antibody reveals the presence of a polypeptide of about 75 kd, the expected molecular weight of the unprocessed influenza virus haemagglutinin (lanes 1 and 2). Again, detectable amounts of gene product are only made in heat-treated oocytes.
  • Anti-human growth hormone serum specifically precipitates a protein of 21 kd in heat-treated but not in untreated oocytes containing p17hGH dhfr DNA (lanes 14 and 15).
  • the molecular weight of the recombinant gene product is identical to that of human growth hormone.
  • Fig. 2b indicates the results obtained with suspension media from the same experiments; it can be seen that lysozyme produced under heteroplogous heat-shock control, either Drosophila or human, is secreted by the oocytes into the suspension medium (lanes 1 to 4). The same holds for human growth hormone ( lanes 5 and 6 ). It may be worth noting that human growth hormone represents a substantial fraction of all newly synthesized proteins secreted into the medium. Aliquots of suspension medium have been analyzed, without immunoprecipitation, in lanes 7 (heat shock) and 8 (control).
  • heat-shock promoter activity does not require the presence of a complete heat shock RNA leader region
  • the haemagglutinin construct contains only about 65 bp of the 250 bp long D .melanogaster hsp70 RNA leader.
  • the presence of foreign non-protein-coding or protein-coding sequences downstream from the hsp RNA leader segment does not appear to affect adversely either promoter function or hybrid product translation: all the different constructs yield significant amounts of protein products. Since neither the D.melanogaster nor the human hsp70 RNA leader segment contains an ATG codon, the translation initiation signals must have been provided by the different protein-coding genes.
  • Lanes 5 to 16 show a similar experiment with cells transfected with p522-lys, p17-lys and p17hGH dhfr.
  • the cells were labeled as indicated in Materials and Methods, and both cell extracts (not shown) and growth media (lanes 5- 16) were analyzed as above with lysozyme- and growth hormonespecific antisera.
  • a labeled protein with a molecular weight identical to that of a lysozyme standard (14.3 kd) is clearly visible in media samples from cells transfected with p522-lys or p17-lys that have undergone a heat shock (lanes 5 to 8). This protein was absent from samples derived from p522-transfected cells (lanes 9 and 10).
  • Example 3 we have described the use of promoter elements derived from both Drosphila and human hsp70 genes for the expression of a number of genes in both homologous and heterologous cell types.
  • both Drosophila and human heat-shock gene constructions can direct the synthesis of, for instance, lysozyme protein in the heterologous Xenopus oocyte system; in addition, both expression units are strictly heat shock-dependent in this system and give rise to comparable levels of lysozyme synthesis.
  • This heterologous system has, in addition, been shown to permit the secretion out of the oocyte of lysozyme and human growth hormone produced after a period of heat shock. Similar studies indicate that a wide range of cell types can be used in an analogous fashion.
  • the system should be acceptable from the points of view of efficiency of gene expression, as well as concerning safety considerations of host cells or vector systems. Finally, since there are frequently problems with cell growth and expansion when such cells are producing large quantities of viral antigens or enzymes, both of which may prove toxic to the cells, a fully inducible expression system is of great advantage. Such an inducible system must, for large-scale application, permit the induction of gene expression by practicable procedures, and these irrespective of the cell culture system employed.
  • SV40, adenovirus, Rous Sarcoma Virus (RSV), mouse mammary tumor virus (MMTV) and metalloghionein promoters appear to fulfill some of the above-mentioned criteria for a generally applicable and acceptable expression system.
  • SV40, adenovirus, and RSV promoters are constitutive, metallothionin promoters appear to be barely inducible (33), and the MMTV promoter although inducible is so in a less than totally practicable fashion.
  • MMTV, RSV, SV40 and adenovirus elements are derived from tumor viruses, and in addition not all of these promoters, or their habitual vector systems, are functional in a wide variety of cell types.

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Abstract

Des éléments de régulation génétique par choc thermique provenant de différentes cellules eucaryotiques sont utilisés pour exprimer des produits génétiques compétitifs dans les mêmes cellules eucaryotiques ou dans des cellules similaires ainsi que dans des cellules hôtes procaryotiques. Les éléments de régulation peuvent être unis à un système de réplication eucaryotique approprié pour former un vecteur d'expression, ou bien ils peuvent être unis au gène à l'étude et introduits directement dans le génome de la cellule hôte.
PCT/US1987/000805 1986-04-04 1987-04-03 Procedes et compositions d'expression de produits genetiques eucaryotiques competitifs WO1987005935A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232845A2 (fr) * 1986-02-06 1987-08-19 The General Hospital Corporation Systèmes d'amplification et de choc thermique inductibles
US5521084A (en) * 1992-11-10 1996-05-28 Biostar, Inc. Bovine heat shock promoter and uses thereof
EP0781848A3 (fr) * 1995-12-28 1997-10-01 Fujirebio Kk Séquence d'ADN fusionnée, protéine de fusion exprimée par ladite séquence d'ADN fusionnée et méthode pour l'expression de la protéine de fusion
WO1998006864A2 (fr) * 1996-08-15 1998-02-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Regulation spatiale et temporelle de l'expression genique au moyen d'un promoteur proteique du stress associe a une source de chaleur locale
US5877398A (en) * 1993-01-29 1999-03-02 University Of British Columbia Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology

Families Citing this family (2)

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ATE67787T1 (de) * 1986-10-15 1991-10-15 Battelle Memorial Institute Verfahren zur herstellung von proteinen mittels induzierbarer expressionssysteme in in-vivovermehrten, genetisch modifizierten eukaryotischen wirtszellen.
PT90657B (pt) * 1988-05-27 1995-03-01 Ortho Pharma Corp Processo para a preparacao de peptidos que bloqueiam a ligacao de hiv-1 a proteina cd4 receptora

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EP0118393A2 (fr) * 1983-02-07 1984-09-12 Battelle Memorial Institute Méthodes et compositions pour l'expression des produits des gènes compétents eucaryotes
EP0159884A2 (fr) * 1984-04-13 1985-10-30 Lubrizol Genetics Inc. Promoteur et gène de choc thermique

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EP0118393A2 (fr) * 1983-02-07 1984-09-12 Battelle Memorial Institute Méthodes et compositions pour l'expression des produits des gènes compétents eucaryotes
EP0159884A2 (fr) * 1984-04-13 1985-10-30 Lubrizol Genetics Inc. Promoteur et gène de choc thermique

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CHEMICAL ABSTRACTS, Volume 101, No. 25, 17 December 1984, (Columbus, Ohio, US), R.E. KINGSTON et al.: "Regulation of Gene Expression by the Adenoviral E1A Region and by C-Myc", see page 198, Abstract 223944r, Cancer Cells 1984, 2 (Oncog. Viral Genes), 539-44 *
CHEMICAL ABSTRACTS, Volume 102, No. 19, 13 May 1985, (Columbus, Ohio, US), R. Lawson et al.: "Expression of Heat Shock-beta-Galactosidase Hybrid Genes in Cultured Drosophila Cells", see page 155, Abstract 161423e, MGG, Mol. Gen. Genet. 1984, 198 (1), 116-24 (cited in the application) *
CHEMICAL ABSTRACTS, Volume 106, No. 19, 11 May 1987, (Columbus, Ohio, US), M. DREANO et al.: "High-Level, Heat-Regulated Synthesis of Proteins in Eukaryotic Cells", see page 183, Abstract 150662p, Gene 1986, 49 (1), 1-8 *
Proceedings of the National Academy of Sciences of the USA, Volume 83, No. 15, 1986, (Washington, US), F.M. WURM et al.: "Inducible Overproduction of the Mouse C-Myc Protein in Mammalian Cells", pages 5414-5418 see Abstract; page 5415, column 1, lines 1-19; page 5417, column 2, line 4 - page 5418, column 1, line 23 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232845A2 (fr) * 1986-02-06 1987-08-19 The General Hospital Corporation Systèmes d'amplification et de choc thermique inductibles
EP0232845A3 (fr) * 1986-02-06 1988-12-14 The General Hospital Corporation Systèmes d'amplification et de choc thermique inductibles
US5521084A (en) * 1992-11-10 1996-05-28 Biostar, Inc. Bovine heat shock promoter and uses thereof
US5733745A (en) * 1992-11-10 1998-03-31 Biostar Inc. Bovine heat shock promoter and uses thereof
US5981224A (en) * 1992-11-10 1999-11-09 Biostar Inc. Bovine heat shock promoter and uses thereof
US5877398A (en) * 1993-01-29 1999-03-02 University Of British Columbia Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology
EP0781848A3 (fr) * 1995-12-28 1997-10-01 Fujirebio Kk Séquence d'ADN fusionnée, protéine de fusion exprimée par ladite séquence d'ADN fusionnée et méthode pour l'expression de la protéine de fusion
US6602689B1 (en) 1995-12-28 2003-08-05 Fujirebio Inc. Fused DNA sequence, fused protein expressed from said fused DNA sequence and method for expressing said fused protein
US7067631B2 (en) 1995-12-28 2006-06-27 Fujirebio Inc. (Fujirebio Kabushiki Kaisha) Fused DNA sequence, fused protein expressed from said fused DNA sequence and method for expressing said fused protein
WO1998006864A2 (fr) * 1996-08-15 1998-02-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Regulation spatiale et temporelle de l'expression genique au moyen d'un promoteur proteique du stress associe a une source de chaleur locale
WO1998006864A3 (fr) * 1996-08-15 1998-05-14 Chrit Moonen Regulation spatiale et temporelle de l'expression genique au moyen d'un promoteur proteique du stress associe a une source de chaleur locale
US7186698B2 (en) 1996-08-15 2007-03-06 The United States Of America As Represented By The Department Of Health And Human Services Spatial and temporal control of gene expression using a heat shock protein promoter in combination with local heat

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