WO1988001296A1 - Essai d'evaluation de l'activite de promoteurs - Google Patents

Essai d'evaluation de l'activite de promoteurs Download PDF

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WO1988001296A1
WO1988001296A1 PCT/US1987/001745 US8701745W WO8801296A1 WO 1988001296 A1 WO1988001296 A1 WO 1988001296A1 US 8701745 W US8701745 W US 8701745W WO 8801296 A1 WO8801296 A1 WO 8801296A1
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cell
plasmid
promoter
host cell
sequence
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PCT/US1987/001745
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Richard F. Selden
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The General Hospital Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • 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

  • This invention relates to a recombinant DNA technique for evaluating the strength and efficiency of promoters in a transient assay for gene expression.
  • plasmid which contains an origin of replication sufficient to permit propagation of the plasmid in a bacterial cell, and a second origin of replication sufficient to permit propagation of the plasmid in CHO, L, or cos-1 eukaryotic cells.
  • the plasmid contains the Escherichia coli gene for beta-galactosidase. The gene encoding beta-galactosidase is normally not expressed when the plasmid is introduced into a eukaryotic cell because there is no promoter present to direct its transcription.
  • a DNA fragment whose promoter activity is to be investigated is introduced by insertion into the plasmid adjacent to but preceeding the gene for beta-galactosidase. If the DNA fragment contains a functional promoter the plasmid will, upon introduction into a eukaryotic cell, transiently direct the expression of betagalactosidase.
  • the expression of beta-galactosidase is determined by treating the eukaryotic cells with toluene followed by the addition of a chromogenic substrate.
  • Lopata M. et al., Nucleic Acids Res. 12(14): 5707-5717(1984) have described an assay for transient expression of DNA introduced into mouse L cells.
  • the bacterial CAT gene is not expressed unless a promoter is provided and inserted into the plasmid adjacent to and preceeding the CAT gene. The expression of CAT is therefore dependent upon the presence of a functional and efficient promoter.
  • the assay for CAT enzymatic activity is one of transient expression (the cat plasmid can be stably expressed in cells by co-transfection). At various times post transfection the cells are washed and vigorously disrupted in order to release CAT activity from them. To quantitate the CAT activity, the cell extract is incubated in the presence of 14 C-chloramphenicol. The formation of the acetylated derivative of chloramphenicol is then detected and quantitated by chromatography. More efficient systems, especially ones that are easy to manipulate, would be desirable for evaluating and testing promoters.
  • the invention there is provided a method of ascertaining whether a particular fragment of DNA contains sequences which are sufficient to serve as a promoter for transcription.
  • the invention also provides a means for quantitatively comparing the relative efficiencies of different promoters. Since the invention can distinguish between sequences of DNA which carry promoters and such sequences that do not carry promoters, the invention is capable of detecting any change in a DNA sequence which would create a promoter in a sequence that formerly had lacked a promoter. Thus, the invention is capable of recognizing novel promoters which had heretofore not existed and have arisen as by mutation or by other means.
  • the invention comprises a method of evaluating a promoter capable of functioning in a given host cell which comprises: providing, on a plasmid vector substantially incapable of replicating in the host cell, a first genetic sequence to be evaluated for promoter activity, operably linked to a genetic sequence which codes for a reporter molecule secretable from the host cell without the lysis of the host, measuring the amount of reportable molecule transiently expressed by the vector and secreted from the cell, and evaluating the strength of the promoter as a function of such amount.
  • the invention further comprises a method of evaluating a promoter capable of functioning in a given host cell which comprises: providing, on a plasmid vector capable of being transiently expressed in the host cell, a first genetic sequence to be evaluated for promoter activity, operably linked to a genetic sequence which codes for a reporter molecule secretable from the host cell without the lysis of the host, measuring the amount of reportable molecule transiently expressed by the vector and secreted from the cell, and evaluating the strength of the promoter as a function of such amount.
  • the invention further comprises a plasmid vector usable in a first host cell, such vector be.ing suitable for assessing whether a DNA sequence, when inserted into the vector, contains a promoter functional in the first host cell, which comprises: a) a replicon sequence sufficient to permit the propagation of the vector in a second host cell different from the first cell host, the replicon sequence being insufficient to permit replication of the vector in the first host cell; b) a genetic sequence encoding a selectable marker; c) a reporter gene sequence lacking a promoter, which is therefore substantially incapable of being expressed in the first host cell unless a DNA sequence containing a promoter is inserted into the plasmid vector in such manner as to operably link the DNA sequence to the reporter gene sequence.
  • a plasmid vector usable in a first host cell, such vector be.ing suitable for assessing whether a DNA sequence, when inserted into the vector, contains a promoter functional in the first host cell, which comprises: a) a re
  • a plasmid as described capable of replication in a second host cell, preferably the bacterium E. coli.
  • the plasmid contains four elements: (1) an origin of replication sufficient to permit the propagation of the plasmid in a second host cell; (2) a selectable marker sequence, preferably a gene whose expression confers an antibiotic resistance to the second host cell, sufficient to enable the maintenance of the plasmid within the host cell and to facilitate the manipulation of the plasmid into new host cells; (3) a DNA sequence which is sufficient, if translated, to encode a secretable polypeptide that is traceable, assayable, or indentifiable; (4) a sequence of DNA containing one or multiple restriction endonuclease cleavage sites and which is positioned adjacent to and upstream from the DNA sequence which encodes the secretable polypeptide.
  • the DNA sequence which contains the restriction endonuclease cleavage site is devoid of any promoter activity so that when introduced into a cell the plasmid fails to direct the synthesis and expression of the secretable polypeptide.
  • that fragment is purified by means known in the art and inserted into the plasmid within the upstream sequence of DNA which encodes the one or multiple restriction endonuclease cleavage sites.
  • the covalently closed circular plasmid molecule is then introduced into a first cell and the presence and level of the secretable protein is monitored by enzyme assay, immunoassay, or by other means.
  • the invention therefore relates to a method which involves the use of two different types of host cells.
  • the first type of host cell is one which permits the replication, propagation, and amplification of the plasmid molecule. Promoter expression is not monitored in this host cell but, rather , is assayed by introducing the plasmid molecule into a second host cell type. Since a means for amplifying the plasmid molecule has already been provided (i.e., growth within the first host cell), it is not necessary that the plasmid molecule be able to replicate within the second host cell type. The invention merely requires that the second host cell type be capable of adsorbing the plasmid DNA molecule and transcribing and translating its genetic information.
  • the ability to separate the replication of the plasmid from the expression of the plasmid-borne genetic information allows one to evaluate a promoter in a given host cell even if no known means of replicating the plasmid within that host cell exists.
  • the invention is especially useful in evaluating promoter expression in novel or unique host cell types.
  • FIG. 1 shows plasmid pOGH, a derivative of plasmid pUCl2.
  • the plasmid is 4,817 base pairs in size.
  • the plasmid contains a 2.1 kb BamHI-EcoRI fragment containing the structural sequences for human growth hormone (HGH). These structural sequences contain the four introns which are normally found in the human hormone gene (depicted as unshaded regions within the HGH structural sequences).
  • the 2.1kb fragment has been inserted into the BamHI-EcoRI sites of pUCl2.
  • Immediately adjacent and preceeding the HGH structural gene are DNA sequences which contain restriction sites for the endonucleases Hindlll, Sail, Hindi, and the Xbal.
  • FIG. 2 shows the time course of HGH expression.
  • the figure shows the expression of HGH in culture medium when the mouse metalliothionein-I promoter has been inserted in front of the HGH gene. Media was not changed during the course of the experiment and therefore HGH levels shown are total accumulation in the extracellular culture media.
  • Figure 3 shows the relationship between amount of transfected pXGH5 and pTKGH DNA and the level of HGH expression.
  • Figure 4 shows the effect of increasing DNA concentration on the concentration of Human Growth Hormone mRNA.
  • Figures 5 and 6 show the utility of the HGH transient expression system for the study of regulation of gene expression.
  • Figure 5 shows the effect of SV40 enhancer sequences on the TK promoter of herpes virus.
  • Figure 6 shows the effects of zinc on the ability of the metalothionein promoter to direct the expression of HGH.
  • plasmid refers to a closed covalently circular extrachromosomal DNA molecule.
  • a plasmid may contain .a replicon which is a sequence of DNA sufficient to enable the replication of a plasmid in a host cell. It is a common feature of replicons that they can be functional - enable plasmid replication - in some host cells but not in other host cells of unrelated species.
  • a plasmid may also contain a selectable marker sequence. Such a sequence is a DNA sequence which is sufficient to encode a selectable marker.
  • a selectable marker is a diffusible product produced by a cell which enables one to identify and preferentially maintain cells which produce said product. Examples of selectable markers useful in the invention are proteins conferring cellular resistance to ampicillin, chloramphenicol, and tetracycline.
  • promoter refers to a region of regulatory DNA sequences which are recognized by a cell as a site adjacent to which to begin the initiation of the transcription of proximal DNA into RNA.
  • a promoter when introduced into a microorganism, is introduced as a DNA sequence present on a plasmid or linear DNA fragment.
  • Proximal DNA refers to DNA on the plasmid which is located proximal to said pro- moter.
  • a promoter is said to be operably linked to a sequence of proximal DNA if upon introduction into a host cell the promoter leads to the transcription of the proximal DNA sequence into RNA.
  • promoters from eukaryotic cells or from viruses which infect eukaryotic cells are the promoter of the mouse metallothionein I gene (Hamer, D. et al., J. Mol. Appl. Gen.1:273-288 (1982)); the TK promoter of herpes virus (McKnight, S., Cell 31:355-365 (1982)); and the SV40 early promoter (Benoist, C. et al., Nature (London) 290:304-310 (1981)).
  • promoters from prokaryotic cells or from viruses which infect prokaryotic cells, which can be evaluated are the E.
  • a sequence is said to be a functional promoter (i.e. to have promoter activity) if, when introduced into a host cell, that sequence can be recognized by the host cell's biosynthetic machinery and lead to to the formation of RNA complementary to the DNA proximal to said promoter site.
  • Transient expression refers to the capacity of a host cell to direct the transcription and translation of genetic sequences. This transcription and translation occurs soon after the genetic sequences are introduced into the host cell. Such expression can occur even when a plasmid which carries a reporter gene sequence operably linked to a functional promoter is introduced into a host cell incapable of replicating or propagating the plasmid.
  • a host cell is any microorganism which may be used either to replicate a plasmid molecule (second host cell) or to evaluate promoter expression (first host cell).
  • useful host cells are bacteria such as Escherichia coli. Bacillus subtilis, yeast such as Saccharomyces cerevisiae, and animal cells such as L cells, AtT-20 cells, XC cells, GC cells, GH 4 cells, JEG cells, CV-1 cells, or primary pituitary cells.
  • lysis refers to an active disruption of cellular membranes in order to effectuate the release of intracellular materials. Lysis requires the active mechanical or chemical disruption of cells.
  • secretable refers to the capacity of the reporter molecule to be transported from within the organism to the external culture medium in such manner as to occur without lysis or loss of viability of said organism.
  • secretable as used in this invention is further limited to reporter molecules which are actually secreted from the host system or cell under the conditions which are required for cell maintenance and viability. Thus, the same reporter molecule may be termed as secretable or not secretable depending upon whether it is actually transported from within a particular organism to the external culture medium in the course of the routine maintenance of the organism.
  • reporter molecule as used herein is a polypeptide whose presence can be detected, assayed or identified.
  • secretable reporter molecules are the human growth hormone, insulin, and somatostatin.
  • the genetic sequence which encodes the reporter molecule is referred to as a reporter gene sequence.
  • the level and extent of synthesis of the reporter molecule is a function and measure of the strength of the promoter which is permitting and directing its transient expression. Measurement of the level of the reporter molecule is therefore correlatable to the strength of the promoter responsible for the expression of the reporter molecule.
  • a "naturally occurring analogue" of a human hormone refers to a molecule which is substantially similar to a human hormone either in amino acid sequence, DNA sequence, or physiological activity, and which can be found to be produced in man.
  • Examples of a naturally occurring analogue of human growth hormone are the placental lactogen hormone produced by all humans, and the mutant human growth hormone produced by individuals who suffer from short stature syndrome where the hormone is chemically or physiologically distinguishable from normal human growth hormone.
  • an example of a naturally occurring analogue of human insulin is the insulin hormone produced by diabetic humans where the hormone is chemically or physiologically distinguishable from normal human insulin.
  • Naturally occurring analogues of human hormones may be encoded by genetic sequences having intervening, non- translated regions.
  • Naturally, occuring analogues of human growth hormone may also be produced as a precursor molecule which is naturally processed into a bio- active hormone.
  • the invention also makes use of a plasmid suitable for measuring the activity of promoters in a microorganism and a procedure for making such measurements.
  • a plasmid suitable for measuring the activity of promoters in a microorganism and a procedure for making such measurements.
  • a wide variety of usable plasmids are known and commonly used in the art. Cohen, S. et al, U.S. Patent 4,468,464, discloses examples of DNA plasmids.
  • the plasmid of the invention also contains genetic sequences which encode for a secretable reporter molecule. These sequences are adjacent to and upstream from a region of the plasmid known as a polylinker region which contains one or more endonuclease restriction cleavage sites.
  • a region is said to be "upstream" from a second region if it precedes the second region (i.e., lies 3' to the second region).
  • These sites may be recognized by any restriction endonuclease such as, for example, EcoRI. Common restriction enzymes and the DNA recognition sequences which these enzymes recognize are disclosed in Molecular Cloning, A Laboratory Manual, Maniatis, T. et al., Cold Spring Harbor Laboratory, pages 100-101, 1982. Although many combina tions of endonuclease restriction cleavage sequence recognition sites are possible, it is preferable to have sequence recognition sites for the endonucleases: Ndel, Hindlll, Sail, Hindi, Xbal, or BamHI. It is preferable that the chosen endonuclease recognition site be present only once in the plasmid.
  • a fragment of DNA, from any source whatsoever, may be purified and inserted into the plasmid at any of the restriction endonuclease cleavage sites which precede the reporter gene sequences.
  • the DNA to be inserted may be from prokaryotes, such as Escherichia coli. Bacillus subtilis, Streptomyces lividans or the like; from viruses which infect prokaryotes, such as lambda, T 4 , C31 or the like; from eukaryotes, such as Saccharomyes cerevisiae, Mus musculus. Homo sapiens, or the like; or from viruses which infect eukaryotes such as Herpes Simplex, SV40, or the like.
  • the termini of both molecules will be complementary), or by treatment of the termini so as to remove any overlapping single stranded regions which may have been produced by endonuclease cleavage (the "blunt" end termini may then be ligated).
  • Techniques used to produce DNA molecules having termini which may be ligated are well known in the art and are described in Maniatis, T. et al. It will be further understood that all such plasmid manipulations and constructions may give rise to plasmids in which the inserted sequences are present as multimers or in an undesirable orientation. The correct orientation and construction of a plasmid is determined by agarose gel electrophoresis as described in Maniatis, T. et al., or by other means that are well known to those skilled in the art of recombinant DNA.
  • coli will take up plasmid DNA if such cells have been previously incubated in a solution of calcium chloride, and, if subsequent to their exposure to the calcium chloride and plasmid DNA, the cells are incubated for a brief period to produce a heat shock.
  • Lopata, M. et al. disclose a method for introducing plasmid DNA into mouse L cells.
  • plasmid DNA will be taken up by the mouse L cells if the cells are incubated in the presence of plasmid and DEAE-Dextran, and subsequently exposed to DMSO.
  • the secretable reporter molecule It is possible to monitor for the expression of the secretable reporter molecule at any time after the plasmid has been introduced into the second host cell. If the plasmid contains a functional promoter sequence capable of directing the expression of the secretable reporter molecule, then the secretable reporter molecule will be synthesized and secreted into the external culture medium. Importantly, the presence of the reporter molecule is monitored by assaying the culture medium without requiring the destruction or rupture of the microorganism host cells. An aliquot of culture medium is evaluated by any means capable of detecting the reporter molecule. Such means may either be, for example, by immunoassay or by other means known to the art. The rate of. accumulation of the reporter molecule in the external culture medium is therefore an indication of the existence and strength of any promoter which was present on the fragment cloned adjacent and preceding the reporter gene sequences on the plasmid.
  • reporter gene sequences are relatively stable (i.e. not completely degraded within the time span of the experiment).
  • the reporter gene sequences must encode a reporter gene messenger RNA which is relatively stable within the host cell.
  • the reporter gene protein translated from the messenger RNA must itself be stable.
  • the DNA sequences must be relatively stable (i.e. not completely degraded) during the time span of the investigation.
  • the messenger RNA transcribed from the reporter gene sequences must also be relatively stable during the time span of the investigation.
  • the reporter gene molecule itself must be relatively stable during the time span of the investigation.
  • the reporter gene molecule must be physically excreted from the cell into the external culture medium.
  • Plasmid mediated gene expression is of great concern in the biotechnology, pharmaceutical and chemical industries. Such expression is used for the production of immunological agents, enzymes, therapeutic agents, amino acids and the like. Because these products are often expensive and difficult to produce, it is important to these industries that the plasmid mediated expression be optimal.
  • the present invention permits one to ascertain the promoter strength which drives gene expression. Moreover, it permits a comparison to be made of different promoters so that one may determine which promoter is optimal. The invention is therefore of significant value and utility to these industries.
  • This plasmid was incubated in the presence of the endonucleases BamHI and EcoRI to produce two linear fragments of plasmid pUC12.
  • One linear fragment so produced known as the plasmid fragment contains all sequences needed for the functions of replication, propagation, maintenance and selection of pUC12.
  • the second fragment produced, known as the BamHI-EcoRI fragment was not required for any of these functions. DeNoto et al. (Nucleic Acid Res.
  • the fragment was capable of being inserted into the plasmid in only one orientation (i.e. such that the beginning of the HGH gene was adjacent to the BamHI site and the end of the HGH gene was adjacent to the EcoRI site of the plasmid.)
  • the reaction mixture was then incubated in the presence of competent bacterial cells, preferably E. coli, in order to effectuate the transformation of such cells with a recombinant plasmid which now contained the human growth hormone gene fragment and the plasmid fragment of plasmid pUC12.
  • Plasmid pOGH was 4.8 kb in size and lacked any known eukaryotic promoter sequences.
  • Plasmid pOGH was designed to readily accept and evaluate DNA sequences for promoter activity when such sequences were inserted into either the Nde 1, Hind ⁇ III, Sail, HindII, Xbal, BamHI endonuclease restriction cleavage sites.
  • Figure 1 is a representation of plasmid pOGH, showing the location of restriction sites. The genetic sequences associated with human growth hormone are shown in double lines. Shaded areas are introns.
  • E. coli strain DKl containing plasmid pOGH was deposited on August 7, 1986, with the American Type Culture Collection under the designation ATCC No. 67180.
  • Bell, G.F. et al. (Nature, 282 : 525 (1979)) have disclosed the cloning of the gene encoding the human insulin hormone. Bell et al. have further disclosed that a 2.6 kb gene fragment contains the structural human insulin hormone sequences from the start of transcription to beyond the poly A addition site. This fragment was incubated in the presence of the linearized fragments of plasmid pUC12 described in Example 1. The reaction mixture was then incubated in the presence of DNA ligase to enable the resealing of a circular closed covalent plasmid molecule. The plasmid molecules so obtained were then transformed into bacterium E. coli and amplified, by means well known in the art.
  • This plasmid lacking any known eukaryotic promoter was referred to as pHINT2 and was suitable for the insertion of eukaryotic promoter sequences of interest. Such sequences will be inserted upstream of the human insulin hormone sequences and within the polylinker region of plasmid pUCl12 fragment. Thus, if a functional promoter is inserted into this polylinker region the plasmid will direct the biosynthesis of human insulin hormone which will then be excreted into the external culture medium, where it can be assayed by means known in the art (as by radioimmunoassay).
  • a mouse metallothionein I (mMT-I)/human growth hormone fusion gene was constructed by ligating a 1.8 kb EcoRI-Bglll fragment which has been reported to contain the promoter sequences of the mMT-I gene (Hamer, D. et al., J. Mol. Appl. Gen. 1:273-288, 1982) to a 2.1 kb Bam HI-Eco RI HGH gene fragment which contains HGH structural sequences from the start of transcription at the BamHI site to 526 bp past the poly A addition site.
  • the ligated material was digested with EcoRI and inserted into the EcoRI site of the plasmid vector pUCl2.
  • the resulting plasmid was referred to as pXG5.
  • the desired amount of DNA was ethanol precipitated and resuspended in 60 ul of tris buffered saline (0.15 M NaCl, 5 mM KCL, 1.5 mM Na 2 HPO 4 , 2.5 mM trisbase, 1 mM CaCl 2 , 0.5 mM MgCl 2 , adjusted to pH 7.5- the CaCl, and MgCl 2 were prepared together as a 100x stock and added slowly to other components to avoid precipitation) and then added to 120 ul of warm (30-40°C) 5 mg/ml DEAE-dextran in tris buffered saline. This mixture was then combined with 3 ml of 10% Nuserum (Collaborative Research, Inc.) and added to the plate.
  • the DNA-DEAE dextran-Nuserum was removed and replaced by 5ml of 10% DMSO in PBS. This was incubated for 1 minute at room temperature, the DMSO aspirated, and 10 ml of the appropriate media added to the dish. The cells were incubated at 37°C in 5% CO 2 for several days, and aliquots of media were taken at various times for assay.
  • the procedure used for batch transfections was similar to that described above for 10 cm dishes. Approximately 3 x 10 6 cells were plated on a 25 cm tissue culture dish (Nunc) and allowed to grow for three days. The desired amount of DNA, in 360 ul of tris buffered saline, was added to 720 ul of warm DEAE-dextran, and this mixture was added to the cells in a total volume of 36 mis of Nuserum. 30 mis of 10% DMSO in PBS was used to shock the cells for one minute at room temperature. After incubation in 60 mis of media for 24 hours, the cells were trypsinized and split equally into 6 ten cm dishes, each of which contained 10 mis of media.
  • Levels of human growth hormone in the media were measured using a solid phase 2-site radioimmunoassay kit under the conditions recommended by the manufacturer (Hybritech Inc.). This assay can detect as little as 0.2 ng/ml of HGH and is linear in the range of 0.2-50 ng/ml. When necessary, samples of concentrations of HGH in excess of 50 ng/ml, were diluted into the linear range of the assay using 10% horse serum in DMEM.
  • the levels of HGH secreted into the media were measured using a commercially available (Hybritech) radioimmunoassay in which the amount of HGH specifically bound by the first monoclonal antibody on a solid support is measured using a second, 125 I-labeled monoclonal antibody directed against a different HGH epitope.
  • Hybritech commercially available radioimmunoassay in which the amount of HGH specifically bound by the first monoclonal antibody on a solid support is measured using a second, 125 I-labeled monoclonal antibody directed against a different HGH epitope.
  • HGH levels were measured two and four days after transfection with various amounts of plasmid pXGH5.
  • the transfected cells were washed twice with PBS, osmotically swelled (2mM MgCl 2 , 20 mM Tris pH 7.5), and lysed (0.1% Triton, 20mM Tris pH 7.5). The lysate was collected, centrifuged to remove cellular debris, and assayed for HGH. Two days after transfection approximately 1.5% of the total HGH was present inside the cells, and four days after transfection, approximately 0.5% was present inside the cells. These values indicate that the L cells did not store the newly synthesized HGH.
  • Mouse L cells were transfected, using the procedures described above, with pOGH, pXGH5, pTKGH and pUC12. Plasmid pOGH, plasmid pTKGH and plasmid pXGH5 were found to mediate the expression of human growth hormone as measured by the presence of human growth hormone in the extracellular culture medium. Cells tranfected with plasmid pOGH, which lacks any known eukaryotic promoter sequence expressed extremely low background level amounts of human growth hormone. Plasmid pUCl2 failed to direct the biosynthesis and secretion of any detectable human growth hormone. Mouse L cells which had been transfected with plasmid pTKGH or plasmid pXGH5 were found to have produced significantly more human growth hormone than was produced by mouse L cells subsequent to transfection with plasmid pOGH.
  • mouse metallothionein promoter (mMT-I) was approximately 3 times ( 200/67 ) more powerful than the Herpes TK promoter. This comparison is based upon transfections using amounts of DNA that fall in the linear range for pxGH5 and pTKGH in mouse L cells.
  • RNA was prepared using the method of Alexander, M. et al. (J. Biol. Chem. 260:11978-11985 (1985)). The RNA was resolved by electrophoresis through 1.2% agarose/formaldehyde gels, (Maniatis, T. et al., supra.) and blotted ont a nitrocellulose fil ter. mRNA which was specific for human growth hormone was visualized by autoradiography after hybridization with a 32 P-labeled human growth hormone cDNA probe.
  • the size of human growth hormone mRNA was estimated relative to stained ribosomal RNA markers of known size which were placed in an adjacent lane. The results of this experiment is shown in Figure 4. A single, predominant human growth hormone mRNA species of 900 nucleotides was observed. mRNA from cells which had received 0, 250, 500, and 1000 ng of pXGH5 DNA.
  • Plasmid pTKGH and its enhancer sequence-containing derivative, pSVTKGH were introduced into XC cells, a rat fibroblast line. Secreted HGH levels were determined using the Hybritech RIA assay described above.
  • HGH levels were determined at times indicated by closed circles. Dashed line corresponds to result from pTKGH; solid line indicates pSVTKGH results.
  • the HGH transient expression system described above was used for studies on gene regulation in order to determine the effect of heavy metals on the induction of a mouse metallothionein-1 promoter.
  • Hamer, D. et al have disclosed that the mouse metallothionein promoter was induced by the presence of zinc or cadmium metal ions.
  • mouse L cells were transfected with either plasmid pXGH5 (which contains the mouse metallothionein promoter operably linked to the genetic sequences which encode human growth hormone) or with plasmid pTKGH (which carries the thymidine kinase promoter of herpes virus operably linked to genetic sequences which encode human growth hormone).
  • HGH levels were determined at times indicated by squares or circles. Lines connected by squares are values obtained from pXGHS-infected cells. Lines connected by circles indicate pTKGH-infected cells. Cells grown in the presence of ZnSO 4 are represented by solid symbols.
  • a promoter such as that from the Herpes virus TK gene, may then be inserted into one of the restriction endonuclease cleavage sites located in the polylinker region of the plasmid.
  • a plasmid is produced which is capable upon transfection into mouse L cells of expressing a mutant analogue of human growth hormone.
  • the growth hormone analogue may then be purified from the external culture medium by means known to the art, and in this manner studied and characterized.
  • a variant human growth hormone (the gene for which is present in the human genome) may be expressed in this manner.
  • mice L and rat XC cells In addition to mouse L and rat XC cells, all other cell types examined so far are able to express significant amounts of HGH after transfection with plasmid pXGH5. These include the rat pituitary cell lines: GC and GH 4 ; the mouse pituitary line: AtT-20; the human choriocarcinoma cell line: JEG; the monkey kidney cell line: CV-1; rat primary pituitary cells; mouse primary fibroblasts; and mouse primary hepatocytes.

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Abstract

La technique d'ADN recombinant décrite permet d'évaluer la résistance et l'efficacité de promoteurs dans un essai transitoire pour l'expression de gènes. Un procédé permettant d'évaluer un promoteur capable de fonctionner dans une cellule hôte donnée consiste à disposer sur un vecteur de plasmide essentiellement incapable de réplication dans ladite cellule hôte une première séquence génétique devant être évaluée pour déterminer l'activité du promoteur et liée de façon active à une seconde séquence génétique qui code une molécule rapporteuse pouvant être sécrétée par ladite cellule hôte sans provoquer la lyse de ladite cellule hôte, à mesurer la quantité de molécule rapportable exprimée par ledit vecteur et sécrétée par ladite cellule et à évaluer l'activité du promoteur en tant que fonction de ladite quantité.
PCT/US1987/001745 1986-08-14 1987-07-20 Essai d'evaluation de l'activite de promoteurs WO1988001296A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289034A2 (fr) * 1987-05-01 1988-11-02 The General Hospital Corporation Implantation transcaryotique
FR2693475A1 (fr) * 1992-07-08 1994-01-14 Rhone Poulenc Rorer Sa Procédé d'identification et/ou de clonage de promoteurs transcriptionnels, et utilisation de ces promoteurs pour l'expression de gènes.
WO1998036097A1 (fr) * 1997-02-14 1998-08-20 Ventana Genetics, Inc. Procedes pour identifier, caracteriser et faire evoluer des elements regulateurs a effet cis specifique de type cellulaire

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0108667A1 (fr) * 1982-10-07 1984-05-16 Institut Pasteur (Fondation) Lignées cellulaires originaires de mammifères, productrices d'hormone de croissance, de préférence humaine, et vecteurs pour leur obtention

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0108667A1 (fr) * 1982-10-07 1984-05-16 Institut Pasteur (Fondation) Lignées cellulaires originaires de mammifères, productrices d'hormone de croissance, de préférence humaine, et vecteurs pour leur obtention

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Title
CHEMICAL ABSTRACTS, Volume 105, No. 17, 27 October 1986, (Columbus, Ohio, US), R.F. SELDEN et al.: "Human growth Hormone as a Reporter Gene in Regulation Studies Employing Transient Gene Expression", see page 193, Abstract 147447w, & Mol. Cell. Biol. 1986, 6(9), 3173-9 *
Gene, Volume 29, Nos. 1/2, July/August 1984, Elsevier Science Publishers, (Amsterdam, NL), O. RAIBAUD et al.: "A Technique for Integrating any DNA Fragment into the Chromosome of Escherichia Coli", pages 231-241 see the whole document *
Gene, Volume 38, 1985, Elsevier Science Publishers, F. PASLEAU et al.: "Growth Hormone Gene Expression in Eukaryotic Cells directed by the Rous Sarcoma Virus Long Terminal Repeat or Cytomegalovirus Immediate-early Promoter", pages 227-232 see the whole document *
Gene, Volume 38, 1985, Elsevier Science Publishers, T.V. RAMABHADRAN et al.: "High-Level Expression of the Bovine growth Hormone Gene in Heterologous Mammalian Cells", pages 111-118 see the whole document *
Gene, Volume 43, 1986, Elsevier Science Publishers B.V. (Biomedical Division), T. IMANAKA: "Construction of High, Intermediate and Low-Copy-Number Promoter-Probe Plasmids for Bacillus Subtilis", pages 231-236 see page 235, column 1, line 4 - page 236, column 1, line 26 *
Gene, Volume 57, 1987, Elsevier Science Publishers B.V. (Biomedical Division), F. PASLEAU et al.: "A Comparison of Bovine growth Hormone Expression directed by bGH Genomic or Intronless DNA in transiently transfected Eukaryotic Cells", pages 47-52 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289034A2 (fr) * 1987-05-01 1988-11-02 The General Hospital Corporation Implantation transcaryotique
EP0289034A3 (fr) * 1987-05-01 1990-01-17 The General Hospital Corporation Implantation transcaryotique
EP0761233A2 (fr) * 1987-05-01 1997-03-12 The General Hospital Corporation Implantation transcaryotique
EP0761233A3 (fr) * 1987-05-01 1998-07-29 The General Hospital Corporation Implantation transcaryotique
FR2693475A1 (fr) * 1992-07-08 1994-01-14 Rhone Poulenc Rorer Sa Procédé d'identification et/ou de clonage de promoteurs transcriptionnels, et utilisation de ces promoteurs pour l'expression de gènes.
WO1998036097A1 (fr) * 1997-02-14 1998-08-20 Ventana Genetics, Inc. Procedes pour identifier, caracteriser et faire evoluer des elements regulateurs a effet cis specifique de type cellulaire

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