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WO1992012635A1 - Procedes de modulation transcriptionnelle de l'expression genetique de genes viraux et d'autres genes - Google Patents

Procedes de modulation transcriptionnelle de l'expression genetique de genes viraux et d'autres genes Download PDF

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WO1992012635A1
WO1992012635A1 PCT/US1992/000424 US9200424W WO9212635A1 WO 1992012635 A1 WO1992012635 A1 WO 1992012635A1 US 9200424 W US9200424 W US 9200424W WO 9212635 A1 WO9212635 A1 WO 9212635A1
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molecule
promoter
cell
gene
expression
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PCT/US1992/000424
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J. Gordon Foulkes
Casey C. Case
Franz Leichtfried
Christian Pieler
John Stephenson
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Oncogene Science, Inc.
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Publication of WO1992012635A1 publication Critical patent/WO1992012635A1/fr

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Definitions

  • Major expression systems common to investigators include bacterial, yeast, insect and mammalian cells (1) .
  • the method of choice is usually dictated by a number of criteria, including size of protein being produced, whether or not the protein is secreted, the presence of host modifying enzymes which can affect the recombinant protein's structure or function, and the specific purpose for which the expressed protein will be used.
  • a number of different expression vectors with which to insert the appropriate genetic material have been developed. These often include segments from viral or yeast promoter regions which, when orientated in the proper context to the gene, or cDNA to be expressed, can be recognized by the host cell's transcriptional machinery and result in the production of protein. The next section of this introduction will deal with some of the more common viral and yeast promoters used for this purpose.
  • a number of viral vectors have been described including those made from various promoters and other regulatory elements derived from virus sources (2) .
  • Promoters consist of short arrays of nucleic acid sequences that interact specifically with cellular proteins involved in transcription. The combination of different recognition sequences and the cellular concentration of the cognate transcription factors determines the efficiency with which a gene is transcribed in a particular cell type (3) .
  • Papovavirus These are small, non-enveloped DNA containing viruses SV-40 and polyo a being two of the best studied papovavirus examples (4) .
  • the viral genome of SV-40 is a covalently closed circular double- stranded DNA molecule of 5243 bp. The genome is divided into early and late regions which are transcribed from the two DNA strands in opposite directions.
  • Various plasmid-based expression vectors contain specific regulatory regions derived from SV-40, the most commonly used being a 300 bp segment which lies between the viral early and late transcription units and containing a number of different cis-acting elements, including the DNA origin of replications and promoters, and sites of initiation of transcription of early and late mRNAs (5) .
  • Use of the strong promoters in the SV-40 regulatory region can result in high levels of expression in transfected host cells.
  • Cytomegalovirus The human CMV immediate early promoters serves as an efficient transcription element with which to express foreign proteins. In combination with the CMV enhancer element, the CMV promoter's transcriptional activity can be increased to 10 to 100- fold. The human CMV enhancers are also active in a wide variety of cells from many species (97) .
  • MMTV Mouse Mammary Tumor Virus
  • LTR long terminal repeat
  • GRE glucocorticoid-responsive element
  • Baculovirus High level expression of foreign proteins in insect cells has been demonstrated using the Baculovirus expression vectors (7,8).
  • the baculovirus vector utilizes the highly expressed and regulated Autographa californica nuclear polyhedrosis virus polyhedron promoter which has been modified for the insertion of foreign genes.
  • the viral genome consists of double-stranded circular, supercoiled DNA 128 kilobases long.
  • Most transfer vectors contain the promoter of the polyhedron gene (which is non-essential for replication or production of extracellular virus in cultured cells) .
  • the foreign gene sequences in the recombinant plasmid can be transferred to the wild type virus by homologous recombination within a cell transfected with both the plasmid and wild-type virus DNAs.
  • yeast Protein expression in yeast offers certain advantages over bacterial expression systems (described below) .
  • Yeast being eucaryotic, possess much of the complex cell biology typical of multicellular organisms, including a highly compartmentalized intracellular organization and an elaborate secretory pathway which mediates the secretion and modification of many host proteins (9) .
  • Using a yeast expression system thus affords a broader range of potential applications than is possible with bacterial expression systems.
  • a number of yeast promoters are available for high level protein expression. Below is a brief description of yeast promoters commonly used in protein expression systems.
  • GAL Galactose-inducible Promoters
  • Copper Metallothionine Promoter Induction of the yeast metallothionine (MT) promoter results in high level expression of recombinant genes.
  • the MT promoter located upstream of the CUPI coding sequence, is rapidly induced during addition of copper ions to the media.
  • the promoter comprises a 450 bp fragment which contains the metal regulatory sequences, the mRNA cap site, the TATA box and associated transcription signals (11).
  • CYC1 The CYC1 gene of Saccharomyces cerevisiae encodes the cytochro e C protein. Two independent upstream activation sequenced (UAS) are found in the CYC1 gene, which appears to function as regulatory sites.
  • Alcohol dehydrogenase 2 (ADH2) promoter The ADH2 gene is regulated by glucose repression. When yeast are grown on glucose, ADH2 transcription is undetectable; however, derepression to a level which ultimately produces about 1% of soluble cellular protein, occurs when yeast are grown on a non-fermentable carbon source (12) . Analysis of the ADH2 promoter reveals two cis- acting regulatory components (upstream activation sequences) which mediates derepression (13,14). Both of these elements act synergistically to confer maximum expression on the promoter. The ADH2 promoter provides a strong transcriptional start signal for heterologous gene expression.
  • ADH2 promoter transcription is highly repressed by glucose, cultures can be grown to a high density in the presence of glucose without overexpressing the protein.
  • glucose becomes depleted by normal cellular metabolism the promote is derepressed alleviating the need for changing the growth medium, adding inducing compounds, or changing the temperature as is required by other the promoters.
  • the most straight forward application of this invention is the use of the transcriptionally modulating compounds described herein to increase the production of proteins whose coding sequence has been put under the transcriptional control of one of the claimed promoters.
  • Examples of this are the use of compounds to increase recombinant protein production in tissue culture application, increase monoclonal antibody production by including active compounds in Ascites injections, increase protein production in fungal fermentations, etc.
  • Fungal and Viral promoters are used in the biotechnology industry for the production of protein because they are considerably stronger than typical mammalian promoters.
  • An additional benefit of screening for compounds which increase viral and fungal promoter transcription is that inevitably compounds will be found which do just the opposite. These compounds could potentially be developed into pharmaceuticals useful for the treatment of viral or fungal infections.
  • Viral Diseases The list of viral-related diseases is extensive. Additionally, a number of different viruses can infect a particular organ or tissue, thereby leading to disease. A few examples:
  • CMV Cytomegalovirus
  • A Cytomegalovirus (CMV) : In vivo, CMV infects a wide range of host tissues (15), causing pneumonitis, retinitis, gastrointestinal disease, hepatitis, renalitis and encephalitis. Most CMV-related diseases occur in immunocompromised patients, either those undergoing immunosuppression to prevent transplant rejection or presenting with HIV infection. CMV is a common infection in high risk populations for HIV infection; nearly all homosexual men have ⁇ erologic evidence of recently acquired or reactivated CMV infection, and 30% shed CMV in the urine intermittently (16) . Current therapy of CMV disease treatment includes discontinuation, if possible, of immunosuppressive therapy or use of a variety of anti-viral agents. This approach has not been shown to be particularly efficacious in patients with serious CMV disease.
  • HPV Human papillomaviruses
  • EBV Epstein-Barr Virus
  • C Epstein-Barr Virus
  • EBV is lymphotropic, infecting and replicating within lymphocytes. It has been identified in other organs (ie. , the salivary gland, the tongue, and in T-cell lymphomas (19) .
  • the paradigm for EBV- induced illness is acute infectious ononucleosis.
  • EBV- associated malignancies have also been described, particularly Burkitt's lymphoma in which 95% of tumors from endemic (African) cases are EBV-positive (20) .
  • Nasopharyngeal carcinoma is also EBV-associated, along with much rarer human cancers including some salivary tumors, malignant thymomas, and squamous tumors of the head, neck and lung (21) .
  • HBV hepatitis B virus
  • HBV hepatitis B virus
  • serum levels of HBV are highest.
  • HCC Primary hepatocellular carcinoma
  • HCC represents a complication of chronic hepatitis (23) .
  • Epidemiological and molecular biological evidence have linked HCC with chronic HBV infection.
  • Chronic HBV infection appears to be the single major etiologic factor in the development of this tumor.
  • HIV Human Immunodeficiency Virus
  • HIV is a retrovirus in which the single-stranded RNA genome is converted into a double-stranded DNA provirus in the host cell by reverse transcriptase (98) .
  • HIV codes for a number of regulatory proteins which act in trans to regulate HIV- transcription. The virus infects and kills CD4 helper T lymphocytes. Infection of monocyte-macrophage and possibly other cells may also be a critical aspect of the pathogenesis of HIV-infection (24) .
  • asymptomatic individuals, seropositive for HIV may maintain their status for a year to decades.
  • clinical manifestations include fever, lymphadenopathy, pharyngitis, aseptic meningitis and a mild erythematous macular exanthem (25).
  • Opportunistic infections or malignancies ie., pneumonia, encephalitis, Kaposi's sarcoma and lymphoma of the brain, or other non-Hodgkin's, non T-cell lymphomas
  • Fungal infections are a critical and rapidly increasing health problem. Typically, fungi are opportunistic pathogens, requiring an immunocompromised host or facilitated entry allowing acute infection (e.g. through catheters) . Multiple factors have contributed to the recent increase in the number of immunosuppressed individuals, the most significant of which is the AIDS epidemic. Nearly 60% of AIDS patients develop life threatening opportunistic fungal infections (in particular Crvptoco ⁇ cus neoformans) . In addition, the increase in transplantations, with the concomitant use of immunosuppressive drugs, has uncovered a critical new need for effective anti-fungal therapies. Fungal infections are also serious problem in patients suffering from neutropenic leukemias or undergoing intensive chemotherapy. Tissue and/or systemic fungal infections occur in 25-40% of persistently febrile granulocytopenic hosts .
  • the expression of a specific gene can be regulated at any step in the process of producing an active protein. Modulation of total protein activity may occur via transcriptional, transcript-processing, translational or post-translational mechanisms. Transcription may be modulated by altering the rate of transcriptional initiation or the progression of RNA polymerase (26) . Transcript-processing may be influenced by circumstances such as the pattern of RNA splicing, the rate of mRNA transport to the cytoplasm or mRNA stability. This invention concerns the use of molecules which act by modulating the in vivo concentration of their target proteins via regulating gene transcription. The functional properties of these chemicals are distinct from previously described molecules which also affect gene transcription.
  • Transcriptional regulation is sufficiently different between procaryotic and eucaryotic organisms so that a direct comparison cannot readily be made.
  • procaryotic cells lack a distinct membrane bound nuclear compartment.
  • the structure and organization of procaryotic DNA elements responsible for initiation of transcription differ markedly from those of eucaryotic cells.
  • the eucaryotic transcriptional unit is much more complex than its procaryotic counterpart and consists of additional elements which are not commonly found in bacteria, including enhancers and other cis-acting DNA sequences (29,30).
  • Procaryotic transcription factors most commonly exhibit a "helix-turn-helix” motif in the DNA binding domain of the protein (31,32).
  • Eucaryotic transcriptional factors frequently contain a "zinc finger” (32,33), a "helix-loop- helix” or a “leucine zipper” (34) in addition to sometimes possessing the "helix-turn-helix” motif (35) .
  • RNA splicing and polyadenylation are not found in procaryotic systems (36,37).
  • modulation of gene transcription in response to extracellular factors can be regulated in both a temporal and tissue specific manner (38) .
  • extracellular factors can exert their effects by directly or indirectly activating or inhibiting tissue-specific transcription factors (38,39).
  • Modulators of transcription factors involved in direct regulation of gene expression have been described, and include those extracellular chemicals entering the cell passively and binding with high affinity to their receptor-transcription factors.
  • This class of direct transcriptional modulators include steroid hormones and their analogs, thyroid hormones, retinoic acid, vitamin D 3 and its derivatives, and dioxins, a chemical family of polycyclic aromatic hydrocarbons (33,40,41).
  • Dioxins are molecules generally known to modulate transcription. Dioxins, however, bind to naturally- occurring receptors which respond normally to xenobiotic agents via transcriptionally activating the expression of cytochrome P450, part of an enzyme involved in detoxifi ⁇ cation. Similarly, plants also have naturally occurring receptors to xenobiotics to induce defense pathways. For example, the fungal pathogen Phytophthora megasperma induces an anti-fungal compound in soybeans. Such molecules which bind to the defined ligand binding domains of such naturally occurring receptors are not included on the scope of this invention.
  • Indirect transcriptional regulation involves one or more signal transduction mechanisms. This type of regulation typically involves interaction with a receptor, the receptor being part of a multistep intracellular signaling pathway, the pathway ultimately modulating the activity of nuclear transcription factors.
  • This class of indirect transcriptional modulators include polypeptide growth factors such as platelet-derived growth factor, epidermal growth factor, cyclic nucleotide analogs, and mitogenic tumor promoters such as PMA (42,43,44).
  • nucleotide analogs in methods to non- specifically modulate transcription.
  • the mechanism involves incorporating nucleotide analogs into nascent mRNA or non-specifically blocking mRNA synthesis.
  • alkylating agents e.g. cyclophosphamide
  • intercalating agents e.g. doxorubicin
  • hydroxymethyl-glutaryl CoA reductase e.g. lovastatin
  • chemical inhibitors of hydroxymethyl-glutaryl CoA reductase are known to indirectly modulate transcription by increasing expression of hepatic low density lipoprotein receptors as a consequence of lowered cholesterol levels.
  • Signal effector type molecules such as cyclic AMP, diacylglycerol, and their analogs are known to non-specifically regulate transcription by acting as part of a multistep protein kinase cascade reaction. These signal effector type molecules bind to domains on proteins which are thus subject to normal physiological regulation by low molecular weight ligands (45,46).
  • PCT/US88/10095 The specific use of sterol regulatory elements from the LDL receptor gene to control expression of a reporter gene has recently been documented in PCT/US88/10095.
  • One aspect of PCT/US88/10095 deals with the use of specific sterol regulatory elements coupled to a reporter as a means to screen for drugs capable of stimulating cells to synthesize the LDL receptor.
  • PCT/US88/10095 describes neither the concept of simultaneously screening large numbers of chemicals against multiple target genes nor the existence of transcriptional modulators which (a) do not naturally occur in the cell, (b) specifically transcriptionally modulate expression of the genes of interest, and (c) binds to DNA or RNA, or bind to a protein through a domain of such protein which is not a defined ligand binding domain which naturally occurs in the cell, the binding of a ligand to which ligand binding domain is normally associated with the production in cell culture of the protein encoded by the gene.
  • the main focus of PCT/US88/10095 is the use of the sterol regulatory elements from the LDL receptor as a means to inhibit expression of toxic recombinant biologicals.
  • reporter gene to analyze nucleotide sequences which regulate transcription of a gene- of-interest.
  • the demonstrated utility of a reporter gene is in its ability to define domains of transcriptional regulatory elements of a gene-of-interest.
  • Reporter genes which express proteins, e.g. luciferase are widely utilized in such studies. Luciferases expressed by the North American firefly, Photinus pyralis and the bacterium. Vibrio fischeri were first described as transcriptional reporters in 1985 (47,48).
  • Reporter genes have not been previously used to identify compounds which (a) do not naturally occur in the cell and (b) specifically transcriptionally modulate expression of the gene, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with the production in cell culture of the protein encoded by the gene.
  • a method to define domains of transcriptional regulating elements of a gene-of-interest typically has also involved use of phorbol esters, cyclic nucleotide analogs, concanavalin A, or steroids, molecules which are commonly known as transcriptional modulators.
  • transcriptional modulators molecules which are commonly known as transcriptional modulators.
  • available literature shows that researchers have not considered using a transcription screen to identify specific transcriptional modulators. Hence, success would be unlikely in doing so, however, we have demonstrated herein that this is not the case.
  • This invention provides a method of obtaining a gene product of interest, which comprises culturing cells capable of expressing a gene encoding the gene product in the presence of a molecule at a concentration effective to directly transcriptionally modulate expression of the gene so as to increase the biosynthesis of the gene product expressed by the cells and recovering the gene product.
  • the molecule (a) does not naturally occur in the cells, (b) specifically transcriptionally modulates expression of the gene, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with the production in cell culture of the gene product.
  • the present invention further includes a method of directly transcriptionally modulating the expression of a gene encoding a viral protein, the expression of which gene is associated with a defined pathological effect within a multicellular organism, which comprises contacting a cell, which is capable of expressing the gene, with a molecule at a concentration effective to transcriptionally modulate expression of the gene and thereby affect the level of the protein encoded by the gene which is expressed by the cell.
  • the molecule (a) does not naturally occur in the cell and (b) specifically transcriptionally modulates expression of the gene encoding the protein, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with a defined pathological effect.
  • this present invention also provides a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a protein of interest which gene is associated with the production in a cell culture of the protein encoded by the gene, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence encoding a polypeptide, which polypeptide is capable of producing a detectable signal, which DNA sequence is coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable detectable signal to be produced by the polypeptide so expressed, quantitatively determining the amount of the signal produced, comparing the amount so determined with the amount of produced signal
  • a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a protein of interest which gene is associated with the production in a cell culture of the protein encoded by the gene which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a reporter gene, which expresses a polypeptide, coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable change in the amount of the polypeptide produced, quantitatively determining the amount of the polypeptide so produced, comparing the amount so determined with the amount of polypeptide produced in the absence of any molecule being tested or upon contacting the sample with any other
  • the present invention includes a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a protein of interest which gene is associated with the production in a cell culture of the protein encoded by the gene, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence transcribable into mRNA coupled to and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable difference in the amount of mRNA transcribed from the DNA sequence, quantitatively determining the amount of the mRNA produced, comparing the amount so determined with the amount of mRNA detected in the absence of any molecule being tested or upon contacting the sample with
  • this invention includes a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a viral protein, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence encoding a polypeptide, which polypeptide is capable of producing a detectable signal, which DNA sequence is coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable detectable signal to be produced by the polypeptide so expressed, quantitatively determining the amount of the signal produced, comparing the amount so determined with the amount of produced signal detected in the absence of any molecule being tested or upon contacting the sample with any other molecule, and
  • this present invention provides a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a viral protein, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a reporter gene, which expresses a polypeptide, coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable change in the amount of the polypeptide produced, quantitatively determining the amount of the polypeptide so produced, comparing the amount so determined with the amount of polypeptide produced in the absence of any molecule being tested or upon contacting the sample with any other molecule, and thereby identifying the molecule as one which causes a change
  • this invention provides a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a viral protein, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and
  • a method for directly transcriptionally modulating in a multicellular organism the expression of a gene encoding a viral gene, the expression of which is associated with a defined physiological or pathological effect in the organism comprises administering to the organism a molecule at a concentration effective to transcriptionally modulate expression of the gene and thus affect the defined pathological effect.
  • the molecule (a) does not naturally occur in the organism, (b) specifically transcriptionally modulates expression of the gene encoding the viral gene, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with a defined pathological effect.
  • Figure 1 is a view of the mammalian expression shuttle vector pUV102 with its features.
  • the mammalian expression shuttle vector was designed to allow the construction of the promoter-reporter gene fusions and the insertion of a neomycin resistance gene coupled to the herpes simplex virus thymidine kinase promoter (TK-NEO) .
  • TK-NEO herpes simplex virus thymidine kinase promoter
  • Figure 2 is a partial restriction enzyme cleavage map of the plasmid pD0432 which contains the luciferase gene from the firefly, Photinus pyralis.
  • Figure 3 is a partial restriction enzyme cleavage map of the plasmid pSVLuci which contains the luciferase gene from the firefly, Photinus pyralis.
  • Figure 4 is a partial restriction enzyme cleavage map of the plasmid pMLuci which contains the luciferase gene of the firefly, Photinus pyralis and the mouse mammary tumor virus long terminal repeat.
  • Figure 5 provides the nucleotide sequences of six oligonucletides, pUV-1 through pUV-6, which were annealed, ligated, and inserted into the Sall/EcoRl sites of the plasmid pTZ18R.
  • Figure 6 is a diagrammatic representation of the construction of the plasmid pUVOOl from the plasmids pTZ18R and pBluescript KS(+).
  • Figure 7 is a diagrammatic representation of the construction of the plasmid pUVlOO from the plasmid pUVOOl and two DNA fragments, the Xbal/Xmal fragment from pMLuci and the Xmal/BamHI fragment from pMSG.
  • Figure 8 is a diagrammatic representation of the construction of the plasmid pUVlOO-3 from the plasmid pUVlOO and a 476 bp fragment containing a dimeric SV40 polyadenylation site.
  • Figure 9 is a diagrammatic representation of the construction of the plasmids pUV102 and pUV103 from the plasmid pUVlOO-3 and D-link oligonucleotides and the plasmid pUV100-3 and R-link oligonucleotides, respectively.
  • Figure 10 provides the nucleotide sequences of oligos 1-4 used for the construction of a synthetic HSV-Thymidine Kinase promoter and provides a diagrammatic representation of the HSV-TK promoter.
  • Figure 11 is a diagrammatic representation of the construction of the plasmid pTKLlOO which contains the luciferase gene from the firefly, Photinus pyralis and the HSV-TK promoter sequence.
  • Figure 12 is a diagrammatic representation of the construction of the plasmid pTKNEO which contains the neo gene, from about 3.5 kb Nhel/X al fragment from pTKLlOO, and the about 0.9 kb BstBI/Bglll fragment containing the neo coding region from pRSVNEO.
  • Figure 13 is a diagrammatic representation of the construction of the plasmid pTKNE02 from the plasmid pTKNEO and the oligonucleotides Neo 1 and 2.
  • Figure 14 is a diagrammatic representation of the construction of the plasmid pTKNE03 from the plasmid PTKNE02 and about 0.9 kb EcoRl/Sall fragment from pMClNEO.
  • Figure 15 is a partial restriction enzyme cleavage map of the plasmid pCM106 which contains CMV upstream sequences fused to the luciferase gene.
  • Figure 16 is a partial restriction enzyme cleavage map of the plasmid pCM106 which contains the Cytomegalovirus immediate early promoter fused to the luciferase gene from the firefly, Photinus pyralis.
  • Figure 17 is a partial restriction enzyme cleavage map of the plasmid pNEU106 which contains neu upstream sequences fused to the luciferase coding region.
  • Figure 18 is a partial restriction enzyme cleavage map of the plasmid pKRASlO ⁇ which contains K-ras upstream sequences fused to the luciferase gene from the firefly, Photinus pyralis.
  • Figure 19 is an autoradiogram of a Southern blot showing the correct luciferase vector integration of independently isolated SV40 reporter vector transfectants.
  • Lane 1 is a plasmid control. The expected result is a single band of the same molecular weight as the control.
  • Figure 20 is an autoradiogram of a Southern blot showing the correct luciferase vector integration of independently isolated CMV reportervector transfectants.
  • Lane l is a plasmid control. The expected result is a single band of the same molecular weight as the control.
  • Figure 21 is a graphical representation of the response of the MMTV reporter cell line to various steroids. Relative light production is compared to an untreated control.
  • Figure 22 is a graphical representation of the decay of reporter gene signal after treatment of cells with Actinomycin D. Plotted is relative intensity of the signal versus time after ActD addition.
  • Figure 23 is a graphical representation of the linearity of response of the yeast luciferase reporter system. Light intensity versus cell number is plotted.
  • Figure 24 is a quality assurance analysis of a high throughput screen measuring the ratios of negative values at various positions within a plate.
  • the expected value is 1.0.
  • Figure 25 is a quality assurance analysis of a high throughput screen measuring a coefficient of variance for the negative controls on a number of plates. Values less than 10 are acceptable.
  • Figure 26 is a quality assurance analysis of a high throughput screen measuring a coefficient of variance for the positive controls on a number of plates. Values less than 10 are acceptable.
  • Figure 27 is a quality assurance analysis of a high throughput screen measuring a response of a reporter cell line to three different concentrations of a compound known to induce transcription.
  • Figure 28 is a bar graph illustrating specific induction of luciferase expression in reporter cell lines for MMTV
  • M10 human growth hormone (532) and human G-CSF (G21) promoters in response to chemicals identified in a high throughput screen and known transcriptional inducers.
  • Figure 29 is a bar graph illustrating specific inhibition of luciferase expression in reporter cell lines for MMTV (M10) , human growth hormone (532) , and human G-CSF (G21) in response to chemicals identified in a high throughput screen.
  • Antisense nucleic acid means an RNA or DNA molecule or a chemically modified RNA or DNA molecule which is complementary to a sequence present within an RNA transcript of a gene.
  • Cell culture means the in vitro growth of either single cells or groups of cells by means of tissue culture or fermentation.
  • Directly transcriptionally modulate the expression of a gene means to transcriptionally modulate the expression of the gene through the binding of a molecule to (1) the gene (2) an RNA transcript of the gene, or (3) a protein which binds to (i) such gene or RNA transcript, or (ii) a protein which binds to such gene or RNA transcript.
  • a gene means a nucleic acid molecule, the sequence of which includes all the information required for the normal regulated production of a particular protein, including the structural coding sequence, promoters and enhancers.
  • Indirectly transcriptionally modulate the expression of a gene means to transcriptionally modulate the expression of such gene through the action of a molecule which cause enzymatic modification of a protein which binds to (1) the gene or (2) an RNA transcript of the gene, or (3) protein which binds to (i) the gene or (ii) an RNA transcript of the gene.
  • a molecule which cause enzymatic modification of a protein which binds to (1) the gene or (2) an RNA transcript of the gene, or (3) protein which binds to (i) the gene or (ii) an RNA transcript of the gene constitutes indirect transcript modulation.
  • Ligand means a molecule with a molecular weight of less than 5,000, which binds to a transcription factor for a gene. The binding of the ligand to the transcription factor transcriptionally modulates the expression of the gene.
  • Ligand binding domain of a transcription f ctor means the cite on the transcription factor at which the ligand binds.
  • Modulatable transcriptional regulatory sequence of a gene means a nucleic acid sequence within the gene to which a transcription factor binds so as to transcriptionally modulate the expression of the gene.
  • Receptor means a transcription factor containing a ligand binding domain.
  • transcriptionally modulate the expression of a gene means to transcriptionally modulate the expression of such gene alone, or together with a limited number of other genes.
  • Transcription means a cellular process involving the interaction of an RNA polymerase with a gene which directs the expression as RNA of the structural information present in the coding sequences of the gene.
  • the process includes, but is not limited to the following steps: (1) transcription initiation, (2) transcript elongation, (3) transcript splicing, (4) transcript capping, (5) transcript termination, (6) transcript polyadenylation, (7) nuclear export of the transcript, (8) transcript editing, and (9) stabilizing the transcript.
  • Transcription factor for a gene means a cytoplasmic or nuclear protein which binds to (1) such gene, (2) an RNA transcript of such gene, or (3) a protein which binds to (i) such gene or such RNA transcript or (ii) a protein which binds to such gene or such RNA transcript, so as to thereby transcriptionally modulate expression of the gene.
  • Transcriptionally modulate the expression of a gene means to change the rate of transcription of such gene.
  • Triple helix means a helical structure resulting from the binding of one or more oligonucleotide to double stranded DNA.
  • This invention provides a method of obtaining a gene product of interest, which comprises culturing cells capable of expressing a gene encoding the gene product in the presence of a molecule at a concentration effective to directly transcriptionally modulate expression of the gene so as to increase the biosynthesis of the gene product expressed by the cells and recovering the gene product.
  • the molecule (a) does not naturally occur in the cells, (b) specifically transcriptionally modulates expression of the gene, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with the production in cell culture of the gene product.
  • the invention further provides a method of directly transcriptionally modulating the expression of a gene encoding a viral protein, the expression of which is associated with a defined pathological effect within a multicellular organism, which comprises contacting a cell, which is capable of expressing the gene, with a molecule at a concentration effective to transcriptionally modulate expression of the gene and thereby affect the level of the protein encoded by the gene which is expressed by the cell.
  • the molecule (a) does not naturally occur in the cell, (b) specifically transcriptionally modulates expression of the gene encoding the protein, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with a defined pathological effect.
  • the molecule does not naturally occur in any cell of a lower eucaryotic organism such as yeast. In a preferred embodiment, the molecule does not naturally occur in any cell, whether of a multicellular or a unicellular organism. Alternatively, the molecule is naturally occurring, but not normally found in the cell. In a presently more preferred embodiment, the molecule is not a naturally occurring molecule, e.g. is a chemically synthesized entity.
  • the cell contacted with the molecule may be a cell of a multicellular organism, for example, an insect cell, an animal cell or a human cell, a hybridoma, a COS cell, a CHO cell, a HeLa cell, a fungal cell. (e.g. a yeast cell) of a plant cell.
  • a multicellular organism for example, an insect cell, an animal cell or a human cell, a hybridoma, a COS cell, a CHO cell, a HeLa cell, a fungal cell. (e.g. a yeast cell) of a plant cell.
  • the method of the invention permits modulating or a transcription of the gene which results in upregulation or downregulation of the expression of the gene (either the gene encoding the gene product or the gene encoding the viral protein) .
  • the methods of the invention are most advantageouslyemployed to specificallytranscriptionally modulate expression of such genes.
  • the molecule may bind to a promoter region upstream of the coding sequence encoding the viral gene.
  • the molecule comprises an antisense nucleic acid which is complementary to a sequence present in a modulatable transcriptional sequence.
  • the molecule may also be a double stranded nucleic acid or a nucleic acid capable of forming a triple helix with a double stranded DNA.
  • the molecule bonds to a modulatable transcription sequence of the gene.
  • the gene is typically associated with amelioration of a disorder caused by the virus such as a virus infection.
  • viruses examples include cytomegalovirus, hepatitis, herpes, HIV, EBV, papilloma virus, cytomegalovirus, rhinovirus, influenza virus, varicella- zoster virus, parainfluenza virus, mumps virus, respiratory syncytial virus, adenovirus, measles virus, rubella virus, human parvovirus, poliovirus, rotavirus, echovirus, arbovirus, human T cell leukemia-lymphoma virus.
  • viruses the genes of which are subject to transcriptional modulation include cytomegalovirus, hepatitis, herpes, HIV, EBV, papilloma virus, cytomegalovirus, rhinovirus, influenza virus, varicella- zoster virus, parainfluenza virus, mumps virus, respiratory syncytial virus, adenovirus, measles virus, rubella virus, human parvovirus, poliovirus, rotavirus, echovirus, arbovirus
  • a gene encoding the gene product is a gene encoding any protein or RNA of industrial of commercial significance.
  • proteins are either already available commercially or are under commercial development.
  • proteins include human and animal growth hormones, tissue plasminogen activators, erythropoietin, and factor VIII.
  • the invention may be employed to augment the production of such protein in cell culture, particularly in animal cell culture such as in CHO cells grown in culture and thereby reduce the substantial costs involved in commercial production of such proteins.
  • the present invention further provides a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene.
  • a method comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence encoding a polypeptide, which polypeptide is capable of producing a detectable signal, which DNA sequence is coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable detectable signal to be produced by the polypeptide so expressed, quantitatively determining the amount of the signal produced, comparing the amount so determined with the amount of produced signal detected in the absence of any molecule being tested or upon contacting the sample with any other molecule, and thereby
  • the invention further includes a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a protein of interest which is associated with production in a cell culture of the protein encoded by the gene, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence transcribable into mRNA coupled to and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable difference in the amount of mRNA transcribed from the DNA sequence, quantitatively determining the amount of the mRNA produced, comparing the amount so determined with the amount of mRNA detected in the absence of any molecule being tested or upon contacting the sample with any other
  • Also provided by this invention is a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a viral protein, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence encoding a polypeptide, which polypeptide is capable of producing a detectable signal, which DNA sequence is coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable detectable signal to be produced by the polypeptide so expressed, quantitatively determining the amount of the signal produced, comparing the amount so determined with the amount of produced signal detected in the absence of any molecule being tested or upon contacting the sample with any other molecule
  • the invention provides a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a viral protein, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a reporter gene, which expresses a polypeptide, coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable change in the amount of the polypeptide produced, quantitatively determining the amount of the polypeptide so produced, comparing the amount so determined with the amount of polypeptide produced in the absence of any molecule being tested or upon contacting the sample with any other molecule, and thereby identifying the molecule as one which causes a change in
  • the invention further provides a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene encoding a viral protein, which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene, (ii) the promoter, and (iii) a DNA sequence transcribable into mRNA coupled to and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene, causes a measurable difference in the amount of mRNA transcribed from the DNA sequence, quantitatively determining the amount of the mRNA produced, comparing the amount so determined with the amount of mRNA detected in the absence of any molecule being tested or upon contacting the sample with any other molecule, and thereby identifying the molecule as one which causes a change in
  • the sample comprises cells in monolayer ⁇ .
  • the sample comprises cells in suspension.
  • Cells may include animal cells (such as human cells) , fungal cells, insect cells, and plant cells.
  • the predefined number of cells may be from about 1 to about 5 X 10 5 cells.
  • the predefined number of cells may be from about 2 X 10 2 to about 5 X 10 4 cells.
  • the predetermined amount of the molecule to be tested is based upon the volume of the sample. In one example of the invention, the predetermined amount is from about 1.0 pM to about 20 ⁇ M. In another example, the predetermined amount is from about 10 nM to about 500 ⁇ M.
  • the contacting is effected from about 1 to about 24 hours. In one example, the contacting is effected from about 2 to about 12 hours. Also, the contacting may be effected with more than one predetermined amount of the molecule to be tested.
  • the molecule to be tested may be a purified molecule.
  • the modulatable transcriptional regulatory sequence may comprise a cloned genomic regulatory sequence.
  • the DNA may consist essentially of more than one modulatable transcriptional regulatory sequence.
  • the polypeptide may be a luciferase, chloramphenicol acetyltransferase, ,9 glucuronidase, ⁇ galactosidase, neomycin phosphotransferase, alkaline phosphatase, or guanine xanthine phosphoribosyltransferase.
  • the polypeptide may be capable of recognizing and binding to an antibody.
  • the polypeptide may be capable of recognizing and binding to biotin.
  • mRNA may be detected by quantitative polymerase chain reaction.
  • the invention further provides the above-described screening method which further comprises separately contacting each of a plurality of substantially identical samples, each sample containing a predefined number of cells under conditions such that contacting is affected with a predetermined amount of each different molecule to be tested.
  • the plurality of samples may comprise more that about 10* samples.
  • the plurality of samples may comprise more than about 5 X 10 4 samples.
  • the present invention provides a method of essentially simultaneously screening molecules to determine whether the molecules are capable of transcriptionally modulating one or more genes.
  • the method comprises essentially simultaneously screening the molecules against the genes encoding the proteins of interest according to the above-describe method.
  • more than about 10 3 samples per week are contacted with different molecules.
  • plasmid designated pUV106 deposited under ATCC Accession No. 40946;
  • SW 480 human breast carcinoma cell line transfected with pKRAS106, designated K-2, deposited under ATCC Accession No. CRL 10662;
  • NIH Swiss mouse embryo cell line NIH 3T3, transfected with the MMTV reporter plasmid, designated M10, deposited under ATCC Accession No. CRL 10659.
  • a cell containing a gene comprising a heterologous regulatory element or a strong promoter may be fused to a coding sequence so as to produce a desired protein encoded by such sequence.
  • a strong promoter such as an immunoglobulin promoter
  • Contacting the cell (which is capable of expressing the gene) with a molecule having the properties described herein may be effective to transcriptionally modulate expression of the gene and increase the level of the protein expressed by the cell.
  • suitable promoters include a viral promoter.
  • adenovirus promoter examples include an adenovirus promoter, an simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter, a mouse mammary tumor virus (MMTV) promoter, a Malony urine leukemia virus promoter, a murine sarcoma virus promoter, and a Rous sarcoma virus promoter.
  • SV40 simian virus 40
  • CMV cytomegalovirus
  • MMTV mouse mammary tumor virus
  • Malony urine leukemia virus promoter a murine sarcoma virus promoter
  • Rous sarcoma virus promoter examples include an adenovirus promoter, an simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter, a mouse mammary tumor virus (MMTV) promoter, a Malony urine leukemia virus promoter, a murine sarcoma virus promoter, and a
  • a suitable promoter is a heat shock promoter.
  • a suitable promoter is a bacteriophage promoter. Examples of suitable bacteriophage promoters include a T7 promoter, a T3 promoter, an SP6 promoter, a lambda promoter, a baculovirus promoter.
  • a promoter is an animal cell promoter such as an interferon promoter, a metallothionein promoter, an immunoglobulin promoter.
  • a fungal promoter is also a suitable promoter. Examples of fungal promoters include an ADC1 promoter, an ARG promoter, an ADH promoter, a CYC1 promoter, a CUP promoter, an EN01 promoter, a GAL promoter, a PHO promoter, a PGK promoter, a GAPDH promoter, a mating type factor promoter.
  • plant cell promoters and insect cell promoters are also suitable for the methods described herein.
  • a method of obtaining a polypeptide which comprises culturing cells capable of expressing a gene encoding the polypeptide in the presence of a molecule at a concentration effective to directly transcriptionally modulate expression of the gene so as to increase the biosynthesis of the polypeptide expressed by the cells and recovering the polypeptide.
  • the molecule (a) does not naturally occur in the cells, (b) specifically transcriptionally modulates expression of the gene, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with the production in cell culture of the polypeptide.
  • the polypeptide is a homologous polypeptide.
  • the polypeptide is a desired product.
  • the desired product is a monoclonal antibody.
  • the DNA is recombinant DNA.
  • the cell is a animal cell, a plant cell, a bacterial cell or a fungal cell.
  • the polypeptide is associated with production of a desired product.
  • desired products include is an antibiotic, citric acid, a desired antigen for the development of a vaccine, tissue plasminogen activator, a growth hormone, a blood clotting factor, erythropoietin, an interleukin, a colony stimulating factor, a transforming growth factor.
  • the following provides a biological method for recovering a substance from a mixture containing the substance which involves contacting the mixture with cells so as to separately recover the substance, which cells (i) comprise DNA encoding, and (ii) are capable of expressing a gene product, which gene product facilitates separating the substance from the mixture so as to recover the substance from the mixture, the improvement comprising (1) treating the cells with a molecule which (a) does not naturally occur in the cell and (b) binds to DNA or RNA or binds to a protein through a domain of such protein which is not a ligand binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand binding domain is normally associated with increased production of the gene product.
  • the substance is a metal.
  • the following provides a biological method for treating a substance with cells so as to effect a biochemical transformation by contacting the substance with cells which (i) comprises DNA encoding, and (ii) is capable of expressing a gene product which permits biochemical transformation, the improvement comprising contacting the cells with a molecule which (a) does not naturally occur in the cell and (b) binds to DNA or RNA or binds to a protein through a domain of such protein which is not a ligand binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand binding domain is normally associated with enhanced production of the gene product.
  • the biochemical transformation is associated with the production of a steroid, an alcohol or with the degradation of petroleum products.
  • this invention would have commercial applications both in the case where the polypeptide itself is commercially important, and in the case where expression of the polypeptide mediates the production of a molecule which is commercially important. Examples include, but are not limited to:
  • Vaccines can be used against viral, bacterial or parasitic infections. Such viral vaccines include poliomyelitis, measles, mumps and rubella. In addition, foot and mouth disease vaccine and rabies vaccine are of major commercial importance. Viable, disease-associated viruses can be subsequently inactivated, live viruses can be attenuated to lose their pathogenicity or genetically engineered. The expression of a viral antigen could be under the control of a heterologous promoter (59) ; 2.
  • Monoclonal antibodies could be produced by growing the hybridoma in tissue culture or in vivo (59) ;
  • heterologous polypeptide by a cell (i.e. a polypeptide introduced into the cell by genetic engineering) , typically, under the control of a strong promoter.
  • promoters include the promoter of the SV40 virus, the immediate early promoter of the cytomegalovirus or the baculovirus promoter.
  • heterologous polypeptides would include tissue plasminogen activator, human or animal growth hormones, blood clotting factors, erythropoietin, interleukins, interferons, the colony stimulating factors G-, GM- and M-CSF, and transforming growth factors-,91, -,92 and -,93.
  • the polypeptide of interest could be expressed by the cell without genetic engineering.
  • One example would be the production of interferon alpha by the lymphoblastoid cell line •Namala' (59);
  • biodegradation is effected by the conversion of petroleum products to emulsified fatty acids.
  • Bacteria useful in this invention include, but are not restricted to, Archromobacter, Arthrobacter, Flavobacteriu , Nocardia, Pseudomonas (e.g. Pseudomonas oleovorans) and Cytophaga.
  • Yeast useful in this invention include, but are not restricted to, Candida (e.g., Candida tropicalis) , Rhodotorula, and Trichosporon (64) ;
  • the invention would include the following steps (i) identification of the protein responsible for controlling the rate limiting step and (ii) screening for molecules capable of increasing the production of that protein using the methods described herein as will be clearly and readily understood by one skilled in the art.
  • the invention provides both for the method of screening for such molecules and for the use of such molecules to regulate expression of a rate limiting polypeptide as described herein.
  • a method for directly transcriptionally modulating in a multicellular organism the expression of a gene encoding a viral gene, the expression of which is associated with a defined physiological or pathological effect in the organism is also included.
  • This method comprises administering to the organism a molecule at a concentration effective to transcriptionally modulate expression of the gene and thus affect the defined pathological effect.
  • the molecule (a) does not naturally occur in the organism, (b) specifically transcriptionally modulates expression of the gene encoding the viral gene, and (c) binds to DNA or RNA, or binds to a protein at a site on such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand-binding domain is normally associated with a defined pathological effect.
  • the molecule may bind to a modulatable transcription sequence of the gene.
  • the method discussed above includes the use of a molecule of an antisense nucleic acid, a double stranded nucleic acid, or a nucleic acid capable of forming a triple helix with double-stranded DNA.
  • the virus may be one which is associated with a pathological effect such as cancer.
  • the cancer may be a hepatocellular carcinoma, a leukemia, or a cervical carcinoma.
  • the pathological effect may also be AIDS, cytomegalovirus infection, influenza, infectious mononucleosis, mumps, poliomyelitis, measles, rubella, herpes or hepatitis.
  • the administering discussed in the preceding methods may comprise topical contact, or oral, transdermal, intravenous, intramuscular or subcutaneous administration.
  • Methods of administration of molecules in the practice of the invention are well known to those skilled in the art as are methods of formulating the molecule for administration depending on the specific route of administration being employed.
  • FCS Fetal calf serum
  • Hep3B A human hepatocellular carcinoma derived cell line, Hep3B (ATCC# HB8064) , was used for transfection of plasmids containing the SV40 and CMV promoters. These cells were maintained on MEM:OptiMEM (1:1) supplemented with 10% FCS.
  • a murine embryonic fibroblast cell line, NIH3T3 (ATCCf CCL92) was used for the transfection of plasmids carrying the MMTV promoter. These cells were maintained on DMEM, supplemented with 10% FCS.
  • a human colon adenocarcinoma cell line, SW480 (ATCC CCL 228) was used for experiments concerning expression of the K-ras proto-oncogene (used as a control) .
  • This cell line was maintained on DMEM, 15% fetal calf serum (FCS) , 1% Nonessential amino acids (NEAA) .
  • Stable transfectants of this cell line were selected in the same medium with the addition of G418 (Geneticin, Gibco) to a final concentration of 0.6 mg/ml.
  • a human breast adenocarcinoma derived cell line, SK-BR-3 (ATCC HTB 30) was used for the experiments concerning expression of the neu (ErbB2) proto-oncogene (also used as a control) .
  • This cell line was maintained on DMEM, 15% FCS and 1 ug/ml insulin. Stable transfectants of this cell line were selected in this same medium with the addition of G418 to a final concentration of 0.4 mg/ml.
  • Oligonucleotides were synthesized by the beta-cyanoethyl phosphoramidite method according to protocols provided by the manufacturer of the
  • a mammalian expression shuttle vector was designed to allow the construction of the promoter-reporter gene fusions to be used in high-throughput screens to identify transcriptionally modulating chemicals. Features of the plasmid are shown in Figure 1. The shuttle vector was constructed in several steps.
  • the firefly luciferase gene was removed from the plant expression plasmid pD0432 (52) ( Figure 2) as a 1.9 kb BamHI fragment and cloned into the BamHI site of pSVL
  • pSVLuci (Pharmacia, Piscataway, NJ) , a mammalian expression vector containing the SV40 promoter.
  • the resulting plasmid (pSVLuci; Figure 3) was digested with Xhol and Sail to produce a 2.4 kb fragment containing the luciferase coding sequences and the SV40 late polyadenylation site. This fragment was inserted into the Xhol site of pMSG (Pharmacia, Piscataway, NJ) , a eucaryotic expression vector containing the MMTV promoter.
  • the resulting MMTV promoter-luciferase fusion plasmid (pMLuci; Figure 4) was used to transfect NIH/3T3 cells as described below. Similar constructs can be made using luciferase vectors from Clontech (Palo Alto, CA) .
  • oligonucleotides (pUV-1 through pUV-6) were synthesized (see Figure 5 for sequence) (SEQ ID NO: 1-6) .
  • the sequences of pUV-1, pUV-2 and pUV-3 correspond to a multicloning site, the beta-globin leader sequence and the first 53 bases of the firefly luciferase coding region.
  • the sequences of pUV-4, pUV-5 and pUV-6 are complementary to the first three oligonucleotides.
  • the pUV oligonucleotides were annealed, ligated and inserted into the Sall/EcoRI sites of pTZ18R (Pharmacia, Piscataway NJ) ( Figure 6) .
  • the resulting vector was then digested with Smal/PvuII and the oligonucleotide containing fragment was cloned into the pBluescriptKS(+) plasmid (Stratagene, La Jolla, CA) , previously digested with PvuII, to yield pUVOOl ( Figure 6) .
  • pUVOOl Figure 6
  • Several fragments were ligated into pUVOOl to create pUVlOO.
  • the luciferase coding sequences (except first 53 bases) and polyadenylation site were obtained as a 1.8 kilobase Xbal/Xmal fragment from pMLuci (section B-l, Figure 4) .
  • the SV40 early splice site and the SV40 late polyadenylation site were obtained as an 871 bp Xmal/BamHI fragment from pMSG (Pharmacia, Piscataway NJ, Figure 7) . Both DNA fragments were cloned into pUVOOl, previously digested with Xbal/BamHI to yield pUVlOO ( Figure 7) .
  • a 476 b fragment containing a dimeric SV40 polyadenylation site was then cloned into the Bell site of pUVlOO ( Figure 8) .
  • a 238 bp BclI/BamHI fragment was obtained from SV40 genomic DNA (BRL) , ligated, digested with BclI/BamHI, gel isolated, and inserted into pUVlOO, resulting in the vector pUV100-3 ( Figure 8) .
  • Linkers containing one Sfil and one NotI restriction site were then cloned into the PvuII/BamHI sites of pUV100-3.
  • the plasmid that contains D-link oligonucleotides was named pUV102 and the plasmid that contains R-link oligonucleotides was named pUV103 ( Figure 9) .
  • HSV-TK Herpes Simplex Virus thymidine kinase
  • the about 3.5 kb Nhel/Smal fragment was isolated from pTKLlOO, and the about 0.9 kb BstBI/Bglll fragment containing the neo coding region was isolated from pRSVNEO (54) . These two fragments were filled in with Klenow polymerase and ligated to form pTKNEO ( Figure 12) .
  • a 352 bp fragment containing the SV40 early promoter (55) was purified and ligated into pUV102 which had previously been digested with NotI (the ends rendered blunt by treatment with Klenow fragment) and HinDIII, generating pUVSV.
  • a 666 bp Nael-Xbal fragment from pUVSV containing the SV40 promoter and a portion of the luciferase open reading frame was purified by preparative gel electrophoresis, and ligated int pUV106 which had previously been digested with SnaBI and Xbal, generating pSV106 ( Figure 15) , the vector used to transfeet the SV40 reporter cell lines.
  • Oligonucleotide probes based on the published sequence of the 5' region of the c-ErbB2 gene were synthesized and used to screen a human leukocyte genomic library (Clontech Inc.). A 3.2 kb Bgll fragment from a positive plaque, containing the upstream regulatory elements, the 5' untranslated leader and exon 1 was then subcloned into pBluscriptKS(+) , generating pNEUOOl.
  • oligonucleotides were annealed to one another, phosphorylated and ligated into Ncol digested pNEU002, generating pNEU103.
  • the synthetic linker fuses the DNA coding for the neu 5' untranslated leader to the luciferase open reading frame such that the AUG utilized for translation initiation of the neu gene forms the first codon of the luciferase gene.
  • the Seal-Xbal fragment of pNEU103 containing vector sequences, the upstream regulatory elements, the 5'untranslated leader and a portion of the luciferase open reading frame, was purified by preparative gel electrophoresis and ligated into pUV106 which had previously been digested with Seal and Xbal, generating pNEU106 ( Figure 17) .
  • Linearized pNEU106 was used in the transfections to generate the neu-luciferase reporter cell lines as described below.
  • Oligonucleotides based on the published K-ras sequence were used to isolate two genomic clones by standard methods from a human leukocyte library (Clontech) . DNA from these two phages was subcloned into pBluscriptKS(+) (Stratagene) generating pKS4 and pKSll.
  • the resulting plasmid was designated PGEM715.
  • a 3 kb HinDIII-XhoI fragment from pKS4, comprising 2.2 kb of K-ras untranscribed upstream DNA and sequences coding for exon 0 and part of intron 1 was purified by preparative gel electrophoresis and ligated into pGEM715 which ad been previously digested with HinDIII and Xhol to generate pGEM7.
  • a 7.7 kb HinDIII-Ncol fragment of pGEM7 comprising 2.2 kb of K-ras upstream regulatory elements, exon 0, intron 1, and part of exon 1 (to the ATG at the Ncol site) , was purified by preparative gel electrophoresis and ligated int pUV102 which had previously been digested with HinDIII and Ncol to generate pKRAS102.
  • the TK-Neo fragment from pTKNeo3 was then ligated into the Sfil site of pKRAS102 to generate pKRAS106 ( Figure 18) , the vector used for transfections to generate the stable K-ras reporter cell lines.
  • D-PBS Dulbecco's phosphate-buffered saline
  • Lysis Buffer 1 50 mM Tris acetate pH 7.9, 1 mM EDTA, 10 mM magnesium acetate, 1 mg/ml bovine serum albumin [BSA], 0.5% Brij 58, 2 mM ATP, 100 mM dithiothreitol [DTT]). All reagents were obtained from Sigma except for DTT which was from Boehringer Mannheim.
  • plasmid DNA was electroporated into approximately 5 million cells.
  • molar ratio of luciferase fusion plasmid to neomycin resistant plasmid was either 10:1 or 20:1.
  • Neomycin resistant clones were selected by growth in media containing G418 (Geneticin, Gibco) .
  • pMluci and pSV2Neo an antibiotic resistance plasmid (112)
  • NIH/3T3 mouse fibroblast cells were co-transfected into NIH/3T3 mouse fibroblast cells using the calcium phosphate precipitation method (103) with a commercially available kit (Pharmacia, Piscataway NJ) . Two days later, cells were transferred to media containing 0.4 mg/ml G 18 and were grown for an additional 10-14 days. G418-resistant clones were isolated by standard methods. Once sufficient cell numbers were obtained, clones were analyzed based on several criteria: constitutive luciferase production, induction of luciferase expression by dexamethasone (1 ⁇ m, Sigma, St.
  • Hep3B hepatocellular carcinoma cells were transfected by electroporation with 75 micrograms of pSV106 which had been linearized by a single Seal cut within the vector backbone. Neomycin resistant colonies were isolated and tested for luciferase activity. Luciferase positive, neomycin resistant clones were subjected to Southern blot analysis (see below) . The best clone, producing the most luciferase activity from a single, correctly integrated vector was selected for use as the SV40 reporter cell line in the high throughput screen (this clone was designated SV12) .
  • Hep3B hepatocellular carcinoma cells were transfected by electroporation with 75 micrograms of pCM106 which had been linearized by a single Seal cut within the vector backbone. Neomycin resistant colonies were isolated and tested for luciferase activity. Luciferase positive, neomycin resistant clones were subjected to Southern blot analysis (see below) . The best clone, producing the most luciferase activity from a single, correctly integrated vector was selected for use as the CMV reporter cell line in the high throughput screen (this clone was designated CM1) .
  • clone N-2 75 micrograms of the pNEU106 plasmid was linearized by a single restriction endonuclease cleavage within the vector backbone and electroporated into HTB30 human breast carcinoma cells.
  • Neomycin resistant clones were isolated and tested for luciferase activity. Clones testing positive for luciferase production were subjected to Southern blot analysis (see below) . The best clone (producing the highest signal and carrying a single intact copy of the transfected DNA) was utilized for high throughput screening (designated clone N-2).
  • clone K-2 75 micrograms of the pKRAS106 plasmid was linearized by a single restriction endonuclease cleavage within the vector backbone and electroporated into SW480 human colon carcinoma cells.
  • Neomycin resistant clones were isolated and tested for luciferase activity. Clones testing positive for luciferase production were subjected to Southern blot analysis (see below) . The best clone (producing the highest signal and carrying a single intact copy of the transfected DNA) was utilized for high throughput screening (designated clone K-2) .
  • J. Construction of a Yeast Expression Vector Plasmid pHZl ⁇ (97,98) contains 2 ⁇ DNA for propagation in S. cerevisiae. the yeast promoter eye 1, which is compatible with expression in yeast cells, and the URA 3 gene for selection.
  • the plasmid was linearized with BamHI, the ends were filled-in using deoxynucleotides and E.coli DNA polymerase Klenow fragment, and then the plasmid was digested with Aat II.
  • a 4.1 kb fragment containing the cycl promoter, URA3 and 2 ⁇ genes was separated by agarose gel electrophoresis and subsequently purified by electroelution onto ion-exchange paper (Whatman, DE81) .
  • Plasmid pBR322 was treated with endonucleases Aat II and Pvu II and a 2.2 kb fragment containing the plasmid's origin of replication and the amp R gene was isolated by agarose gel electrophoresis and eluted onto DE ⁇ l paper.
  • the 2.2 kb pBR322 and 4.1 kb pHZl ⁇ fragments were ligated using T4 DNA ligase according to standard procedures (94).
  • the resulting 6.3 kb vector pHZBR was digested with BamHI for subsequent insertion of the luciferase coding sequence downstream of the cycl promoter.
  • Neo I - Sal I fragment of pUV102 containing the luciferase gene starting at the second ATG was made blunt-ended by filling in and ligated into the filled-in BamHI site of pHZBR. Clones of the correct orientation were identified via restriction mapping to yield plasmid pHZluci24. This plasmid was used to transform S.cerevisiae strain DB745.
  • 1-2 x 10 4 yeast cells were seeded into 96-well plates which have been custom designed to allow filtration of the media while retaining the cells, and which are opaque to permit analysis using a luminometer (Millipore) .
  • a luminometer Millipore
  • lOO ⁇ l of lysis buffer 50 mM Tris/acetate pH 7.9, l mM EDTA, 10 mM Mg-acetate, 0.5% Brij 5 ⁇ , 100 mM DTT and 4 mM ATP, 0.2 mM Luciferin, ⁇ OO U/ml lyticase
  • lysis buffer 50 mM Tris/acetate pH 7.9, l mM EDTA, 10 mM Mg-acetate, 0.5% Brij 5 ⁇ , 100 mM DTT and 4 mM ATP, 0.2 mM Luciferin, ⁇ OO U/ml lyticase
  • stably transfected cell clones were subjected to Southern blot analysis (57) .
  • Genomic DNA was prepared of each clone to be tested and restriction-cut with Dra I.
  • transfer to nylon filters and immobilization by UV irradiation using a Stratalinker UV device (Stratagene, La Jolla, California) integrated promoter/lueiferase fusion constructs were visualized by probing with radioactively labelled Xbal-EcoRI fragments of the luciferase coding region. Probes were labelled using the random primer method (5 ⁇ ) .
  • Dra I cuts in the SV40 polyadenylation sites located in the OSI mammalian expression shuttle vector just upstream the inserted promoter sequences as well as downstream of the luciferase coding region, but not in any of the promoter sequences used for generating stably transfected cell clones, a single fragment should be visualized by the probe used. The size of that fragment should be characteristic for each of the three promoter sequences analyzed.
  • Dynatech Microliter 96 well plates were custom pretreated for cell attachment by Dynatech Laboratories, Inc. (Chantilly, VA) .
  • the 96 well plates were treated with 50 ⁇ l per well of human fibronectin (hFN, 15 ⁇ g/ml in PBS, Collaborative Research, Bedford, MA) overnight at 37°C.
  • hFN-treated plates were washed with PBS using an Ultrawash 2 Microplate Washer (Dynatech Labs) , to remove excess hFN prior to cell plating.
  • M10 and G21 cells maintained in their respective serum media were washed with PBS, harvested by trypsinization, and counted using a hemocytometer and the Trypan Blue exclusion method according to protocols provided by Sigma, St. Louis, MO Chemical Company.
  • Cells were then diluted into serum free defined media (with 0.2 mg/ml G41 ⁇ ) , and 0.2 ml of cell suspension per well was plated onto Dynatech treated plates (G21) or hFN-treated plates (M10) using a Cetus Pro/Pette (Cetus, Emeryville CA) . Plates were incubated overnight at 37oC in a humidified 5% C02 atmosphere.
  • Bioluminescence Assay After incubation with OSI-file chemicals, cell plates were washed 3 times with PBS using an Ultrawash 2 Microplate Washer (Dynatech Labs) and 75 ul of Lysis Buffer 2 were added to each well (Lysis Buffer 2 is the same as Lysis buffer 1 except that the ATP and DTT concentrations were changed to 2.67 mM and 133 mM, respectively) . Bioluminescence was initiated by the addition of 25 ul 0.4 ⁇ m Luciferin in Buffer B to each well, and was measured in a Dynatech ML 1000 luminometer following a 1 minute incubation at room temperature. Data were captured and analyzed using Lotus-Measure (Lotus) software.
  • the cell lysis buffer was modified to also contain the luciferin. Therefore, lysis of cells and the bioluminescence reaction begin simultaneously and the production of bioluminescent light reaches a maximum at about 5 min. The level of light output declines by about 20% within further 30 min.
  • bovine serum albumin has been omitted. This improved lysis buffer has been shown to remain fully functional for at least 12 hours, when kept on ice and protected from direct light.
  • the size of that fragment should be characteristic for each of the three promoter sequences analyzed.
  • the Southern blots were hybridized with either an end-labelled luciferase- specific 40 nucleotide oligomer (OSI, ON227) , or a random primer labelled DNA fragment corresponding to the 5' third of the luciferase open reading frame (an Xbal-EcoRI fragments of pUV106) .
  • Figure 19 and 20 show the resulting autoradiograms. All but one of the luciferase expressing clones show the correct characteristic fragment.
  • One of the clones has an extra unexpected fragment that may be the result of two insertion events, one resulting in an intact vector and the other rearranged. We selected a single, correctly integrated clone from each transfection for use in the high throughput screen.
  • MMTV reporter cell line M10
  • the response to steroid hormones is well documented. M10 cells were incubated with several different steroids for 6 hours. The cells were then harvested and assayed for luciferase as described above. The data are shown graphically in figure 21. Compared to an untreated control, the MMTV LTR was induced over 10 fold by the steroids known to have active steroid receptors in this cell line. The concentrations of steroids required for maximal induction of luciferase expression were approximately the same as those reported in the literature.
  • the half-life of the reporter molecule becomes a crucial parameter in determining the minimal incubation time that would be necessary to allow enough decay of reporter molecules so that the inhibition of their synthesis became visible.
  • the CMV reporter cell line was therefore tested for the time dependency of luciferase activity after treatment of the cells with Actinomycin D, an inhibitor of transcription. This experiment measured the combined half-life of luciferase mRNA and of the luciferase protein and compares the rate of signal decay of the CMV reporter to three other well characterized genes; H-ras, K-ras and c-erbB2.
  • CMl CMl
  • K-2 K-ras
  • H21 H- ras
  • N-2 c-erbB2
  • Actinomycin D 25 ⁇ g/ml
  • cells were washed with PBS and luciferase activity of Actinomycin-treated cells determined as described in Materials and Methods. The signal from the treated cells was compared to the luciferase activity of untreated controls. The logarithm of the treated/untreated ratio was plotted versus time, this data is shown in figure 22.
  • the calculated half- life of the signal from each of the four cell lines is shown in table 1.
  • the half-lives were found to range 5 from about 3 to 10 hours indicating that a 24 hour incubation with a 100% efficient inhibitor of transcription would be sufficient to reduce luciferase levels to 6% of the control in the tested cell lines.
  • yeast expression plasmid carrying the luciferase gene under control of a yeast promoter was transfected into 5 . appropriate yeast cells. Using these cells as a model system, a format for a 96-well luciferase expression assay in yeast cells was developed as described in more detail in Materials and Methods.
  • Luciferase activity was assayed essentially as described 0 by De Wet et al. (1967) using a modified single step lysis-assay buffer which contained Lyticase, to break down the yeast cell wall, and the detergent Brij 58, to help effect lysis.
  • Cells were grown in custom made, 96 well microtiter plates equipped with membrane-type filter bottoms. These plates retain liquid until vacuum is applied. Medium was removed by filtration, and the cells were washed with PBS. The lysis/assay buffer was added and resulting luminescence measured in a luminometer. 5. Representative data are shown in figure 23. This simple assay is reliable and sensitive. Small changes in luciferase expression from as few as 5,000 yeast cells was easily detected.
  • Figure 24 shows an analysis of the consistency of the luciferase signal on various areas of each plate. The ratios of negative control values from three different areas within each plate are calculated and plotted versus plate number. The expected value is 0 1.0. Values greater than 1.5 or less than 0.4 indicate uneven signal generation across the plate.
  • 240 plates, representing 1440 compounds, tested against three cell lines, are shown.
  • the coefficient of variance for the 12 negative control values from each of the same 240 plates are represented by the data shown in Figure 25. Values less than 20% are considered acceptable. Similar data for the 12 positive control values of the same plates are shown in figure 26.
  • FIG 27 shows the transcription induction ratio (TIR) for the positive controls of one cell line represented in the same set of 240 plates.
  • the TIR is the ratio of the experimental values to the untreated controls.
  • the cell line is the K-ras reporter and the positive control is Actinomycin D a potent general inhibitor of transcription.
  • Three values are shown for each plot, representing three different concentrations of 5 . Actinomycin D.
  • the expected value for such an analysis depends on the half life of the signal and the incubation time (here 24 hours) , but for this combination, typical values range from 0.4 to 0.3 fold.
  • Aqueous fractions >2.0 7 l ⁇ O total ⁇ 0.6 >0. ⁇ >0. ⁇ 0
  • Menthanol extracts >2.0 ⁇ 1.8 ⁇ 1.8 0
  • Aqueous fractions >2.0 ⁇ 1.8 ⁇ l. ⁇ 0
  • Table 5 shows a summary of the results of a one-week, high-throughput screen of 2,000 chemicals to identify those chemicals specifically stimulating or inhibiting transcription from the MMTV, G-CSF or human Growth Hormone (the last two as controls for specificity) promoters.
  • This screen as with the other screens, concurrently tested chemicals at three concentrations on quadruplicate samples of the M10, 532 and G21 cell lines.
  • a minimum stimulation of one promoter, to the degree indicated, and less than 50% activation of the other promoter was required for a chemical to be considered a selective activator.
  • a minimum inhibition of 3 fold of one promoter and less than 20% inhibition of the other promoter was required for a chemical to be considered a selective inhibitor.
  • Chemicals which scored as positive in this screen are identified in Table 6.
  • Figure 28 illustrates the transcriptional stimulation and Figure 29 the transcriptional inhibition observed with some of the lead chemicals.
  • G-CSF lead chemicals #1760, #58, #1783, #1374 were subjected to 48 independent luciferase assays performed on the same day.
  • Compounds #58, #1780 and #1374 scored as positives in every single one of these assays inducing luciferase expression between 2 and 28 fold (#5 ⁇ ) , 20 and 80 fold (#1780) and 5 and 40 fold (#1374).
  • Compound #1783 scored as positive only in half of the 48 repeat assays.
  • Table 7 presents the data from one more high throughput screen. In this case the data are from a three week high throughput screen of 2334 compounds. Three cell lines were utilized; CMl (the CMV reporter cell line) . N-2
  • transcription induction ratio which is the median of the samples quadruplicate values divided by the median of the negative control values.
  • transcriptional inhibitors and inducers are sought, so the selection criteria for lead compounds is that the test promoter be inhibited to 0.4 or induced to 1.8X of the negative control while the other cell lines remain within 0.8X of the control value.
  • 10 compounds scored positive for the specific inhibition of the K-ras promoter 19 scored as leads for the inhibition of the c-erbB2 promoter and 39 compounds inhibited nonspecifically. Compounds scoring as leads in the primary screen are repeated and then subjected to secondary analysis such as effects on the minigene transfectant phenotypes (see above) .
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  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:7:
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Abstract

Cette invention concerne un procédé de modulation transcriptionnelle directe de l'expression d'un gène codant un produit génique, l'expression dudit gène étant associée à la production du produit génique. L'invention concerne également un procédé de modulation transcriptionnelle directe de l'expression d'un gène codant une protéine d'un virus, dont l'expression est associée à un effet pathologique défini provoqué par le virus au sein d'un organisme multicellulaire. Des procédés de criblage sont également décrits, y compris des procédés de criblage simultané de molécules servant à déterminer si les molécules sont capables de moduler par transcription un ou plusieurs gènes viraux ou autres associés à la production de polypeptides ou d'autres produits recherchés. En dernier lieu, cette invention concerne un procédé de modulation transcriptionnelle directe, dans un organisme multicellulaire, de l'expression d'un gène codant un gène viral, dont l'expression est associée à un effet pathologique ou physiologique défini provoqué par le virus, dont le génome comprend un tel gène dans l'organisme.
PCT/US1992/000424 1991-01-18 1992-01-17 Procedes de modulation transcriptionnelle de l'expression genetique de genes viraux et d'autres genes WO1992012635A1 (fr)

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WO1997038139A1 (fr) * 1996-04-10 1997-10-16 Signal Pharmaceuticals, Inc. Systeme de lignee cellulaire de marquage permettant de detecter la presence du cytomegalovirus et d'identifier des modulateurs de l'expression de genes viraux
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US6288091B1 (en) 1995-12-29 2001-09-11 Boehringer Ingelheim Ltd. Antiherpes virus compounds and methods for their preparation and use
US6525094B1 (en) 1999-06-01 2003-02-25 The University Of Texas Southwestern Medical Center Method of treating hair loss using diphenylether derivatives
US6646005B1 (en) 1999-06-01 2003-11-11 The University Of Texas Southwestern Medical Center Method of treating hair loss using sulfonyl thyromimetic compounds
US6680344B1 (en) 1999-06-01 2004-01-20 The University Of Texas Southwestern Medical Center Method of treating hair loss using diphenylmethane derivatives
WO2007007781A1 (fr) * 2005-07-12 2007-01-18 Japan Science And Technology Agency Nouvelle utilisation d'un produit transformé à base de feuilles de myrtillier
JP2016047817A (ja) * 2014-08-25 2016-04-07 住友化学株式会社 化合物、樹脂、レジスト組成物及びレジストパターンの製造方法
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