WO1998012337A1 - Integration aleatoire et ciblee d'un promoteur pour l'analyse de genes - Google Patents

Integration aleatoire et ciblee d'un promoteur pour l'analyse de genes Download PDF

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Publication number
WO1998012337A1
WO1998012337A1 PCT/US1997/016893 US9716893W WO9812337A1 WO 1998012337 A1 WO1998012337 A1 WO 1998012337A1 US 9716893 W US9716893 W US 9716893W WO 9812337 A1 WO9812337 A1 WO 9812337A1
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Prior art keywords
promoter
expression
gene
target gene
cell
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PCT/US1997/016893
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English (en)
Inventor
Klaus Giese
John Tamkun
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Chiron Corporation
Regents Of The University Of California
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Application filed by Chiron Corporation, Regents Of The University Of California filed Critical Chiron Corporation
Priority to AU46485/97A priority Critical patent/AU4648597A/en
Publication of WO1998012337A1 publication Critical patent/WO1998012337A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • the invention relates to the area of eukaryotic gene function. More particularly, the invention relates to methods of analyzing and modulating the function of eukaryotic genes by genetic manipulation.
  • One embodiment of the invention provides a eukaryotic cell for observing altered expression of a gene.
  • the cell comprises a first and a second DNA construct.
  • the first DNA construct comprises an ectopic promoter.
  • the ectopic promoter controls transcription of a gene in the eukaryotic cell.
  • the second DNA construct comprises a regulatable promoter and a coding sequence for a foreign transcriptional transactivator.
  • the regulatable promoter controls the expression of the transcriptional transactivator.
  • the transcriptional transactivator activates the ectopic promoter to alter expression of the gene.
  • Another embodiment of the invention provides a method of altering the expression of a gene in a eukaryotic cell.
  • a first and a second DNA construct are introduced into the eukaryotic cell.
  • the first DNA construct comprises an ectopic promoter.
  • the ectopic promoter controls transcription of a gene in the eukaryotic cell.
  • the second DNA construct comprises a regulatable promoter and a coding sequence for a foreign transcriptional transactivator.
  • the regulatable promoter controls the expression of the transcriptional transactivator.
  • the transcriptional transactivator activates the ectopic promoter to alter expression of the gene.
  • Still another embodiment of the invention provides a transgenic eukaryotic organism. Cells of the transgenic eukaryotic organism comprise a first and a second DNA construct.
  • the first DNA construct comprises an ectopic promoter.
  • the ectopic promoter controls transcription of a gene in the cells of the organism.
  • the second DNA construct comprises a regulatable promoter and
  • the regulatable promoter controls the expression of the transcriptional transactivator.
  • the transcriptional transactivator activates the ectopic promoter to alter expression of the gene.
  • the insect cell for observing diminished expression of a target gene.
  • the insect cell comprises a first and a second DNA construct.
  • the first DNA construct comprises a first promoter.
  • the first promoter controls reverse orientation transcription of the target gene in the insect cell.
  • the second expression construct comprises a second promoter and a coding sequence for a transcriptional transactivator.
  • the second promoter controls the expression of the transcriptional transactivator.
  • the transcriptional transactivator activates the first promoter whereby expression of the target gene is diminished.
  • a first and a second DNA construct are introduced into the insect cell.
  • the first DNA construct comprises a first promoter.
  • the first promoter controls reverse orientation transcription of the target gene in the insect cell.
  • the second DNA construct comprises a second promoter and a coding sequence for a transcriptional transactivator.
  • the second promoter controls the expression of the transcriptional transactivator.
  • the transcriptional transactivator activates the first promoter, whereby expression of the target gene is diminished.
  • Still another embodiment of the invention provides a transgenic insect.
  • Cells of the transgenic insect comprise a first and a second DNA construct.
  • the first DNA construct comprises a first promoter.
  • the first promoter controls reverse orientation transcription of a target gene in the insect cell.
  • the second DNA construct comprises a second promoter and a coding sequence for a transcriptional transactivator.
  • the second promoter controls the expression of the transcriptional transactivator.
  • the transcriptional transactivator activates the first promoter, whereby expression of the target gene is diminished.
  • Another embodiment of the invention provides an insect cell for observing diminished expression of a target gene.
  • the insect cell comprises a DNA construct comprising a promoter.
  • the promoter controls reverse orientation transcription of the target gene in the insect cell.
  • Yet another embodiment of the invention provides a method of diminishing the expression of a target gene in an insect cell.
  • a DNA construct comprising a promoter is introduced into the insect cell.
  • the promoter controls reverse orientation transcription of the target gene in the insect cell, whereby expression of the target gene is diminished.
  • Even another embodiment of the invention provides a transgenic insect.
  • Cells of the transgenic insect comprise a DNA construct.
  • the DNA construct comprises a promoter.
  • the promoter controls reverse orientation transcription of a target gene.
  • the present invention thus provides the art with a novel method of altering expression of a gene in a eukaryotic cell.
  • the method can be used, inter alia, to observe the effects of altered and ectopic gene expression during development.
  • the inventors have discovered a method of selectively altering the expression of a gene in a eukaryotic cell. This method is particularly useful for observing the effect of altering the expression of an unknown gene or a gene whose function is unknown.
  • the method can also be used for screening test compounds for the ability to alter gene expression in eukaryotes, for example, for agricultural purposes.
  • the method can be used in eukaryotic cells in vitro or in vivo, for example by constructing transgenic eukaryotic organisms.
  • the method can be used to alter the expression of any gene in a eukaryotic cell, including the cells of protozoa, algae and other plants, fungi, sponges, coelenterates, worms, cephalopods, starfish, gastropods, arthropods, including insects, spiders, and crustaceans, and chordates, including vertebrates, especially amphibians, reptiles, birds, and mammals.
  • a eukaryotic cell including the cells of protozoa, algae and other plants, fungi, sponges, coelenterates, worms, cephalopods, starfish, gastropods, arthropods, including insects, spiders, and crustaceans, and chordates, including vertebrates, especially amphibians, reptiles, birds, and mammals.
  • Genes involved in development are particularly suited for use with the present method.
  • Other eukaryotic genes such as those which control apoptosis, signal transduction, enzymatic function, oncogenesis and metastasis, hormone function and regulation, motility, reproduction, and neuronal development and function, can also be observed effectively using the invention.
  • expression of the gene can be manipulated at various points in development or in particular tissues. Alterations in gene expression induced by the method can be, for example, decreased or increased transcription, or temporal or spatial (tissue- or cell-specific) changes in transcription.
  • an ectopic promoter is integrated randomly into the genome of a eukaryotic cell.
  • the ectopic promoter is a promoter which is adjacent to a gene which it does not normally regulate in the eukaryotic cell.
  • the exogenous promoter is preferably a strong, regulatable promoter which can be controlled by a foreign trans-acting transcriptional transactivator, which is also introduced into the eukaryotic cell.
  • the foreign transcriptional transactivator is one which is not normally present in the cell. Transcription of the transcriptional transactivator is also preferably regulatable. Thus, when the transcriptional transactivator is not transcribed, the ectopic promoter is silent.
  • the ectopic promoter In the presence of the transcriptional transactivator, however, the ectopic promoter is activated. Alternatively, the ectopic promoter is regulated by its tissue or developmental milieu. Thus, the promoter may be selectively activated or repressed in certain tissues or at certain stages of development.
  • the ectopic promoter is integrated within the coding sequence of a gene, transcription of that gene can be disrupted and the phenotypic effects of that disruption observed.
  • the ectopic promoter is integrated near a gene (either 5' to an initiation codon or 3' to a termination codon) such that its presence does not interrupt the coding sequence of the gene, expression of the gene can be altered by controlling the expression of the transcriptional transactivator which regulates the ectopic promoter.
  • the ectopic promoter is tissue or developmentally regulated, it will be activated only under appropriate conditions in the appropriate tissue or developmental stage.
  • the first DNA construct comprises an ectopic promoter.
  • the ectopic promoter is a strong, regulatable promoter which can be controlled by a foreign trans-acting transcriptional activator.
  • the ectopic promoter can integrate randomly into a coding sequence of a gene and interrupt its transcription, as discussed above.
  • the ectopic promoter is spatially or temporally regulated, as discussed above.
  • ectopic promoter If integrated into the cell's DNA (v ⁇ thin or 3' to the coding sequence) so that the orientation of the ectopic promoter is opposite to the orientation of the gene sequence and its native promoter, activation of the ectopic promoter can result in diminished expression of the gene. Diminished expression of the affected gene can be achieved by either or both of two mechanisms. First, because transcription from the regulated promoter proceeds from the 3' end of the coding sequence, and transcription of the affected gene from its native promoter proceeds from the 5' end, concurrent transcription from the two promoters results in physical disruption of transcription (collision).
  • an antisense transcript is produced.
  • This antisense transcript can bind to the coding sequence and prevent transcription of the gene.
  • the antisense transcript can also bind to a pre-mRNA or mature mRNA transcript of the gene and prevent its translation.
  • the foreign transcriptional transactivator can be any protein which is not normally present in the eukaryotic cell and which is capable of binding the ectopic promoter and activating transcription of a gene which has randomly come under the regulatory control of that promoter.
  • transcriptional transactivator is LexA, Gal4, or a Tet-repressor-derived transcriptional activator such as the Tet-OnTM "gene switch" (CLONTECH).
  • the second DNA construct comprises a regulatable promoter and a coding sequence for the foreign transcriptional transactivator.
  • the regulatable promoter can be a naturally occurring promoter or can be genetically engineered.
  • the regulatable promoter can be a constitutively active promoter or can be a tissue-specific promoter, such as a promoter functional only in eye, muscle, heart, brain, lung, thymus, colon, stomach, liver, lymph node, blood, bladder, or ovary tissue.
  • a cell type-specific promoter for example, a promoter functional only in glia, neurons, T-cells, B-cells, or epithelial cells, can also be used to control the transcription of the transcriptional transactivator.
  • the regulatable promoter can be a conditionally regulatable promoter, such as a temperature sensitive promoter or a promoter which is sensitive to a chemical inducer.
  • the DNA constructs can be fabricated as is known in the art, using standard recombinant DNA techniques.
  • the DNA constructs can be flanked at each end by nucleotide sequences complementary to sequences in a cell's genomic DNA to facilitate integration of the expression constructs into the genome by homologous recombination.
  • at least the first DNA construct is integrated into the genome.
  • the DNA constructs can be introduced into a eukaryotic cell in vitro by any methods available in the art including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, and calcium phosphate-mediated transfection.
  • the DNA constructs are introduced into cells of a eukaryotic organism to create a transgenic organism. Techniques for constructing transgenic animals or plants are well known in the art.
  • the method is used to inactivate or diminish the expression of a target gene in a cell, particularly an insect cell.
  • the insect can be any member of the class Insecta in which further information regarding expression of a target gene is desired, including cate ⁇ illars, water nymphs and larvae, aphid-like insects, flealike insects, earwigs and silverfish, diving beetles and water bugs, hopperlike insects, plant bugs, toad bugs, and cockroaches, weevils, beetles, grasshoppers, crickets, and cicadas, mantids and walkingsticks, ants and termites, lacewings, dragonflies and damselflies, flies, bees, wasps and hornets, moths, butterflies, ticks, mites, and sco ⁇ ions.
  • the insect is a fly, more preferably a Drosophila melanogaster.
  • the target gene can be any eukaryotic gene whose coding sequence is known and about whose expression more information is desired. Genes involved in insect development, such as homeotic genes, polycomb group genes, trithorax group genes, and genes involved in metamo ⁇ hosis, are particularly suited for use with the present method. Other eukaryotic genes, such as those which control apoptosis, signal transduction, enzymatic function, oncogenesis and metastasis, hormone function and regulation, motility, reproduction, and neuronal development and function, can also be observed effectively using the invention. The method is especially useful for observing the effects of altered expression of embryonic lethal genes in insects, such as Ras.
  • an endogenous or exogenous copy of the target gene coding sequence can be placed under the control of two promoters.
  • One promoter is preferably the native promoter for the target gene and controls its transcription in the normal 5 1 to 3' direction.
  • the other promoter is a regulatable promoter which controls reverse orientation transcription of a gene (i.e., transcription from the 3* end of the gene).
  • the regulatable promoter is regulated by a transcriptional transactivator.
  • the transcriptional trar_sactivator is one which is not normally present in the cell. Transcription of the transcriptional transactivator is also preferably regulatable. Thus, when the transcriptional transactivator is not transcribed, the regulatable promoter is silent and expression of the targeted gene proceeds normally.
  • the regulated promoter is activated and, because of its reverse orientation with respect to the targeted gene, initiates transcription of the affected gene from the 3' end of the gene.
  • the target gene with upstream and downstream promoters can be introduced as a single cassette.
  • one promoter is weaker and one is stronger.
  • gene expression can be turned on or off, depending on whether the stronger promoter is 5' or 3'.
  • Regulation of the promoter can be by either endogenous tissue-specific or stage-specific cues, or by introduced foreign transactivators.
  • one or two DNA constructs can be introduced into the eukaryotic cell.
  • the first DNA construct comprises a first promoter.
  • the first promoter is a strong, regulatable promoter which can be controlled by a trans-acting transcriptional activator, or a tissue- or stage-specific cue.
  • the DNA construct can be flanked at each end by nucleotide sequences complementary to sequences of the target gene or its flanking sequences, to integrate the first DNA construct at a desired site at, near, or in the target gene, as described below. If the orientation of the first promoter is opposite to the orientation of the target gene sequence and its normal orientation promoter, activation of the first promoter can result in diminished expression of the gene, as described above.
  • the first DNA construct may also comprise the target gene coding sequence and a promoter 5' to it.
  • the 5' promoter may be constitutive or regulatable.
  • the transcriptional transactivator can be any protein which is not normally present in the eukaryotic cell and which is capable of binding the first promoter and activating reverse orientation transcription of the target gene under the regulatory control of the first promoter.
  • a variety of transcriptional transactivators are known to those of skill in the art.
  • the transcriptional transactivator is LexA, Gal4, or a Tet-repressor-derived transcriptional activator such as the Tet-OnTM "gene switch” (CLONTECH).
  • the second DNA construct comprises a second promoter and a coding sequence for the tians ⁇ iptional transactivator.
  • the second promoter is regulatable so that activation of the first promoter by the transcriptional transactivator can be controlled as desired.
  • the second promoter can be a naturally occurring promoter or can be genetically engineered.
  • the second promoter can be a constitutiv y active promoter or can be a tissue-specific promoter, such as a promoter functional only in eye, muscle, heart, brain, lung, thymus, colon, stomach, liver, lymph node, blood, bladder, or ovary tissue.
  • a cell type-specific promoter for example, a promoter functional only in glia, neurons, T-cells, B-cells, or epithelial cells, can also be used to control the transcription of the transcriptional transactivator.
  • the second promoter is an insect developmental stage- specific promoter, such as a nymph-, larval instar- or molt-specific promoter.
  • the second promoter can be a conditionally regulatable promoter, such as a temperature sensitive promoter or a promoter which is sensitive to a chemical inducer.
  • the first DNA ⁇ nstruct can further comprise a coding sequence for the target gene and a third promoter.
  • the third promoter is the native promoter for the target gene in situ.
  • the third promoter may also be regulatable, as discussed for the first promoter.
  • the third promoter is located 5* to the coding sequence of the target gene and controls transcription of the target gene from 5* to 3'.
  • the distance between the first and third promoters in the first DNA construct can be, for example, 10, 25, 40, 50, 75, 85, 100, 125, 150, 200, 250, 300, 500, 750, 1000, 1500, or 2000 nucleotides.
  • a DNA construct comprising a promoter is introduced into the cell such that the promoter integrates in or near a target gene in reverse orientation to the orientation of the target gene and its native promoter.
  • the promoter can be a naturally occurring promoter or can be genetically engineered. It can be a constitutively active promoter or can be regulatable, for example, a tissue-specific, cell type-specific promoter, or a developmental stage-specific promoter.
  • a conditionally regulatable promoter such as a temperature sensitive promoter or a promoter which is sensitive to a chemical inducer, can also be used to initiate reverse orientation transcription of the target gene.
  • the DNA constructs can be fabricated as is known in the art, using standard recombinant DNA techniques.
  • the DNA constructs can be flanked at each end by nucleotide sequences complementary to sequences in a the target gene, to facilitate integration of the expression constructs into the target gene by homologous recombination.
  • the length of the flanking sequences can vary, for example, from 6, 8, 10, 15, 25, or 50 nucleotides. Longer sequences may also be used.
  • DNA construct can be flanked by P elements, as is known in the art (see, for example, U.S. Patent 4,670,388, inco ⁇ orated herein by reference).
  • at least the first DNA construct is integrated into the genome.
  • the DNA constructs can remain episomal or on a minichromosome.
  • Transgenic insects can also be constructed according to the method of the invention.
  • the transgenic insect is a Drosophila melanogaster. Techniques for constructing transgenic flies are also well known.
  • the first DNA construct comprising a promoter which will control reverse orientation transcription of a gene, can be injected into a Drosophila melanogaster embryo, resulting in a mosaic embryo which contains the first
  • Drosophila will be diminished, as described above.
  • the eukaryotic cell can have one or two wild-type endogenous copies of the target gene or can lack wild-type endogenous copies of the target gene.
  • endogenous copies of the target gene can be mutated or deleted using standard genetic manipulations, including targeted mutagenesis, successive matings of heterozygous strains, and other techniques known in the art. In such cells lacking any wild- type copies of the gene, only the collision mechanism is required to diminish expression of the target gene.
  • the first DNA construct can comprise an identifying marker for distingwshing expression of the target gene in the first
  • DNA construct from expression of the endogenous copy of the target gene For example, a sequence encoding an epitope which can be specifically bound by an antibody, such as a hemagglutinin tag, can be included in the first DNA construct so that expression of the target gene sequence in the construct can be detected immunocytochemically, as is known in the art.
  • an antibody such as a hemagglutinin tag
  • This example illustrates the construction of brahma transgenes containing promoters which can be regulated by Gal4.
  • Promoters which can be regulated by Gal4 can be inserted into a P-element vector carrying the white gene and a coding sequence for the brahma gene.
  • the brahma gene sequence used for these constructs encodes a hemagglutinin (HA)-epitope tagged brahma protein under the control of the native brahma promoter.
  • HA hemagglutinin
  • Three constructs can be made: (1) a construct in which the regulated promoter is placed in opposing orientation 3' to the endogenous brahma promoter within an intron which is less than 1 kilobase from the endogenous brahma promoter, (2) a construct in which the regulated promoter is placed in the same orientation as the endogenous brahma promoter and located 5' to the endogenous promoter, and (c) a construct in which the regulated promoter is placed in opposite orientation to the endogenous brahma promoter and 3' to the brahma polyadenylation site.
  • Brahma gene expression in insect cells can be detected using antibodies to brahma protein.
  • Expression of the brahma transgene can be detected using antibodies to HA.
  • This example demonstrates the generation of transgenic strains of Drosophila melanogaster.
  • a DNA construct for example, one of the brahma constructs described in Example 1, above, can be injected into Drosophila melanogaster embryos, as is known in the art, resulting in mosaic embryos. These mosaic embryos mature after two to three weeks into mosaic adults. Individual adults are thai back crossed to a white strain of fly. The resulting heterozygous transformants can be mapped to the X, second, or third chromosomes within one to two generations. Expression of the tagged brahma proton can be measured throughout the developing embryo. Transformants can be crossed to various Gal4 driver lines of flies, as are known in the art, such as lines which express Gal4 in stripes in the developing embryo, under regulatory control of the engrailed or even-skipped promoter. These flies express Gal4 along the anterior-posterior axis of the developing embryo in fourteen and seven stripes, respectively.
  • Gal4 Expression of Gal4 can be monitored using antibodies against Gal4.
  • the resulting effect of expression of the brahma transgene can be measured with a monoclonal antibody against the HA protein, as previously described.
  • Endogenous brahma expression can be measured using antibodies which specifically bind to the brahma protein.
  • Double-label immunofluoresence can reveal if activation of Gal4 interferes with brahma expression.
  • a convenient internal control for this experiment is that expression of brahma should not be altered in the regions between the stripes of Gal4-expressing cells.
  • a eukaryotic cell for observing altered expression of a gene comprising: a first DNA construct comprising an ectopic promoter, wherein the ectopic promoter controls transcription of a gene in the eukaryotic cell; and a second DNA construct comprising a regulatable promoter and a coding sequence for a foreign transcriptional transactivator, wherein the regulatable promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transactivator activates the ectopic promoter, whereby expression of the gene is altered.
  • the eukaryotic cell of item 1 wherein the foreign transcriptional transactivator is selected from the group consisting of Gal4, LexA, and a Tet-repressor- derived transactivator.
  • a method of altering the expression of a gene in an eukaryotic cell comprising the steps of: introducing into the eukaryotic cell a DNA construct comprising an ectopic promoter, wherein the ectopic promoter controls transcription of a gene in the eukaryotic cell; and introducing into the eukaryotic cell a DNA construct comprising a regulatable promoter sequence and a coding sequence for a foreign transcriptional transactivator, wherein the regulatable promoter controls transcription of the transcriptional transactivator and the transcriptional transactivator activates the ectopic promoter, whereby expression of the gene is altered.
  • the ectopic promoter is integrated 3' to a termination codon of a gene and wherein the ectopic promoter controls reverse orientation transcription of the gene.
  • a transgenic eukaryotic organism wherein cells of the transgenic eukaryotic organism comprise: a first DNA construct comprising an ectopic promoter, wherein the ectopic promoter controls transcription of a gene in the cells of the transgenic eukaryotic organism; and a second DNA construct comprising a regulatable promoter and a coding sequence for a foreign transcriptional transactivator, wherein the regulatable promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transactivator a ⁇ ivates the e ⁇ opic promoter, whereby expression of the target gene is altered.
  • the transgenic eukaryotic organism of item 9 wherein the ectopic promoter is integrated 3' to a termination codon of a gene and wherein the e ⁇ opic promoter controls reverse orientation transcription of the gene.
  • transgenic eukaryotic organism of item 9 wherein the ectopic promoter is integrated 5' to an initiation codon of a gene and wherein the ectopic promoter controls transcription of the gene.
  • transgenic eukaryotic organism of item 9 wherein the foreign transcriptional transa ⁇ ivator is selected from the group consisting of Gal4, LexA, and a Tet-repressor-derived transa ⁇ ivator.
  • An insect cell for observing diminished expression of a target gene comprising: a first DNA constru ⁇ comprising a first promoter, wherein the first promoter controls reverse orientation transcription of the target gene in the insect cell; and a second DNA constru ⁇ comprising a second promoter and a coding sequence for a transcriptional transactivator, wherein the second promoter controls the expression of the transcriptional transa ⁇ ivator and wherein the transcriptional transactivator a ⁇ ivates the first promoter, whereby expression of the target gene is diminished.
  • a method of diminishing the expression of a target gene in an insect cell comprising the steps of: introducing into the insect cell a first DNA construct comprising a first promoter, wherein the first promoter controls reverse orientation transcription of the target gene in the insect cell; and introducing into the insect cell a second DNA construct comprising a second promoter sequence and a coding sequence for a transcriptional transactivator, wherein the second promoter controls transcription of the transcriptional transactivator and the transcriptional transactivator activates the first promoter, whereby expression of the target gene is diminished.
  • the first DNA construct further comprises a coding sequence of the target gene and a third promoter, wherein the first promoter is located 3 1 to the coding sequence and wherein the third promoter is located 5' to the coding sequence, and wherein the orientation of the first promoter is opposite to the orientation of the third promoter and the coding sequence.
  • the inse ⁇ cell is a fly cell.
  • a transgenic insect wherein cells of the transgenic inse ⁇ comprise: a first DNA construct comprising a first promoter, wherein the first promoter controls reverse orientation transcription of a target gene in the insect cell; and a second DNA construct comprising a second promoter and a coding sequence for a transcriptional transa ⁇ ivator, wherein the second promoter controls the expression of the transcriptional transa ⁇ ivator and wherein the transcriptional transa ⁇ ivator activates the first promoter, whereby expression of the target gene is diminished.
  • the transgenic inse ⁇ of item 31 wherein the first DNA constru ⁇ further comprises a coding sequence for the target gene and a third promoter, wherein the first promoter is located 3' to the coding sequence and wherein the third promoter is located 5' to the coding sequence, and wherein the orientation of the first promoter is opposite to the orientation of the third promoter and the coding sequence.
  • transgenic inse ⁇ of item 31 wherein the transcriptional transa ⁇ ivator is selected from the group consisting of Gal4, LexA, and a Tet-repressor-derived transa ⁇ ivator.
  • An insect cell for observing diminished expression of a target gene comprising a DNA constru ⁇ comprising a promoter, wherein the promoter controls reverse orientation transcription of the target gene in the insect cell.
  • a method of diminishing the expression of a target gene in an insect cell comprising the step of introducing into the insect cell a DNA construct comprising a promoter, wherein the promoter controls reverse orientation transcription of the target gene in the insect cell, whereby expression of the target gene is diminished.
  • transgenic insect of item 50 wherein the fly is a Drosophila melanogaster.
  • transgenic insect of item 49 wherein the DNA constru ⁇ is integrated into the genome of cells of the transgenic insect.
  • the promoter is a regulatable promoter.
  • a DNA constru ⁇ for altering expression of a target gene in a eukaryotic cell comprising: a first promoter, a coding sequence of the target gene, and a second promoter, wherein the first promoter controls reverse orientation transcription of the target gene in the cell, wherein the first promoter is located 3' to the coding sequence, and wherein the second promoter is located 5' to the coding sequence, and wherein the orientation of the first promoter is opposite to the orientation of the third promoter and the coding sequence; wherein the first promoter is regulated.
  • the DNA constru ⁇ of item 54 wherein the first promoter is regulated in a tissue-specific manner.
  • a eukaryotic cell comprising the DNA construct of item 54.
  • the eukaryotic cell of item 58 further comprising a second DNA construct comprising a third promoter and a coding sequence for a transcriptional transa ⁇ ivator, wherein the third promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transa ⁇ ivator a ⁇ ivates the first promoter, whereby expression of the target gene is altered.
  • a DNA constru ⁇ for altering expression of a target gene in a eukaryotic cell comprising: a first promoter, a coding sequence of the target gene, and a second promoter, wherein the first promoter controls reverse orientation transcription of the target gene in the cell, wherein the first promoter is located 3* to the coding sequence, and wherein the second promoter is locate 5' to the coding sequence, and wherein the orientation of the first promoter is opposite to the orientation of the third promoter and the coding sequence; wherein the second promoter is regulated.
  • a eukaryotic cell comprising the DNA constru ⁇ of item 61.
  • the eukaryotic cell of item 65 further comprising a second DNA construct comprising a third promoter and a coding sequence for a transcriptional transa ⁇ ivator, wherein the third promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transactivator a ⁇ ivates the second promoter, whereby expression of the target gene is altered.
  • the second promoter is a stronger promoter than the first promoter.

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Abstract

L'invention concerne une méthode permettant de modifier sélectivement l'expression d'un gène dans une cellule eucaryote. L'expression du gène peut être manipulée à différents points du développement ou dans des tissus particuliers. Cette méthode est particulièrement utile pour observer les effets produits par la modification de l'expression d'un gène inconnu ou d'un gène dont la fonction est inconnue. Elle peut être utilisée chez les cellules eucaryotes in vitro ou in vivo, par exemple pour construire des organismes eucaryotes transgéniques.
PCT/US1997/016893 1996-09-23 1997-09-23 Integration aleatoire et ciblee d'un promoteur pour l'analyse de genes WO1998012337A1 (fr)

Priority Applications (1)

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AU46485/97A AU4648597A (en) 1996-09-23 1997-09-23 Random and targeted promoter integration for gene analysis

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US2688896P 1996-09-23 1996-09-23
US60/026,888 1996-09-23
US2675696P 1996-09-25 1996-09-25
US60/026,756 1996-09-25
US3379396P 1996-12-30 1996-12-30
US60/033,793 1996-12-30

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EP1210875A1 (fr) * 2000-12-04 2002-06-05 Aventis CropScience GmbH Modification de l'expression des gnes par l'utilisation d'ARN double brin (dsRNA)
US6602686B1 (en) 1997-09-26 2003-08-05 Athersys, Inc. Compositions and method for non-targeted activation of endogenous genes
FR3093897A1 (fr) 2019-03-20 2020-09-25 Michel Gigoux Boucle déployante avec longerons rigides parallèles

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7419828B2 (en) 1997-09-26 2008-09-02 Abt Holding Company Compositions and methods for non-targeted activation of endogenous genes
US7033782B2 (en) 1997-09-26 2006-04-25 Athersys, Inc. Compositions and methods for non-targeted activation of endogenous genes
US7842792B2 (en) 1997-09-26 2010-11-30 Abt Holding Company Compositions and methods for non-targeted activation of endogenous genes
US7785831B2 (en) 1997-09-26 2010-08-31 Abt Holding Company Compositions and methods for non-targeted activation of endogenous genes
US6897066B1 (en) 1997-09-26 2005-05-24 Athersys, Inc. Compositions and methods for non-targeted activation of endogenous genes
US6602686B1 (en) 1997-09-26 2003-08-05 Athersys, Inc. Compositions and method for non-targeted activation of endogenous genes
US7569220B2 (en) 1997-09-26 2009-08-04 Abt Holding Company Compositions and methods for non-targeted activation of endogenous genes
US6670185B1 (en) 1997-09-26 2003-12-30 Athersys, Inc. Compositions and methods for non-targeted activation of endogenous genes
WO2000049162A3 (fr) * 1999-02-19 2000-12-28 Athersys Inc Compositions et methodes permettant l'activation non ciblee de genes endogenes
WO2000049162A2 (fr) * 1999-02-19 2000-08-24 Athersys, Inc. Compositions et methodes permettant l'activation non ciblee de genes endogenes
WO2002046432A3 (fr) * 2000-12-04 2002-10-17 Aventis Cropscience Gmbh Identification de molecules cibles insecticides au moyen d'arn bicatenaire
WO2002046432A2 (fr) * 2000-12-04 2002-06-13 Bayer Cropscience Gmbh Identification de molecules cibles insecticides au moyen d'arn bicatenaire
EP1210875A1 (fr) * 2000-12-04 2002-06-05 Aventis CropScience GmbH Modification de l'expression des gnes par l'utilisation d'ARN double brin (dsRNA)
FR3093897A1 (fr) 2019-03-20 2020-09-25 Michel Gigoux Boucle déployante avec longerons rigides parallèles

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