WO1998012337A1

WO1998012337A1 - Random and targeted promoter integration for gene analysis

Info

Publication number: WO1998012337A1
Application number: PCT/US1997/016893
Authority: WO
Inventors: Klaus Giese; John Tamkun
Original assignee: Chiron Corporation; Regents Of The University Of California
Priority date: 1996-09-23
Filing date: 1997-09-23
Publication date: 1998-03-26
Also published as: AU4648597A

Abstract

A method of selectively altering the expression of a gene in a eukaryotic cell is disclosed. Expression of the gene can be manipulated at various points in development or in particular tissues. 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 be used in eukaryotic cells in vitro or in vivo, for example by constructing transgenic eukaryotic organisms.

Description

RANDOM AND TARGETED PROMOTER INTEGRATION FOR GENE ANALYSIS

This application claims the benefit of the following copending provisional applications: Serial No. 60/026,888, filed September 23, 1996, Serial No. 60/026,756, filed September 25, 1996, and Serial No. 60/033,793, filed December 30, 1996, each of which is incorporated by reference herein.

TECHNICAL AREA OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Analysis of eukaryotic gene function is typically accomplished by examining the biological effects of defined mutations in known genes. Such mutations can be identified by phenotypic selection of mutant organisms or can be created by site-directed mutagenesis or homologous recombination targeting mutations to a particular gene of interest. For most rriarnmalian genes, the rarity of targeted recombinants and phenotypically evident mutants limits the utility of these approaches. Thus, there is a need in the art for methods which can be used to observe eukaryotic, particularly mammalian, gene function. SUMMARY OF THE INVENTION

It is an object of the invention to provide a eukaryotic cell for observing altered expression of a gene. It is another object of the invention to provide a method of altering the expression of a gene in a eukaryotic cell.

It is yet another object of the invention to provide a transgenic eukaryotic organism.

It is an object of the invention to provide an insect cell for observing diminished expression of a target gene. It is another object of the invention to provide a method of diminishing the expression of a target gene in an insect cell.

It is yet another object of the invention to provide a transgenic insect.

These and other objects of the invention are provided by one or more of the embodiments described below. 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 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.

One embodiment of the invention provides an 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.

Another embodiment of the invention provides a method of diminishing the expression of a target gene in an insect cell. 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.

n TATT,F,D DESCRIPTION OF THE PREFERRED EMBODIMENTS

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.

Genes involved in development, such as homeotic genes, 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. According to the method of 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.

In order to alter expression of a gene, 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. 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.

If 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. Alternatively, if 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. Similarly, if the ectopic promoter is tissue or developmentally regulated, it will be activated only under appropriate conditions in the appropriate tissue or developmental stage.

According to one method, two DNA constructs are introduced into the eukaryotic cell. 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. Alternatively, the ectopic promoter is spatially or temporally regulated, as discussed above.

If integrated into the cell's DNA (vΛthin 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). While not wishing to be bound by any particular theory, we believe that both physical collision of the transcription machineries and other mechanisms, such as the generation of positive superhelical tension by the RNA polymerase which imtiates at the promoters, contribute to effect diminished expression of the affected gene.

Second, to the extent that reverse orientation transcription of the gene proceeds without disruption, 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. A variety of transcriptional transactivators are known to those of skill in the art. Preferably, the transcriptional transactivator is LexA, Gal4, or a Tet-repressor-derived transcriptional activator such as the Tet-On™ "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. Alternatively, 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. Preferably, 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. In one embodiment of the invention 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.

In another embodiment of the invention, 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 cateφillars, 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 scoφions. Preferably 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 metamoφhosis, 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.

In order to control expression of the target gene, 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 5¹ 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. Preferably 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. In the presence of the transcriptional transactivator, however, 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. Preferably one promoter is weaker and one is stronger. By regulating transcription from the stronger promoter, 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. According to one method, 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.

Preferably, the transcriptional transactivator is LexA, Gal4, or a Tet-repressor-derived transcriptional activator such as the Tet-On™ "gene switch" (CLONTECH).

The second DNA construct comprises a second promoter and a coding sequence for the tiansαiptional transactivator. Preferably, 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. In one embodiment, the second promoter is an insect developmental stage- specific promoter, such as a nymph-, larval instar- or molt-specific promoter. Alternatively, 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. Preferably, 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. By using combinations of stronger and weaker promoters as first and third prmoters, the gene can be selectively activated or repressed according to a desired cue.

According to another method, 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. For integration into insect cell DNA, the

DNA construct can be flanked by P elements, as is known in the art (see, for example, U.S. Patent 4,670,388, incoφorated herein by reference). Preferably, at least the first DNA construct is integrated into the genome. Alternatively, the DNA constructs can remain episomal or on a minichromosome. Transgenic insects can also be constructed according to the method of the invention. Preferably the transgenic insect is a Drosophila melanogaster. Techniques for constructing transgenic flies are also well known. For example, 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

DNA construct in some of its cells. This method is described in U.S. Patent 4,670,388. Additionally after the embryo matures into a mosaic adult, it can be mated with a strain of I osophila melanogaster which has been genetically engineered using standard techniques to express a transcriptional transactivator, for example, Gal4, LexA, or a Tet repressor- derived activator, under the control of a second, regulatable promoter. As disclosed above, activation of the second, regulatable promoter can result in the transcriptional transactivator' s expression in either temporally or spatially restricted patterns. The offspring of the two strains ofDrosophila will express the targeted gene in tissues or cells or at stages when the transcriptional transactivator is not expressed. In the presence of the transcriptional transactivator, the expression of the targeted gene in these transgenic

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. Those of skill in the art will recognize that 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.

If the cell has an endogenous copy 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.

The following are provided for exemplification puφoses only and are not intended to limit the scope of the invention which has been described in broad terms above.

EXAMPLE 1

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. 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.

Depending on the specificity of the promoter which is used to regulate the expression of Gal4 in these cells, the effects of altered patterns of brahma gene expression in various cell types and at various times during the life of the fly can be studied.

EXAMPLE 2

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.

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.

SYNOPSIS OF THE INVENTION

1. 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. 2. The eukaryotic cell of item 1 wherein the ectopic promoter is integrated 3' to a teπnination codon of a gene and wherein the ectopic promoter controls reverse orientation transcription of the gene.

3. The eukaryotic cell of item 1 wherein the ectopic promoter is integrated 5' to an initiation codon of a gene and wherein the ectopic promoter controls transcription of the gene.

4. 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. 5. 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. 6. The method of item 5 wherein 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.

7. The method of item 5 wherein the ectopic promoter is integrated 5' to an initiation codon of a gene and wherein the ectopic promoter controls transcription of the gene.

8. The method of item 5 wherein the foreign transcriptional transactivator is selected from the group consisting of Gal4, LexA, and a Tet-repressor-derived transactivator.

9. 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 aαivates the eαopic promoter, whereby expression of the target gene is altered. 10. The transgenic eukaryotic organism of item 9 wherein the ectopic promoter is integrated 3' to a termination codon of a gene and wherein the eαopic promoter controls reverse orientation transcription of the gene.

11. The 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.

12. The transgenic eukaryotic organism of item 9 wherein the foreign transcriptional transaαivator is selected from the group consisting of Gal4, LexA, and a Tet-repressor-derived transaαivator.

13. An insect cell for observing diminished expression of a target gene, comprising: a first DNA construα comprising a first promoter, wherein the first promoter controls reverse orientation transcription of the target gene in the insect cell; and a second DNA construα comprising a second promoter and a coding sequence for a transcriptional transactivator, wherein the second promoter controls the expression of the transcriptional transaαivator and wherein the transcriptional transactivator aαivates the first promoter, whereby expression of the target gene is diminished.

14. The inseα cell of item 13 wherein the first DNA construα further comprises a coding sequence of 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. 15. The insect cell of item 13 wherein the inseα is a fly.

16. The inseα cell of item 15 wherein the fly is a Drosophila melanogaster.

17. The inseα cell of item 13 wherein the inseα cell does not contain a native wild-type copy of the tφget gene.

18. The insect cell of item 13 wherein the first DNA construα is integrated into the genome of the inseα cell.

19. The inseα cell of item 14 wherein the first DNA construα further comprises an identifying marker for distinguishing expression of the target gene in the first DNA construct from expression of an endogenous copy of the target gene.

20. The inseα cell of item 13 wherein the transcriptional transaαivator is seleαed from the group consisting of Gal4, LexA, and a Tet-repressor-derived transaαivator.

21. The inseα cell of item 13 wherein the second promoter is a regulatable promoter.

22. 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.

23. The method of item 22 wherein the first DNA construct further comprises a coding sequence of the target gene and a third promoter, wherein the first promoter is located 3¹ 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. 24. The method of item 22 wherein the inseα cell is a fly cell.

25. The method of item 24 wherein the fly cell is a Drosophila melanogaster cell.

26. The method of item 22 wherein the inseα cell does not contain a native wild-type copy of the target gene. 27. The method of item 22 wherein the first DNA construct is integrated into the genome of the insect cell.

28. The method of item 23 wherein the first DNA construα further comprises an identifying marker for distinguishing expression of the target gene in the first DNA construα from expression of an endogenous copy of the target gene. 29. The method of item 22 wherein the transcriptional transaαivator is seleαed from the group consisting of Gal4, LexA, and a Tet-repressor-derived transaαivator.

30. The method of item 22 wherein the second promoter is a regulatable promoter. 31. A transgenic insect wherein cells of the transgenic inseα 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 transaαivator, wherein the second promoter controls the expression of the transcriptional transaαivator and wherein the transcriptional transaαivator activates the first promoter, whereby expression of the target gene is diminished.

32. The transgenic inseα of item 31, wherein the first DNA construα 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. 33. The transgenic inseα of item 31 wherein the transgenic inseα is a fly.

34. The transgenic inseα of item 33 wherein the fly is a Drosophila melanogaster.

35. The transgenic inseα of item 31 wherein the inseα cell does not contain a native wild-type copy of the target gene. 36. The transgenic inseα of item 32 wherein the first DNA construα further comprises an identifying marker for distinguishing expression of the target gene in the first DNA construα from expression of an endogenous copy of the target gene.

37. The transgenic inseα of item 31 wherein the transcriptional transaαivator is selected from the group consisting of Gal4, LexA, and a Tet-repressor-derived transaαivator.

38. The transgenic insect of item 31 wherein the second promoter is a regulatable promoter.

39. An insect cell for observing diminished expression of a target gene, comprising a DNA construα comprising a promoter, wherein the promoter controls reverse orientation transcription of the target gene in the insect cell.

40. The inseα cell of item 39 wherein the insect is a fly.

41. The inseα rail of item 40 wherein the fly is a Drosophila melanogaster.

42. The inseα cell of item 39 wherein the DNA construct is integrated into the genome of the insect cell. 43. The inseα cell of item 39 wherein the promoter is a regulatable promoter.

44. 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.

45. The method of item 44 wherein the insect is a fly.

46. The method of item 45 wherein the fly is a Drosophila melanogaster.

47. The method of item 44 wherein the DNA construα is integrated into the genome of the insect cell.

48. The method of item 44 wherein the promoter is a regulatable promoter.

49. A transgenic inseα wherein cells of the transgenic inseα comprise a DNA construct comprising a promoter, wherein the promoter controls reverse orientation transcription of a target gene. 50. The transgenic insect of item 49 wherein the insect is a fly.

51. The transgenic insect of item 50 wherein the fly is a Drosophila melanogaster.

52. The transgenic insect of item 49 wherein the DNA construα is integrated into the genome of cells of the transgenic insect. 53. The transgenic insect of item 49 wherein the promoter is a regulatable promoter.

54. A DNA construα 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. 55. The DNA construα of item 54 wherein the first promoter is regulated in a tissue-specific manner.

56. The DNA construα of item 54 wherein the first promoter is regulated in a developmental stage-specific manner.

57. The DNA construct of item 54 wherein the first promoter is regulated by a transcriptional transaαivator which is not endogenous to the eukaryotic cell.

58. A eukaryotic cell comprising the DNA construct of item 54.

59. The eukaryotic cell of item 58 further comprising a second DNA construct comprising a third promoter and a coding sequence for a transcriptional transaαivator, wherein the third promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transaαivator aαivates the first promoter, whereby expression of the target gene is altered.

60. The eukartoic cell of item 58 wherein the first promoter is a stronger promoter than the second promoter. 61. A DNA construα 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.

62. The DNA construα of item 61 wherein the first promoter is regulated in a tissue-specific manner. 63. The DNA construα of item 61 wherein the first promoter is regulated in a developmental stage-specific manner.

64. The DNA construα of item 61 wherein the first promoter is regulated by a transcriptional transaαivator which is not endogenous to the eukaryotic cell.

65. A eukaryotic cell comprising the DNA construα of item 61. 66. The eukaryotic cell of item 65 further comprising a second DNA construct comprising a third promoter and a coding sequence for a transcriptional transaαivator, wherein the third promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transactivator aαivates the second promoter, whereby expression of the target gene is altered. 67. The eukaryotic cell of item 65 wherein the second promoter is a stronger promoter than the first promoter.

Claims

1. An eukaryotic cell for observing altered expression of a gene, comprising: a first DNA construα comprising an eαopic promoter, wherein the ectopic promoter controls transcription of a gene in the eukaryotic cell; and a second DNA construα comprising a regulatable promoter and a coding sequence for a foreign transcriptional transactivator, wherein the regulatable promoter controls the expression of the transcriptional transaαivator and wherein the transcriptional transaαivator aαivates the eαopic promoter, whereby expression of the gene is altered.

2. A method of altering the expression of a gene in an eukaryotic cell, comprising the steps of: integrating into the DNA of the eukaryotic cell a first 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 second 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.

3. 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 construα comprising a regulatable promoter and a coding sequence for a foreign transcriptional transaαivator, wherein the regulatable promoter controls theexpression of the transcriptional transaαivator and wherein the transcriptional transaαivator aαivates the eαopic promoter, whereby expression of the gene is altered.

4. An inseα cell for observing diminished expression of a target gene, comprising: a first DNA construα comprising a first promoter, wherein the first promoter controls reverse orientation transcription of the target gene in the inseα cell; and a second DNA construα comprising a second promoter and a coding sequence for a transcriptional transactivator, wherein the second promoter controls the expression of the transcriptional transaαivator and wherein the transcriptional transaαivator aαivates the first promoter, whereby expression of the target gene is diminished.

5. 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.

6. A transgenic inseα wherein cells of the transgenic insect 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 construα comprising a second promoter and a coding sequence for a transcriptional transactivator, wherein the second promoter controls the expression of the transcriptional transactivator and wherein the transcriptional transaαivator aαivates the first promoter, whereby expression of the target gene is diminished.

7. An inseα cell for observing diminished expression of a target gene, comprising a DNA construα comprising a promoter, wherein the promoter controls reverse orientation transcription of the target gene in the inseα cell.

8. 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.

9. A transgenic inseα wherein cells of the transgenic inseα comprise a DNA construct comprising a promoter, wherein the promoter controls reverse orientation transcription of a target gene.

10. DNA construα 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 5¹ 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.

11. A DNA construα for altering expression of a target gene in a eukaryotic cell, comprising: a first promoter, a coding sequence of the targα 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 second promoter is regulated.