WO2001077350A2 - Compositions and methods for inhibiting gene expression - Google Patents

Abstract

The present invention provides an eukaryotic recombinant vector suited for bi-directional transcription of a transgene to yield both sense and antisense RNA transcripts of the transgene in an eukaryotic cell. The invention vectors are particularly suited for mediating gene silencing in a variety of biological systems. The present invention also provides host cells and transgenic plants comprising the invention vectors. Further provided by the invention are methods of inhibiting expression of an endogenous gene present in an eukaryotic cell. Also included is a method of identifying a biological function(s) of an endogenous gene of interest in an eukaryotic cell by selectively inhibiting the expression of the endogenous gene.

Description

COMPOSITIONS AND METHODS FOR INHIBITING GENE EXPRESSION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. Patent Application 09/545,574, filed April 7, 2000, pending, which is hereby incorporated herein by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

TECHNICAL FIELD This invention is in the field of genetic analysis. Specifically, the invention relates to the generation of a eukaryotic vector that allows bi-directional transcription of a transgene to yield both sense and antisense RNA transcripts from the same transgene. The compositions and methods embodied in the present invention are particularly useful for targeted inhibition of gene expression in a eukaryotic cell.

BACKGROUND OF THE INVENTION

The structure and biological behavior of a cell is determined by the pattern of gene expression within that cell at a given time. Perturbations of gene expression have long been acknowledged to account for a vast number of diseases including, numerous forms of cancer, vascular diseases, neuronal and endocrine diseases. Abnormal expression patterns, in form of amplification, deletion, gene rearrangements, and loss or gain of function mutations, are now known to lead to aberrant behavior of a disease cell. Aberrant gene expression has also been noted as a defense mechanism of certain organisms to ward off the threat of pathogens. One of the major challenges of genetic engineering has been to regulate the expression of targeted genes that are implicated in a wide diversity of physiological responses. While overexpression of an exogenously introduced transgene in a eukaryotic cell is relatively straightforward, targeted inhibition of specific genes has been more difficult to achieve. Traditional approaches for suppressing gene expression, including site-directed gene disruption, antisense RNA or co-suppressor injection, require complex genetic manipulations or heavy dosages of suppressors that often exceeds the toxicity tolerance level of the host cell.

Recently, a new technique, "double- stranded RNA interference" has emerged in the study of gene silencing. Several research groups have demonstrated a marked inhibition of a specific nuclear gene expression in a wide range of eukaryotes by introduction into cells of dsRNA fragments that bear sequence homo logy with the nuclear gene. For instance, Fire et al. (1998) Nature 395: 854 reported the success of gene-specific interference in C. elegans that was mediated by ingested E. coli carrying a prokaryotic vector capable of producing both sense and antisense RNAs of the selected C. elgans genes. Misquitta et al. demonstrated the targeted disruption of nautilus gene in Drosophila melanogaster by injecting into the Drosophila embryo multiple copies of nautilus dsRNA. See Misquitta et al. (1999) PNAS U.S.A. 96:1451-1456. Studies by Ngδ et al. (1998) Proc. Natl. Acad. of Sci. U.S.A., 96: 1451-1456 confirmed that dsRNA interference also occurs in certain protozoan species. Earlier studies by Cogoni et al. and Hamilton et al. suggested that formation of dsRNA play a pivotal role in gene silencing in fungi Neurospora crassa and other plants. See Cogoni et al. (1999) Nature 399: 166-169; Hamilton et al. (1999) Science 286: 950-952; and Waterhouse et al. (1999) PNAS U.S.A. 95: 13959-13964. More recent investigations by Wargelius et al. revealed that this phenomenon is also conserved in vertebrates such as the zebrafish. Wargelius et al. Biochem. Biophys. Res. Commun. 263: 156-161.

Current techniques for achieving RNA mediated gene silencing include: (a) use of prokaryotic vectors capable of transcribing both sense and antisense RNA (Fire et al. (1998) Nature 395: 854; (b) in vitro transcription of individual strands of a selected gene followed by annealing the transcribed sense and antisense RNAs (see, e.g. Misquitta et al. (1999) PNAS U.S.A. 96: 1451-1456); and possibly (c) viruses induced gene silencing (see, e.g. Angell et al. (1997) EMBO Journal 16: 3675-3684; Angell et al. (1999) Plant Journal 20: 357-362). However, these methods bear a number of intrinsic limitations. First, none of these methods employs gene delivery vehicles that are applicable for consistent and persistent inhibition of gene expression in a eukaryote. Second, these existing methods do not necessarily result in production of a substantially homogenous population of dsRNAs. Notably, the in vitro preparation of double-stranded RNAs by transcribing and annealing sense RNA transcripts to antisense transcripts is time consuming, labor intensive, and not amenable for mass production or high-throughput analyses. Thus, there remains a considerable need for compositions and methods to effect dsRNA-mediated gene silencing. An ideal reagent would be a self-replicating vector that is (a) capable of autonomous replication and expression of a selected transgene in a eukaryotic cell; and (b) capable of yielding both sense and antisense RNA transcripts from the same transgene, so as to effect production of dsRNA transcripts in a eukaryotic host cell. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION A principal aspect of the present invention is the design of a eukaryotic recombinant vector to effect gene silencing in a eukaryotic cell that is susceptible to dsRNA-mediated reduction of gene expression. Such a vector allows bi-directional transcription of a transgene to yield both sense and antisense RNA transcripts of the same transgene in a eukaryotic cell. While not being bound to any one theory, the production of dsRNAs induces transcriptional and/or post- transcriptional gene silencing in the host cell. Accordingly, the present invention provides a recombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene fragment in a eukaryotic host cell. In one aspect of this embodiment, each of the overlapping transcription units of the vector comprises a promoter and a terminator that are arranged in one of the configurations shown in Figure 2(a)-(d). The promoter can be constitutive or inducible; it can be active in all tissues and cell types of an organism or operative only in selected tissues (i.e. tissue-specific).

In another aspect, the recombinant vector comprises a viral replicon that is derived from a DNA virus. Such DNA viruses can be selected from the group consisting of Geminivirus, Caulimoviridae, Badnaviridae, Circoviridae,

Circinoviridae, Parvoviridae, Papovaviridae, Polyomaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae, Baculoviridae, Hepadnaviridae, Retroviridae, Gyrovirus, Nanovirus, and African Swine Fever virus.

In yet another aspect, the subject vector is capable of autonomous replication in a eukaryotic cell.

In still another aspect, the subject vector is capable of inhibiting expression of genes endogenous to a eukaryotic host cell. Non- limiting representative eukaryotic cells whose gene expression can be inhibited upon introduction of the subject vectors are fungi, yeast cells, plant cells, inset, avian, mammalian or other animal cells. Preferably, the vectors effect a reduced expression of an endogenous gene that is substantially homologous to the transgene contained in the overlapping transcription units of the vectors. More preferably, delivery of the vectors into a suitable host cell results in a phenotypic change of the host cell. In certain preferred embodiments, the endogenous gene is native to the host cell. The endogenous gene can also be heterologous to the host cell. In some embodiments, the endogenous gene is a pathogenic gene derived from one or more members of the group consisting of virus, bacterium, fungus, and protozoa. The transgene carried in the vector can be a nucleotide sequence that encodes a membrane protein, a cytosolic protein, a secreted protein, a nuclear protein, or a chaperon protein. The present invention also provides host cells transformed with the invention vectors. The present invention further provides a transgenic plant comprising a eukaryotic recombinant vector of the present invention.

Also provided by the present invention is a kit for generating a double- stranded RNA transcript in a eukaryotic cell that contains the subject vectors in suitable packaging.

Further embodied in the present invention is a method of inhibiting expression of an endogenous gene present in a eukaryotic cell. The method involves: (a) providing a eukaryotic recombinant vector containing a transgene that is substantially homologous to the endogenous gene; (b) introducing the eukaryotic recombinant vector into the eukaryotic cell; and (c) culturing the eukaryotic cell of (b) under conditions favorable for expression of both sense and antisense RNA transcripts from the transgene that is contained in the transcription units of the vector, and thereby inhibiting expression of the corresponding endogenous gene in the eukaryotic cell.

Also included in the present invention is a method of identifying a biological function(s) of an endogenous gene of interest in a eukaryotic cell by selectively inhibiting the expression of the endogenous gene. The method comprises: (a) providing a eukaryotic recombinant vector containing a transgene that is substantially homologous to the endogenous gene; (b) introducing the eukaryotic recombinant vector of (a) into the eukaryotic cell; (c) culturing the eukaryotic cell of (b) under conditions favorable for expression of both sense and antisense RNA transcripts from the transgene contained in the eukaryotic recombinant vector and thereby inhibiting expression of the endogenous gene in the eukaryotic cell; and (d) determining one or more phenotypic changes in the eukaryotic cell that correlate with the inhibited expression of the endogenous gene, thereby identifying the biological function(s) of the endogenous gene in the eukaryotic cell. In essence, the subject methods allow the creation of a transient or more long-term gene-specific knock-out system for analyzing the biological function of any endogenous gene of interest.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the process for production of dsRNA transcripts by a subject vector containing two overlapping transcription units.

Figure 2 (a)-(d) depict four different configurations of the overlapping transcription units of the subject vectors.

Figure 3 is a schematic representation of an exemplary construct MSVLSB- 6.

Figure 4 depicts the nucleotide sequence of the vector pMSVLSB-1 (SEQ ID NO: 9) described in Examples 1-2. Figure 5 depicts the nucleotide sequence of the vector pMSVLSB-2 (SEQ ID NO: 10) described in Examples 1-2.

Figure 6 depicts the nucleotide sequence of the vector pMSVLSB-3 (SEQ ID NO:l 1) described in Examples 1-2. Figure 7 depicts the nucleotide sequence of the vector pMSVLSB-4 (SEQ ID

NO:12) described in Examples 1-2.

Figure 8 depicts the nucleotide sequence of the vector pMSVLSB-5 (SEQ ID NO: 13) described in Examples 1-2.

Figure 9 depicts the nucleotide sequence of the vector pMSVLSB-6 (SEQ ID NO: 14) described in Examples 1-2.

MODES FOR CARRYING OUT THE INVENTION Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

General Techniques: The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See, e.g., Matthews, PLANT VIROLOGY, 3^rd edition (1991); Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2^nd edition (1989); CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. Definitions:

A "plant cell" refers to the structural and physiological unit of plants, consisting of a protoplast and the cell wall. A "protoplast" is an isolated cell without cell walls, having the potency for regeneration into cell culture, tissue or whole plant.

A "host cell" includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

The terms "polynucleotide", "nucleotides" and "oligonucleotides" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. "Genes of a specific developmental origin" refer to genes expressed at certain but not all developmental stages. For instance, a gene may be of embryonic or adult origin depending on the stage during which the gene is expressed.

A "disease-associated" or "disease-causing" gene refers to any gene which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease- associated gene also refers to gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at normal or abnormal level.

A gene "database" denotes a set of stored data which represent a collection of sequences including nucleotide and peptide sequences, which in turn represent a collection of biological reference materials.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as "transcript") is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectedly referred to as gene product. Ifthe polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

"Differentially expressed", as applied to nucleotide sequence or polypeptide sequence in a subject, refers to over-expression or under-expression of that sequence when compared to that detected in a control. Underexpression also encompasses absence of expression of a particular sequence as evidenced by the absence of detectable expression in a test subject when compared to a control.

"Differential expression" refers to alterations in the abundance or the expression pattern of a gene product. A "primer" is a short polynucleotide, generally with a free 3' -OH group, that binds to a target or "template" potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. The term "hybridize" as applied to a polynucleotide refers to the ability of the polynucleotide to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues in a hybridization reaction. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. Hybridization can be performed under conditions of different "stringency".

Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide, and the washing procedure. Higher stringency conditions are those conditions, such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 C in 1 x SSC.

When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called "annealing" and those polynucleotides are described as "complementary". A double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. "Complementarity" or "homology" (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules. In the context of polynucleotides, a "linear sequence" or a "sequence" is an order of nucleotides in a polynucleotide in a 5' to 3' direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polynucleotide. A "partial sequence" is a linear sequence of part of a polynucleotide which is known to comprise additional residues in one or both directions.

The terms "cytosolic", "nuclear" and "secreted" as applied to cellular proteins specify the extracellular and or subcellular location in which the cellular protein is mostly localized. Certain proteins are "chaperons", capable of translocating back and forth between the cytosol and the nucleus of a cell.

A "subject" as used herein refers to a biological entity containing expressed genetic materials. The biological entity is preferably can be plant, animal, or microorganisms including bacteria, viruses, fungi, and protozoa. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative". For example, where the purpose of the experiment is to detect a differentially expressed transcript or polypeptide in cell or tissue affected by a disease of concern, it is generally preferable to use a positive control (a subject or a sample from a subject, exhibiting such differential expression and syndromes characteristic of that disease), and a negative control (a subject or a sample from a subject lacking the differential expression and clinical syndrome of that disease). "Heterologous" means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

A "cell line" or "cell culture" denotes bacterial, plant, insect or higher eukaryotic cells grown or maintained in vitro. The descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell.

A "vector" is a nucleic acid molecule, preferably self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of a DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.

An "expression vector" is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An "expression system" usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

A "replicon" refers to a polynucleotide comprising an origin of replication (generally referred to as an on sequence) which allows for replication of the polynucleotide in an appropriate host cell. Examples of replicons include episomes (such as plasmids), as well as chromosomes (such as the nuclear or mitochondrial chromosomes).

A "transcription unit" is a DNA segment capable of directing transcription of a gene or fragment thereof. Typically, a transcription unit comprises a promoter operably linked to a gene or a DNA fragment that is to be transcribed, and optionally regulatory sequences located either upstream or downstream of the initiation site or the termination site of the transcribed gene or fragment.

Vectors of the present invention

A central aspect of the present invention is the design of a recombinant vector suited for bi-directional transcription of a transgene to yield both sense and antisense RNA transcripts of the transgene in a eukaryotic cell. The invention vectors are particularly suited for mediating nuclear gene silencing in a variety of biological systems. Distinguished from the previously described DNA vectors, the subject vectors have the following unique characteristics: (a) the vector replicates and directs expression of a transgene in a eukaryotic cell; and (b) the vector comprises a replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene in a eukaryotic host cell. Several factors apply to the design of vectors having the above-mentioned characteristics. First, the vector comprises a replicon having an origin of replication (generally referred to as an on sequence) which permits replication of the vector in a eukaryotic host cell. A preferred replicon is one comprising viral sequences capable of directing autonomous replication of the vector in an appropriate host cell. Non- limiting examples of viral replicons include sequences derived from DNA viruses such as Geminivirus, Cauhmoviridae, Badnaviridae; Circovindae, Circinovindae, Parvoviridae, Papovavirtdae, Polyomavirtdae, Adenovindae, Herpesviridae, Poxviridae, Iridoviridae, Baculoviridae, Hepadnaviridae, Retrovindae, Gyrovirus,

Nanovirus, and African Swine Fever virus, or the like. In addition to the replication ongin, a replicon typically carries a transcπption unit that directs transcription of a transgene or a fragment thereof to yield a plurality of RNA transcripts.

A second consideration in designing the subject vector is to select two overlapping transcription units By "overlapping" is meant that the two transcπption units directs transcription of both DNA strands of the same transgene to yield a plurality of partially or perfectly double stranded RNA transcnpts. The two overlapping transcription units are typically arranged in an opposing orientation so that each unit can drive transcnption of one of the complementary strands from the same transgene, and thus facilitate the generation of double stranded RNA transcripts Elements within a transcription unit include but are not limited to promoter regions, enhancer regions, repressor binding regions, transcription initiation sites, πbosome binding sites, translation initiation sites, protein encoding regions and introns, and termination sites for transcription and translation. Preferred transcription units are arranged in a configuration shown m Figure 2(a)-(d).

As used herein, a "promoter" is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region located downstream (in the 3' direction) from the promoter. It can be constitutive or inducible. In general, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcnption at levels detectable above background Within the promoter sequence is a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes

The choice of promoters will largely depend on the host cells in which the vector is introduced Commonly employed plant promoters include but are not limited those from agrobactenum, nopahne synthase gene, octopine synthase gene, mannopine synthase, rbcS (small subunit of ribulose bis-phosphate carboxylase). In addition, the promoter sequences may be provided by viral material. Any RNA virus subgenomic promoters described in Dawson et al. Advances in Virus Research. 38:307-342 and WO93/03161 can thus be employed. For animal cells, a variety of robust promoters, both viral and non-viral promoters, are known in the art.

Non-limiting representative viral promoters include CMV, the early and late promoters of SV40 virus, promoters of various types of adenoviruses (e.g. adenovirus 2) and adeno-associated viruses. It is also possible, and often desirable, to utilize promoters normally associated with a desired transgene sequence, provided that such control sequences are compatible with the host cell system. See Goeddel et al., Gene Expression Technology Methods in Enzymology Volume 185, Academic Press, San Diego, (1991), Ausubel et al, Protocols in Molecular Biology, Wiley Interscience (1994).

Suitable promoter sequences for other eukaryotic cells such as yeast cells include the promoters for 3-phosphoglycerate kinase, or other glycolytic enzymes, such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. To optimize the yield of double-stranded RNAs formed from the sense and anti-sense strands transcribed by the overlapping units, it is preferable to use two promoters of comparable strength. The relative strength of the promoters can be determined or ascertained by any convention recombinant techniques and methods exemplified herein. Representative techniques are Northern blot hybridization and DNA array-based technologies. An illustrative promoter pair comprises MSV mp promoter and CaMV 35S RNA promoter.

Where desired, heterologous promoters that are removed from their native coding sequences and operatively linked to a transgene which it is not naturally found linked, can be used in constructing the invention vectors. As such, any viral promoters described above can be used to drive the transcription of a non-viral transgenes; promoters of one class of genes can be employed to direct transcription of transgenes coding for other related or unrelated classes of proteins. In certain embodiments of the invention, it is preferable to employ inducible promoters to control the transcription of a transgene. A diverse variety of inducible promoters have been described in the art. Promoters of any endogenous genes whose expressions are inducible by internal or external factors can be employed. Factors applicable for transcription induction include but are not limited to hormones, heat shock, oxygen deficiency, light, stress and various chemicals. Commonly employed inducible promoters are β-gal promoter that is activated upon addition of IPTG; hps70 promoter that is inducible by heat shock; and ribulose-l,5-biphosphate carboxylase (RUBISCO) promoter that is regulated by light.

Tissue-specific promoters may also be used. A vast diversity of tissue specific promoters have been described and employed by artisans in the field.

Representative plant tissue promoters include that of legumin (or other seed storage protein promoters), patatin and the like. Exemplary promoters operative in selective animal tissue include hepatocyte-specifϊc promoters and cardiac muscle specific promoters. Depending on the intended use of the subject vectors, those skilled in the art will know of other suitable tissue-specific promoters applicable for non- constitutive bi-directional transcription.

In constructing the subject vectors, the termination sequences associated with the transgene are also inserted into the 3' end of the sequence desired to be transcribed to provide polyadenylation of the mRNA and/or transcriptional termination signal. The terminator sequence preferably contains one or more transcriptional termination sequences (such as polyadenylation sequences) and may also be lengthened by the inclusion of additional DNA sequence so as to further disrupt transcriptional read-through. Prefened terminator sequences (or termination sites) of the present invention have a gene that is followed by a transcription termination sequence, either its own termination sequence or a heterologous termination sequence. Examples of such termination sequences, including stop codons coupled to various polyadenylation sequences that are known in the art, widely available, and exemplified below. Where the terminator comprises a gene, it can be advantageous to use a gene which encodes a detectable or selectable marker; thereby providing a means by which the presence and/or absence of the terminator sequence (and therefore the corresponding inactivation and/or activation of the transcription unit) can be detected and/or selected. Alternatively, a terminator may simply be a second promoter, arranged in inverted orientation to the promoter described above.

The terminators and promoters of the two overlapping transcription units may take a variety of configurations. In one aspect, terminators 1 and 2 of the overlapping transcription units are arranged to immediately flank the transgene as shown in Figure 2(a). In another aspect, the two terminators are placed at the 5' end or the 3' end of their respective promoters as depicted in Figure 2(b). In other aspects, terminator 1 and promoter 1 are flanked by terminator 2 and promoter 2 as shown in Figure 2(c), or vice versa (see Figure 2(d)). Any other variations in configuring the two overlapping transcription units that permit bi-directional transcription are encompassed by the present invention.

The transgene transcribed by an invention vector can be any gene expressed in a eukaryotic cell. The selection of transgene is determined largely by the intended purpose of the vector. Where the vector is used to inhibit expression of an endogenous gene present in a host cell, the transgene selected are substantially homologous to the target endogenous gene. In general, substantially homologous nucleotide sequences are at least about 60% identical with each other, after alignment of the homologous regions. Preferably, the sequences are at least about 75% identical; more preferably, they are at least about 80% identical; more preferably, they are at least about 90% identical; still more preferably, the sequences are 95% identical.

Sequence alignment and homology searches are often determined with the aid of computer methods. A variety of software programs are available in the art. Non-limiting examples of these programs are Blast (http://www.ncbi.nlm.nih.gov/BLAST/), Fasta (Genetics Computing Group package, Madison, Wisconsin), DNA Star, MegAlign, and GeneJocky. Any sequence databases that contains DNA sequences corresponding to a target gene or a segment thereof can be used for sequence analysis. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS. Sequence similarity can be discerned by aligning the transgene sequence against a target endogenous gene sequence. Common parameters for determining the extent of homology set forth by one or more of the aforementioned alignment programs include p value and percent sequence identity. P value is the probability that the alignment is produced by chance. For a single alignment, the p value can be calculated according to Karlin et al. (1990) Prco.Natl. Acad. Sci 87: 2264. For multiple alignments, the p value can be calculated using a heuristic approach such as the one programmed in Blast. Percent sequence identity is defined by the ratio of the number of nucleotide matches between the query sequence and the known sequence when the two are optimally aligned. A selected transgene and target endogenous sequences are considered to be substantially homologous when the regions of alignment exhibit the aforementioned range of percentage of identity using Fasta or Blast alignment program with the default settings. Sequence homology can also be determined by functional analyses. A sequence that preserves the functionality of the nucleic acid with which it is being compared is particularly prefened. Functionality may be established by different criteria, such as ability to hybridize with a target polynucleotide, ability to effectively amplify a target sequence to yield a substantially homogenous multiplicity of products, and the ability to extend the 3' end sequence complementary to a target sequence in a nucleotide sequencing reaction.

Where desired, the transgene may comprise heterologous sequences that facilitate detection of the expression and purification of the gene product. Examples of such sequences are known in the art and include those encoding reporter proteins such as β-galactosidase, β -lactamase, chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (GFP) and their derivatives. Other heterologous sequences that facilitate purification may code for epitopes such as Myc, HA (derived from influenza virus hemagglutinin), His-6, FLAG, glutathione S- transferase (GST), maltose-binding protein (MBP), or the Fc portion of immunoglobulin.

The target endogenous genes whose expression is to be inhibited encompass native and heterologous genes present in the host cell. "Native" genes are nucleic acid sequences originated from the host cell. Non-limiting illustrative native genes include those encode membrane proteins, cytosolic proteins, secreted proteins, nuclear proteins and chaperon proteins. Heterologous genes are sequences acquired exogenously by the host cell. Exogenous sequences can be either integrated into the host cell genome, or maintained as episomal sequences. An exemplary class of heterologous genes includes pathogenic genes derived from viruses, bacteria, fungi, and protozoa.

The endogenous genes suitable for the present invention may also be characterized based on one or more of the following features: ability to induce a phenotypic change in a host cell or organism, species origin, developmental origin, primary structural similarity, involvement in a particular biological process, association with or resistance to a particular disease or disease stage, tissue, sub- tissue or cell-specific expression pattern, and subcellular location of the expressed gene product. In one aspect, the endogenous gene may be any gene expressed in a eukaryote cell, such as a plant cell, animal cell or a yeast cell. In another aspect, the endogenous gene confers a phenotypic characteristic detectable by visual, microscopic, genetic, or chemical means. Within this class of genes, of particular interest are plant genes involved in growth phenotypes, e.g. stunting, hyperbranching, vein banding, ring spot, etching, and those responsible for color characteristics including bleaching and chlorosis. Also, of particular relevance are genes which upon inhibition provide an enhanced resistance to pathogens (e.g. bacteria, fungi, viruses, insects, and protozoa), and resistance to adverse environmental factors (e.g. temperature fluctuation, nutritional deficiency, adverse soil conditions, moisture, dryness, etc.).

In another aspect, the endogenous genes are of a specific developmental origin, such as those expressed in an embryo or an adult organism, during ectoderm, mesoderm, or endoderm formation in a multi-cellular animal, or during development of leaves, tubers, bud of a plant. In yet another aspect, the endogenous genes belong to a family of genes, or a sub-family of genes that share primary structural similarities. Structural similarities can be discerned with the aid of computer software described above. Non-limiting examples of gene families include those encoding proteinase, proteinase inhibitors, cell surface receptors, protein kinases (e.g. tyrosine, serine/threonine or histidine kinases), trimeric G-proteins, cytokines, PH-, SH2-, SH3-, PDZ-domain containing proteins, and any of those gene families published by the Institute for Genomic Research (TIGR), Incyte Pharmaceuticals, Inc., Human Genome Sciences Inc., Monsanto, and PE-Celera.

In yet another aspect, the endogenous genes are involved in a specific biological process, including but not limited to cell cycle regulation, cell differentiation, chemotaxsis, apoptosis, cell motility and cytoskeletal rearrangement.

In still another aspect, the endogenous genes embodied in the invention are associated with a particular disease or with a specific disease stage. Such genes include but are not limited to those associated with autoimmune diseases, obesity, hypertension, diabetes, neuronal and/or muscular degenerative diseases, cardiac diseases, endocrine disorders, any combinations thereof. In yet still another aspect, the endogenous genes encompass those exhibiting restricted expression patterns. Non-limiting exemplary gene transcripts of this class include those that are not ubiquitously expressed, but rather are differentially expressed in one or more of the plant tissues including leaf, seed, tuber, stems, root, and bud; or expressed in animal body tissues including heart, liver, prostate, lung, kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, and selected tissues that are affected by various types of cancer (malignant or non-metastatic), affected by cystic fibrosis or polycystic kidney disease. Additional examples of non-ubiquitously expressed genes are those whose gene products are localized to certain subcellular locations: extracellular matrix, nucleus, cytoplasm, cytoskeleton, plasma and/or intracellular membranous structures which include but are not limited to coated pits, Golgi apparatus, endoplasmic reticulum, endosome, lysosome, and mitochondria.

In addition to the above-described elements, the vectors may contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode protein(s) that (a) confer resistance to antibiotics or other toxins substances, e.g., ampicillin, neomycyin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. The vectors embodied in this invention can be obtained using recombinant cloning methods and/or by chemical synthesis. A vast number of recombinant cloning techniques such as PCR, restriction endonuclease digestion and ligation are well known in the art, and need not be described in detail herein. One of skill in the art can also use the sequence data provided herein or that in the public or proprietary databases to obtain a desired vector by any synthetic means available in the art.

Host cell and transgenic organisms of the present invention: The invention provides eukaryotic host cells transformed with the recombinant DNA vectors described above. The recombinant vectors containing the transgene of interest can be introduced into a suitable eukaryotic cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is coupled to an infectious agent). The choice of introducing vectors will often depend on features of the host cell.

For most animal cells, any of the above-mentioned methods is suitable for vector delivery. For plant cells, a variety of techniques derived from these general methods is available in the art. The host cells may be in the form of whole plants, isolated cells or protoplasts. Preferably, the cells are "intact" in that the cell comprises an outer layer of cell wall, typically composed of cellulose for protection and maintaining the rigidity of the plant cell. Illustrative procedures for introducing vectors into plant cells include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs and injection into immature embryos. As is evident to one skilled in the art, each of these methods has distinct advantages and disadvantages. Thus, one particular method of introducing genes into a particular plant species may not necessarily be the most effective for another plant species. Agrobacterium tumefaciens-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated expression vectors to introduce DNA into plant cells is well known in the art. This technique makes use of a common feature of Agrobacterium which colonizes plants by transferring a portion of their DNA (the T-DNA) into a host cell, where it becomes integrated into nuclear DNA. The T-DNA is defined by border sequences which are 25 base pairs long, and any DNA between these border sequences is transferred to the plant cells as well.

The insertion of a recombinant plant viral nucleic acid between the T-DNA border sequences results in transfer of the recombinant plant viral nucleic acid to the plant cells, where the recombinant plant viral nucleic acid is replicated, and then spreads systemically through the plant. Agro-infection has been accomplished with potato spindle tuber viroid (PSTV); CaV; and Lazarowitz, S., Nucl. Acids Res. 16:229

(1988)) digitaria streak virus (Donson et al., Virology 162:248 (1988)), wheat dwarf and tomato golden mosaic virus (TGMV). Therefore, agro-infection of a susceptible plant could be accomplished with a virion containing a recombinant plant viral nucleic acid based on the nucleotide sequence of any of the above viruses. Particle bombardment or electrosporation or any other methods known in the art may also be used.

Because not all plants are natural hosts for Agrobacterium, alternative methods such as transformation of protoplasts may be employed to introduce the subject vectors into the host cells. For certain monocots, transformation of the plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. See, for example, Potrykus et al., Mol. Gen. Genet., 199:167-177 (1985); Fromm et al, Nature, 319:791 (1986); Callis et al., Genes and Development, 1:1183 (1987). Applicability of these techniques to different plant species may depend upon the feasibility to regenerate that particular plant species from protoplasts.

In addition to protoplast transformation, particle bombardment is an alternative and convenient technique for delivering the invention vectors into a plant host cell. Specifically, the plant cells may be bombarded with microparticles coated with a plurality of the subject vectors. Bombardment with DNA-coated microprojectiles has been successfully used to produce stable transformants in both plants and animals (see, for example, Sanford et al. (1993) Methods in Enzymology, 217:483-509). Microparticles suitable for introducing vectors into a plant cell are typically made of metal, preferably tungsten or gold. These microparticles are available for example, from BioRad (e.g., Bio-Rad's PDS-1000/He). Those skilled in the art will know that the particle bombardment protocol can be optimized for any plant by varying parameters such as He pressure, quantity of coated particles, distance between the macrocarrier and the stopping screen and flying distance from the stopping screen to the target.

Vectors can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymology, 101 :433 (1983); Luo et al., Plant Mol. Biol. Reporter, 6:165 (1988). Alternatively, the vectors can be injected into reproductive organs of a plant as described by Pena et al., Nature, 325:2 '4

(1987).

Other techniques for introducing nucleic acids into a plant cell include:

(a) Hand Inoculations. Hand inoculations are performed using a neutral pH, low molarity phosphate buffer, with the addition of celite or carborundum (usually about 1%). One to four drops of the preparation is put onto the upper surface of a leaf and gently rubbed.

(b) Mechanized Inoculations of Plant Beds. Plant bed inoculations are performed by spraying (gas-propelled) the vector solution into a tractor- driven mower while cutting the leaves. Alternatively, the plant bed is mowed and the vector solution sprayed immediately onto the cut leaves.

(c) High Pressure Spray of Single Leaves. Single plant inoculations can also be performed by spraying the leaves with a narrow, directed spray (50 psi, 6-12 inches from the leaf) containing approximately 1% carborundum in the buffered vector solution. (d) Vacuum Infiltration. Inoculations may be accomplished by subjecting a host organism to a substantially vacuum pressure environment in order to facilitate infection.

Once introduced into a suitable host cell, expression of the transgene can be determined using any assay known in the art. For example, the presence of transcribed sense or anti-sense strands of the transgene can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis), amplification procedures (e.g. RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934). In conducting these analytical procedures, it is preferable to induce transcription of one strand of the transgene at a time. As is apparent to one skilled in the art, the simultaneous transcription of both sense and anti-sense strands facilitates formation of double stranded RNA molecules, which may obscure the accurate determination of the levels of sense and anti-sense RNA transcripts.

Expression of the transgene can also be determined by examining the protein product. A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using e.g. , colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunoflourescent assays, and PAGE-SDS.

In general, determining the protein level involves (a) providing a biological sample containing polypeptides; and (b) measuring the amount of any immunospecific binding that occurs between an antibody reactive to the transgene product and a component in the sample, in which the amount of immunospecific binding indicates the level of expressed proteins. Antibodies that specifically recognize and bind to the protein products of the transgene are required for immunoassays. These may be purchased from commercial vendors or generated and screened using methods well known in the art. See Harlow and Lane (1988) supra. and Sambrook et al. (1989) supra. The sample of test proteins can be prepared by homogenizing the eukaryotic transformants (e.g. plant cells) or their progenies made therefrom, and optionally solubilizing the test protein using detergents, preferably non-reducing detergents such as triton and digitonin. The binding reaction in which the test proteins are allowed to interact with the detecting antibodies may be performed in solution, or on a solid tissue sample, for example, using tissue sections or solid support that has been immobilized with the test proteins. The formation of the complex can be detected by a number of techniques known in the art. For example, the antibodies may be supplied with a label and unreacted antibodies may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed. Results obtained using any such assay on a sample from a plant transformant or a progeny thereof is compared with those from a non-transformed source as a control.

The eukaryotic host cells of this invention are grown under favorable conditions to effect transcription of the transgene. Non- limiting examples of eukaryotic hosts are fungus, yeast, plant cells, insect, avian, mammalian or other animal cells. The host cells can be used, inter alia, as repositories of the transgene and/or vehicles for production of the transgene-specific double stranded RNAs. The host cells may also be employed to generate transgenic organisms such as transgenic animals and plants comprising the recombinant DNA vectors of the present invention. Preferred host cells are those having the propensity to regenerate into tissue or a whole organisms. Examples of these preferred host cells are oocytes, blastocytes, and certain plant cells exemplified herein.

Accordingly, this invention provides transgenic plants carrying the subject vectors. In a preferred embodiment, the trangenic plant exhibits a reduced expression (when compared to a control plant) of an endogenous gene that is substantially homologous to the transgene carried in the subject vector.

The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, Mary A. Shuler and Raymond E. Zielinski, Academic Press, Inc., San

Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil.

The regeneration of plants containing the subject vector introduced by Agrobacterium tumefaciens from leaf explants can be achieved as described by

Horsch et al., Science, 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transformant shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil to allow the production of roots. These procedures will vary depending upon the particular plant species employed, as is apparent to one of ordinary skill in the art.

A population of progeny can be produced from the first and second transformants of a plant species by methods well known in the art including cross fertilization and asexual reproduction. Transgenic plants embodied in the present invention are useful for production of desired proteins, and as test systems for analysis of the biological functions of a gene.

Uses of the vectors of the present invention: The subject vectors provide specific reagents for inhibiting expression of an endogenous gene present in a host cell. The expression inhibition methods may be used in a wide variety of circumstances including suppression of a gene associated with a particular disease or disease stage; delineating the biological functions of a gene by analyzing a phenotypic change in the host cell that correlates with the selective suppression of gene expression; and facilitating drug screening by rendering the host cell more susceptible or resistant to a therapeutic agent of interest. Accordingly, this invention provides a method of inhibiting expression of an endogenous gene present in a eukaryotic cell. The method comprises the steps of: (a) providing a subject vector containing a transgene that is substantially homologous to an endogenous gene of a eukaryotic cell; (b) introducing the recombinant vector into the eukaryotic cell; (c) culturing the eukaryotic cell of (b) under conditions favorable for expression of both sense and antisense RNA transcripts from the transgene, and thereby inhibiting expression of the corresponding endogenous gene in the eukaryotic cell. In a separate embodiment, the invention provides a method of identifying a biological function(s) of an endogenous gene of interest in a eukaryotic cell by selectively inhibiting the expression of the endogenous gene. The method involves: (a) providing a recombinant vector of the present invention, wherein the transgene contained in the vector is substantially homologous to the endogenous gene; (b) introducing the recombinant vector of (a) into the eukaryotic cell; (c) culturing the eukaryotic cell of (b) under conditions favorable for expression of both sense and antisense RNA transcripts from the transgene contained in the recombinant vector and thereby inhibiting expression of the endogenous gene in the eukaryotic cell; and (d) determining one or more phenotypic changes in the eukaryotic cell that correlate with the inhibited expression of the endogenous gene, thereby identifying the biological function(s) of the endogenous gene in the eukaryotic cell.

The host cells encompassed by these embodiments are eukaryotic cells susceptible to dsRNA-mediated "genetic interference". dsRNA induced gene silencing has been observed in a variety of multi-cellular organisms including but not limited to worms, fruitflies, protozoa, fungi, mammals, and zebrafish. Thus, cells from any of these exemplary organisms can be employed. Suitable host cells may be derived from primary cultures or subcultures generated by expansion and/or cloning of primary cultures. Any cells capable of growth in culture can be used as host cells. Of particular interest is the type of cell that differentially expresses (over- expresses or under-expresses) a disease-causing gene. As is apparent to one skilled in the art, various cell lines may be obtained from public or private repositories. The largest depository agent is American Type Culture Collection (http://www.atcc.org), which offers a diverse collection of well-characterized cell lines derived from a vast number of organisms and tissue samples.

Upon delivery of the subject vectors, the host cells are cultured under conditions favorable for gene transcription. The parameters governing eukaryotic cell survival are generally applicable for induction of gene transcription. The culture conditions are well established in the art. Physicochemical parameters which may be controlled in vitro are, e.g., pH, CO_2ι temperature, and osmolarity. The nutritional requirements of cells are usually provided in standard media formulations developed to provide an optimal environment. Nutrients can be divided into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives and vitamins. Apart from nutrients for maintaining cell metabolism, most cells also require one or more hormones from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones to survive or proliferate (Sato, G.H., et al. in "Growth of Cells in Hormonally Defined Media", Cold Spring Harbor Press, N.Y., 1982; Barnes and Sato (1980) Anal. Biochem., 102:255. Given the vast wealth of information on the nutrient requirements, medium conditions optimized for cell survival, one skilled in the art can readily fashion various culture conditions using any one of the aforementioned methods and compositions, alone or in any combination.

The inhibition of expression of the endogenous gene sharing substantial sequence homology with the transgene carried in the vectors can be determined by assaying for a difference, between the host cell and the control cell, in the level of mRNA transcripts of the endogenous gene. Alternatively, a suppression in expression is determined by detecting a difference in the level of the polypeptide(s) encoded by the endogenous gene. A preferred method is to detect a phenotypic change resulting from the decrease in expression of the endogenous gene of interest. In assaying for an alteration in mRNA level, nucleic acid contained in the host cells is first extracted according to standard methods in the art. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989), supra or extracted by nucleic- acid-binding resins following the accompanying instructions provided by manufacturers. The mRNA contained in the extracted nucleic acid sample is then detected by hybridization (e.g. Northern blot analysis) and or amplification procedures according to methods widely known in the art or based on the methods exemplified herein.

Reduction in expression of the endogenous gene can also be determined by examining the protein product of the endogenous gene. A variety of techniques is available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunoflourescent assays, and SDS-PAGE. In addition, cell sorting analysis can be employed to detect cell surface antigens. Such analysis involves labeling target cells with antibodies coupled to a detectable agent, and then separating the labeled cells from the unlabeled ones in a cell sorter. A sophisticated cell separation method is fluorescence-activated cell sorting (FACS). Cells traveling in single file in a fine stream are passed through a laser beam, and the fluorescence of each cell bound by the fluorescently labeled antibodies is then measured. Antibodies that specifically recognize and bind to the protein products of interest are required for conducting the aforementioned protein analyses. These antibodies may be purchased from commercial vendors or generated and screened using methods well known in the art. See Harlow and Lane (1988) supra, and Sambrook et al. (1989) supra.

Inhibition of gene expression can also result in phenotypic change(s) in a host cell. As used herein, phenotypic change refers to any non-genotypic change that can be detected visually, or analyzed biochemically or genetically. The choice of detection methods will largely depend on the nature of the phenotypic characteristics that are under investigation. For instance, certain phenotypic features of a plant cell can be detected microscopically or macroscopically. These features include improved tolerance to herbicides, improved tolerance to extremes of heat or cold, drought, salinity or osmotic stress; improved resistance to pests (insects, nematodes or arachnids) or diseases (fungal, bacterial or viral), production of enzymes or secondary metabolites; male or female sterility; dwarfhess; early maturity; improved yield, vigor, heterosis, nutritional qualities, flavor or processing properties, and the like. Other detectable phenotypic changes are morphological alterations including but not limited to stunting, hyperbranching, vein banding, ring spot, etching, and those responsible for color characteristics including bleaching and chlorosis.

For animal cells, detectable phenotypic changes may encompass alterations in cell cycle regulation, cell differentiation, apoptosis, chemotaxsis, cell motility and cytoskeletal rearrangement. Methods for detecting these phenotypic changes are well-established in the art and hence are not detailed herein. Other phenotypic changes commonly observed in both plant and animal cells involve differential expression (over-expression or under-expression) of a particular protein due to the selective inhibition of the endogenous gene of interest. Differential gene expression may be analyzed by any chemical means available in the art or those disclosed herein. As is also apparent to artisans, altering expression of one endogenous gene may lead to changes in gene expression profile of a host of genes mapped to the same or related signal transduction pathways. As used herein, "signal transduction" refers to the process by which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response. Any fluctuation in intracellular response of a eukaryotic host cell is also considered as a type of phenotypic change.

Alteration in intracellular response is often determined with the aid of reporter molecules. For example, when examining a signaling cascade involving a fluctuation of intracellular pH condition, pH sensitive molecules such as fluorescent pH dyes can be used as the reporter molecules. In another example where the signaling pathway of a trimeric G_q protein is analyzed, calcium-sensitive fluorescent probes can be employed as reporters. As is apparent to artisans in the field of signal transduction, trimeric G_q protein is involved in a classic signaling pathway, in which activation of G_q stimulates hydrolysis of phosphoinositides by phospholipase C to generate two classes of well-characterized second messengers, namely, diacylglycerol and inositol phosphates. The latter stimulates the mobilization of calcium from intracellular stores, and thus resulting in a transient surge of intracellular calcium concentration, which is a readout measurable with a calcium- sensitive probe.

Another exemplary class of reporter molecules is a reporter gene operably linked to an inducible promoter that can be activated upon the stimulation or inhibition of a signaling pathway. Reporter proteins can also be linked with other proteins whose expression is dependent upon the stimulation or suppression of a given signaling cascade. Commonly employed reporter proteins can be easily detected by a colorimetric or fluorescent assay. Non-limiting examples of such reporter proteins include : β-galactosidase, β -lactamase, chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (GFP) and their derivatives. Those skilled in the art will know of other suitable reporter molecules for assaying changes in a specific signaling transduction readout, or will be able to ascertain such, using routine experimentation.

To discern inhibition of gene expression, one typically conducts a comparative analysis of the subject and appropriate controls. Preferably, a test includes a positive control sample exhibiting a decrease in gene expression and a negative control having an unaltered expression level. The selection of an appropriate control cell or tissue is dependent on the sample cell or tissue initially selected and its phenotype which is under investigation. In one aspect, the invention methods can be employed to selectively inhibit expression of an endogenous gene that is native to the eukaryotic host cell. Such a gene may encode encodes a protein selected from the group consisting of a membrane protein, a cytosolic protein, a secreted protein, a nuclear protein and a chaperon protein. Of particular interests are endogenous genes that confer phenotypic changes as a result of inhibition of the expression and/or function of the endogenous genes. In another aspect within this embodiment, the endogenous gene is heterologous to the host cell. As used herein, heterologous genes are acquired exogenously by the host cell. Non-limiting examples of heterologous genes are those derived from virus, bacterium, fungus, and protozoa.

In a separate embodiment, the invention methods are used to identify a biological function(s) of an endogenous gene in a eukaryotic cell by examining a phenotypic change associated with the inhibition in its expression and thus loss of biological function. In essence, the subject methods allow the creation of a transient or more long-term gene-specific knock-out system for analyzing the biological function of any endogenous gene of interest.

Kits comprising the vectors of the present invention

The present invention also encompasses kits containing the vectors of this invention in suitable packaging. Kits embodied by this invention include those that allow generation of a double-stranded RNA transcript in a eukaryotic cell.

Each kit necessarily comprises the reagents which render the delivery of vectors into a eukaryotic host cell possible. The selection of reagents that facilitate delivery of the vectors may vary depending on the particular transfection or infection method used. The kits may also contain reagents useful for generating labeled polynucleotide probes or proteinaceous probes for detection of gene silencing. Each reagent can be supplied in a solid form or dissolved/suspended in a liquid buffer suitable for inventory storage, and later for exchange or addition into the reaction medium when the experiment is performed. Suitable packaging is provided. The kit can optionally provide additional components that are useful in the procedure. These optional components include, but are not limited to, buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information. The kits can be employed to generate eukaryotic cells whose endogenous genes are selectively inhibited, and transgenic organisms comprising these eukaryotic cells.

Further illustration of the development and use of vectors and assays according to this invention are provided in the Example section below. The examples are provided as a guide to a practitioner of ordinary skill in the art, and are not meant to be limiting in any way.

EXAMPLES

Example 1: Construction of recombinant vectors comprising two opposing transcription units

We have designed a recombinant vector construct useful for silencing nuclear genes in many of the agriculturally-important cereal crops. The vector comprises sequences derived from maize streak geminivirus, isolated MSV-Kom (genbank accession number AF003952. classification: Family Geminiviridae, genus Mastrevirus, species maize streak virus, designated MSV-Komatipoort. Maize streak virus has a broad host range that encompasses all agriculturally important cereal crops, including but not limited to corn, wheat, rice, barley, rye, sorghum and millet. The methods for construction of infectious geminiviruses are well known to those skilled in the art, and are described in European patent application 8687015.5 as well as in US Patent No. 5,569,597.

We have synthesized a 1618 base pair synthetic DNA that contains the MSV-Kom repA and repB, long intergenic region (LIR) and short intergenic region (SIR) and thus all sequences that are required for viral replication. Palmer et al.(1999,) Archives of Virology 144: 1345-1360. This fragment was cloned into the pZeRO-2 vector (Invitrogen) as an EcoRl-Xbal fragment, to create the plasmid pMSVLSB-1, the sequence of which is shown in Figure 4. A 171 base pair fragment containing the movement protein (mp) promoter of MSV-Kom is synthesised and cloned into the pZeRO-2 vector as an HmdIII-EcoRI fragment to create pMSVLSB-2 (sequence shown in Figure 5). The Apal fragment containing the mp promoter is inserted between the two Apal sites in pMS VLSB- 1 , to create pMSVLSB-3 (sequence shown in Figure 6).

The cauliflower mosaic virus 35S RNA promoter (CaMV 35S promoter) sequence is amplified with a vector containing this sequence (pBI121, from Clontech) as template DNA, using the following PCR primers containing the following restriction sites (shown in italicized): EcoRI in CaMV35SF and Sail in CaMV35SR.

CaMV35SF: TTTG Λ7TCGTCAACATGGTGGAGCAC (SEQ ID NO:l)

CaMV35SR:

TTTGrCG CGTCCTCTCCAAATGAAATGAAC (SEQ ID NO:2)

The CaMV 35S promoter PCR product yielded is digested with EcoRl and Sail and the restricted fragments are purified.

The zeocin resistance gene is amplified by PCR with the vector pZeRO-1 (Invitrogen) as template, using the following primers containing the following restriction sites shown in italicized: Sail, Pad and Notl in ZeoF and Xhol, Pad and Notl in ZeoR:

ZeoF: CCCGrCGv4C7 4v rr^4GCGGCCGCGTTTACAATTTCGCCTGATGC

(SEQ ID ΝO:3)

ZeoR:

CCCCΓCG G7T^ΓΓ^4GCGGCCGCCTCAAAAAGGATCTTCACCTA G (SEQ ID NO:4)

The zeocin resistance gene product yielded is digested with Xhol and Sad and purified.

The nopaline synthase (nos) terminator sequence is amplified by PCR with the vector pBI121 (Clontech) as template, using the following primers, with restriction sites Xhol in nosF and Spel in nosR italicized:

NosF: TTTCrCG GCGAATTTCCCCGATCGTTCAAAC (SEQ ID NO:5)

NosR:

TTTΛCΣ4G7€CCGATCTAGTAACATAGATGAC (SEQ ID NO:6) The nos terminator product yielded is digested with ΛTioI and Spel and purified.

The digested CaMV35S promoter, zeocin resistance gene and nos terminator sequences are ligated together with T4 DNA ligase. The ligated product is diluted 1:100 in sterile water and the whole ligation product is re-amplified with the CaMV35SF and nosR primers. The resulting PCR product is digested with EcoRI and Spel, purified and ligated with pMSVLSB-3 that is pre-digested with EcoRI and Spel. The ligation reaction is used to transform E. coli competent cells.

Transformants are selected on Luria Agar plates containing both kanamycin (100 μg/ml) and zeocin (50 μg ml) to select for colonies containing the CaMV35S promoter-zeocin resistance gene-nos terminator cassette inserted into pMSVLSB-3 (Figure 6 and SΕQ ID NO:l 1). Colonies putatively containing the coπect plasmid are chosen, plasmid DNA isolated and screened by digestion with EcoRI and Spel.

One plasmid designated pMSVLSB-4 (Figure 7 and SΕQ ID NO: 12) is selected.

One of the methods in the art of construction of infectious clones of geminivirus genomes is to clone tandemly duplicated sequences of the geminivirus genome, with at least the LIR duplicated. This allows the virus sequence to escape from the cloning vector in planta by a replicative release mechanism. The virus Rep protein is transiently expressed in transfected cells, and induces a nick at each of the stem loop sequences contained within the origin of replication in the LIR. Rolling circle replication is initiated at each nick point, and this results in release of a ssDNA copy of the virus replicon, which is circularized by the Rep protein, and which then replicates autonomously in the plant cell nucleus. The Xbal-Spel fragment from pMSVLSB-3, containing the viral LIR and Rep genes is inserted into the unique Spel site in pMSVLSB-4 to create pMSVLSB-5 (Figure 8 and SΕQ ID NO: 13). The zeocin resistance gene is deleted by digestion with Notl; the DΝA is recircularized and used to transform E.coli to kanamycin resistance with a new vector, pMSVLSB-6 (Figure 9 and SΕQ ID NO: 14). When the vector is introduced into plant cells, a monomeric copy of the insert is released by replicative release (described above) and replicates autonomously as construct MSVLSB-6 in the nuclei of infected cells. The restriction map of construct MSVLSB-6 is shown in Figure 3; this genetic construct possesses the following features: (a) the rep genes and origins of replication from maize streak geminivirus that are necessary and sufficient for the autonomous replication of the viral construct and its associated foreign DNA in the host plant cell; (b) two overlapping transcription units present in the DNA replicon.

The two overlapping transcription units are arranged according to the configuration shown in Figure 2. With reference to Figure 2, "promoter 1" and "terminator 1" in MSVLSB-6 are the MSV mp promoter and transcription termination signals present in the SIR, respectively, and "promoter 2" and "terminator 2" are the CaMV 35S RNA promoter and nos terminator sequences, respectively. The two overlapping transcription units share three unique restriction sites (Sail, Pad and Notl) and one non-unique restriction site (Xhol) where foreign DΝA may be inserted so that it may be transcribed by both promoters to yield at least a partially double stranded RΝA duplex of the foreign DΝA sequence.

Example 2: Use of recombinant vectors to inhibit or silence gene expression in cereal crops:

Application ofpMSVLSB-6 in inhibition of Dwarf 1 gene expression in rice

The vector pMSVLSB-6 exemplified above can be employed to inhibit expression of any endogenous gene in a variety of plant host cells. By way of illustration, the rice gene Dwarf 1 is inhibited to duplicate known mutant phenotype using a pMSVLSB-6 containing a fragment of the coding sequence oϊDwarfl (Genbank accession number AB028602). The gene is amplified from cDΝA isolated from rice seedlings. Primer sequences are designed to have homology with the published sequence of Dwarfl. Ashikari et al. (1999) RNΛS U.S.A. 96:10284- 10289. The primer sequences contain Notl restriction sites at their 5' ends. The PCR product is digested with Notl and cloned into the Notl site of pMSVLSB-6 to generate pMSVLSB-6: :dwarfl s and pMSVLSB-6: :dwarfl a, with the insert cloned in the sense and antisense orientation with respect to the MSV mp promoter, respectively. The Xbal-Spel fragment from each of these plasmids is transferred into an Agrobacterium binary vector that is commonly used for rice transformation. This vector is used to transform electrocompetent Agrobacterium strain LBA4404 (Life Technologies). Agrobacterium cultures containing the appropriate plasmids are used in transformation of rice. Transgenic rice is generated by standard protocols (see, e.g. US Patent 5,591,616). The transgenic rice plants display similar phenotypes to the dwarfλ mutant described by Ashikari et al. (1999) supra: they are giberellin-insensitve, dwarfed in comparison with un-silenced transgenic controls, and having broad, dark green leaves, compact pannicles and short, round grains.

Application ofpMSVLSB-6 in inhibition ofphytoene desaturase expression in maize seedlings

The coding sequence for the maize phytoene desaturase gene (pds), having the Genbank accession number U37285, is amplified from cDNA made from RNA isolated from four-day-old maize seedlings, of the cultivar "Golden Cross Bantam". The primers used for amplification of this cDNA have the following sequences containing the Pad sites (italicized) at the 5' ends:

zeapdsl330:

TT7TrA47Tv4AGGTCCGCCTGAATTCTCG (SEQ ID NO:7)

zeapdsl873

TTTΓΓΛΛΓΓΛΛCGGCAAGGCTCACAGTTTG (SEQ ID NO:8)

PCR amplification with these primers and cDNA made from RNA isolated from maize seedlings yields a product of 565 base pairs, which is then digested with Pad. The progenitor plasmid to pMSVLSB-6, pMSVLSB-5 is digested with Xba and Spel to release the MSV and associated overlapping transcription unit sequences from the pZeRO-2 cloning vector as a single 4816 base pair fragment. This fragment is cloned into the Agrobacterium binary vector pBinl9 (Genbank: U09365) digested with Xbal to yield pMSVLSB-7. The plasmid pMSVLSB-7 is digested with Pad and the pds PCR fragment is inserted into this position, generating plasmid pMSVLSB-7::/« (cloned in the sense orientation with respect to the MSV mp promoter) and pMSVLSB-7::/?Js2 (cloned in the antisense orientation with respect to the MSV mp promoter. These two plasmids are each introduced into Agrobacterium strain C58Cl(pMP90) (Koncz and Schell, 1985) by electroporation. The Agrobacterium containing the binary vector plasmids is grown overnight in Luria Bertani medium containing appropriate selective antibiotics. The bacterial suspension is loaded into a 100 μl Hamilton syringe and injected into three day old maize seedlings (cultivar Golden Cross Bantam) according to methods described by Escudero et al. (1994) in the chapter "Agroinfection" of The Maize Handbook, Freelings M, Walbot V (eds). Plants that are successfully agroinfected display a photobleaching phenotype on the first three leaves, similar to that induced by spraying the plants with the phytoene desaturase-inhibitor norfluorazon.

Claims

CLAIMS What is claimed is:

1. A eukaryotic recombinant vector comprising a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene in a eukaryotic host cell.

2. The eukaryotic recombinant vector of claim 1 , wherein each of the overlapping transcription units comprises a promoter and a terminator.

3. The eukaryotic recombinant vector of claim 2, wherein the promoter is a constitutive promoter.

4. The eukaryotic recombinant vector of claim 2, wherein the promoter is an inducible promoter.

5. The eukaryotic recombinant vector of claim 2, wherein the promoter is a tissue-specific promoter.

6. The eukaryotic recombinant vector of claim 1, wherein the promoter and the terminator of the overlapping transcription units are arranged in a configuration shown in Figure 2(a).

7. The eukaryotic recombinant vector of claim 1, wherein the promoter and the terminator of the overlapping transcription units are arranged in a configuration shown in Figure 2(b).

8. The eukaryotic recombinant vector of claim 1, wherein the promoter and the terminator of the overlapping transcription units are arranged in a configuration shown in Figure 2(c).

9. The eukaryotic recombinant vector of claim 1, wherein the promoter and the terminator of the overlapping transcription units are arranged in a configuration shown in Figure 2(d).

10. The eukaryotic recombinant vector of claim 1 that inhibits gene expression of the eukaryotic host cell.

11. The eukaryotic recombinant vector of claim 1, wherein the eukaryotic host cell is selected from the group consisting of fungus, yeast cell, plant cell and animal cell.

12. The eukaryotic recombinant vector of claim 1 that inhibits expression of an endogenous gene present in the host cell, wherein the endogenous gene is substantially homologous to the transgene contained in the overlapping transcription units.

13. The eukaryotic recombinant vector of claim 12, wherein the endogenous gene is native to the host cell.

14. The eukaryotic recombinant vector of claim 12, wherein the endogenous gene is heterologous to the host cell.

15. The eukaryotic recombinant vector of claim 12, wherein the endogenous gene is a pathogenic gene derived from one or more members of the group consisting of virus, bacterium, fungus, and protozoa.

16. The eukaryotic recombinant vector of claim 1, wherein expression of the transgene to yield double-stranded RNA transcripts confers a phenotypic change in the eukaryotic host cell.

17. The eukaryotic recombinant vector of claim 1, wherein the transgene encodes a protein selected from the group consisting of a membrane protein, a cytosolic protein, a secreted protein, a nuclear protein, and a chaperon protein.

18. The eukaryotic recombinant vector of claim 1 that is an autonomously replicating vector.

19. The eukaryotic recombinant vector of claim 1, wherein the viral replicon is derived from a DNA virus.

20. The eukaryotic recombinant vector of claim 19, wherein the DNA virus is selected from the group consisting of Geminivirus, Caulimoviridae, Badnaviridae; Circoviridae, Circinoviridae, Parvoviridae, Papovaviridae,

Polyomaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae, Baculoviridae, Hepadnaviridae, Retroviridae, Gyrovirus, Nanovirus, and African Swine Fever virus.

21. A host cell transformed with a vector of claim 1 or 10.

22. The host cell of claim 21 that is a eukaryotic cell selected from the group consisting of fungus, yeast cell, plant cell and animal cell.

23. A transgenic plant comprising a eukaryotic recombinant vector of claim 1 or 10.

24. The transgenic plant of claim 23 exhibiting reduced expression of an endogenous gene that is substantially homologous to the transgene contained in the eukaryotic recombinant vector.

25. A kit for generating a double-stranded RNA transcript in a eukaryotic cell comprising a eukaryotic recombinant vector of claim 1 in suitable packaging.

26. A method of inhibiting expression of an endogenous gene present in a eukaryotic cell, comprising:

(a) providing a eukaryotic recombinant vector of claim 12; (b) introducing the eukaryotic recombinant vector into the eukaryotic cell;

(c) culturing the eukaryotic cell of (b) under conditions favorable for expression of both sense and antisense RNA transcripts from the transgene that is contained in the transcription units of the vector, and thereby inhibiting expression of the corresponding endogenous gene in the eukaryotic cell.

27. The method of claim 26, wherein the endogenous gene is native to the host cell.

28. The method of claim 26, wherein the endogenous gene is heterologous to the host cell.

29. The method of claim 26, wherein the endogenous gene is a pathogenic gene derived from one or more members of the group consisting of virus, bacterium, fungus, and protozoa.

30. The method of claim 26, wherein inhibition of the endogenous gene confers a phenotypic change in the host cell.

31. The method of claim 26, wherein the host eukaryotic cell is selected from the group consisting of fungus, yeast cell, plant cell, and animal cell.

32. The method of claim 26, wherein the eukaryotic recombinant vector is an autonomously replicating vector.

33. The method of claim 26, wherein the eukaryotic recombinant vector comprises a viral replicon derived from a DNA virus.

34. The method of claim 26, wherein the DNA virus is selected from the group consisting of Geminivirus, Caulimoviridae, Badnaviridae; Circoviridae, Circinoviridae, Parvoviridae, Papovaviridae, Polyomaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae, Baculoviridae, Hepadnaviridae, Retrovirida, Gyrovirus, Nanovirus, and African Swine Fever virus.

35. The method of claim 26, wherein the eukaryotic recombinant vector comprises two overlapping transcription units, wherein each transcription unit comprises a promoter and a terminator.

36. The method of claim 26, wherein the promoter is a constitutive promoter.

37. The method of claim 26, wherein the promoter is an inducible promoter.

38. The method of claim 26, wherein the promoter is a tissue-specific promoter.

39. The method of claim 35, wherein the promoter and the terminator of the overlapping transcription units are aπanged in a configuration shown in Figure 2(a).

40. The method of claim 35, wherein the promoter and the terminator of the overlapping transcription units are arranged in a configuration shown in Figure 2(b).

41. The method of claim 35, wherein the promoter and the terminator of the overlapping transcription units are aπanged in a configuration shown in Figure 2(c).

42. The method of claim 35, wherein the promoter and the terminator of the overlapping transcription units are arranged in a configuration shown in Figure 2(d).

43. A method of identifying a biological function(s) of an endogenous gene of interest in a eukaryotic cell by selectively inhibiting the expression of the endogenous gene, the method comprising: (a) providing a eukaryotic recombinant vector of claim 12;

(b) introducing the eukaryotic recombinant vector of (a) in to the eukaryotic cell; (c) culturing the eukaryotic cell of (b) under conditions favorable for expression of both sense and antisense RNA transcripts from the transgene contained in the eukaryotic recombinant vector and thereby inhibiting expression of the endogenous gene in the eukaryotic cell; and

(d) determining one or more phenotypic changes in the eukaryotic cell that correlate with the inhibited expression of the endogenous gene, thereby identifying the biological function(s) of the endogenous gene in the eukaryotic cell.

44. The method of claim 43, wherein the eukaryotic cell is selected from the group consisting of fungus, yeast cell, plant cell, and animal cell.

45. The method of claim 43, wherein the eukaryotic cell is a plant cell.

46. The method of claim 43, wherein the eukaryotic cell is an animal cell.