HK1078105B - Sirna expression system and process for producing a cell which functional gene has been knocked down or the like using the same - Google Patents

Description

siRNA expression system and method for preparing cell containing knocked down functional gene by using same

Technical Field

The present invention relates to in vivo siRNA expression systems capable of silencing expression of a target gene, and methods of using the expression systems to produce knockdown cells.

Background

RNA interference (hereinafter, abbreviated as "RNAi") is a phenomenon (process) in which the expression of a target gene is silenced by introducing double-stranded RNA (hereinafter, abbreviated as "dsRNA") comprising a sense RNA having a sequence homologous to the mRNA of the target gene and an antisense RNA having a sequence complementary to the sense RNA into a cell to induce degradation of the mRNA of the target gene. Since RNAi can silence target gene expression, efforts have been made to make it a simple gene knock-over method, to replace conventional gene disruption methods that rely on tedious and inefficient homologous recombination, or to make it a way of gene therapy. Initially, the RNAi was found in nematodes (Fire, A et al, patent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature391, 806-. Subsequently, RNAi was also observed in a variety of organisms including plants, nematodes, Drosophila and protozoa (Fire, A.RNA-triggered gene cloning. trends Gene.15, 358-. Furthermore, Zefen Wang et al, j.biol.chem., 275 (51): 40174-40179, 2000 an interfering RNA expression vector is prepared by inserting a target DNA sequence consisting of several hundred bases encoding an interfering RNA against a plurality of target genes of Trypanosoma brucei, and the phenotype of Trypanosoma brucei transfected by the vector is changed. The actual introduction of exogenous dsRNA into these organisms further confirmed silencing of target gene expression. This technique has been used as a method of producing knockdown individuals.

Similar to the above organisms, attempts have been made to introduce exogenous dsRNA in mammalian cells to induce RNAi. However, at this time, the host anti-viral infection protection mechanism triggered by the transfected dsRNA acted, inhibiting protein synthesis, so RNAi was not observed.

Recently, it has been reported by Tuschl et al that RNAi can also be induced in mammalian cells by transducing cells with short dsRNA of 21 or 22 nucleotides in length, which have single-stranded 3' overhangs of 2 or 3 nucleotides (Elbashir, S.M et al, duplex of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells 411, 494-498 (2001); Caplen, N.J. et al, specificity of gene expression by double-stranded RNAs in transformed and transformed systems, Proc. Natl. Acad. Sci. U S A98, 9742-9747 (2001)). Elbashir, S.M et al, Nature 411, 494-498(2001) also indicate that it is not possible to detect RNA interference in mammalian cells with synthetic dsRNA of more than 30bp, since the interferon reaction degrades dsRNA in mammalian cells.

As described above, RNAi has been successfully induced in mammalian cells using short interfering double stranded RNA (hereinafter referred to simply as "siRNA"). In order to perform functional analysis and gene therapy based on gene silencing induced by RNAi, siRNA must be efficiently introduced into cells and stably maintained in the cells.

The efficiency of exogenous siRNA introduction into cells varies from cell type to cell type, and can be as low as 1% to 10% in some cells. In addition, exogenous siRNA introduced into mammalian cells disappears after several days of introduction, and does not have sufficient stability for analyzing gene function. In addition, in gene therapy, it is necessary to administer siRNA at regular time intervals, which increases the physiological burden on patients.

In addition, it is difficult to induce RNAi only in a specific tissue or at a specific development/differentiation stage by introducing exogenous siRNA. Furthermore, although siRNA is small, RNA synthesis is much more expensive than DNA synthesis, and RNAi induced directly by siRNA is not economical.

Since most of the primary DNA sequences of the human genome have been determined, systematic and efficient methods for searching functional genes have been developed, which can rapidly elucidate the functions of genes. Based on phenotypic changes of cells or individuals, systemic functional gene searches can be conducted using gene silencing by RNAi to expedite the discovery and analysis of new functional genes.

Disclosure of Invention

An object of the present invention is to provide an intracellular siRNA expression system capable of more efficiently, stably and economically producing RNAi in a cell, a method of producing knockdown cells using the siRNA expression system, and a method of searching for functional genes using the siRNA expression system.

In view of the above problems, the present inventors studied an in vivo siRNA expression system and succeeded in developing the system. More specifically, the present invention relates to:

(1) an intracellular siRNA expression system comprising antisense coding DNA encoding antisense RNA directed to a region of the mRNA of a target gene, sense coding DNA encoding sense RNA directed to the same region of the mRNA of said target gene, and one or more promoters capable of expressing said antisense and sense RNAs from said antisense and sense coding DNAs, respectively;

(2) the siRNA expression system according to (1), wherein the final transcript length of siRNA expressed by the system is 15 to 49 bp;

(3) the siRNA expression system according to (1), wherein the final transcript length of siRNA expressed by the system is 15 to 35 bp;

(4) the siRNA expression system according to (1), wherein the final transcript length of siRNA expressed by the system is 15 to 30 bp;

(5) an siRNA expression system according to (1), wherein the double-stranded RNA region in which the two RNA strands pair contains a mismatch or a bulge in the siRNA;

(6) an siRNA expression system according to (5), wherein one nucleotide of the mismatch is guanine and the other is uracil;

(7) the siRNA expression system according to (5), wherein the siRNA contains 1 to 7 mismatches;

(8) an siRNA expression system according to (5), wherein the siRNA comprises 1 to 7 lobes;

(9) the siRNA expression system according to (5), wherein the siRNA contains 1 to 7 mismatches and bulges;

(10) an siRNA expression system according to any one of (1) to (9), wherein said promoter is pol II or pol III promoter;

(11) an siRNA expression system according to any one of (1) to (10), wherein said pol III promoter is a U6 promoter;

(12) the siRNA expression system according to any one of (1) to (11), wherein the promoter is an inducible promoter;

(13) the siRNA expression system according to any one of (1) to (12), wherein said promoter is separately located upstream of said antisense and sense coding DNAs;

(14) an siRNA expression system according to any one of (1) to (13), wherein said system comprises a loxP sequence in any one of the following forms (a) to (c) so that expression can be controlled:

(a) the promoter contains a Distal Sequence Element (DSE) and a Proximal Sequence Element (PSE) with a spacer region therebetween, wherein the spacer region contains two loxP sites, one located near the DSE and the other located near the PSE;

(b) the promoter comprises DSE and PSE, which are positioned to maintain promoter activity, and a loxP site is positioned between them, and another loxP site is positioned upstream of the DSE or downstream of the PSE; and

(c) the two loxP sites are located in such a position that they can interfere with the antisense coding DNA or sense coding DNA;

(15) the siRNA expression system according to any one of (1) to (14), wherein the antisense and sense coding DNAs are located on the same carrier DNA molecule or separately located on different carrier DNA molecules;

(16) the siRNA expression system according to any one of (1) to (13), wherein the promoter is located on one side of a unit in which the antisense and sense coding DNAs are linked in opposite directions via a linker;

(17) the siRNA expression system according to (16), wherein said system comprises a loxP sequence in any one of the following forms (a) to (d) so that expression can be controlled:

(a) the promoter comprises DSE and PSE, a spacer is arranged between the DSE and the PSE, and two loxP sites are arranged in the spacer, one is positioned near the DSE, and the other is positioned near the PSE;

(b) the promoter comprises DSE and PSE, which are positioned to maintain promoter activity, and a loxP site is positioned between them, and another loxP site is positioned upstream of the DSE or downstream of the PSE;

(c) the two loxP sites are located in such a position that they can interfere with the antisense coding DNA or sense coding DNA; and

(d) two loxP sites in the position to allow it to contain termination sequence (such as TTTTT) joint;

(18) the siRNA expression system according to (16) or (17), wherein the antisense and sense coding DNAs are located in a vector molecule;

(19) the siRNA expression system according to (15) or (18), wherein said vector is a plasmid vector;

(20) the siRNA expression system according to (15) or (18), wherein said vector is a viral vector;

(21) the siRNA expression system according to (15) or (18), wherein said vector is a dumbbell-shaped DNA vector;

(22) a cell maintaining the siRNA expression system according to any one of (1) to (21);

(23) the cell according to (22), wherein the cell is a mammalian cell;

(24) a single organism maintaining an siRNA expression system according to any one of (1) to (21);

(25) a composition comprising an siRNA expression system according to any one of (1) to (21);

(26) the composition according to (25), wherein the composition is a pharmaceutical composition;

(27) a method of producing a cell with silenced expression of a target gene, wherein said method comprises the steps of: introducing an siRNA expression system according to any one of (1) to (21) into a cell, and selecting a cell into which the siRNA expression system has been introduced;

(28) an intracellular siRNA library expression system comprising a double-stranded DNA encoding siRNA and two promoters placed opposite to each other, said double-stranded DNA comprising an arbitrary sequence of a length as long as the expressed siRNA and being located between the two promoters, said promoters being capable of expressing RNAs complementary to each other from each strand of said double-stranded DNA;

(29) an intracellular siRNA library expression system comprising a stem-loop siRNA producing unit in which an antisense encoding DNA and a sense encoding DNA complementary to said antisense encoding DNA are linked in opposite directions via a linker and a promoter capable of expressing the stem-loop siRNA on either side of said unit;

(30) the siRNA library expression system according to (28) or (29), wherein the siRNA final transcript expressed by the system has a length of 15 to 49 bp;

(31) the siRNA library expression system according to (28) or (29), wherein the siRNA final transcript expressed by the system has a length of 15 to 35 bp;

(32) the siRNA library expression system according to (28) or (29), wherein the siRNA final transcript expressed by the system has a length of 15 to 30 bp;

(33) an siRNA library expression system according to any one of (28) to (32), wherein a region of the double-stranded RNA in which two RNA strands are paired contains a mismatch or a bulge;

(34) an siRNA library expression system according to any one of (28) to (33), wherein said promoter is a pol II or pol III promoter;

(35) the siRNA library expression system according to any one of (28) to (33), wherein the promoter is an inducible promoter;

(36) the siRNA library expression system according to any one of (28) to (33), wherein the siRNA expressed by the system consists of a random RNA strand;

(37) an siRNA library expression system according to any one of (28) to (33), wherein said system is a collection of siRNA expression vectors, wherein each vector targets a gene sequence containing coding and/or non-coding regions;

(38) an siRNA library expression system according to any of (28) to (33), wherein the siRNA expressed by the system consists of an RNA strand encoded by a DNA fragment of any cDNA or genomic DNA, said fragment being of equal length to the siRNA expressed;

(39) a collection of siRNA library expression systems according to any one of (28) to (38), wherein each system in the collection expresses a different siRNA;

(40) a method of searching for a functional gene, the method comprising the steps of:

(a) introducing into a cell an siRNA library expression system according to any one of (28) to (38) or a collection of siRNA library expression systems according to (39);

(b) selecting cells that have been introduced into said siRNA library expression system or said collection; and

(c) analyzing the phenotype of the selected cells;

(41) the method for searching a functional gene according to (40), wherein the method further comprises the steps of: screening for functional genes based on the DNA sequence encoding siRNA in cells found to have an altered phenotype by phenotypic analysis; and

(42) a method of selecting high activity siRNA, said method comprising the steps of:

(a) introducing the siRNA library expression system according to any one of (28) to (38) or the collection of siRNA library expression systems according to (39) into a cell, and

(b) determining the expression level of a particular gene or protein in cells that have been introduced into said siRNA library expression system or said collection.

In one aspect, the invention relates to an intracellular siRNA expression system. The siRNA expression system comprises antisense encoding DNA encoding antisense RNA directed to a region of the target gene mRNA, sense encoding DNA encoding sense RNA directed to the same region of the target gene mRNA, and one or more promoters capable of expressing the antisense and sense RNAs from the antisense and sense encoding DNAs, respectively.

"siRNA" refers to short interfering RNA, which is a short double-stranded RNA that is not toxic to mammalian cells. The length is not limited to 21 to 23bp as reported by Tuschl et al (supra). There is no particular limitation on the length of the siRNA as long as it does not exhibit toxicity. The length of the "siRNA" may be, for example, 15 to 49bp, preferably 15 to 35bp, more preferably 21 to 30 bp. Alternatively, the length of the double stranded RNA portion of the final transcription product of the expressed siRNA may be, for example, 15 to 49bp, preferably 15 to 35bp, more preferably 21 to 30 bp. The double-stranded RNA portion of the siRNA in which the two RNA strands are paired is not limited to perfect pairing, but may contain unpaired portions caused by mismatches (corresponding nucleotides are not complementary), bulges (lack of corresponding complementary nucleotides on one strand), and the like. The amount of unpaired portion should be formed so as not to interfere with the siRNA. As used herein, a "bulge" preferably contains 1 to 2 unpaired nucleotides, and a double-stranded RNA region in which two RNA strands in the siRNA are paired preferably contains 1 to 7, more preferably 1 to 5 bulges. In addition, the double stranded RNA region in which the two RNA strands in the siRNA pair preferably contains 1 to 7, more preferably 1 to 5 "mismatches" as used herein. In a preferred mismatch, one nucleotide is guanine and the other nucleotide is uracil. The mismatch is caused by, but not limited to, a C to T, G to A mutation or a combination thereof in the DNA encoding the sense RNA. In addition, in the present invention, the double-stranded RNA region where the two RNA strands in the siRNA pair may contain both bulges and mismatches, and preferably they may amount to 1 to 7 in total, more preferably 1 to 5 in total.

The unpaired portion (mismatch or bulge, etc.) can inhibit recombination between antisense and sense coding DNAs described below and can stabilize an siRNA expression system described below. In addition, although it is difficult to sequence stem-loop DNA that does not contain unpaired portions in the double-stranded RNA region where the two RNA strands in siRNA pair, sequencing can be performed by introducing mismatches or bulges as described above. In addition, siRNAs with mismatches or bulges in the paired double stranded RNA regions have the following advantages: can stably exist in Escherichia coli or animal cells.

The terminal structure of siRNA may be blunt end or sticky end (overhang) as long as siRNA is capable of silencing the expression of a target gene due to its RNAi effect. It has been reported by Tuschl et al, supra, that the sticky-end (overhang) structure is not limited to 3 'overhangs, but may also include 5' overhang structures, as long as it induces RNAi effects. In addition, the number of overhang nucleotides is not limited to the reported 2 or 3, but may be any number as long as the overhang can induce RNAi effect. For example, the overhang consists of 1 to 8, preferably 2 to 4 nucleotides. Herein, the total length of the siRNA having the sticky end structure is expressed as the sum of the length of the paired double-stranded portion and the length of the pair having the protruding single strands at both ends. For example, when the double-stranded RNA portion is 19bp and both ends are extended by 4 nucleotides, the total length can be expressed as 23 bp. In addition, since the overhang sequence has low specificity for the target gene, it does not have to be complementary (antisense) or identical (sense) to the target gene sequence. In addition, the siRNA may contain a low molecular weight RNA (which may be a natural RNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNA molecule) at, for example, a protruding portion of one end of the siRNA so long as the siRNA retains its gene silencing effect on the target gene.

Further, the end structure of the "siRNA" must be a double-end cleavage structure as described above, which may have a stem-loop structure in which the ends on one side of the double-stranded RNA are linked by an adaptor RNA. The length of the double stranded RNA region (stem-loop portion) may be, for example, 15 to 49bp, preferably 15 to 35bp, more preferably 21 to 30 bp. Alternatively, the length of the double-stranded RNA region as the final transcription product of the expressed siRNA is, for example, 15 to 49bp, preferably 15 to 35bp, more preferably 21 to 30 bp. In addition, the length of the joint is not particularly limited as long as its length does not interfere with the stem assignment pair. For example, the linker moiety may have a clover tRNA structure in order to allow stable pairing of the stem moieties and to inhibit recombination between the DNA encoding the moieties. Even if the linker length prevents pairing of the stem portions, the linker portion may be constructed, for example, to contain an intron, which is cleaved off during processing of the precursor RNA to mature RNA, thereby enabling pairing of the stem portions. For stem-loop sirnas, either end (head or tail) of an RNA without a loop structure can have a low molecular weight RNA. As mentioned above, the low molecular weight RNA may be a natural RNA molecule, such as tRNA, rRNA or viral RNA, or an artificial RNA molecule.

The term "target gene" refers to a gene whose expression is silenced by the siRNA expressed by the system of the present invention, and the target gene can be arbitrarily selected. For example, preferred target genes are genes whose sequences are known but whose functions are yet to be elucidated, and genes whose expression is considered to be causative of a disease. The target gene may be a gene whose genomic sequence has not been completely elucidated, as long as a partial sequence of at least 15 nucleotides or longer in length of mRNA of the gene has been determined, the length being a length capable of binding to one strand of siRNA (antisense RNA strand). Therefore, even if the full-length sequence is not determined, a portion of Expressed Sequence Tags (ESTs) and mRNAs of genes whose sequences have been elucidated can be selected as the "target gene".

"antisense RNA" is an RNA strand having a sequence complementary to the mRNA of a target gene, and it is thought that it can induce RNAi by binding to the mRNA of the target gene. A "sense RNA" has a sequence complementary to an antisense RNA that anneals to its complementary antisense RNA to form an siRNA. These antisense and sense RNAs can be routinely synthesized using an RNA synthesizer. In the present invention, these RNAs are expressed intracellularly from DNA encoding antisense and sense RNAs (antisense and sense coding DNA), respectively, using an siRNA expression system.

The siRNA expression system of the present invention contains a "promoter" for the purpose of expressing antisense and sense RNAs from antisense and sense coding DNAs, respectively. The type, number and position of the promoter may be arbitrarily selected so long as it can express the antisense and sense coding DNAs. A simple method of constructing a siRNA expression system is to form a tandem expression system in which a promoter is located upstream of the antisense and sense coding DNA. The tandem expression system can generate siRNA having the above cleavage structure at both ends. In the stem-loop siRNA expression system (stem expression system), antisense and sense coding DNAs are placed in opposite directions, and these DNAs are connected by linker DNA to construct a unit. The promoter was ligated to one side of the unit to construct a stem-loop siRNA expression system. The length and sequence of the linker DNA are not particularly limited herein, and the linker DNA may have any length and sequence as long as its sequence is not a termination sequence, and its length and sequence do not interfere with the pairing of stem portions during the production of mature RNA as described above. For example, DNA encoding the above tRNA or the like can be used as the linker DNA.

For both tandem and stem-loop expression systems, the 5' end may have sequences that facilitate transcription initiated by a promoter. More specifically, for tandem siRNA, the efficiency of siRNA production can be improved by adding a sequence that promotes transcription initiated by a promoter to the 5' end of the antisense and sense coding DNA. For stem-loop siRNAs, the sequence may be added at the 5' end of the above unit. The transcript of the sequence may be used in a state of being bound to the siRNA as long as it does not interfere with silencing of the target gene by the siRNA. If this state prevents gene silencing, the transcript is preferably trimmed using a trimming tool (e.g., a ribozyme as described below).

In the above tandem or stem expression system, pol II or pol III promoters may be used, provided that the promoters are capable of producing the corresponding RNA from the above DNA. Preferably, a pol III promoter suitable for expression of short RNAs, such as siRNAs, is used. Pol III promoters include the U6 promoter, tRNA promoter, retroviral LTR promoter, adenoviral VA1 promoter, 5Sr RNA promoter, 7SK RNA promoter, 7SL RNA promoter, and H1 RNA promoter. The U6 promoter adds 4 uridine nucleotides at the 3 ' end of the RNA, thus, by providing 0, 1, 2, 3 or 4 adenines to the 5 ' end sequence of the antisense and sense coding DNA, the 3 ' overhang of the resulting siRNA can be changed to 4, 3, 2, 1 or 0 nucleotides without hindrance. When other promoters are used, the number of 3' overhang nucleotides can be varied as desired.

When the pol III promoter is used, it is preferable to further provide a terminator at the 3' end of the sense and antisense encoding DNA in order to express only a short RNA and appropriately terminate transcription. Any terminator sequence may be used so long as it can terminate transcription initiated by the promoter. A sequence consisting of 4 or more consecutive adenine nucleotides, a sequence capable of forming a palindrome, or the like can be used.

Pol II promoters include cytomegalovirus promoter, T7 promoter, T3 promoter, SP6 promoter, RSV promoter, EF-1 alpha promoter, beta-actin promoter, gamma-globulin promoter, and SR alpha promoter. The RNA produced by Pol II promoter is not as short as that produced by Pol III promoter, but rather is somewhat longer. Thus, when using pol II promoters, it is necessary to truncate somewhat longer RNAs using means for cleaving the RNA by self-processing, such as ribozymes, to produce antisense or sense RNAs. The unit for producing antisense or sense RNA using a ribozyme may have the following structure. As shown in FIG. 2A, the antisense or sense RNA-producing unit has an antisense or sense coding DNA, a region coding for an RNA sequence recognized by a ribozyme at its 5 '-and 3' -ends (recognition sequence-coding region), and a region coding for a 5 '-and 3' -end-cleaving ribozyme to cleave off the recognition sequence, which are located outside and adjacent to the respective recognition sequence-coding regions. The antisense and sense RNA-producing units can be linked in tandem and operably linked downstream of the same pol II promoter (FIG. 2B), or can be separately and operably linked downstream of the respective promoters (FIG. 2C). Although FIG. 2B shows an example in which two units are connected in series, an arbitrary spacer sequence may be inserted between the antisense and sense RNA-producing units as necessary to adjust the distance between the two RNAs expressed by the respective units, thereby making it easy for the ribozyme to act on the RNA.

The ribozyme that cleaves the 5 '-and 3' -ends of the antisense and sense coding DNA may be the hammerhead ribozyme (biochem. Biophys. Res. Commun., Vol.186, pp.1271-1279 (1992); Proc. Natl.Acad. Sci. USA, Vol.90, pp.11302-11306 (1993)). Hammerhead ribozymes may have any other sequence as long as they are capable of self-processing (BIO mediaca, Vol.7, pp.89-94 (1992)). In addition, ribozymes are not limited to hammerhead ribozymes, and, for example, hairpin ribozymes, HDV ribozymes, and ribozymes derived from tetrahymena may be used, provided that they are capable of self-processing (Gene, Vol.122, pp.85-90 (1992)). Ribozyme recognition sequences are sequences that can be recognized by 5 '-and 3' -cleaving ribozymes. For example, hammerhead ribozymes cleave the phosphodiester bond of the NUH sequence (N is A, G, C or U and H is A, C or U). Although any combination of nucleotides may be used, it is preferred to use "GUC" as the most effective cleavage sequence in the 3' -direction. Thus, when hammerhead ribozymes are used, NUH, preferably GUC, can be used as the recognition sequence. FIG. 2D shows an example of an mRNA construction process for producing antisense and sense RNA to which the combination of hammerhead ribozyme and recognition sequence GUC has been added at the 5 '-and 3' -ends of the antisense and sense RNA. In FIG. 2D, the 5' -end of the antisense and sense RNA is made to be "C" in order to form a 2-nucleotide overhang. When it is desired to form a 3-nucleotide overhang, the 5' -end is not limited to "C". In the construction process shown in FIG. 2D, the sequence GUC is added in the 3' -direction.

If an inducible promoter is used as the promoter of the present invention, siRNA can be expressed at any desired timing. The inducible promoter includes the tetracycline-inducible U6 promoter (Ohkawa, J. & Taiira, K.control of the functional activity of an antisense RNA by atrocerine-responsive activity of the human U6 snRNA promoter. hum. Gene Ther.11, 577-functional 585 (2000); FIG. 12). In addition, tissue-specific promoters or DNA recombination systems, such as the Cre-loxP system, can be used to induce siRNA expression tissue-specifically.

In addition, in addition to the promoter which can be induced by a drug and the promoter as described above, a recombinase, for example, can be used to control siRNA production. An example of using the CRE-loxP recombinase system will be described in the examples (FIG. 17). In the promoter, there is a spacer between the Distal Sequence Element (DSE) and the Proximal Sequence Element (PSE), and in the spacer there is one loxP site located near the DSE and another loxP site located near the PSE. Typically, due to the distance between the DSE and the PSE, the promoter activity is in the off state to suppress siRNA expression. The action of CRE protein on this expression system induces recombination between loxP sites located in the vicinity of DSE and PSE, resulting in DNA replacement between the loxP sites. Then, DSE and PSE are brought close to each other to turn on the promoter activity to a state where siRNA is expressed. This example describes an siRNA production system in which promoter activity is switched to an on state by the action of CRE. In contrast, a system (not shown) that inhibits siRNA expression by the action of CRE can also be constructed. For example, one loxP can be placed between the DSE and the PSE, while the other loxP can be placed upstream of the DSE or downstream of the PSE, provided that promoter activity is maintained. In the absence of Cre protein, the promoter activity is in the on state, and in the presence of Cre protein, DSE or PSE is replaced by recombination between loxps, thereby shifting the promoter activity to the off state, resulting in suppression of siRNA production. Although this is an example of loxP placement in the promoter region, two loxPs can be provided to interfere with the antisense or sense coding DNA by providing CRE protein to inhibit siRNA production.

In addition, for stem-loop siRNA expression systems, two loxP sites can be provided in the linker moiety to intervene in the termination sequence (e.g., TTTTT). Transcription initiated from the promoter can be terminated by a termination sequence in the linker moiety without the CRE protein, resulting in suppression of siRNA production. The CRE protein induces recombination between loxps to replace the termination sequence, resulting in transcription of the antisense and sense coding DNA to produce stem-loop siRNA (see figure 28).

An siRNA expression system comprising the above-described "promoter", "antisense coding DNA" and "sense coding DNA" can be integrated into a chromosome to express the antisense and sense RNAs in a cell, thereby producing the siRNA. The siRNA expression system is preferably introduced into a target, such as a cell, using a vector carrying the expression system to efficiently transport the system. Vectors that can be used in the present invention can be selected according to the target to be transfected, such as cells, and include, for mammalian cells, viral vectors such as retrovirus vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, lentivirus vector, herpes virus vector, alphavirus vector, EB virus vector, papilloma virus vector and foamy virus vector, and non-viral vectors including cationic liposome, ligand DNA complex, gene gun, etc. (Y. Niitsu et al, Molecular Medicine 35: 1385-. Dumbbell-shaped DNA (Zanta M.A et al, Gene delivery: a single nuclear localization peptide is present to the card DNA to the cell nucleus. Proc Natl Acad Sci U S.1999 Jan 5; 96 (1): 91-6), modified DNA having ribozyme resistance, or naked plasmid (Liu F, Huang L.Improviding plasmid DNA-mediated plasmid transfer in the viral vector. J.Gene Med.2001 Nov-Dec; 3 (6): 569-76) may also be preferably used as a substitute for the viral vector. As shown in examples described below, the present inventors found that the expression of a target gene can be effectively silenced by including the siRNA expression system of the present invention in dumbbell-shaped DNA. Therefore, in a preferred embodiment of the present invention, it is preferred to use an siRNA expression system contained in a dumbbell-shaped DNA molecule. The dumbbell-shaped DNA may be linked to an antibody, a peptide, or the like so that it can be introduced into cells.

The antisense and sense RNA can be expressed in the same vector or in different vectors. For example, constructs for expressing antisense and sense RNA from the same vector can be prepared by ligating upstream of the antisense and sense coding DNA a promoter capable of expressing short RNA, such as a pol III promoter, to form an antisense and sense RNA expression cassette, and inserting the cassette into the vector in the same or opposite orientation. FIG. 1A shows an example of such a structure in which the expression cassettes are inserted in the same orientation. An expression system may also be constructed as shown in FIG. 1B, in which the antisense and sense coding DNAs are placed on opposite strands for pairing. The construct may comprise a double stranded DNA comprising the coding strands of the antisense and sense RNAs (DNA encoding the siRNA) and promoters positioned at both ends and opposite to each other such that the antisense and sense RNAs are expressed from each DNA strand. In this case, in order to avoid adding an excessive amount of sequence downstream of the sense and antisense RNAs, it is preferable to place a terminator at the 3' -end of each strand (the strand encoding the antisense and sense RNAs). The terminator may be a sequence of 4 or more consecutive adenine (a) nucleotides. In this palindromic expression system, it is preferred to use two different promoters. For the present, in the expression system as shown in FIG. 1A, siRNA having a cleavage structure at both ends is generated.

In addition, as another alternative structure capable of expressing the above-mentioned stem-loop siRNA, a unit may be formed in which antisense and sense coding DNAs are placed in opposite directions on the same DNA strand via a linker, and the resulting unit is ligated downstream of a single promoter. In this case, the order of expression is not necessarily limited to "DNA- > linker- > DNA encoding antisense RNA" and may be "DNA- > linker- > DNA encoding sense RNA" encoding antisense RNA. The RNA produced by the expression system having such a structure has a stem-loop structure in which a linker portion forms a loop, and sense and antisense RNA pairs (stem structure) flank it. Then, the loop portion in the palindrome is cleaved by an intracellular enzyme, thereby producing siRNA. In this case, the length of the stem portion, the length and type of the linker, etc. may be selected as described above.

In the system using ribozymes shown in FIG. 2, the system shown in FIG. 2B can be inserted into a vector, or the two expression cassettes shown in FIG. 2C can be inserted into the same vector in the same direction or in opposite directions. A system capable of expressing multiple sirnas (sirnas to different target gene mrnas, sirnas to different target sites of the same target gene mRNA, or sirnas to the same target site of the same target gene mRNA) can also be contained in a single vector.

Systems for expressing antisense and sense RNA in different vectors can be constructed by ligating, for example, a pol III promoter capable of expressing short RNA upstream of the antisense and sense coding DNA to construct antisense and sense RNA expression cassettes, and introducing the expression cassettes into different vectors. In addition, an expression system using a ribozyme can be constructed by introducing the two expression cassettes shown in FIG. 2C into different vectors.

If desired, the vector may further carry a sequence, such as a selectable marker, which enables selection of cells transfected with the vector. Examples of the selection marker include drug resistance markers such as neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene; markers selected for enzyme activity as indicators, such as galactosidase; markers selected for fluorescent emission as indicators, such as GFP; markers selected with cell surface antigens such as EGF receptor, B7-2 and CD4 as indicators, and the like. The selectable marker can only select for cells transfected with the vector, i.e., cells transfected with the siRNA expression system. Therefore, the low transfection efficiency of conventional delivery of exogenous siRNA fragments to cells can be improved, and only cells expressing siRNA can be concentrated. In addition, the use of the vector can prolong the maintenance time of the siRNA expression system. Vectors such as retroviral vectors induce systemic integration into the chromosome, thereby enabling the siRNA expression system in cells to stably provide siRNA.

The present invention relates to a cell maintaining the above siRNA expression system. The cell transduced by this siRNA expression system is preferably a mammalian cell because siRNA can induce RNAi in mammalian cells, which is generally difficult to induce. In addition, cells that are difficult to maintain long-term stable expression of long-stranded dsRNA, such as plant cells, are also preferred as cells transduced by the siRNA expression system of the present invention. However, the above-mentioned cells used in the present invention are not particularly limited to mammalian and plant cells, but may be, for example, cells of other non-mammals, yeast cells, fungal cells, and the like.

The method for introducing the siRNA expression system into the cell can be arbitrarily selected depending on the cell. For example, for transduction of mammalian cells, the following methods may be selected: calcium phosphate method (Virology, vol.52, p.456(1973)), electroporation (Nucleic Acids res., vol.15, p.1311(1987)), lipofection method (j.clin.biochem.nutr., vol.7, p.175(1989)), virus infection-mediated method (sci.am., p.34, March (1994)), particle gun method, and the like. Plant cells can be transduced by electroporation (Nature, Vol.319, p.791(1986)), polyethylene glycol method (EMBO J., Vol.3, p.2717(1984)), particle gun method (Proc. Natl.Acad.Sci.USA, Vol.85, p.8502(1988)), Agrobacterium-mediated method (Nucleic Acids Res., Vol.12, p8711(1984)), and the like.

Cells transduced by the above-described siRNA expression system can be selected by known techniques such as hybridization and PCR using DNA sequences specific for the siRNA expression system as probes or primers. However, when the siRNA expression system is maintained in a vector containing a selectable marker, the selection can be made using a phenotype since the marker can serve as an indicator.

Cells transduced by the siRNA expression system become knockdown cells in which expression of the target gene is silenced. In this context, "knocked down cells" include cells in which expression of the target gene is completely suppressed, as well as cells in which expression of the target gene is not completely suppressed, but is reduced. By deleting or modifying the target gene or its regulatory region, knockdown cells can be routinely generated. In contrast, by introducing the siRNA expression system of the present invention into cells and selecting transduced cells, the use of the siRNA expression system enables simple generation of cells in which expression of the target gene is inhibited without any modification of the target gene on the chromosome. The knockdown cell of the present invention can be used as a research tool for functional analysis of a target gene, and a cell in which a causative gene as a target gene has been silenced can be used as a disease model cell or the like. Further, by introducing the siRNA expression system into germ cells and producing each organism from germ cells maintaining the system, target gene knock-down animals, disease model animals, and the like can be produced.

The method for producing a target gene knock down animal using the above-mentioned siRNA expression system is not particularly limited, and any known method can be used. For example, the siRNA expression vector is injected into a fertilized egg obtained by mating F1 female mice (e.g., CBA/JxC57BL/6J) with male mice (e.g., C57 BL/6J). Peripheral blood DNA was obtained from the tail of a mouse developed from the fertilized egg, and genomic Southem blot analysis was performed on the DNA using a part of the expression vector as a probe, thereby identifying a positive progenitor animal having the siRNA expression vector integrated in the chromosome. Contacting said progenitor mice with C57BL/6J or F₁(CBA/JxC57BL/6J) the heterozygous mice were repeatedly backcrossed to obtain their offspring mice. Then, genomic Southern blotting and PCR analysis were performed to identify progeny that were positive for gene recombination.

In addition, although the case where the siRNA expression system is introduced into a mammal is mainly described above, the system can also be applied to plants. RNAi induced by direct introduction of conventional double-stranded RNA into plant cells is difficult to maintain due to the loss of dsRNA during cell passage. By integrating the siRNA producing system into the chromosome of a plant cell using the RNA expression system of the present invention, RNAi effect can be maintained in the plant cell. Transgenic plants that stably maintain the RNAi effect can also be produced from these cells. Transgenic plants can be produced by methods known to those skilled in the art.

The invention also relates to a composition containing the siRNA expression system. Since the siRNA expression system of the present invention can use siRNA to suppress the expression of any desired target gene, the system can silence a pathogenic gene. The siRNA expression system can be used as a pharmaceutical composition or the like to which an appropriate carrier is added.

Another embodiment of the invention relates to a system for intracellular expression of a library of siRNAs. The siRNA expressed by the "siRNA library" of the present invention consists of an RNA strand containing adenine, guanine, cytosine or uracil arranged in any order and having the same length as the expressed siRNA, or an RNA strand encoded by a (random) cDNA or genomic DNA fragment having the same length as the expressed siRNA. Herein, the siRNA as described above is also referred to as "random siRNA". That is, "random siRNA" as used herein consists of any sequence, or any sequence selected from the group consisting of a particular cDNA sequence, a sequence contained in a particular cDNA library, or a genomic sequence. The above-mentioned siRNA expression system is capable of silencing the expression of a specific target gene, and at the same time, the system of this embodiment can also be used to search for a new functional gene by expressing an siRNA library and silencing an arbitrary gene, for example, a gene whose function and sequence are unknown. One example of an siRNA library expression system has the structure shown in FIG. 1B. The system comprises a DNA encoding a double-stranded siRNA wherein a DNA encoding a random antisense RNA is paired with a DNA complementary to a DNA encoding a sense RNA (hereinafter referred to as "siRNA-encoding DNA"), and two promoters placed opposite to each other and intervening the siRNA-encoding DNA and capable of separately expressing the antisense RNA or the sense RNA.

The "random siRNA" described above is identical to the siRNA expression system described above except that it contains any sequence, or any sequence selected from a specific cDNA sequence, a sequence contained in a specific cDNA library, or a genomic sequence, and consists of double-stranded RNA having a strand short enough not to exhibit toxicity to mammalian cells. It has been reported by Tuschl et al (supra) that the short chain length is not limited to 21 to 23bp, but may be, for example, 15 to 49bp, preferably 15 to 35bp, more preferably 21 to 30bp, as long as it does not exhibit toxicity. In addition, the terminal structure of the random siRNA may be a blunt end or a sticky end (overhang) as long as they can silence the target gene by RNAi action. In addition, the sticky end (overhang) structure includes not only 3 '-overhangs but also 5' -overhangs as long as they can induce the above-mentioned RNAi effect. In addition, the number of overhang nucleotides is not limited to 2 or 3, and may be any number that induces an RNAi effect, such as 1 to 8 nucleotides, preferably 2 to 4 nucleotides. Further, as described above, the siRNA may contain low molecular weight RNA at an overhang at one end thereof. In addition, as described above, the siRNAs expressed by the siRNA library expression system may contain mismatches or bulges, or both, in the regions of the RNA paired on double stranded RNA.

In addition, the siRNA library expression system is not limited to the above-described structure (having two promoters placed opposite to each other intervening in siRNA-encoding DNA), but may also have a structure capable of expressing stem-loop siRNA. That is, the present invention also includes a structure in which a promoter is ligated upstream of a unit (hereinafter referred to as "stem-loop siRNA library producing unit") formed by ligating a DNA encoding an antisense RNA (e.g., any random sequence, or any sequence selected from a specific cDNA sequence, a sequence contained in a specific cDNA library, or a genomic sequence) and a DNA encoding a sense RNA complementary to the above-mentioned antisense RNA in opposite directions with a linker DNA. FIG. 18 shows an example of a method for preparing the above-described stem-loop siRNA library-generating unit. That is, a single-stranded DNA containing a DNA encoding an antisense RNA having a random sequence (antisense encoding DNA) and further containing a sequence capable of forming an arbitrary palindrome at the 3' -end thereof is synthesized using a DNA synthesizer or the like. A primer complementary to the 5 '-side of the single-stranded DNA is prepared, and the primer is annealed thereto to form a palindrome at the 3' -end of the single-stranded DNA. A DNA polymerase and a DNA ligase are allowed to act on the structure to synthesize a sense-encoding DNA strand complementary to the antisense-encoding DNA, while forming an elongated palindromic structure of the stem portion. The palindrome is rendered single-stranded by denaturation, and PCR is performed using primers complementary to sequences flanking both the antisense encoding DNA and the sense encoding DNA to produce double-stranded DNA containing stem-loop siRNA library-producing units. If necessary, one strand of the double-stranded DNA containing the stem-loop siRNA library producing unit is trimmed with a restriction enzyme or the like, and the thus obtained double-stranded DNA is ligated downstream of an appropriate promoter to produce a stem-loop siRNA library expression system.

The stem-loop siRNA is produced by the stem-loop siRNA library expression system described above. In the stem-loop siRNA as described above, the length of the double-stranded RNA portion (stem portion) produced may be, for example, 15 to 49bp, preferably 15 to 35bp, more preferably 21 to 30 bp. In addition, there is no particular limitation on the length and sequence of the linker, as long as they do not interfere with the stem assignment pair, and low molecular weight RNA, such as clover tRNA, can be used as the linker.

The above-mentioned "random antisense encoding DNA" is composed of any sequence which can be arbitrarily selected from, for example: a sequence of four nucleotides "A, G, C and T" in any combination, of equal length to the expressed siRNA. Alternatively, a "random antisense encoding DNA" consists of any sequence selected from a particular cDNA sequence, a sequence contained in a particular cDNA library, or a genomic sequence. Promoters useful herein may be pol II or pol III promoters. Preferably, a pol III promoter suitable for expression of short RNAs, such as siRNAs, is used. In addition, the two promoters may be the same or different, and it is preferable that the promoters are different in view of expression efficiency. Examples of pol II and pol III promoters that may be used at this time are the same as those described above.

In addition, when the pol III promoter is used, in order to appropriately terminate transcription after expression of a complementary short RNA, it is preferable to provide a terminator between the promoter and the DNA encoding siRNA as shown in FIG. 1B. A sequence consisting of 4 or more consecutive adenines as shown in fig. 1B, any terminator known to those skilled in the art, or the like may be used.

When an inducible promoter is used, the library of siRNAs can be expressed at a predetermined timing. Thus, genes that function at a particular developmental/differentiation stage of an organism can be analyzed. In addition, the use of a promoter having tissue-specific transcriptional activity can induce tissue-specific expression of siRNA, thereby enabling analysis of functional genes in specific tissues. The inducible promoter and the tissue-specific promoter that can be used at this time are the same as those described above. The siRNA library expression system described above can be integrated into the chromosome of a cell as a DNA insert. For efficient introduction into cells and the like, it is preferable to maintain the siRNA library expression system in a vector. The "vector" usable herein is the same as described above. The efficiency of screening for functional genes can also be improved by introducing an siRNA library expression system capable of expressing multiple siRNAs into a single vector. If necessary, the vector carrying the siRNA library expression system may further contain a selection marker or the like. The selectable markers that may be used at this time are the same as those described above. Thus, the use of a selectable marker allows for the selection of cells transfected with a vector carrying an expression system for an siRNA library, thereby improving the efficiency of screening for a functional gene.

Another embodiment of the siRNA expression system of the present invention is an siRNA library expression system comprising a collection of siRNA expression vectors, each vector targeting a gene sequence comprising a coding region and/or a non-coding region.

siRNA library expression systems that express different siRNAs can also be pooled and a pool constructed. For example, an siRNA encoding DNA and stem-loop siRNA library expression system can be constructed to generate an RNA strand containing a sequence of four nucleotides "A, G, C and U" in any combination, as long as the expressed siRNA, as the siRNA expressed from this pool. Alternatively, the siRNA encoding DNA may comprise any cDNA fragment, or any sequence selected from the group consisting of sequences contained in any cDNA library or genomic sequences. Therefore, a collection containing multiple siRNA library expression systems can be used to search for functional genes more efficiently.

The method for searching for functional genes can be performed using the random siRNA library expression system described above or a collection of these siRNA library expression systems by the following steps: introducing an siRNA library expression system or a collection of siRNA library expression systems as described above into a cell, selecting cells transduced with the siRNA library expression system or collection, and analyzing the phenotype of the cells so selected.

As described above, the method of introducing the siRNA library expression system or the like into the cell may be changed depending on the type of the cell. Specifically, the method for introducing the system into mammalian cells may be selected from the group consisting of calcium phosphate method (Virology, vol.52, p.456(1973)), electroporation method (Nucleic Acids Res., vol.15, p.1311(1987)), lipofection method (J.Clin.biochem.Nutr., vol.7, p.175(1989)), viral infection transduction method (Sci.am.p.34, March (1994)), particle gun method, etc., and plant cells may be introduced by electroporation method (Nature Vol.319, p.791(1986)), polyethylene glycol method (EMBO J.vol.3, p.2717(1984)), particle gun method (Proc.Natl.Acad.Sci.USA.871.85, p.8502(1988)), Agrobacterium-mediated method (Nucleic Acids Res.Vol.12, p.1984), etc.

When an siRNA library expression system or the like is introduced into a vector carrying a selection marker, cells transduced by the system or pool can be selected by collecting cells having a phenotype caused by the selection marker. When the vector does not contain a selection marker, transduced cells can be selected by detecting the transduced cells using a specific sequence shared by the siRNA library expression systems as a probe or primer using known hybridization methods, PCR, or the like.

After selecting cells transduced with the siRNA library expression system or pool described above, the phenotype of the transduced cells is analyzed by comparing the phenotype of the transduced cells to control cells not transduced with the siRNA library expression system or pool. These phenotypes are not limited to cell surface-exhibited phenotypes, but include, for example, intracellular changes and the like.

Cells that are considered to have phenotypic changes as analyzed above may contain an siRNA library expression system that can silence any functional gene. Therefore, in order to screen for functional genes, probes and primers can be constructed based on the DNA sequence encoding siRNA contained in the cell, and they can be used for hybridization or PCR to perform cloning of functional genes. Database searches for functional genes can also be performed based on the DNA sequences encoding the sirnas.

The effect of the siRNA expression system of the present invention will generally vary greatly depending on the location of the target site of the target gene. For example, when targeting HIV, siRNA is expected to achieve high gene silencing by targeting the priming site. The siRNA library expression system of the present invention is effective even in cases where the preferred target site is unknown. That is, the above-mentioned siRNA library expression system of the present invention is useful as a system for searching for an optimal target site of mRNA efficiently degraded by siRNA. The invention provides a method for selecting high-activity siRNA, which comprises the following steps: the siRNA library expression system of the present invention or the collection of said siRNA library expression systems is introduced into cells and the expression level of a specific gene or protein in the cells transduced with the siRNA library expression system or the collection thereof is determined. The expression level of any desired gene or protein can be readily determined by methods known to those skilled in the art, such as Northern blot hybridization or Western blot hybridization.

The cells into which the siRNA expression system and siRNA library expression system of the present invention are introduced are not particularly limited to mammalian cells, and may include cells of other animals, plants, yeasts, fungi, and the like.

The siRNA expression library of the present invention can be used, for example, to search for genes associated with viral infection. Genes associated with viral infection can be readily identified by introducing the siRNA expression library into cells, infecting the cells with a virus, and examining the surviving cells. All genes associated with viral infection can be identified using an siRNA expression library containing 40,000 human cDNAs. Randomized siRNA expression libraries or siRNA expression libraries of genomic fragments can identify genes other than cDNA. The two libraries can be used in combination.

Drawings

FIG.1 depicts an siRNA expression system using the U6 promoter and a method for generating siRNA using the system. (A) The production process of siRNA is shown. Two U6 promoters produced sense and antisense short RNAs with 4 uridines (U) added to the 3' -end of the RNA. The sense and antisense RNAs so expressed anneal to form an siRNA duplex having 4-nucleotide 3' overhangs. (B) A palindromic structural siRNA expression system is shown comprising double-stranded DNA encoding siRNA and promoters at both ends, said DNA comprising sense and antisense encoding DNA, with sense and antisense RNA expressed from said promoters.

FIG. 2 depicts an example of the structure of an siRNA expression system generated using ribozymes.

FIG. 3 depicts the EGFP gene silencing effect of a siRNA expression system against EGFP when introduced into hygromycin/EGFP expressing cells. The left set of experiments (A, D, G and J) showed hygromycin/EGFP expression; the middle set of experiments (B, E, H and K) showed expression of DsRed; the right set of experiments (C, F, I and L) shows the results of the combined expression of hygromycin/EGFP and DsRed.

FIG. 4 depicts the gene silencing effect of the siRNA expression system against either sea pansy (Renilla) or firefly luciferase when introduced into HeLa S3 cells with luciferase activity. In fig. 4a, the ordinate values refer to: luciferase activity of cells into which a siRNA expression system for firefly luciferase has been introduced normalized based on Renilla luciferase activity, or luciferase activity of cells into which a siRNA expression system for Renilla luciferase has been introduced normalized based on firefly luciferase activity. FIG. 4b shows the concentration-dependent silencing effect of siRNA expression systems on firefly or Renilla luciferase activity when different amounts of siRNA expression systems for each luciferase were introduced into the cells.

FIG. 5 depicts gene silencing using a series of siRNAs or siRNA expression systems directed against different target sites on the same target gene (firefly luciferase). FIG. 5a shows the gene silencing effect of siRNA expression vectors directed against different target sites when the vectors are introduced into cells. FIG. 5b shows the results obtained when different concentrations of exogenous siRNA directed against different target sites were introduced directly into cells.

FIG. 6a depicts gene silencing of siRNA 3' overhangs of varying lengths. FIG. 6b shows gene silencing effect of siRNA expression system against two target genes or two target sites. In FIG. 6b, the values of luciferase activity were normalized based on the beta-galactosidase activity introduced as an internal control.

FIG. 7 depicts the ability of the siRNA expression system to silence the endogenous β -catenin gene. The experimental groups A, B and C were groups transduced with siRNA expression vector (pHygEGFP/i. beta. -catenin) against beta-catenin (catenin), while the experimental groups D, E and F were groups transduced with empty vector (pHygEGFP). All groups were stained with anti- β -catenin antibody. The experimental groups on the left (A and D) show the expression of hygromycin/EGFP; the middle panel (B and E) shows the expression of β -catenin; while the right experimental groups (C and F) show the merged images of these two expressions.

FIG. 8 depicts a comparison of RNAi effects between tandem and stem-loop siRNAs. siRNA expression vectors pU6tandem19 and pU6stem19 are tandem and stem-loop, respectively. Cont. control (blank vector).

FIG. 9 depicts gene silencing effects of multiple siRNA expression vectors.

FIG. 10 depicts gene silencing of siRNA expression vectors containing a cytomegalovirus-derived promoter (CMV promoter) and a tRNA promoter.

FIG. 11 depicts RNAi induction of double stranded siRNA containing mismatches or bulges.

Fig.12 illustrates the principle of the Tet-ON system. In the absence of tetracycline, the tetracycline repressor binds to the U6 promoter, resulting in inhibition of transcription, while in the presence of tetracycline, the tetracycline repressor binds to tetracycline, which is released from the U6 promoter and transcription is initiated.

FIG. 13 is a schematic depiction of RNAi induction of siRNA expression vectors with tetracycline-inducible promoters. U6Teti represents the siRNA expression vector containing tetracycline manipulation sequence in the U6 promoter, U6i represents the siRNA expression vector without this sequence.

FIG. 14 illustrates auto-cleavage in RNA transcripts containing tailored ribozymes.

FIG. 15 is a graph depicting siRNA production resulting from self-processing of tailored ribozymes. Nucleotide cleavage occurs at the position indicated by the black arrow to generate siRNA.

FIG. 16 is an electrophoretogram showing siRNA production resulting from RNA self-processing. The arrow indicates the band corresponding to 21nt siRNA.

FIG. 17 is a diagram illustrating an example of a structure for controlling siRNA expression using the Cre-lox system.

FIG. 18 shows an example of the preparation of stem-loop siRNA library expression systems.

FIG. 19 illustrates an example of the preparation of an siRNA library expression system.

Shows random DNA fragments with dephosphorylated blunt ends of 19 to 29bp in length.

② a random DNA fragment (i) having 5' -phosphorylated hairpin-type DNA linkers (1) ligated to both ends thereof.

Fig. 20 is a continuation of fig. 19.

③ shows the strand displacement from the Nick site by Bst DNA polymerase.

(iv) represents a fragment ligated to the DNA linker 2.

Fig. 21 is a continuation of fig. 20.

Fifthly, shows the strand displacement from the Nick site by Bst DNA polymerase.

Sixthly, cracking of the fifth component by the AscI.

Fig. 22 is a continuation of fig. 21.

And (c) representing the siRNA library expression pre-library.

(viii) shows the BspMI cleavage of the siRNA library expression pre-library.

When the loop sequence TTCG is inserted between the sense and antisense encoding DNAs, cleavage proceeds to step viii-2 of fig. 23.

Ninthly, represents a completed siRNA library expression system obtained by blunting with Klenow fragment, removing the DNA linker 1 and performing self-ligation.

Fig. 23 is a continuation of fig. 22.

And (b) -2 represents the case where a loop sequence TTCG is inserted between the sense and antisense encoding DNAs in (b). The siRNA library was expressed by cleaving with BsgI.

The eighty-3 shows the expression of the pre-library by cleaving the siRNA library with BspMI. The cleavage site of BsgI is not attacked by BspMI.

Ninthly-2 represents a completed siRNA library expression system obtained by blunting with T4DNA polymerase, removing the DNA linker 1 and performing self-ligation.

FIG. 24 illustrates the preparation of EGFP cDNA fragments of about 20 to 25bp in length. Random EGFP cDNA fragments approximately 20 to 25bp long with dephosphorylated blunt ends the final product was used as random DNA fragment in FIG. 19 (r).

FIG. 25 illustrates the preparation of a cloning vector. The promoter is the human U6 promoter or the human tRNA promoter. BspMI and Klenow were used to prepare cloning vectors containing the U6 promoter, and BseRI and T4DNA polymerase were used to prepare cloning vectors containing tRNA promoters.

Fig. 26 is a micrograph showing the result of observing the fluorescence intensity of EGFP with a confocal microscope.

FIG. 27 is a graph showing the relative EGFP fluorescence intensity in pUC18, U6 GFP25s iRNAlib-loop-, U6 GFP25 siRNA lib TTCG, tRNA GFP25 siRNA lib loop-, and tRNAGFP25 siRNA lib TTCG, measured 24 and 48 hours after transfection.

FIG. 28 is a diagram illustrating a stem-loop siRNA expression system containing two loxPs capable of hybridizing to linker moieties containing termination sequences.

FIG. 29 is a graph depicting the gene silencing effect of siRNA expressing adenoviral vectors.

FIG. 30 is a graph depicting the gene silencing effect of siRNA expressing HIV vectors.

FIG. 31 is a graph showing the gene silencing effect of siRNA expressing dumbbell-type vectors.

FIG. 32 is a graph showing the gene silencing effect of siRNA expression systems containing mismatches or bulges in the double stranded RNA regions of the siRNAs. The numbers in parentheses in front of each sequence represent SEQ ID NO: .

Best mode for carrying out the invention

Example 1 Induction of RNAi Using siRNA expression vector

And detecting whether the siRNA expression vector can silence a target gene encoding exogenous hygromycin/EGFP fusion protein.

hygromycin/EGFP expression vector (pHygEGFP) and internal control DsRed expression vector (pDsRed2) were purchased from Clontech. An siRNA expression vector was constructed using plasmid pU6(Ohkawa, J. & Taira, K.control of the functional activity of an antisense RNA by a tetracycline-responsive activity of the human U6 snRNA promoter. hum Gene ther.11, 577-cell 585(2000)) carrying the human U6 promoter. A fragment containing DNA encoding the hygromycin/EGFP sense and antisense RNA portions was synthesized using a DNA synthesizer and subcloned directly into pU6 downstream of the U6 promoter. In order to insert these synthetic fragments into pU6 downstream of the U6 promoter, a BspMI recognition site was provided downstream of the U6 promoter of the vector used for subcloning, and a BspMI site was provided further downstream. After cleavage with BspMI, a 4-nucleotide sticky end was formed. A vector capable of expressing sense RNA is constructed by inserting synthetic sense coding DNA having a terminal complementary to the sticky end.

DNA (19 nucleotides) encoding antisense RNA was synthesized in a similar manner and subcloned directly into pU6 downstream of the U6 promoter.

An antisense RNA expression cassette containing the U6 promoter was excised from the vector, and inserted into pU6 vector containing the sense RNA expression cassette to construct an siRNA expression vector (pU6 iHyg/EGFP). Herein, since it is reported that when the U6 promoter is used, 4 uridine (U) is added to the 3 '-end of the expressed mRNA, siRNA expressed by the siRNA expression vector and formed inside the cell has an overhang of 4 nucleotides at both 3' -ends. That is, the siRNA expression vector expresses 23-nucleotide long siRNA having a 19-nucleotide duplex with 4-nucleotide overhangs at both 3' ends (FIG. 1A).

Human HeLa S3 cells were co-transfected with pHygEGFP (1g), pDsRed2(0.5g) and pU6iHyg/EGFP (1g) as described above by lipofection (using Lipofectamine 2000). 48 hours after transfection, the cells were placed at 37 ℃ and observed under a focusing microscope. As a control experiment, similar procedure was performed using pU6 instead of siRNA expression vector.

In FIG. 3, the upper experimental group describes the results of a control experiment using pU6, while the lower experimental group shows the results obtained by introducing pU6 iHyg/EGFP. As shown in the middle column of FIG. 3, in the cells transduced with pDsRed, which emits red fluorescence, as an internal control, there was no significant difference in fluorescence intensity between the control and the pU6 iHyg/EGFP-transduced group, indicating that there was no difference in vector transfer efficiency between the two experimental groups, and that the siRNA expression vectors did not exhibit non-specific gene silencing effects on gene expression. On the other hand, as shown in the left column of FIG. 3, the number of cells emitting green fluorescence due to pHygEGFP was reduced, and the fluorescence intensity of green fluorescent cells was also reduced in the pU6 iHyg/EGFP-transduced group as compared with the control group. Similarly, as shown in the right column of fig. 3, even when red and green fluorescence were combined, the number of green-fluorescent cells and yellow-fluorescent cells due to the combination of red and green were reduced in siRNA expression vector-transduced cells compared to the control. These results confirm that: introduction of the siRNA expression vector induces RNAi, resulting in silencing of the target gene. In addition, the results of a similar analysis using mouse COS7 cells showed: expression of pDsRed was not affected at all, while expression of pHygEGFP was specifically silenced (not shown).

EXAMPLE 2 quantitative determination of Gene silencing Activity of siRNA expression vectors

To quantify the RNAi effect, siRNA gene expression silencing activity against firefly and renilla luciferase genes (as another reporter gene) was analyzed as follows.

HeLa S3 and COS7 cells were cultured in Dulbecco 'S modified Eagle' S medium supplemented with 10% fetal bovine serum. The respective cultured cells (3X 10)⁴Individual cells/well) were placed in each well of a 48-well culture plate. For luciferase reporter gene analysis, RSV-Renilla luciferase expression vector (pRL-RSV) was used by lipofection using Lipofectamine 2000(Life technologies)¹⁵(30ng), firefly luciferase expression vector pGL3(Promega) (30ng) and various amounts of siRNA expression vectors against firefly or Renilla luciferase transcripts were co-transfected into cells in each well.

FIG. 4 shows the results of luciferase assay in HeLa S3 cells. FIG. 4A shows the results obtained by introducing a fixed amount (300ng) of siRNA expression vector, indicating that the introduction of siRNA can silence the expression of the corresponding target gene. In addition, both plasmids did not contribute at all to the expression of firefly and renilla luciferase in luciferase activity assays with plasmids expressing only sense or antisense RNA. FIG. 4B shows the results obtained by introducing different amounts of siRNA. The siRNA expression vector directed against the firefly luciferase gene dose-dependently reduced firefly luciferase activity without affecting renilla luciferase activity. On the other hand, in cells transfected with siRNA expression vectors directed against the renilla luciferase gene, the renilla luciferase activity decreased dose-dependently. These results clearly show that: the siRNA expression system using the U6 promoter can specifically and effectively silence target genes.

[ example 3] target site-dependent Gene silencing

Next, whether siRNA expression vectors directed against different target sites in the same transcript have different gene silencing effects is examined. In this assay, individual siRNA expression vectors directed against 4 different target sites on the firefly luciferase transcript, together with the firefly luciferase expression vector and the renilla luciferase expression vector used as internal controls, were co-transfected into HeLa S3 cells under conditions similar to example 2. The sequences of the sense and antisense encoding DNAs in the siRNA expression vectors for 4 different target sites are as follows:

firefly luciferase site O sense strand: 5'-GCTATGAAACGATATGGGC-3' (SEQ ID NO: 1);

site O antisense strand: 5'-GCCCATATCGTTTCATAGC-3' (SEQ ID NO: 2);

site a sense strand: 5'-GTTCGTCACATCTCATCTAC-3' (SEQ ID NO: 3);

site a antisense strand: 5'-GTAGATGAGATGTGACGAA-3' (SEQ ID NO: 4);

site B sense strand: 5'-GTGCGCTGCTGGTGCCAAC-3' (SEQ ID NO: 5);

site B antisense strand: 5'-GTTGGCACCAGCAGCGCAC-3' (SEQ ID NO: 6);

site C sense strand: 5'-ATGTACACGTTCGTCACAT-3' (SEQ ID NO: 7);

site C antisense strand: 5'-ATGTGACGAACGTGTACAT-3' (SEQ ID NO: 8);

control renilla luciferase

(sense strand with nucleotide overhang):

5’-GTAGCGCGGTGTATTATAC-3’(SEQ ID NO：9)；

(antisense strand with nucleotide overhang):

5’-GTATAATACACCGCGCTAC-3’(SEQ ID NO：10)。

as shown in FIG. 5A, luciferase gene silencing activity varied depending on the target site. That is, the gene silencing activity of the siRNA expression vector directed against site B was the highest, resulting in a decrease of firefly luciferase activity to 14% of that of the control. The siRNA expression vectors directed to positions A, C and D reduced the enzyme expression levels to 44%, 38% and 36% of the control, respectively. At this time, the siRNA expression vector directed to site O showed no gene expression silencing activity, similar to the two control expression vectors Hyg-U6siRNA and pU 6.

It was also examined whether the above-mentioned difference in gene silencing activity depending on the target site is caused by only the difference in the target site or the difference in transcription efficiency of each siRNA. For detection, the above sense and antisense RNA oligonucleotides were synthesized separately. The sequences of these siRNA oligonucleotides are as follows:

firefly luciferase site O sense strand:

5’-GCUAUGAAACGAUAUGGGCUU-3’(SEQ ID NO：11)；

site O antisense strand: 5'-GCCCAUAUCGUUUCAUAGCUU-3' (SEQ ID NO: 12);

site a sense strand: 5'-GUUCGUCACAUCUCAUCUACUU-3' (SEQ ID NO: 13);

site a antisense strand: 5'-GUAGAUGAGAUGUGACGAAUU-3' (SEQ ID NO: 14);

site B sense strand: 5'-GUGCGCUGCUGGUGCCAACUU-3' (SEQ ID NO: 15);

site B antisense strand: 5'-GUUGGCACCAGCAGCGCACUU-3' (SEQ ID NO: 16);

site N sense strand: 5'-AUGUACACGUUCGUCACAUUU-3' (SEQ ID NO: 17);

site N antisense strand: 5'-AUGUGACGAACGUGUACAUUU-3' (SEQ ID NO: 18);

control renilla luciferase

(sense strand with nucleotide overhang):

5’-GUAGCGCGGUGUAUUAUACUU-3’(SEQ ID NO：19)；

(antisense strand with nucleotide overhang):

5’-GUAUAAUACACCGCGCUACUU-3’(SEQ ID NO：20)。

the RNA oligonucleotides were synthesized using a model 394 RNA synthesizer (Applied Biosystems). The synthesized RNA was deprotected and purified by denaturing acrylamide gel electrophoresis. After elution from the gel, each RNA fraction was loaded on a NAP-10 column (Pharmacia) and eluted with RNase-free water for desalting. The resulting eluate was dried under vacuum and resuspended in annealing buffer (phosphate-buffered saline (PBS) pH6.8, 2mM MgCl₂). Then, in order to anneal RNA oligonucleotides, 10. mu.M RNA was prepared, incubated at 95 ℃ for 1 minute, then cooled to 70 ℃ and slowly cooled to 4 ℃ over 2 hours. The resulting siRNA oligonucleotides were introduced into HeLa S3 cells to detect luciferase activity according to a similar method to that described above (FIG. 5B).

The luciferase gene silencing profiles caused by siRNA oligonucleotides directed against each target site exhibited patterns similar to those obtained when introduced into 1 and 0.1nM siRNA expression vectors, except for the weak gene silencing activity exhibited by the siRNA oligonucleotide directed against site O. These results show that: the difference in gene silencing activity is not caused by the difference in expression efficiency of each siRNA but depends on the difference in target sites (e.g., their secondary structures) and the presence of RNA-binding proteins.

EXAMPLE 4 Effect of siRNA 3' overhang Length

The siRNA generated by the U6 promoter had a 4 uridine nucleotide overhang at the 3' -end. On the other hand, Elbashir et al reported that in vitro experiments with Drosophila (Drospilia), the gene silencing efficiency of siRNA decreased when the 3' overhang was longer than 2 to 3 nucleotides (Elbashir, S.M., Lendeckel, W. & Tuschl, T.RNA interference is meditated by 21-and 22-nucleotide RNAs. genes Dev.15, 188- "200 (2001)). Therefore, we examined whether the 4-nucleotide 3' overhang of the above siRNA would affect the efficiency of siRNA to induce RNAi. siRNA oligonucleotides directed against the same target site on the renilla luciferase transcript described above were prepared by chemical synthesis similar to example 3 above, with a 3' overhang set to 2, 3 or 4 uridine nucleotides in number. These siRNA oligonucleotides directed against the renilla luciferase transcript, as well as the firefly luciferase expression vector as an internal control were introduced into HeLa S3 cells at different concentrations by lipofection to detect luciferase activity (fig. 6A).

The expression of renilla luciferase was not silenced with the internal control alone, but was dose-dependently inhibited in the cell groups transduced with the siRNA oligonucleotide described above. In addition, it was observed that there was no significant difference in gene silencing activity when the length of the 3 'overhang was changed from 2 nucleotides to 4 nucleotides, thereby revealing that siRNA with a 4-nucleotide 3' overhang produced from the U6 promoter was also able to effectively silence the expression of a target gene as was the case with siRNA with a 2-or 3-nucleotide overhang.

[ example 5] Simultaneous silencing of multiple genes

We examined whether siRNA can simultaneously silence different target genes when two different genes comprising the target gene are simultaneously expressed. Plasmids containing two siRNA expression cassettes for firefly and renilla luciferase were constructed and co-transfected into cells.

Firefly luciferase expression vector (30ng), renilla luciferase expression vector (30ng), vector expressing siRNA against both luciferase transcripts (300ng), along with β -galactosidase expression vector (100ng) as an internal control were co-transfected into HeLa S3 cells. As a control, similar experiments were performed using vectors expressing siRNA against either firefly and renilla luciferase transcripts.

As shown in FIG. 6B, transfection with siRNA expression vectors for both luciferases (U6 i-Finefly/Renilla) simultaneously silenced the expression of Firefly and Renilla luciferases to the same level as the result of introducing siRNA expression vectors for either luciferase (U6i-Renilla or U6 i-Finefly).

As described above, it has been clarified that by placing a plurality of siRNA expression cassettes in the same plasmid to express a plurality of sirnas simultaneously, the corresponding target gene can be silenced without causing interference in each promoter.

[ example 6] endogenous Gene silencing

All of the above examples relate to silencing of exogenous genes introduced into cells. In this experiment, whether siRNA expression vectors can silence endogenous gene expression was examined.

Endogenous genes encoding beta-catenin were selected as targets. Beta-catenin is a membrane-bound cytoplasmic protein known to be a factor associated with cadherin in intercellular adhesion, and is also an important oncogene (Peifer, M. & Polakis, p. wnt signaling in oncogenesis and endogene-a lok outings the nuclear. science 287, 1606-1609 (2000)).

An EGFP expression plasmid (pEGFP/i β -catenin) containing a siRNA expression cassette against β -catenin was introduced into SW480 cells expressing β -catenin. As a control, an EGFP expression plasmid (pEGFP) without siRNA expression cassette against β -catenin was introduced into 60% confluent cells in a similar manner. These plasmids are introduced into cells fixed on slides using reagents such as Effectene (Qiagen) or Fugene 6(Roche Molecular Biochemicals). After 48 hours of transduction, cells were fixed in PBS containing 4% paraformaldehyde for 20 minutes, permeabilized in 0.1% Triton X100, and then stained with anti- β -catenin antibody (UBI) and Cy 3-labeled secondary antibody. The stained cells were analyzed for fluorescence using a focusing microscope (fig. 7).

The results show that: the expression level of β -catenin was much lower in green fluorescent cells containing pEGFP/i β -catenin compared to cells not transduced with the plasmid. In addition, there was no difference in the expression level of β -catenin in green fluorescent cells transduced with pEGFP and cells transduced without plasmid.

Example 7 comparison of Gene silencing Effect between tandem and Stem-Loop siRNA expression vectors

This example examined the gene silencing effect of siRNA expression vector pU6tandem19, in which DNAs encoding sense and antisense RNAs were placed in tandem, and siRNA expression vector pU6stem19, in which the latter vector was capable of expressing stem-loop RNA molecules. The siRNAs expressed by the above respective vectors are referred to as tandem siRNA and stem-loop siRNA. The sequence of the stem-loop siRNA transcribed in pU6stem19 was 5'-GTGCGCTGCTGGTGCCAACgugugcuguccGTTGGCACCAGCAGCGCAC-3' (SEQ ID NO: 21). Gene silencing activity was quantified by luciferase assay as described in the above examples.

The results are shown in FIG. 8. Both tandem and stem-loop sirnas are able to concentration-dependently reduce luciferase activity. These results indicate that both tandem and stem-loop siRNA expression systems are effective in inhibiting the expression of target genes.

EXAMPLE 8 Gene silencing Effect of various siRNA expression vectors

This example examines the gene silencing effect of siRNA expression vectors containing various promoters. Luciferase assays were performed under conditions similar to those of the above examples. The following siRNA expression vectors were used.

● expression of the vector of tandem siRNA (pU6tandem19) through the human U6 promoter,

● the vector (p5Standem19) expressing tandem siRNA through the human 5S rRNA promoter,

● vector (pH1tandem19) expressing tandem siRNA through human H1 promoter, and

● expression of stem-loop siRNA through human H1 promoter (pH1stem 19).

As shown in fig. 9, various expression vectors used herein showed luciferase inhibitory activity, indicating that promoters usable in siRNA expression vectors are not limited to specific promoters, and various promoters such as human U6 promoter, human 5S rRNA promoter and human H1 promoter can be used.

EXAMPLE 9 Gene silencing Effect of siRNA expression vectors containing CMV promoters or tRNA promoters

This example examined gene silencing of siRNA expression vectors (pCMV-TRz and ptRNA-TRz, respectively) containing either a cytomegalovirus-derived promoter (CMV promoter) or a tRNA promoter and a trimming ribozyme. A tRNA molecule is added to the 5' end of the tRNA promoter transcript. The 3' terminal excess RNA molecules not necessary for siRNA formation were cleaved off by the action of a trimming ribozyme.

As shown in FIG. 10, both siRNA expression vectors containing CMV promoters or tRNA promoters were able to reduce luciferase activity, indicating that it is preferable to use CMV promoters or tRNA promoters as promoters in the siRNA expression vectors of the present invention, and that even expression vector transcripts bound to a molecule such as 5' terminal tRNA have a target gene silencing effect.

Example 10 RNAi Induction by double stranded siRNA containing mismatches or bulges

This example examines the effect of the presence of mismatches or bulges in double stranded siRNA on RNAi. Luciferase assays were performed under conditions similar to those of the above examples. The following RNA sequences were used in the experiments.

● control RNA sequence

5′-GUGCGCUGCUGGUGCCAACCCgugugcuguccGGGUUGGCACCAGCAGCGCAC-3′(SEQ ID NO：22)

● RNA sequences containing mismatches

5′-GUGCGCUGuUGGUGuCAACCCgugugcuguccGGGUUGGCACCAGCAGCGCAC-3′(SEQ ID NO：23)

● contains a raised RNA sequence

5′-GUGCGCUGCUGGUGCuCAACCCgugugcuguccGGGUUGGCACCAGCAGCGCAC-3′(SEQ ID NO：24)

As shown in fig. 11, a plurality of expression vectors used herein showed luciferase inhibitory activity. The presence or absence of mismatches or bulges in double-stranded siRNA does not produce significant differences in gene silencing.

In addition, this example examined the RNAi effect induced by various siRNAs containing mismatches or bulges. FIG. 32 shows a DNA sequence encoding one siRNA strand and RNAi effect (luciferase activity) induced by the sequence.

These results show that: even an siRNA containing a mismatch or a bulge in its double strand can effectively suppress the expression of a target gene. That is, each strand constituting the siRNA duplex need not be completely complementary to each other.

Example 11 RNAi induced by siRNA expression vector having tetracycline-inducible promoter

A system capable of controlling the transcriptional activity of an RNA promoter by tetracycline (Tet-ON system) is known (Ohkawa, J. & Taira, K.control of the functional activity of an antisense RNA bya tetracycline-responsive activity of the human U6 snRNA promoter. HumGene Ther.11, 577-585 (2000)). The tetracycline operator sequence of the tetracycline-resistant transposon has been inserted into the human U6 promoter used in this system (fig. 12). Binding of the tetracycline repressor to this sequence results in suppression of promoter activity. In cells expressing tetracycline repressor (HeLa cells), the expression vector whose transcription is controlled by this promoter is in a state in which the transcription activity is suppressed. The possible reasons are: the tetracycline repressor in the cell binds to the human U6 promoter, thereby inhibiting transcriptional activity. When tetracycline (or a tetracycline derivative) is added to the cell, it binds to the tetracycline repressor to release the repressor from the U6 promoter, resulting in transcriptional activation.

The present inventors constructed an siRNA expression vector having a human U6 promoter into which a tetracycline operator sequence of a tetracycline resistance transposon was inserted, and examined the gene silencing effect of the siRNA expression vector using the TetON system. Luciferase assays were performed under conditions similar to those of the above examples.

As shown in FIG. 13, the siRNA expression vector containing the human U6 promoter, into which the tetracycline operator sequence of the tetracycline resistance transposon was inserted, was able to reduce luciferase activity by the addition of tetracycline. On the other hand, siRNA expression vectors that do not contain a tetracycline operator sequence, with or without tetracycline, can reduce luciferase activity. These results show that: the tetracycline inducible U6 promoter described above is preferably used as a promoter for inducing expression of siRNA.

EXAMPLE 12 production of siRNA by RNA self-processing

The use of pol II promoter in siRNA expression vectors results in transcription of slightly longer RNAs. Thus, when using the polII promoter, it is necessary to cleave the RNA by, for example, self-processing to produce antisense or sense RNA. Regardless of whether self-processing actually occurs, the region encoding an RNA sequence recognized at its 5 'and 3' ends by a ribozyme (recognition sequence-encoding region), and the regions flanking the recognition sequence-encoding region flanking the 5 'and 3' end cleavage ribozyme encoding the region encoding the cleavage recognition sequence, are examined using an RNA-producing unit (FIG. 2) containing either antisense-encoding DNA or sense-encoding DNA. FIGS. 14 and 15 illustrate RNA self-processing. The transcripts of the above units were subjected to gel electrophoresis.

The results of gel electrophoresis are shown in FIG. 16. In FIG. 16, the left side of the electrophoretic band shows the putative structures of RNA molecules corresponding to the respective bands. Several bands corresponding to different lengths of RNA were observed, which are thought to result from self-processing of RNA. Also observed was 21 nucleotide long siRNA bands. These results show that: the use of the RNA-producing unit shown in FIG. 2 results in efficient production of siRNA due to the RNA self-processing effect.

EXAMPLE 13 preparation of EGFP mRNA-targeting siRNA library expression System

And (3) preparing an expression system of the siRNA library targeting EGFP mRNA random site. Fig. 19 to 23 show an outline of the preparation.

(a) Preparation of about 20 to 25bp EGFP cDNA fragment

"about 20 to 25bp random EGFP cDNA fragment with dephosphorylated blunt ends" was prepared as follows, which was the starting material for preparing the siRNA expression library shown in FIG. 19-r. Fig. 24 shows an outline of the preparation process.

The EGFP coding region was amplified by PCR from pEGFP-N1 and a random EGFP cDNA fragment of approximately 20 to 25bp was obtained by treating the amplified product with DNase I. To prepare the fragments on a large scale, the resulting fragments were blunted with the Klenow fragment and subcloned into the pSwaI vector constructed by modifying pUC 18. The vector contains recognition sites for the restriction enzyme BseRI in the regions upstream and downstream of the cloning site (SwaI recognition site) to allow the DNA insert to be excised by BseRI.

Approximately 20 to 25bp EGFP cDNA fragments of the subclones were excised with BseRI. The two nucleotide excess sticky ends formed after cleavage with BseRI were blunt-ended with T4DNA polymerase and then dephosphorylated by CIAP treatment. The above method produced a large number of random EGFP cDNA fragments of about 20 to 25bp with dephosphorylated blunt ends.

(b) DNA fragments synthesizing sense-encoding DNA and antisense-encoding DNA with siRNA targeting EGFP mRNA

The 5' -phosphorylated hairpin linker 1 was ligated to both ends of the "about 20 to 25bp dephosphorylated blunt-ended EGFP cDNA fragment prepared in (a) (FIG. 19-c). The hairpin linker contains recognition sites for the type II restriction enzymes BspMI and BsgI such that they can be cleaved at or near the ligation site. The strand displacement reaction was performed by reacting Bst DNA polymerase with Nick sites present in the ligation sites of the ligation products of about 20 to 25bp cDNA fragments having hairpin linkers. Thus, a DNA product was synthesized in which about 20 to 25bp EGFP cDNA fragments and hairpin linkers were ligated to each other in a ratio of 1:1 (FIG. 20-C). Then, a 5' -end phosphorylated DNA linker 2 was ligated to the blunt end side (EGFP cDNA fragment side) of the product (FIG. 20-4). The DNA linker has an AAAAA/TTTTT sequence at one end, which signals transcription from the pol III promoter, and an AscI recognition site at the other end. Only the AAAAA/TTTTTTT side is 5' -phosphorylated.

The ligation product of EGFP cDNA, linker 1 and linker 2 was reacted again with Bst DNA polymerase to perform a strand displacement reaction from the Nick site at the ligation site (FIG. 21-c). Thus, a DNA fragment was synthesized in which DNA encoding sense and antisense RNA of siRNA targeted to EGFP mRNA was placed in tandem. In this fragment, recognition sites for the type II restriction enzymes BspMI and BsgI are present between the sense coding DNA and the antisense coding DNA.

(c) Cloning of DNA fragments with DNA encoding sense and antisense RNA of siRNA targeting EGFP mRNA

Cleaving the DNA fragment having the DNA encoding the sense and antisense RNAs of the siRNA targeting the EGFP mRNA synthesized in (b) with AscI to prepare a DNA fragment having a sticky end and a blunt end (fig. 21-sixty). To clone this fragment, a cloning vector containing the human U6 promoter was constructed (fig. 25). Downstream of this promoter there are recognition sites for the restriction enzymes BspMI, BseRI and AscI. After cleavage with BspMI at a position immediately downstream of the promoter, the cleavage site was blunted with Klenow, and the AscI cleavage product was used to prepare a cloning vector having a sticky end and a blunt end. This vector was ligated to DNA fragments with blunt and cohesive ends with DNA encoding sense and antisense RNA of siRNA targeting EGFP mRNA, followed by cloning (anti-EGFP U6siRNA expression pre-library) (fig. 22-c).

A cloning vector containing the human tRNA-val promoter was also constructed (FIG. 25). Recognition sites for the restriction enzymes BspMI, BseRI, and AscI are similarly present downstream of the human tRNA-val promoter. The cloning vector was prepared by cleavage with BseRI instead of BspMI. The vector was cleaved with BseRI immediately downstream of the promoter, the cleavage site was blunted with T4DNA polymerase, and the product was cleaved with AscI and cloned in a similar manner (anti-EGFP tRNA siRNA pre-library).

The siRNA expression pre-library plasmid DNA was cleaved at the BspMI site between the siRNA sense and antisense encoding DNAs (fig. 22-b). The cleavage fragments were blunt-ended and self-ligated with Klenow to construct an anti-EGFP siRNA expression library, in which the promoter, antisense DNA, sense DNA and TTTTT were ligated in series (FIG. 22-ninthly). Sequence analysis confirmed the production of the desired product. Table 1 shows examples of DNA sequences encoding sirnas from anti-EGFP U6siRNA expression libraries.

TABLE 1

5 '-U6 promoter- (antisense encoding DNA) (acyclic) (sense encoding DNA) TTTTT AscI-3'

Clone number	Antisense encoding DNA	Ring (C)	Sense coding DNA
				1	GFP 24	Is free of	GFP 24
	CCCGTGCCCTGGCCCACCCTCGTG(SEQ ID NO：39)		CACGAGGGTGGGCCAGGGCACGGG(SEQ ID NO：40)
				2	GFP 24	Is free of	GFP 24
	ACCAGGATGGGCACCACCCCGGTG(SEQ ID NO：41)		CACCGGGGTGGTGCCCATCCTGGT(SEQ ID NO：42)

In table 1, the orientation of the antisense and sense codons may be reversed in some cases from that in EGFP mRNA, which does not affect RNAi induction.

A siRNA expression library having a siRNA loop sequence TTCG between antisense and sense DNAs was constructed by cleaving pre-library plasmid DNA with BsgI and then BspMI (FIG. 23-b-2), blunting the cleavage site with T4DNA polymerase, and then self-ligating. By changing the sequence of hairpin linker 1 and the type and site of restriction enzyme, the loop sequence between antisense and sense coding DNA can be changed at will, and promoter fragments, selection markers, etc. can be added.

[ example 14] evaluation of siRNA library expression System targeting EGFP mRNA

The above anti-EGFP U6siRNA expression library (1g) and pEGFP-N1(0.01g) were co-transfected into human HeLa S3 cells by lipofection (Lipofectamine 2000). The cells were left at 37 ℃ for 48 hours and then observed under a confocal microscope. As a control experiment, pUC18(1g) was used in place of the siRNA library expression system for similar operation.

As shown in FIG. 26, the number of cells emitting green fluorescence due to pEGFP-N1 was reduced in the group transduced with the anti-EGFP U6siRNA library expression system, as compared with the control group (pUC 18-introduced group). In addition, the results of FACS analysis revealed a decrease in the fluorescence intensity of the cells (fig. 27). These results show that: introduction of an anti-EGFP U6siRNA library expression system into cells induces RNAi to silence target genes.

Thus, clones that induce RNAi and silence the target gene will be present in the anti-EGFP U6siRNA library expression system.

Similar results were obtained in the anti-EGFP tRNA siRNA library expression system (fig. 26 and 27).

EXAMPLE 15 Gene silencing Effect of siRNA-expressing Adenoviral vectors and HIV vectors

The luciferase gene (pGL 3-Control: Promega) was used as a marker gene for evaluation. The siRNA was expressed in tandem using the human U6 promoter. The target sequence is site B chain: 5'-GTGCGCTGCTGGTGCCAAC-3' (SEQ ID NO: 43). An adenovirus vector was prepared according to the method of Mizuguchi et al (Nippon Rinsho, 58, 1544-1553 (2000)).

1) Incorporation of RNAi expression cassettes into shuttle plasmids

Although 3 HincII sites were present in the PShuttle sequence from Clontech, the results of the sequencing confirmed: only one HincII site is present between the I-CeuI and PI-SceI sites.

After the expression plasmid (pU6i-FGLB) was cleaved with HindIII, the ends were blunt-ended by Klenow treatment and then cleaved with EcoRI. The prepared expression cassette (about 600bp) was incorporated into a Shuttle vector (pShuttle) which had been treated with EcoRI and HincII to construct pU6 i-FGLB/Shuttle.

2) The expression cassette from pShuttle was incorporated into an Ad vector plasmid and an Ad vector was prepared

According to the method of Mizuguchi et al, an RNAi expression cassette was incorporated into an Ad vector plasmid (pAdHM15-RGD) having RGD fiber to construct pU 6-FGLB/RGD.

As a control, after digesting pAdHM15-RGD and pU6-FGLB/RGD without the insert with PacI, lipofection was performed using TransIT293 (TaKaRa). Ad vectors were prepared from cells in which CPE was observed according to the method of Mizuguchi et al.

Ad vectors prepared by cesium chloride ultracentrifugation were purified by dialysis overnight in PBS (-) containing 1% BSA. The Titer of the purified Ad vector was determined using the Adeno-X Rapid Titer kit (Clontech). Titers determined for each Ad vector were as follows:

RGD/Ad (control): 6.76 x10 ^10ifu/ml

U6-FGLB/Ad：5.27×10^10ifu/ml

3) Preparation of HeLa-S3 cells

HeLa-S3 cells were prepared to a density of 5x10⁵One cell per ml and seeded at 1ml per well in 6-well plates. The results were then evaluated at Moi for 1, 10,amounts of 20, 50 and 100 were added to each well for each Ad vector. After 24 hours, medium (1.5ml) was added, followed by lipofection.

4) Lipofefection of luciferase plasmids

24 hours after Ad transduction, cells were lipofected with luciferase expression plasmids in each well composition described below.

Opti-MEM (250. mu.l) was placed in the A tube, to which pGL3-Control (0.02. mu.g), pRL-Tk (0.1. mu.g) and pUC19 (1.0. mu.g) were added. Opti-MEM (250. mu.l) was placed in the B tube, LipofectAmine 2000 (5. mu.l; Invitrogen) was added thereto, and the mixture was left at room temperature for 5 minutes. The full amount of tube B was poured into tube A and the mixture was thoroughly mixed. After the mixture was left at room temperature for 20 minutes, the whole amount was added to each well and incubated at 37 ℃ for 48 hours.

5) Luciferase assay

Luciferase detection was performed using the Dual-Luciferase Reporter Assay System (Promega).

After 48 hours of incubation following lipofection, each well in the plate was washed once with (500. mu.l). After PBS (-) was removed, 1 XPLB (500. mu.l) was added to each well and left at room temperature for 15 minutes, with occasional shaking of the plates to lyse the cells. The cell lysate was transferred to a 1.5ml tube, centrifuged at 14000rpm for 1 minute and the supernatant transferred to a new 1.5ml tube (PLB lysate).

Luciferase activity was measured using autolumat plus LB953 (Berthold). Luminescence of firefly luciferase and Renilla luciferase was measured for 10 seconds using PLB lysate (101). The value of cells transduced with control RGD/Ad was taken as 100%, and relative luciferase silencing of Ad expressed by each RNAi was expressed based on the RLU value of firefly luciferase/Renilla luciferase (FIG. 29).

Similarly, as shown in fig. 30, luciferase inhibition of RNAi was observed from the expression HIV vector.

siRNA expressing HIV vectors were prepared by inserting a siRNA expression cassette targeting firefly luciferase into an HIV shuttle vector according to essentially the same method as for the preparation of siRNA expressing adenovirus vectors. In this assay, the target sequence is the site B chain: 5'-GTGCGCTGCTGGTGCCAAC-3' (SEQ ID NO: 43), using the U6 promoter, the expressed RNA has a stem-loop including 5 ' -GUGCGCUGCUGGUGCCAACCCgugugcuguccGGGUUGGCACCAGCAGCGCAC (SEQ ID NO: 22), 5'-GUGCGCUGuUGGUGuCAACCCgugugcuguccGGGUUGGCACCAGCAGCGCAC-3' (SEQ ID NO: 23) and 5'-GUGCGuUGuUGGUGuuAAuCCgugugcuguccGGGUUGGCACCAGCAGCGCAC-3' (SEQ ID NO: 57). After introduction of the siRNA expressing shuttle plasmid into 293T cells, viral particles were harvested by standard methods, concentrated, and transfected into 293T cells at a moi of 5 to 8. Then, RNAi effect was examined in terms of luciferase activity according to a method similar to that when an adenovirus vector was used.

EXAMPLE 16 Gene silencing Effect of siRNA-expressing dumbbell vectors

The siRNA-expressing dumbbell vectors were examined for gene silencing. Luciferase assay was performed under similar conditions to the above examples using the following siRNA expression vectors.

● use human U6 promoter, a vector for expressing stem-loop siRNA (pU6stem), and

● Dumbbell shaped vector for expressing siRNA (Dumbbell)

As shown in fig. 31, luciferase silencing was observed in the siRNA-expressing dumbbell vectors, indicating that the siRNA expression system maintained in the dumbbell vectors exhibited potent gene silencing.

Industrial applicability

As described above, the expression of functional genes can be silenced using an intracellular siRNA expression system. In addition, expression of multiple target genes can also be silenced as a result of introducing a single vector in a cell that has been transformed to maintain an siRNA expression system for multiple target genes. By using such an intracellular siRNA expression system, siRNA can be provided inside the cell, so that stable and long-term siRNA expression, i.e., target gene silencing, can be performed. In addition, by using a viral vector or the like, the efficiency of transferring the siRNA expression system to cells can be improved, and RNAi induction can be successfully performed in mammalian cells. Thus, the system of the present invention can be used for gene therapy and knock down animal production via RNAi.

In addition, in order to apply the system of the present invention to a method of searching for functional genes, the present invention provides an siRNA library expression system and a collection thereof. The use of these systems and the like makes it possible to make the search for functional genes simple and efficient, so that the system of the present invention comprising an expression system of siRNA library can be used for the rapid elucidation of functional genes.

Sequence listing

<110> Duobiliang Hecheng (TAIRA, Kazunari)

Palace bank true (MIYAGISHI, Makoto)

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<211>195

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(39)..(58)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(139)..(158)

<223> "n" ═ one of the bases a, t, g, or c

<400>30

<210>31

<211>152

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(39)..(55)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(137)..(152)

<223> "n" ═ one of the bases a, t, g, or c

<400>31

<210>32

<211>152

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(1)..(16)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(98)..(113)

<223> "n" ═ one of the bases a, t, g, or c

<400>32

<210>33

<211>142

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(24)..(42)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(124)..(142)

<223> "n" ═ one of the bases a, t, g, or c

<400>33

<210>34

<211>138

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(1)..(19)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(101)..(119)

<223> "n" ═ one of the bases a, t, g, or c

<400>34

<210>35

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(6)..(43)

<223> "n" ═ one of the bases a, t, g, or c

<400>35

<210>36

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(1)..(38)

<223> "n" ═ one of the bases a, t, g, or c

<400>36

<210>37

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(6)..(24)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(29)..(47)

<223> "n" ═ one of the bases a, t, g, or c

<400>37

<210>38

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<220>

<221>misc_feature

<222>(1)..(19)

<223> "n" ═ one of the bases a, t, g, or c

<220>

<221>misc_feature

<222>(24)..(42)

<223> "n" ═ one of the bases a, t, g, or c

<400>38

<210>39

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>39

<210>40

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>40

<210>41

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>41

<210>42

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>42

<210>43

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>43

<210>44

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>44

<210>45

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>45

<210>46

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>46

<210>47

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>47

<210>48

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>48

<210>49

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>49

<210>50

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>50

<210>51

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>51

<210>52

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>52

<210>53

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>53

<210>54

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>54

<210>55

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>55

<210>56

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>56

<210>57

<211>53

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400>57

Claims (33)

1. An intracellular siRNA expression system, said siRNA capable of silencing expression of a mammalian target gene, said system comprising antisense encoding DNA encoding an antisense RNA directed to a region of an mRNA of a mammalian target gene, sense encoding DNA encoding a sense RNA directed to the same region of said mRNA of a mammalian target gene, and one or more pol III promoters capable of expressing said antisense and sense RNAs from said antisense and sense encoding DNAs, respectively, wherein said antisense and sense encoding DNAs, and said one or more pol III promoters are located in the same vector DNA molecule, wherein the final transcript length of the siRNA expressed by the system is from 15 to 30 bp.

2. An siRNA expression system according to claim 1 wherein the region of double stranded RNA in which the two RNA strands mate contains a mismatch or bulge.

3. An siRNA expression system according to claim 2 wherein one nucleotide in said mismatch is guanine and the other is uracil.

4. An siRNA expression system according to claim 2 wherein the siRNA contains between 1 and 7 mismatches.

5. An siRNA expression system according to claim 2 wherein the siRNA comprises 1 to 7 lobes.

6. An siRNA expression system according to claim 2 wherein the siRNA contains 1 to 7 mismatches and bulges.

7. An siRNA expression system according to any of claims 1 to 6 wherein said promoter is the U6 promoter.

8. An siRNA expression system according to any of claims 1 to 6, wherein said promoter is an inducible promoter.

9. An siRNA expression system according to any of claims 1 to 6 wherein said promoter is located separately upstream of said antisense and sense coding DNAs.

10. An siRNA expression system according to claim 1 wherein said system comprises loxP sites in any one of the following forms (a) to (c) so that expression of said siRNA can be controlled:

(a) the promoter comprises a distal sequence element DSE and a proximal sequence element PSE, with a spacer region therebetween, wherein the spacer region comprises two loxP sequences, one located near the DSE and the other located near the PSE;

(b) the promoter contains DSE and PSE, they are in the position of maintaining promoter activity, have a loxP site located between them, another loxP site is located in DSE upstream or PSE downstream; and

(c) the two loxP sites are in positions intervening in the antisense coding DNA or sense coding DNA.

11. An siRNA expression system according to any of claims 1 to 6 wherein the promoter is located on one side of a unit in which the antisense and sense coding DNAs are linked in opposite directions via a linker.

12. An siRNA expression system according to claim 11, wherein said system comprises loxP sequences in any one of the following forms (a) to (d) so that expression of said siRNA can be controlled:

(a) the promoter comprises DSE and PSE, a spacer is arranged between the DSE and the PSE, and two loxP sites are arranged in the spacer, one is positioned near the DSE, and the other is positioned near the PSE;

(b) the promoter contains DSE and PSE, they are in the position of maintaining promoter activity, have a loxP site located between them, another loxP site is located in DSE upstream or PSE downstream;

(c) two loxP sites at positions intervening in the antisense coding DNA or sense coding DNA; and

(d) the alignment of the two loxP sites results in the possibility of intervening the linker containing the termination sequence.

13. An siRNA expression system according to claim 1 wherein said vector is a plasmid vector or a viral vector.

14. An siRNA expression system according to claim 1 wherein said vector is a dumbbell-shaped DNA vector.

15. A transformed mammalian cell which maintains an siRNA expression system according to any one of claims 1 to 6, 10, 13 or 14.

16. The cell according to claim 15, wherein the cell is a human cell that has been transformed.

17. A method of producing a transformed non-human mammal having silenced expression of a target gene, the method comprising:

a) introducing the siRNA expression system of any of claims 1-6, 10, 13, or 14 into a fertilized egg of a non-human mammal; and

b) obtaining a non-human mammalian progenitor developed from the fertilized egg, wherein the siRNA expression system has been integrated into the chromosome of the progenitor.

18. A composition comprising an siRNA expression system according to any one of claims 1-6, 10, 13 or 14.

19. The composition according to claim 18, wherein the composition is a pharmaceutical composition.

20. A method of producing a mammalian cell that has been transformed and has silenced expression of a target gene, wherein said method comprises the steps of: introducing an siRNA expression system according to any of claims 1-6, 10, 13 or 14 into a mammalian cell, and selecting a mammalian cell into which said siRNA expression system has been introduced.

21. An intracellular siRNA library expression system, said siRNA capable of silencing expression of a mammalian target gene, said system comprising a double stranded DNA encoding an siRNA and two pol III promoters positioned relative to each other, said double stranded DNA comprising an arbitrary sequence of equal length as the expressed siRNA and positioned between the two pol III promoters, said pol III promoters capable of expressing RNAs complementary to each other from each strand of said double stranded DNA,

wherein the siRNA is directed against a region of the mRNA of a mammalian target gene,

wherein said double stranded DNA encoding siRNA and said two pol III promoters are maintained in the same vector DNA molecule, and

wherein the system expresses siRNA with a final transcript length of 15 to 30 bp.

22. An intracellular siRNA library expression system, said siRNA capable of silencing expression of a mammalian target gene, said system comprising a stem-loop siRNA producing unit in which an antisense encoding DNA and a sense encoding DNA complementary to said antisense encoding DNA are linked in opposite directions via a linker and a pol III promoter capable of expressing a stem-loop siRNA on either side of said unit

Wherein the stem-loop siRNA is directed against a region of the mRNA of a mammalian target gene,

wherein said stem-loop siRNA producing unit and said pol III promoter are maintained in the same vector DNA molecule, and

wherein the system expresses siRNA with a final transcript length of 15 to 30 bp.

23. An siRNA library expression system according to claim 21 or 22 wherein the double stranded RNA region of the two RNA strands in the siRNA pair contains a mismatch or a bulge.

24. An siRNA library expression system according to claim 21 or 22, wherein said promoter is the U6 promoter.

25. An siRNA library expression system according to claim 21 or 22, wherein said promoter is an inducible promoter.

26. An siRNA library expression system according to claim 21 or 22 wherein said system expresses siRNA consisting of random RNA strands.

27. An siRNA library expression system according to claim 21 or 22 wherein said system is a collection of a plurality of siRNA expression vectors wherein each vector targets a gene sequence comprising coding and/or non-coding regions.

28. An siRNA library expression system according to claim 21 or 22 wherein the siRNAs expressed by the system consist of RNA strands encoded by DNA fragments of any cDNA or genomic DNA, said fragments being of equal length to the expressed siRNAs.

29. A collection of siRNA library expression systems according to claim 21, wherein each system in said collection expresses a different siRNA.

30. A collection of siRNA library expression systems according to claim 22, wherein each system in said collection expresses a different siRNA.

31. A method for searching for a functional gene in a mammal in vitro, the method comprising the steps of:

(a) introducing into an isolated mammalian cell an siRNA library expression system according to claim 21 or 22 or a collection of siRNA library expression systems according to claim 29 or 30;

(b) selecting a mammalian cell that has been transformed and introduced into said siRNA library expression system or said collection; and

(c) the phenotype of the selected cells is analyzed.

32. The in vitro method according to claim 31, wherein said method further comprises the steps of: screening mammalian functional genes for DNA sequences encoding siRNA in transformed mammalian cells which have been phenotypically analyzed to find a change in phenotype.

33. A method for selecting in vitro a highly active siRNA capable of silencing expression of a mammalian target gene, said method comprising the steps of:

(a) introducing an siRNA library expression system according to claim 21 or 22 or a collection of siRNA library expression systems according to claim 29 or 30 in an isolated mammalian cell, and

(b) determining the expression level of a particular gene or protein in the mammalian cells transformed and introduced into the siRNA library expression system or the collection.