WO2010071206A1 - DNA CONSTRUCT FOR TRANSCRIPTION/EXPRESSION OF siRNA, AND USE THEREOF - Google Patents

Abstract

Disclosed is a DNA construct which is adaptable to different supply forms of siRNA to cells. The DNA construct comprises a hybrid promoter. In the DNA construct, the hybrid promoter comprises a nucleotide sequence produced by using a first promoter activity sequence and a second promoter activity sequence and by substituting, inserting or adding the second promoter activity sequence in the first promoter activity sequence, wherein the first promoter activity sequence has a promoter activity of a first RNA polymerase that is DNA-dependent RNA polymerase III capable of acting in animal cells and the second promoter activity sequence has a promoter activity of a second RNA polymerase that is a DNA-dependent RNA polymerase capable of acting in vitro. The hybrid promoter has both the promoter activity of the first RNA polymerase and the promoter activity of the second RNA polymerase.

Description

siRNA transcription / expression DNA construct and use thereof

This application claims priority based on Japanese Patent Application No. 2008-322846 filed on Dec. 18, 2008, the entire contents of which are incorporated herein by reference. Incorporated in the description.
The present invention allows RNAi (RNA interference) and other RNAs that exhibit gene regulatory functions to be expressed in vivo by a transcription reaction in animal cells and obtained in vitro by a transcription reaction in a test tube. The present invention relates to a DNA construct for RNA preparation and use thereof.

It is known that RNA that is not translated into protein in the cell plays a part in the gene regulatory mechanism. Among these, various non-coding RNAs (ncRNAs) such as antisense RNAs, siRNAs, microRNAs (miRNAs) and the like are known as such functional RNAs.

For example, RNAi is a short double-stranded RNA (short interfering RNA, siRNA) consisting of a sense RNA comprising a sequence homologous to the mRNA of the target gene and an antisense RNA comprising a complementary sequence thereto. It is a phenomenon that induces the destruction of gene mRNA and suppresses the expression of the target gene. RNAi is expected to be applied to nucleic acid medicine and a drug discovery target gene search method in addition to gene function analysis by producing knockdown cells and animals. In addition, siRNA degrades mRNA in a sequence-specific manner, but its expression suppression ability varies greatly depending on the sequence. For this reason, it is important to specify a sequence having a high inhibitory effect in the target gene.

In generating such RNAi in a cell, there are two main modes for supplying siRNA into the cell. One is a method of introducing siRNA prepared outside the cell into the cell. In this case, there are a case of chemical synthesis and a case of enzymatic synthesis by in vitro transcription reaction using T7 RNA polymerase or the like.

The other is a method in which a siRNA expression vector using U6 RNA polymerase or the like is introduced into a cell and siRNA is expressed by a transcription reaction in the cell (Patent Document 1). In this case, a form using a plasmid as a vector and a form using a virus are further included.

In the former method, synthetic RNA is expensive and it is difficult to prepare various target sequences, and it is necessary to prepare a vector for transcription of a large number of target sequences even in the case of in vitro transcription reaction. . In addition, it is difficult to design a target sequence having an inhibitory effect in advance. Furthermore, although the former method is a simple and rapid method as long as siRNA can be obtained, the effect of suppressing gene expression is transient, and the introduction efficiency may be a problem.

On the other hand, the latter method can be expected to have a continuous RNAi effect by siRNA expression in cells, but the difficulty in designing the target sequence is the same as the former method.

Furthermore, in recent years, a method for constructing a comprehensive siRNA library from DNA has also been developed (Patent Document 2). This method describes that an siRNA expression construct is comprehensively obtained from a desired target gene, and this construct is connected to an appropriate vector to form an siRNA library. According to such a library, there is a possibility that a target sequence can be searched exhaustively.

Republished 2003-04186 Republished 2005-063980

As described above, researchers and others prepared siRNA by different techniques depending on the effective siRNA supply form for the purpose of siRNA research and the like. This has generated a great deal of effort in developing various uses of RNAi.

That is, since it is difficult to predict a sequence with a high inhibitory effect so far, in order to search for a sequence with a high expression inhibitory effect for a specific target gene, an siRNA that covers the possible target sequences is prepared. It is desirable to do. In this case, it is reasonable to perform primary screening of sequences with a high inhibitory effect based on a transient expression inhibitory effect and secondary screening based on a continuous expression inhibitory effect.

However, enormous costs and labor are required to comprehensively produce a large number of siRNAs by the former method for the primary screening. In addition, a separate siRNA expression construct must be prepared for the secondary screening based on the primary screening results.

Furthermore, screening based on the continuous expression suppression effect can be performed from the beginning using an exhaustive siRNA expression vector included in the siRNA library, but since it is assumed to be expressed in cells, it is still rapid. In some cases, it is not suitable for selecting an effective target sequence or confirming the knockdown effect.

The same problem is considered to exist in antisense RNA and miRNA, which are RNAs that are considered to exert gene regulatory functions in a base pair-dependent manner.

Therefore, an object of the present invention is to provide a DNA construct for RNA preparation which is more practical, that is, can easily cope with different supply forms of RNA to cells, and use thereof.

In order to solve the above-mentioned problems, the present inventors have prepared RNA that can be transcribed with RNA polymerase in in vivo (in a cell) and can be transcribed by RNA polymerase in in vitro (in vitro). We aimed to construct a DNA construct. The present inventors synthesize siRNA according to the supply form of functional ncRNA, that is, in vitro, according to a hybrid promoter capable of transcription reaction by DNA-dependent RNA polymerase operating in in vivo and in vitro. At the same time, it was found that siRNA can be expressed in vivo or in cells, and the present invention was completed. That is, according to the present invention, the following means are provided.

According to the present invention, a DNA-dependent RNA polymerase that operates in vitro against a first promoter active sequence having the promoter activity of the first RNA polymerase that is a DNA-dependent RNA polymerase III that operates in animal cells. A promoter sequence of the second RNA polymerase having the promoter activity of the second RNA polymerase is substituted, inserted or added, and the promoter activity of the first RNA polymerase and the second RNA polymerase There is provided a DNA construct for RNA production comprising a hybrid promoter having promoter activity.

In the DNA construct of the present invention, the first promoter active sequence can be either a sequence having U6 promoter activity or a sequence having H1 promoter activity. More preferably, it is a sequence having U6 promoter activity.

In the DNA construct of the present invention, the second promoter active sequence may be selected from the group consisting of a sequence having T7 promoter activity, a sequence having T3 promoter activity, and a sequence having SP6 promoter activity. It is preferable to use an array having

In the DNA construct of the present invention, the hybrid promoter has a TATA box box sequence in the first promoter active sequence, and the substitution or insertion of the second promoter active sequence is 3 'to the TATA box sequence. And may have a base sequence to which any of addition is made.

In the DNA construct for RNA preparation of the present invention, the hybrid promoter is a sequence in which the first promoter active sequence has U6 promoter activity, and the second promoter active sequence has T7 promoter activity. More preferably, the first promoter active sequence has a TATA box sequence, and the sequence having the second promoter activity is substituted, inserted or added 3 ′ to the TATA box sequence. It is preferable.

In the DNA construct of the present invention, the hybrid promoter has one of the following sequences:
(A) a base sequence represented by SEQ ID NO: 1 (b) a base sequence represented by SEQ ID NO: 1 comprising a base sequence having any one of substitution, deletion, addition and insertion of one or more bases be able to.

The DNA construct for RNA preparation of the present invention may be a plasmid vector or a virus vector. The DNA construct of the present invention can further comprise a sense code DNA and an antisense code DNA that are linked to be transcribed under the control of the hybrid promoter.

According to the present invention, there is provided a method for producing RNA, wherein RNA is produced by in vitro transcription reaction or expression in cells using any of the above-mentioned DNA constructs for RNA production.

According to the present invention, there is provided a method for suppressing the expression of a target gene by supplying siRNA to a cell, wherein either or both of in vitro transcription reaction using the above RNA construct DNA construct and expression in the cell. Through which a siRNA derived from a sense code DNA and an antisense code DNA of any region of the target gene is supplied into a cell expressing the target gene.

According to the present invention, there is provided a method for regulating the expression of a target gene by supplying RNA to a cell, wherein either or both of an in vitro transcription reaction using the above-described DNA construct for RNA production and expression in the cell are performed. Thus, a method is provided comprising the step of supplying RNA into a cell expressing the target gene.

According to the present invention, there is provided a method for analyzing RNA function, wherein RNA is introduced into an animal cell through either or both of an in vitro transcription reaction using the above-described DNA construct DNA construct and expression in a cell. There is provided a method comprising: a supplying step; and evaluating the function of the RNA by comparing the animal cell supplied with the RNA and a cell not supplied with the RNA.

According to the present invention, a DNA-dependent RNA polymerase that operates in vitro against a first promoter active sequence having the promoter activity of the first RNA polymerase that is a DNA-dependent RNA polymerase III that operates in animal cells. A promoter sequence of the second RNA polymerase having the promoter activity of the second RNA polymerase is substituted, inserted or added, and the promoter activity of the first RNA polymerase and the second RNA polymerase A cell is provided that retains a sense code DNA and an antisense code DNA for any region of a target gene in a transcribable manner under the control of a hybrid promoter having promoter activity. This type of cell is preferably an animal cell.

According to the present invention, a DNA-dependent RNA polymerase that operates in vitro against a first promoter active sequence having the promoter activity of the first RNA polymerase that is a DNA-dependent RNA polymerase III that operates in animal cells. A promoter sequence of the second RNA polymerase having the promoter activity of the second RNA polymerase is substituted, inserted or added, and the promoter activity of the first RNA polymerase and the second RNA polymerase Also provided are non-human mammals that retain sense code DNA and antisense code DNA for any region of the target gene in a transcribable manner under the control of a hybrid promoter having promoter activity.

According to the present invention, a DNA-dependent RNA polymerase that operates in vitro against a first promoter active sequence having the promoter activity of the first RNA polymerase that is a DNA-dependent RNA polymerase III that operates in animal cells. A promoter sequence of the first RNA polymerase, and a second RNA polymerase having a nucleotide sequence substituted, inserted or added to the second promoter active sequence having the promoter activity of the second RNA polymerase A hybrid promoter having the promoter activity of

The hybrid promoter of the present invention has one of the following sequences:
(A) a base sequence represented by SEQ ID NO: 1 (b) a base sequence represented by SEQ ID NO: 1 comprising a base sequence having any one of substitution, deletion, addition and insertion of one or more bases Can do.

According to the present invention, there is provided a method for preparing an siRNA library, which is a sense code DNA and an antisense code that respectively encode a sense strand and an antisense strand of a double-stranded RNA that may function as an siRNA for a target gene. A step of preparing DNA and linking each of the DNA strands so as to allow expression of a siRNA or a precursor thereof that may be transcribed under the control of the hybrid promoter of the DNA construct according to any one of claims 1 to 9. And a manufacturing method is provided.

According to the present invention, the DNA strand preparation step can be a step including enzymatic degradation of the target gene.

According to the present invention, there is provided a siRNA library, wherein each of a plurality of types of sense code DNA and antisense code DNA for one or more target genes is the DNA according to any one of claims 1 to 9. A library is provided that includes a construct that is transcribed under the control of the hybrid promoter of the construct.

In the library of the present invention, the sense code DNA and the antisense code DNA can include a DNA fragment obtained by enzymatic degradation of the target gene.

According to the present invention, there is provided a method for screening a target sequence useful for RNAi, comprising the step of preparing any of the above siRNA libraries, an in vitro transcription reaction using the DNA construct of the prepared siRNA library, and / or Supplying the sense code DNA and the siRNA siRNA derived from the antisense code DNA into the cell expressing the target gene via either or both of the expression in the cell, and the target in the cell Measuring the expression of the gene, and a screening method is provided.

According to the present invention, there is provided a method for screening a target sequence useful for prevention or treatment of a disease, comprising the step of preparing any one of the above siRNA libraries for one or more target genes related to the disease, The sense-coding DNA and the sense-coding DNA in the cell expressing the target gene through either or both of in vitro transcription reaction using the DNA construct of the prepared siRNA library and / or expression in the cell. There is provided a screening method comprising a step of supplying siRNA derived from the antisense-encoding DNA and a step of measuring expression of the target gene in the cell.

According to the present invention, there is provided a method for screening drug target genes useful for the prevention or treatment of a disease, wherein the siRNA library according to claim 15 or 16 is prepared for a target gene that may be related to the disease. And the sense in the cell expressing the target gene through either or both of an in vitro transcription reaction using the DNA construct of the prepared siRNA library and / or expression in the cell. There is provided a screening method comprising a step of supplying an siRNA derived from a coding DNA and the antisense coding DNA, and a step of measuring a change before and after the supply of the siRNA in the cell.

According to the present invention, there is provided a method for producing a model animal useful for disease prevention or treatment, the step of preparing the siRNA library described above for a target gene that may be related to the disease, and the preparation An embryo capable of expressing RNA derived from the sense code DNA and the antisense code DNA through either or both of in vitro transcription reaction using the DNA construct of the RNA library and / or expression in cells And a method of producing an individual from the embryo is provided.

It is a figure which shows an example of the hybrid promoter of this invention. It is a figure which shows the example of arrangement | positioning (tandem type and stem loop type) of sense code DNA and antisense code DNA in the DNA construct of this invention. It is a figure which shows a retrovirus vector. It is a figure which shows a lentiviral vector. It is a figure which shows the construction scheme of a retrovirus vector. It is a figure which shows the construction scheme of a lentiviral vector. It is a figure which shows the insertion method of the ireco promoter, the sequence immediately under it, and a siRNA coding sequence. It is a figure which shows the RNAi effect of irecoU6 promoter and U6 promoter, the vertical axis | shaft of a graph shows the relative reduction | decrease degree (average value +/- standard deviation n = 4), and a horizontal axis | shaft shows the arrangement | sequence of siRNA integrated in irecoU6 promoter or U6 promoter. . It is a figure which shows the result of the PAGE analysis of siRNA produced by in-vitro transcription reaction, M shows a marker. It is a figure which shows the knockdown effect of in-vitro transcription | transfer siRNA, and the vertical axis | shaft of a graph shows the relative reduction | decrease degree (mean value +/- standard deviation, n = 4), and a horizontal axis shows the arrangement | sequence and density | concentration of siRNA. It is a figure which shows the RNAi effect by irecoU6 promoter and U6 promoter which were introduce | transduced using the retroviral vector, The vertical axis | shaft of a graph shows the average value of a relative reduction degree, and a horizontal axis shows the arrangement | sequence of siRNA. FIG. 11 (a) shows the RNAi effect due to multicopy infection (estimated average 3-5 copies), and FIG. 11 (b) shows the RNAi effect of 1 copy infection. It is a figure which shows the RNAi effect of the irecoU6 promoter introduce | transduced using the lentiviral vector. The vertical axis of the graph represents the relative decrease (average value), and the horizontal axis represents the siRNA sequence.

The present invention relates to a hybrid promoter that can supply RNA to cells as needed in both in vitro transcription reaction and in vivo expression and its use. Specifically, the hybrid promoter and the hybrid promoter The present invention relates to a DNA construct for RNA preparation, an RNA production method using the DNA construct, an siRNA library production method, an siRNA library, a screening method, and the like.

The hybrid promoter of the present invention is a first promoter activity that is located upstream of the transcription initiation sequence of the first RNA polymerase that is dependent on RNA polymerase III that operates in animal cells and has the promoter activity of the first RNA polymerase. A base sequence in which a second promoter active sequence having a promoter activity of a second RNA polymerase, which is a DNA-dependent RNA polymerase that operates in a test tube, is substituted, inserted or added to the sequence; It has the promoter activity of the first RNA polymerase and the promoter activity of the second RNA polymerase.

According to the hybrid promoter of the present invention, the sense code D name and the antisense code DNA that are recognized by each of the first RNA polymerase and the second RNA polymerase and are linked downstream thereof are transcribed by the respective RNA polymerases. The For this reason, by linking DNA encoding RNA that may act depending on the base sequence of any region of the target gene to the downstream side of this hybrid promoter, such RNA is mediated by in vitro transcription reaction and It can be supplied into cells either via intracellular expression. Therefore, by linking the sense code DNA and the antisense code DNA in any region of the target gene, siRNA can be supplied into the cell either via in vitro transcription reaction or via intracellular expression.

Moreover, the supply form of siRNA can be easily switched by using the hybrid promoter of the present invention. That is, RNA can be obtained by in vitro transcription reaction or intracellular expression by ligating sense code DNA and antisense code DNA under the control of the hybrid promoter of the present invention. As for knockdown and target sequence screening, efficient and continuous knockdown by expressing RNA in cells can be easily performed as needed. Conventionally, it is possible to improve efficiency by omitting or reducing the labor for supplying such different RNAs.

Furthermore, by using the hybrid promoter of the present invention, in addition to the siRNA production DNA construct according to the supply form of siRNA to the cell, provision of an siRNA library including siRNA that may have a comprehensive suppression effect, and A high-throughput screening method can be provided.

In the present specification, RNA means non-coding RNA (ncRNA) other than tRNA and rRNA, and preferably ncRNA involved in gene expression regulation in a base sequence-dependent manner. More specifically, antisense RNA, siRNA and miRNA (including pri-miRNA and pre-miRNA), and anti-gene RNAi (agRNA) are included. “SiRNA” includes RNA that functions as siRNA in a cell, and includes, for example, short hairpin RNA (shRNA). The lengths (base lengths) of these various RNAs differ depending on the type, and may vary greatly even with the same type of RNA. For example, miRNA often has about 22 bases, pri-miRNA: sometimes about 1000 bases, pre-miRNA often has about 70 bases, and antisense RNA has several hundred to several bases. It may be as long as 1000 bases.

Hereinafter, various embodiments of the present invention will be described with reference to the drawings as appropriate. FIG. 1 is a diagram showing an example of the hybrid promoter of the present invention, and FIG. 2 is a diagram showing the arrangement of sense code DNA and antisense code DNA in the DNA of the present invention.

(Hybrid promoter)
The hybrid promoter of the present invention is a DNA-dependent RNA that operates in vitro against a first promoter active sequence having the promoter activity of the first RNA polymerase, which is a DNA-dependent RNA polymerase III that operates in animal cells. It can have a base sequence in which a second promoter active sequence having the promoter activity of the second RNA polymerase, which is a polymerase, is substituted, inserted or added.

Animal cells include both human and non-human animals. Examples of non-human animals include mammals such as mice, rats, cows, pigs, sheep, goats, cats, dogs and monkeys. Examples of vertebrates include fish, amphibians, reptiles, and birds. RNA interference has been confirmed in insects, nematodes, Drosophila, mice, hamsters, humans, and the like.

Examples of the first promoter active sequence include known RNA polymerase III promoters. Typically, H1 promoter, U6 promoter, tRNA promoter, retroviral LTR promoter, adenovirus VA1 promoter, 5S rRNA promoter, 7SK RNA promoter, 7SL RNA promoter are exemplified.

Preferably, it is an RNA polymerase III promoter that does not have a promoter sequence downstream from the transcription initiation sequence. That is, it is an RNA polymerase promoter having a promoter sequence only upstream of the transcription initiation sequence. According to such RNA polymerase III, an arbitrary sequence can be inserted and transcribed immediately after the promoter. Examples of the promoter having a promoter sequence only upstream of the transcription initiation sequence include H1 promoter and U6 promoter.

The U6 promoter and the H1 promoter are promoters for small nuclear RNA (U6) and human RNase P (human RNase P) RNA H1, respectively, and belong to Type III of the PolIII transcription system. The type III promoter has a proximal sequence element (PSE), a staf-binding site, a distal sequence element (DSE), and a TATA box as conserved sequences necessary for transcription. . The first promoter active sequence in the hybrid promoter of the present invention is preferably a U6 promoter sequence.

For example, the human H1 promoter sequence includes the sequence disclosed in GenBank Accession No. S68670. Further, examples of the mouse U6 promoter sequence include the sequence disclosed in the same accession number X07425. Furthermore, examples of the human U6 promoter sequence include the sequence disclosed in the same accession number X06980. The first promoter active sequence is not limited to these, and one or more base substitutions, deletions, insertions and additions, or two or more types of these sequences are made. There may be. For such base substitution on the base sequence, a known method such as Kunkel method or Gapped-duplex method or a method equivalent thereto can be employed. Alternatively, mutation may be introduced using a mutation introduction kit (for example, Mutan-K (manufactured by TAKARA) or Mutan-G (manufactured by TAKARA)) using site-directed mutagenesis.

The second promoter active sequence only needs to have a DNA-dependent RNA polymerase promoter activity operable in a test tube. Examples of such a promoter include an RNA polymerase II promoter or a promoter similar thereto. Examples of such promoters include T3 promoter, T7 promoter, SP6 promoter, cytomegalovirus promoter, RSV promoter, EF-1α promoter, β-actin promoter, γ-globulin promoter, SR-α promoter and the like. Among these, T3 promoter, T7 promoter and SP6 promoter which are advantageous for transcription in a test tube are preferable. In the hybrid promoter of the present invention, a T7 promoter sequence is preferably used as the second promoter active sequence.

The hybrid promoter of the present invention has a sequence in which any of substitution, insertion, and addition with a sequence having the second promoter activity is made with respect to the sequence having the first promoter activity. All of these promoters are arranged in the same direction, and are configured to transcribe sense code DNA and / or antisense code DNA arranged downstream of both sequences. By hybridizing two types of sequences having promoter activity in the same direction, and downstream of sense code DNA and / or antisense code DNA, RNA can be easily obtained from a single DNA construct in in vitro or in vivo. it can.

The hybrid form of the sequence having the first promoter activity and the sequence having the second promoter activity is not particularly limited as long as the first promoter activity and the second promoter activity can be exhibited. For example, the first promoter When the active sequence is outside the gene, that is, in the case of the Pol III type Type III promoter, regions other than various elements necessary for transcription (proximal sequence element, Staf-binding site, distal sequence element and TATA box) In the above, it is preferable that the second promoter active sequence is substituted, inserted or added. More preferably, it is preferably substituted in the vicinity of the 3 'end of the first promoter active sequence. Especially, it is preferable to substitute by the sequence | arrangement which has 2nd promoter activity in 3 'side from the TATA box sequence of 1st promoter active sequence. In the substitution on the downstream side of the TATA box sequence, it is more preferred that the substitution is performed while maintaining the distance from the TATA box sequence to the transcription start point of the sequence having the first promoter activity.

Also, it is preferable that the transcription start point for the first promoter active sequence and the transcription start point for the second promoter active sequence are matched. For example, it is often preferred that the transcription initiation point for the first promoter active sequence (such as U6 promoter or H1 promoter) is a purine base (preferably G). Moreover, it is often preferred that the transcription initiation point for the second promoter active sequence (T7 promoter, T3 promoter, etc.) is also a purine base (A / G). Therefore, a purine base can be arranged as a common transcription start point for the first promoter active sequence and the second promoter active sequence. By matching the transcription start sites, transcripts with the same sequence can be easily obtained in vitro or in vivo. For this reason, screening accuracy is also improved.

Preferred combinations of the first promoter active sequence and the second promoter active sequence include combinations shown in Table 1 below. Of these, a combination of the U6 promoter and the T7 promoter is preferable. In the hybrid promoter of the U6 promoter and the T7 promoter, the guanine base serving as the transcription start point of the T7 promoter coincides with the guanine at the transcription start point (+1) of the U6 promoter on the 3 ′ side of the TATA box sequence of the U6 promoter. Is preferably substituted. An example of the sequence of such a hybrid promoter is shown in FIG.

As long as it has the first promoter activity and the second promoter activity, one or a plurality of base substitutions, deletions, additions and insertions have been made to the hybrid promoter sequence represented by SEQ ID NO: 1. Can be used as the hybrid promoter sequence of the present invention. It should be noted that such a modification on the base sequence can employ a known method such as the Kunkel method, the Gapped duplex method, or a similar method. Alternatively, mutation may be introduced using a mutation introduction kit (for example, Mutan-K (manufactured by TAKARA) or Mutan-G (manufactured by TAKARA)) using site-directed mutagenesis.

In addition, the hybrid promoter may be further modified. For example, RNA can be expressed at a desired timing by using an inducible promoter sequence as the first promoter active sequence. Such inducible promoters include the tetracycline-inducible U6 promoter (Ohkawa, J. & Taira, K. Control of the function of antiterｅｎRNA t eRNA ｃ － －. Gene. Ther. 11, 577-585 (2000)). Alternatively, the expression of RNA may be induced in a tissue-specific manner using a tissue-specific promoter or a DNA recombination system such as the Cre-LoxP system.

Also, for example, the production of RNA may be controlled using a recombinant enzyme. Using the CRE-loxP system as a recombination enzyme, a loxP sequence is provided in the vicinity of each of DSE and PSE in the promoter in the second promoter active sequence such as the H1 promoter or U6 promoter, and between DSE and PSE The distance can be set so that the promoter activity is turned off, and recombination occurs between loxP sequences by the action of the CRE protein, so that the promoter activity can be turned on for the DSE-PSE distance. The promoter activity can also be turned off by the action of the CRE protein.

(DNA construct)
The DNA construct of the present invention comprises the hybrid promoter of the present invention. The DNA construct of the present invention is a construct that can be conveniently switched between in vitro transcription reaction and RNA supply by intracellular expression as needed. Therefore, once the construct of the present invention is constructed, it can be used for both forms of RNA supply, and various applications based on gene expression regulation by various functional RNAs such as siRNA, miRNA and antisense RNA efficiently. Can be implemented. In particular, since RNA supply in vitro and in cells (in vivo) can be easily switched, a wide range of applications from cell level to animal level can be covered.

The DNA construct of the present invention may be in any form as long as it comprises the hybrid promoter of the present invention. A plurality of the hybrid promoters of the present invention may be provided. The DNA construct is preferably in the form of a vector. The vector form of the DNA construct of the present invention can be selected depending on the cell type to be applied to the RNAi library described later and the cell to be introduced.

The DNA construct of the present invention in the form of a vector can have a plasmid backbone that can be amplified by bacteria such as E. coli. Examples of such plasmids include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. The vector can be selected according to the cell to be introduced, the expression mode of the RNAi effect, and the like. For example, in mammalian cells, viruses such as retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, lentivirus vectors, herpes virus vectors, alphavirus vectors, EB virus vectors, papilloma virus vectors, foamy virus vectors, etc. A vector etc. are mentioned.

These vectors can hold a drug selection marker or the like as necessary. Examples of drug selection markers include neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene and the like. In addition, a marker that can be selected with an enzyme activity such as galactosidase as an index, or a marker that can be selected with a fluorescence emission such as GFP as an index.

The DNA construct of the present invention comprises a known RNA polymerase III transcription termination sequence (terminator sequence) that performs a transcription reaction depending on the first promoter active sequence of the hybrid promoter downstream of the hybrid promoter of the present invention. You can also. Even in vitro, in order to terminate the transcription reaction by RNA polymerase II, it is preferable to provide a transcription termination sequence of the RNA polymerase. In addition, in order to terminate the transcription reaction by RNA polymerase II or the like with the transcription termination sequence of RNA polymerase III, the transcription termination sequence in RNA transcription III or the rear end thereof is a cleavage site immediately below the transcription termination sequence of RNA polymerase III. Nucleotides are linked to a base sequence that forms a recognition sequence by a specific restriction enzyme. Prior to In vitro transcription, the treatment expressed by a predetermined restriction enzyme cuts the DNA in the RNA polymerase III transcription termination sequence or at the rear end thereof, so that the in vitro transcription reaction is surely stopped. A transcript with the correct chain length can be transcribed.

For example, by connecting AAA to a transcription termination sequence (TTTT etc.) of RNA polymerase III to form a DraI recognition sequence (TTTAAA), if treated with DraI before in vitro transcription reaction, T7 polymerase Transcription can be terminated accurately.

The DNA construct of the present invention can be provided with a recognition sequence for one or more restriction enzymes into which a coding DNA encoding RNA can be introduced inside or directly under the hybrid promoter. A desired RNA-encoding DNA can be inserted downstream (preferably immediately below) of the hybrid promoter using these restriction enzyme sites and oligonucleotide linkers.

The DNA construct of the present invention can be provided with RNA-encoding DNA by utilizing the restriction enzyme site as described above. Such a DNA construct can be immediately used for in vitro transcription reaction and intracellular expression.

When the DNA construct of the present invention is for producing siRNA, the DNA construct can comprise sense code DNA and / or antisense code DNA. One DNA construct may include a sense code DNA and an antisense code DNA, and two DNA constructs may include a sense code DNA and an antisense code DNA, respectively. When the RNA is siRNA, the sense code DNA provided in the DNA construct may be DNA that encodes the sense strand of any region of the target gene and that forms the sense strand of RNA by transcription. In the same case, the antisense coding DNA may be any DNA that encodes the antisense strand of any region of the target gene and that forms the antisense strand of RNA by transcription.
In addition, when the DNA construct of the present invention is for producing an antisense RNA, the antisense RNA only needs to have an antisense strand of RNA, so that it is sufficient to have an antisense code DNA. Furthermore, when the DNA construct of the present invention is for producing miRNA, for example, there are cases where two miRNAs are encoded in one pre-miRNA, and neither is sense and which is not antisense. It suffices to have a DNA encoding mRNA or pre-mRNA.

The arrangement form of the sense code DNA and the antisense code DNA with respect to the hybrid promoter is not particularly limited as long as the sense code DNA and / or the antisense code DNA can be transcribed under the control of the hybrid promoter, and finally RNA can be transcribed. Typically, a tandem type and a stem loop type are mentioned. In addition, a bidirectional type is also known (BMC-Biotechnology-3, 21, 21, 2003, etc.).

As shown in FIG. 2, the tandem type is usually a form in which a sense code DNA and an antisense code DNA are linked under the control of two hybrid promoters arranged in a construct. Note that the two hybrid promoters may be arranged in the same direction or in opposite directions. In the case of the tandem type, the sense code DNA and the antisense code DNA do not need to be on the same construct, and may be transcribed from two DNA constructs.

In addition, the stem loop type is a form in which a sense code DNA and an antisense code DNA are arranged with a loop sequence between them under the control of one hybrid promoter. The sense code DNA and the antisense code DNA are configured to form a stem via a loop sequence after transcription. As the loop sequence, siRNA can be obtained by in vitro transcribing and then trimming with ribonuclease or the like, and preferably has an effective length for inducing RNA interference in the cell. For example, it can be 5 to 50 bases, preferably 6 to 20 bases. However, a loop sequence having a length longer than that shown here can also be used. It should be noted that the hairpin RNA portion is trimmed in the cell and siRNA having an appropriate length by coding a base sequence, tRNA or the like that can be appropriately cleaved in the cell in the loop portion, or in combination with a hammerhead ribozyme or the like. It is also possible to design so that, etc. can be generated. The loop sequence is not particularly limited and may be an artificial sequence or a sequence derived from microRNA, and can be appropriately selected from known sequences and used.

The DNA construct of the present invention when preparing the sense code DNA and the antisense code DNA is preferably a stem loop type. For example, as a template for in vitro transcription reaction, the stem type becomes double stranded by intramolecular annealing of the transcription product, whereas the tandem type shows intermolecular annealing of the transcription product of the RNA sense strand and RNA antisense strand. Because it is required, it tends to reduce the yield of RNA that correctly forms a double strand. Further, when constructing a construct, the tandem type usually requires two operations for incorporating a template DNA strand into a plasmid or the like, whereas the stem loop type has an advantage that one integration operation is sufficient. Furthermore, there is a report that the stem loop type is more effective than the tandem type (Oligonucleotides 13, 325-333, 2003).

In addition, when the sense code DNA and the antisense code DNA are introduced into the DNA construct of the present invention in a tandem form, each double-stranded DNA is controlled under the control of the hybrid promoter using an appropriate oligonucleotide linker, restriction enzyme or the like. What is necessary is just to connect. In addition, when the sense code DNA and the antisense code DNA are introduced into the DNA construct in a stem loop type, the sense code DNA and the antisense code DNA are arranged in the opposite direction through the hairpin loop sequence. A heavy chain DNA is prepared, and this double-stranded DNA is ligated under the control of a hybrid promoter using an appropriate oligonucleotide linker and a restriction enzyme.

The sense code DNA and antisense code DNA used for the tandem type may be obtained, for example, by DNA synthesis or by a conventional transcription method, or may be obtained from an available cDNA or siRNA library. Furthermore, you may acquire by the random enzymatic degradation with respect to a target gene. In addition, as the sense code DNA and the antisense code DNA used for the stem loop type, a DNA including the sense code DNA and the antisense code DNA via a loop sequence is prepared. Such DNA may be obtained by conventional transcription methods by DNA synthesis, or may be obtained from available cDNA and siRNA libraries. Furthermore, you may acquire by the random enzymatic degradation with respect to a target gene.

Note that the DNA construct of the present invention does not necessarily take the form of a vector. For example, a chromosomally-introduced DNA construct comprising a sense-coding DNA and / or an antisense-coding DNA that can be transcribed under the control of the hybrid promoter of the present invention, and a DNA strand necessary for chromosomal introduction of animal cells, etc. You can also

(Use of DNA construct)
(Production of RNA by in vitro and intracellular expression)
The DNA construct of the present invention, which comprises a predetermined coding DNA that can be transcribed under the control of a hybrid promoter (hereinafter referred to as transcription / expression construct), is expressed in RNA by both in vitro transcription reaction and expression in cells. Can be produced.

(Production and supply of various RNAs by in vitro transcription reaction)
Various RNAs obtained by in vitro transcription reaction include, for example, a transcription / expression construct that is chained with an appropriate restriction enzyme as necessary, and then depends on the necessary NTP and the second promoter activity in the hybrid promoter. It can be obtained by performing an in vitro transcription reaction by acting an element necessary for transcription including RNA polymerase that acts on the protein. For such in vitro transcription reaction, various commercially available in vitro RNA synthesis kits can be used as appropriate. In addition, DNA used as a template can be decomposed with DNase by a conventional method. In addition, the shRNA obtained by the stem-loop type construct may be directly transfected into cells or the like, or a loop sequence that is unnecessary as siRNA may be treated with RNase or the like before transfection. .

The RNA (siRNA, shRNA, miRNA, antisense RNA, etc.) thus obtained can be transfected into animal cells by various methods. For transfection into animal cells, 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)), infection introduction method by virus (Sci. Am., P. 34, March (1994)), gene gun, etc. it can.

(Production and supply of various RNAs by intracellular expression)
In addition, RNA can be produced by expression in cells by transcribing transcription / expression constructs constructed as vectors or the like into animal cells and introducing them into cells, or by preparing virus particles and infecting cells. It can be implemented by introducing. Transfection into animal cells can be appropriately selected from methods similar to those used for introducing RNA into cells.

Further, when the transcription / expression construct of the present invention is a viral vector, viral particles are prepared by incorporating a vector plasmid into a virus, packaging or the like, and these viral particles are introduced into a cell to introduce it.

In order to select RNA produced in vitro from the transcription / expression construct of the present invention and a cell into which the construct has been introduced, known methods such as hybridization and PCR using a DNA sequence specific to the construct as a probe or primer In addition, when a selection marker included in the construct is provided, a phenotype based on the selection marker may be used as an index.

It should be noted that the transcription / expression construct of the present invention may be directly transfected or infected to cells in vitro of humans and non-human animals, or directly to cells in vivo of humans and non-human animals. It may be transfected or infected. Cells outside the living body may be collected from the living body and returned to the collected individual again after transfection or the like.

In addition, the DNA construct for RNA preparation of the present invention is introduced into various germ cells such as sperm, unfertilized egg, fertilized egg and somatic cell for constructing an embryo, and finally an embryo holding the DNA construct of the present invention is obtained. It can also be produced. In addition, living individuals can be produced from such embryos. Various methods well known to those skilled in the art can be used to produce embryos.

(Cells and non-human animals holding the DNA construct of the present invention)
The cell of the present invention can be obtained by introducing the DNA construct of the present invention. That is, a cell retaining the transcription / expression construct of the present invention in the cell can be obtained. When the DNA construct has a target sequence effective for suppressing the expression of the target gene relative to the target gene retained in the cell, a cell in which the expression of the target gene is suppressed can be obtained. In addition, the cell in which the expression of the target gene is suppressed may be a cell in which the expression level of the target gene is partially suppressed, and may not be completely suppressed. Such cells are cells whose expression was suppressed by functional RNA (eg, knockdown in the case of siRNA), and were used by measuring the expression of the target gene in these cells as described below. A target sequence having a high inhibitory effect can be screened from the effect of suppressing the expression of functional RNA such as siRNA derived from the DNA strand held in the DNA construct for RNA preparation. In addition, when knockdown cells expressing siRNA are used to suppress gene function analysis or expression of disease-related genes, they can also be used for drug discovery screening.

In addition, a non-human animal (organism individual) can be obtained from an embryo holding the DNA of the present invention. Such individual organisms can express or selectively express various functional RNAs. For example, the non-human animal (organism individual) produced from an embryo carrying the DNA construct of the present invention capable of expressing siRNA may be a knockdown animal. Knockdown animals are useful for gene function analysis and disease model animals. A technique for producing a knockdown animal in which the expression of the target gene is suppressed is not particularly limited, and a known technique can be used. For example, using a DNA construct for producing a siRNA having a disease-causing gene or the like as a target gene, expressing a siRNA in a cell to produce an embryo that expresses the siRNA; and an individual from the embryo And a manufacturing step.

(Kit containing the DNA construct of the present invention)
The kit of the present invention contains the DNA construct of the present invention. The DNA construct of the present invention can be transcribed both in vitro and in vivo. Therefore, it is useful to be provided as a kit together with reagents that can be used for both transcription reactions. Reagents necessary for the in vitro transcription reaction are well known to those skilled in the art, but preferably contain at least RNA polymerase depending on the second promoter active sequence in the hybrid promoter of the DNA construct of the present invention and various NTPs. In addition, an oligonucleotide linker for introducing a desired DNA chain, various restriction enzymes, restriction enzymes for fragmentation, enzymes such as DNase and RNase, and buffers can also be included. Also, reagents necessary for in vitro transcription reactions are well known to those skilled in the art.

(Use of the DNA construct of the present invention as a medicine)
The DNA construct of the present invention can be used as a medicine based on the function of the RNA to be produced. For example, the case where the said RNA is siRNA which suppresses the expression targeting a disease causative gene etc. is mentioned. In this case, the DNA construct of the present invention can be used properly according to the form of RNA supplied to the patient, that is, the usage of siRNA or the like. For example, when siRNA is administered directly, a transcription / expression construct is produced by in vitro transcription reaction, and siRNA is prepared with a suitable excipient on a cell collected from a tissue or a patient or a cell in a patient. Can be administered. In this case, examples of the administration form include various conventionally known administration forms, but a method used for transfection of siRNA into cells can also be used. In addition, in the case of gene therapy for siRNA expression in a cell, a transcription / expression construct is supplied to the cell at the disease site, and a cell that stably expresses siRNA in the cell is expressed by the expression in the cell. Can be administered to patients. Moreover, siRNA may be similarly expressed in cells or tissues and cells taken out from the patient and returned to the patient.

(SiRNA library and production method thereof)
The siRNA library of the present invention includes a construct comprising a plurality of types of sense code DNAs and antisense code DNAs for one or more types of target genes so that they can be transcribed under the control of the DNA construct hybrid promoter of the present invention. be able to. According to the library of the present invention, siRNA can be prepared by both in vitro and in vivo transcription reactions. Therefore, this library is advantageous for screening target sequences. High target sequences can be efficiently screened. In addition, various applications using the RNAi effect (production of useful disease model cells, disease model animals, drug discovery screening using these, drug discovery target gene screening, etc.) can be performed efficiently.

The siRNA library of the present invention is composed of the transcription / expression construct of the present invention comprising a sense code DNA and an antisense code DNA for one or more target genes that can be transcribed under the control of a hybrid promoter. In the siRNA library of the present invention, the sense code DNA and the antisense code DNA are preferably provided in one transcription / expression construct, more preferably a stem-loop type. The sense code DNA and the antisense code DNA preferably include a DNA fragment obtained by enzymatically degrading the target gene. The sense code DNA and the antisense code DNA are preferably set to such a length that siRNA as a transcript expresses an effective RNAi effect.

The target gene of the siRNA library of the present invention may be a gene coding region in an animal or a non-coding region such as a regulatory region. It may also be another region of chromosomal DNA. The target gene may be one type or two or more types. It is set as appropriate according to the purpose of the siRNA library. The number of DNA pairs is not particularly limited. For example, it may be several tens or more, 100 or more, or 1000 or more DNA pairs per gene. As will be described later, when a DNA pair is prepared by a technique of enzymatically degrading a target gene at random, any kind of DNA pair can be prepared for the gene.

The method for producing an siRNA library of the present invention comprises a pair of DNAs (sense code) that respectively encode a sense strand and an antisense strand of a double-stranded RNA that may function as siRNA against one or more target genes. A plurality of DNA and antisense-encoding DNA), and each of the DNA strands constituting the DNA pair is transcribed under the control of the hybrid promoter of the DNA construct according to any one of the above. Or a step of connecting the precursors in a transferable manner. According to the method for producing an siRNA library of the present invention, a DNA pair capable of exerting an RNAi effect is introduced in a transcribable manner while targeting any region of mRNA transcribed from the target gene under the control of the hybrid promoter of the present invention. Thus, it is possible to construct a library of both a DNA construct for in vitro transcription and a DNA construct for in vivo expression.

In the library preparation method of the present invention, first, a DNA pair comprising a sense code DNA and an antisense code DNA for any region of one or more target genes is prepared. The method for preparing the DNA pair is not particularly limited. A target sequence that has an RNAi effect may be chemically synthesized based on a certain theoretical prediction. It may also be obtained from available cDNA and siRNA libraries. Furthermore, you may acquire by the random enzymatic degradation with respect to a target gene.

It is preferable that the DNA pair is prepared through a step of enzymatically degrading the target gene. By enzymatically degrading the target gene, sense code DNA and antisense code DNA corresponding to any region of the target gene can be comprehensively obtained. As the enzyme that degrades the target gene, it is preferable to use one or more restriction enzymes that can be fragmented to such a length that the transcript functions as a sense strand and an antisense strand of siRNA.

The DNA pair may be processed as follows. That is, a single-stranded hairpin DNA is prepared by linking a double-stranded DNA fragment generated by enzymatic degradation with a hairpin adapter and a truncated adapter at each end. This single-stranded hairpin DNA can be converted into a double-stranded DNA in which sense code DNA and antisense code DNA are arranged in the reverse direction via a hairpin loop sequence derived from a hairpin adapter by a primer extension reaction. . By doing so, a stem-loop siRNA library can be easily constructed. This method is disclosed in detail in the above-mentioned Patent Document 2 (Republication No. 2005-063980), and can be carried out in accordance with the publication.

Next, such a DNA pair is linked to be capable of transcription under the control of the hybrid promoter of the DNA construct of the present invention. In the ligation method, as described for the DNA construct of the present invention, ligation is performed under the control of a hybrid promoter such as by using an oligonucleotide linker and a restriction enzyme. At this time, it is preferable to link the transcription start point depending on the first promoter activity and the transcription start point depending on the second promoter activity sequence so as to coincide with each other.

(Method of screening target sequence useful for RNAi)
The method for screening a target sequence useful for RNAi of the present invention comprises the steps of preparing the siRNA library of the present invention, in vitro transcription reaction using the DNA construct constituting the prepared siRNA library, and expression in a cell. Supplying the siRNA derived from the sense code DNA and the antisense code DNA into the cell expressing the target gene via either or both, and measuring the expression of the target gene in the cell And a process. According to the screening method of the present invention, siRNA can be supplied to cells by in vitro and / or intracellular expression as required. When the expression of a target gene obtained by supplying siRNA to a cell is measured and the suppression of the expression of the target gene can be affirmed, the sense code DNA from which the supplied siRNA is derived is the target gene of the cell, that is, the actual siRNA Can be selected as a target sequence on mRNA that is the target of or a candidate thereof. Further, by using the siRNA library, it is possible to screen for target sequences having a high expression suppression effect for the target genes.

In the screening method of the present invention, the method for measuring the expression of the target gene is not particularly limited, and a known method can be adopted. For example, it is possible by Northern blot hybridization or Western blot hybridization.

For comprehensive screening of target sequences, it is preferable to carry out both siRNA supply by in vitro transcription reaction and siRNA supply by intracellular expression. A more preferable target sequence can be obtained by these two types of target sequence screening. The in vitro transcription reaction and siRNA supply experiment by intracellular expression may be combined in any way. The in vitro transcription reaction may be used in the primary screening, and the in vitro transcription reaction may be used in the secondary screening. The primary screening is based on cell-level screening and uses either or both transcription reactions in in vitro and in vivo, and the secondary screening is animal-level screening in both in vitro or in vivo. Such a transcription reaction may be used.

(Screening method of siRNA useful for prevention or treatment of disease)
The siRNA screening method useful for the prevention or treatment of the disease of the present invention comprises a step of preparing the siRNA library of the present invention for one or more target genes related to the disease, and the preparation of the siRNA library prepared above. Derived from the sense code DNA and the antisense code DNA in cells expressing the target gene through either or both of in vitro transcription reaction using the DNA construct and / or expression in cells Supplying the siRNA to be performed, and measuring the expression of the target gene in the cell. According to the screening method of the present invention, a target sequence capable of effectively suppressing the expression of a disease-related gene can be efficiently and comprehensively screened. That is, siRNA useful as an RNAi drug can be screened. The cells that supply siRNA may be cells or tissues derived from disease model cells, model animals, cells collected from affected individuals (patients), or the like. There may be cells that are artificially constructed to express a phenotype useful for the prevention or treatment of disease.

(Screening method of drug discovery target genes useful for disease prevention or treatment)
The screening method for drug discovery target genes useful for the prevention or treatment of the disease of the present invention comprises the steps of preparing the siRNA library of the present invention for a target gene that may be related to the disease, and the preparation of the siRNA library prepared above. Derived from the sense code DNA and the antisense code DNA in cells expressing the target gene through either or both of in vitro transcription reaction using the DNA construct and / or expression in cells A step of supplying the siRNA to be performed, and a step of measuring a change in the cell before and after the supply of the siRNA. According to the screening method of the present invention, by measuring changes before and after siRNA supply, a target gene related to a disease, and thus a target gene can be screened. That is, by introducing siRNA, if there is a change in the phenotype of the cell, the target DNA may be related to the onset, progression, treatment, prognosis, etc. of the disease, and may be a drug discovery target Because there is. According to the present invention, since comprehensive screening is possible, drug discovery targets can be efficiently screened.

The cells that supply siRNA may be cells or tissues derived from disease model cells, model animals, or cells or tissues collected from affected individuals (patients). It may also be a cell artificially constructed to express a phenotype useful for disease prevention or treatment.

According to the present invention, not only siRNA but also RNA in a cell expressing a target gene through either or both of in vitro transcription reaction using the DNA construct of the present invention and expression in the cell. A method for regulating the expression of the target gene can also be carried out by providing a supplying step. This is because ncRNA is considered to contribute to various gene expression regulation including suppression of gene expression. Similarly, RNA function analysis methods can be implemented in which RNA is supplied into animal cells and RNA functions are evaluated by comparing cells that have supplied RNA with cells that have not supplied RNA. is there. The method for functional analysis is not particularly limited. It may be an expression analysis of mRNA or protein, or an assay of a specific function of a cell.

Hereinafter, the present invention will be described with specific examples. However, the following examples do not limit the present invention.

(Construction of plasmid with hybrid promoter)
1. Sequence of Hybrid Promoter The irecoU6 promoter, which is a hybrid promoter of the present invention, is a part of the mouse U6 promoter (hereinafter referred to as U6 promoter) which is an RNA polymerase III-dependent promoter widely used for intracellular transcription of siRNA. It was prepared by replacing with a widely used T7 RNA polymerase-dependent promoter (hereinafter referred to as T7 promoter). Various forms of insertion position or orientation of T7 RNA polymerase can be considered, but (1) the same transcript is expected when either promoter is functioned, (2) an extra sequence is added to the transcribed siRNA The T7 promoter sequence was inserted so that the transcription start point (+1) of the T7 promoter is aligned with the transcription start point (+1) of the U6 promoter (FIG. 1).

In addition, as shown in FIG. 1, an RNA polymerase III transcription termination sequence (TTTTTT) is added to the siRNA encoding sequence downstream of the irecoU6 promoter, and AAA is added to this transcription termination sequence for “run-off”. In this way, a DraI recognition sequence (TTTAAA) was prepared and treated with DraI before in vitro transcription reaction, so that the transcription of T7 RNA polymerase was designed to stop at this position accurately.

2. Plasmid vector construction Plasmid construction was performed using standard molecular biology techniques. As plasmid vectors, the retroviral vector pNAMA-irecoU6 shown in FIG. 3 and the lentiviral vector plasmid pNAMAh-irecoU6 shown in FIG. 4 were constructed. The construction methods of these plasmid vectors are shown in FIGS. 5 and 6, respectively. The restriction enzymes used in each step are illustrated by the following abbreviations and the restriction enzyme sites are indicated by lollipops (B: BamHI, P: PstI, D: Drai, G: BglII, N: NotI, H: HindIII, K: KpnI, S: SpeI, A: AvrII, M: NgoMIV, Z: XbaI, O: XhoI). Table 2 shows the primers and oligonucleotides (on_01 to 12) used.

3. Retroviral vector First, as shown in FIG. 5, a plasmid pBsk-U63-LTR plasmid based on pBluescriptSK (−) (Stratagene) having a mouse U6 promoter sequence derived from pSilencer 1.0-U6 (Ambion) and a termination sequence (Stratagene) Nature Genet. 36, 190-196, 2004) is used as a template, PCR (primers: on_01, on_02) is performed, the amplification product is treated with BamHI, and self-circularized. Incorporated intermediate 1 was obtained.

Subsequently, this intermediate was cleaved with BamHI and PstI, and intermediate 2 was obtained by linker ligating oligonucleotides (on_03, on_04). This operation incorporated a DraI recognition sequence immediately after the transcription termination sequence of siRNA. On the other hand, a fragment containing puromycin N acetyltransferase coding (PAC) sequence amplified by PCR (primers: on_05, on_06) using pIRES-puro2 (Clontech) as a template was treated with BglII and NotI, and pda5LTR-DsRed2 (Nature Genet. 36: 190-196, 2004), and inserted between BamHI-NotI to obtain Intermediate 3. Furthermore, a fragment containing LTR and irecoU6 sequence extracted from Intermediate 2 with NotI and HindIII was inserted between NotI-HindIII of Intermediate 3 to obtain pNAMA-irecoU6.

4). Lentiviral vector plasmid As shown in FIG. 6, pLKO.1 (addgene) was cleaved with AvrII and NgoMIV, and oligonucleotides (on_07, on_08) were linker-ligated to obtain intermediate 4. This operation removed the T7 promoter sequence (black box) from that present in pLKO.1. Next, pLKO.1 was cleaved with KpnI and AvrII to extract the 3′LTR, and inserted between KpnI-SpeI of pBluescriptSK (−) to obtain Intermediate 5. PCR was performed using this intermediate 5 as a template (primers: on_09, on_10), and the amplified product was treated with NheI, and then self-circulated to obtain intermediate 6. This operation removed the BbsI recognition sequence from SIN3'LTR. Next, a fragment containing 3′LTR extracted from intermediate 6 with KpnI and XbaI was inserted between KpnI-XbaI of intermediate 4 to obtain intermediate 7. PCR was performed using this intermediate 7 as a template (primers: on_11, on_12), the amplified product was treated with BbsI, and then inserted into intermediate 7 cleaved with XhoI to obtain intermediate 8. By this operation, the U6 promoter incorporated in pLKO.1 was removed. Furthermore, a fragment containing the irecoU6 sequence extracted from the already constructed pNAMA-irecoU6 by NheI digestion was inserted into the intermediate 8 digested with AvrII to obtain pNAMAh-irecoU6.

(Confirmation of RNA polymerase III-dependent promoter activity)
In this example, a model experiment (expression in a cell) using GFP was performed in order to examine whether the modification of the U6 promoter had an influence on the original transcriptional activity. In the experiment, three types of siRNA coding sequences with different strengths targeting GFP were used in a stem-loop form. Table 3 shows the sequence of the DNA strand for stem-loop type construct including the sense code DNA, loop sequence and antisense code DNA.

(Insertion of siRNA coding sequence)
The siRNA coding sequence was inserted into the plasmid constructed in Example 1 (pNAMA-irecoU6 retroviral vector plasmid) by oligonucleotide linker ligation. As a control, pNAMA-U6 plasmid ((Nature Genet. 36, 190-196, 2004) carrying U6 promoter was used.

The plasmids constructed in Example 1 are all designed to allow linker ligation by the same operation. That is, FIG. 7 shows the T7 promoter sequence common to each vector and the sequence immediately below it. As shown in FIG. 7, an oligonucleotide is prepared in which the siRNA coding sequence to be inserted is divided by a loop sequence into half of the upstream side and the downstream side. The portions indicated by (N... N) in FIG. 7 correspond to the upstream sequence and the downstream sequence of the siRNA coding sequence, respectively. After annealing 5 ′ phosphorylated oligonucleotides (upstream: A and a, downstream: B and b), respectively, and then performing a ligation reaction with a vector plasmid cleaved with BbsI, the siRNA coding sequence is converted into a plasmid. Inserted into.

(Cell culture)
293T cells were cultured in DMEM medium (Nacalai Tesque) containing 10% inactivated fetal calf serum.
(Transfection)
All transfections were performed in a 96 well format. For siRNA expression plasmid transfection, 200 ng of siRNA expression plasmid per well was transfected with Lipofectamine 2000 together with 100 ng of GFP expression plasmid. Note that pMX-d2EGP was used as the GFP expression plasmid. This plasmid was prepared by inserting a fragment containing the coding sequence of d2EGFP excised with BamHI and NotI from pd2EGFP-1 (Clontech) between BamHI-NotI of pMX (provided by Dr. Toshio Kitamura of the University of Tokyo).

After transfection, the expression level of GFP was analyzed after maintaining for 2 days. In the analysis of the expression level of GFP, the relative decrease degree was evaluated as the RNAi effect based on the fluorescence intensity measured by GENios (Tecan). For example, when the relative reduction degree is 2, it indicates that the fluorescence intensity of GFP is reduced by 50%, and when it is 10, it indicates that it is reduced by 90%. The results are shown in FIG.

As shown in FIG. 8, the RNAi effect (relative decrease in the fluorescence intensity of GFP) by siRNA expressed by intracellular expression using the irecoU6 promoter is slightly enhanced rather than under the control by the promoter before modification. It was shown that the function of RNA polymerase III as a promoter is sufficiently retained.

(Synthesis of siRNA by in vitro transcription reaction using irecoU6 promoter)
In this example, it was examined whether a plasmid having an irecoU6 promoter functions as a template for an in vitro transcription reaction. The pNAMA-irecoU6 retroviral vector plasmid constructed in Example 1 was cleaved with DraI and then subjected to a transcription reaction with T7 RNA polymerase. The reagents necessary for the transcription reaction were appropriately used from CUGA7 in vitro transcription kit (Nippon Gene Co., Ltd.), and shRNA was prepared and purified according to the attached protocol.
Briefly, a reaction solution containing 2 uL of CUGA 7 Enzyme Solution per 1.5 pmol of DraI-digested plasmid was incubated at 37 ° C. for 2 hours, and then added with 4 uL of DNase Enzyme Solution and further incubated for 30 minutes. The dephosphorylation of siRNA was performed by incubating with 10 u SAP (TaKaRa) per μg for 1 hour at 37 ° C. The transcribed RNA was analyzed by polyacrylamide electrophoresis. The results are shown in FIG.

As shown in FIG. 9, a single band was observed around about 25 bp, indicating that the target siRNA was transcribed with high purity. In general, it is said that efficient transcription of T7 RNA polymerase requires three guanine bases at the transcription start point. The transcription start point of the irecoU6 promoter has only one guanine base, but the T7 promoter was found to function well.

In this example, the function of three types of siRNA synthesized from the pNAMA-irecoU6 retrovirus vector plasmid by in vitro transcription was examined by a model experiment using GFP. Since the triphosphate group added to the 5 'end of RNA synthesized by in vitro transcription reaction has been reported to be toxic to cells (Nature Biotechnology 22, 321-325, 2004), it is introduced into cells. A dephosphorylation treatment was performed before. These siRNAs were transfected into 293T cells with a GFP expression plasmid and maintained for 2 days. For siRNA transfection, siRNA was transfected with Lipofectamine 2000 (Invitrogen) together with 160 ng of GFP expression plasmid so that the final concentration was 50 nM, 10 nM, 2 nM. After maintaining for 2 days after transfection, the expression level of GFP was analyzed. Analysis of the expression level of GFP was performed in the same manner as in Example 2. The results are shown in FIG.

As shown in FIG. 10, concentration-dependent expression suppression was observed for any of the siRNAs used. Further, the characteristics of the three sequences were similar to the effect of the plasmid transfection shown in FIG. From these results, it was confirmed that RNA synthesized by in vitro transcription reaction functions as siRNA.

(Cell introduction of irecoU6 promoter using retroviral vector)
In this example, the RNAi effect when the irecoU6 promoter was introduced into cells by a retroviral vector was examined. As a subject, a retroviral vector pNAMA-U6 having a U6 promoter before modification was also used. Three types of siRNA coding sequences targeting GFP shown in Table 3 were inserted into the pNAMA-irecoU6 retroviral vector plasmid constructed in Example 1 directly under the ireco promoter by the linker ligation described in Example 2. Retroviruses were prepared from these plasmids and infected with stable GFP expressing Jurkat T cells. Retrovirus preparation and cell culture were performed as follows.

(Preparation of retro virus)
For retrovirus, GP293 cells seeded in a 35 mm dish were transfected with 4 μg of a retrovirus vector plasmid together with 0.4 μg of pVSVG (Clontech) using Lipofectamine 2000, and a culture solution containing virus particles was collected two days later. .
(Cell culture)
Jurkat T cells were cultured in RPMI 1640 medium (Nacalai Tesque) containing 10% inactivated fetal calf serum (Invitrogen). When drug selection was performed, 0.4 g / ml puromycin (SIGMA) was added to the medium. GP293 cells were cultured in DMEM medium (Nacalai Tesque) containing 10% inactivated fetal calf serum.

After infecting Jurkat T cells with virus particles and maintaining for 3 days, the fluorescence intensity of GFP was measured with a flow cytometer. In the experiment using a virus, the fluorescence intensity was measured by analyzing the fluorescence intensity of GFP measured by FACS calibur (BD) using Cell Quest software (BD), and the relative decrease degree against the virus non-infected cells was evaluated as an RNAi effect. . The relative reduction degree 2 indicates that the fluorescence intensity of GFP is reduced by 50%, and if it is 10, the reduction is 90%. The results are shown in FIG.

As shown in FIG. 11 (a), the expression level of GFP was lowered in any sequence. Furthermore, in order to compare the ireco U6 promoter with the U6 promoter again, in order to examine the RNAi effect of one copy of the virus, the cells infected with the limiting dilution virus were subjected to drug selection with Puromycin, and then the expression level of GFP Was measured. As a result, as shown in FIG. 11B, a sufficient RNAi effect was confirmed even with one copy.

(Cell introduction of irecoU6 promoter using lentiviral vector)
Retroviral vectors are widely used for gene transfer, but their range of use has been limited because of their low efficiency in infecting non-dividing cells. In recent years, lentiviral vectors capable of efficient infection regardless of cell proliferation have been used in place of retroviral vectors. The inventor constructed a lentiviral vector plasmid having an irecoU6 promoter in Example 1. In order to investigate the knockdown effect of this viral vector, in the same manner as in Example 2, three types targeting GFP shown in Table 3 immediately below the ireco promoter of the pNAMAh-irecoU6 lentiviral vector plasmid constructed in Example 1 Of the siRNA coding sequence was inserted. Lentiviruses were prepared from these plasmids and infected with stable GFP expressing Jurkat T cells. In addition, preparation of lentivirus and cell culture were performed as follows.

(Preparation of virus particles)
The lentivirus was prepared by adding 2 ug of lentiviral vector plasmid, 1.8 ug of psPAX2 (addgene) and pMD2. Transfection was performed using Lipofectamine 2000 together with 0.2 ug of G (addgene), and the culture solution containing virus particles was recovered after 2 days.
(Cell culture)
Jurkat T cells were cultured in RPMI 1640 medium (Nacalai Tesque) containing 10% inactivated fetal calf serum (Invitrogen). When drug selection was performed, 0.4 g / ml puromycin (SIGMA) was added to the medium. 293T cells were cultured in DMEM medium (Nacalai Tesque) containing 10% inactivated fetal calf serum.

After infecting stable GFP-expressing Jurkat T cells and maintaining for 3 days, the fluorescence intensity of GFP was measured using a flow cytometer. The fluorescence intensity was measured in the same manner as in Example 5. The results are shown in FIG.

As shown in FIG. 12, it was confirmed that RNAi can be efficiently induced with any DNA. Compared with retroviruses, RNAi effects of the same degree were obtained with a virus amount of about 1/10.

SEQ ID NO: 1: Hybrid promoter operating as U6 promoter and T7 promoter SEQ ID NO: 2-11: Primer SEQ ID NO: 12-13: siRNA coding DNA

Claims (15)

A second RNA that is a DNA-dependent RNA polymerase that operates in vitro against a first promoter active sequence that has the promoter activity of the first RNA polymerase that is a DNA-dependent RNA polymerase III that operates in animal cells A hybrid having a base sequence in which substitution, insertion or addition of a second promoter active sequence having the promoter activity of polymerase has been made, and having the promoter activity of the first RNA polymerase and the promoter activity of the second RNA polymerase A DNA construct comprising a promoter. The construct according to claim 1, wherein the first promoter active sequence is any one of a sequence having U6 promoter activity and a sequence having H1 promoter activity. The construct according to claim 2, wherein the first promoter active sequence is a sequence having U6 promoter activity. 4. The construct according to claim 1, wherein the second promoter active sequence is selected from the group consisting of a sequence having T7 promoter activity, a sequence having T3 promoter activity, and a sequence having SP6 promoter activity. The construct according to claim 4, wherein the second promoter active sequence is a sequence having T7 promoter activity. The hybrid promoter has a base sequence in which any one of substitution, insertion and addition of the second promoter active sequence is made 3 'to the TATA box sequence of the first promoter active sequence. The construct according to any one of the above. The construct according to claim 1, wherein in the hybrid promoter, the first promoter active sequence is a sequence having U6 promoter activity, and the second promoter active sequence is a sequence having T7 promoter activity. The construct according to claim 7, wherein a sequence having the second promoter activity is substituted, inserted or added 3 'to the TATA box sequence of the first promoter active sequence. The hybrid promoter is one of the following sequences:
(A) a base sequence represented by SEQ ID NO: 1 (b) consisting of a base sequence having any one of substitution, deletion, addition and insertion of one or more bases in the base sequence represented by SEQ ID NO: 1, The construct according to claim 8. The construct according to any one of claims 1 to 9, which is a plasmid vector or a virus vector. The method according to any one of claims 1 to 10, further comprising a sense code DNA and an antisense code DNA of any region of the target gene, which is linked to be transcribed under the control of the hybrid promoter, and is for the production of siRNA. Constructs as described in A second RNA polymerase that is a DNA-dependent RNA polymerase that operates in vitro against a first active sequence that has the promoter activity of the first RNA polymerase that is a DNA-dependent RNA polymerase III that operates in animal cells A second promoter active sequence having a promoter activity of: a base sequence that has been substituted, inserted or added, and having the promoter activity of the first RNA polymerase and the promoter activity of the second RNA polymerase; Hybrid promoter. One of the following sequences:
(A) a base sequence represented by SEQ ID NO: 1 (b) having a base sequence having any one of substitution, deletion, addition and insertion of one or more bases in the base sequence represented by SEQ ID NO: 1. The hybrid promoter according to claim 12. A method for producing RNA, comprising:
A method for producing RNA by in vitro transcription reaction or expression in a cell using the DNA construct according to claim 10 or 11. A method of suppressing the expression of a target gene by supplying siRNA to a cell,
11. The cell expressing the target gene in either or both of in vitro transcription reaction using the RNA construct DNA construct according to claim 1 and expression in the cell, or both. Providing a siRNA derived from a sense code DNA and an antisense code DNA in any region of the target gene.

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