JP2014103905A - KNOCK-DOWN METHOD OF TARGET GENE, AND EXPRESSION VECTOR FOR RNAi FOR THE SAME - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for modulating knock-down effect of a target gene by an RNAi.SOLUTION: An expression vector for RNAi comprises an expression cassette including paired promoter (P) and polyA-addition signal (A) which control transcription of a region (I) coding for an RNAi-related transcriptional product, a region (R) including a predetermined site-specific recombinant enzyme-recognition sequence or a transposase recognition sequence, and an optional sequence interposed between those sequences, and a region including both the regions (I) and (R). A knock-down method of a target gene comprises steps of: introducing the expression vector for RNAi into cells; making the site-specific recombinant enzyme or the transposase corresponding to the sequence included in the region (R) express; and changing a base length of the expression cassette. Thus, the knock-down levels of the target gene before and after the whole steps are changed from each other.

Description

  The present invention relates to a method for knocking down a target gene using an expression vector for RNAi (RNA interference). More specifically, the present invention relates to a method for regulating the knockdown effect of a target gene in the method, and an expression vector for RNAi having a suitable mechanism therefor.

  RNAi is known as a mechanism of gene silencing that occurs in a wide range of eukaryotes. RNAi has a pathway involving miRNA (micro RNA) and a pathway involving siRNA (small interfering RNA). A relatively short double-stranded RNA is formed by Dicer, a kind of RNase III, from a specific precursor consisting of a base sequence that is complementary to the target gene mRNA (usually containing several mismatches in the case of miRNA). Is done. After this double-stranded RNA is incorporated into RISC (RNA-Induced Silencing Complex) to become a single-stranded RNA, it binds together with the RISC to a predetermined site of the target gene mRNA where the corresponding base sequence exists. It is thought that the effect of RNAi is produced by suppressing the translation of mRNA or cleaving / degrading the mRNA.

  In order to suppress (knock down) the expression of target genes in eukaryotic cells using such RNAi, a vector containing a nucleotide sequence that generates a precursor of miRNA or siRNA is introduced into eukaryotic cells. ing. In general, miRNA vectors contain a base sequence that uses pre-miRNA (miRNA precursor) or pri-miRNA (primary miRNA), which is a precursor of pre-miRNA, as a transcription product, and siRNA vectors contain sense strands. And shRNA (short hairpin RNA) in which the antisense strand is integrated, or a base sequence having each of the sense strand and the antisense strand as a transcription product. When pri-miRNA is transcribed from a miRNA vector, it is first converted into pre-miRNA by Drosha in the nucleus. These miRNA or siRNA precursors are transported outside the nucleus (in the cytoplasm), where they become mature miRNA or siRNA by the action of Dicer as described above.

  The knockdown method performed as described above is currently widely used as a gene function loss type analysis tool, and is expected to be applicable to gene therapy. In order to clearly see the effects of knocking down a gene, it is first desirable to use a construct that exhibits a stronger knockdown effect. On the contrary, depending on the purpose, it is also useful to weaken the knockdown effect.

However, there are almost no examples in which knockdown effects are comparatively examined for a large number of knockdown constructs.
Patent Document 1 refers to “on / off control of RNA interference” (conditional targeting method), and the following is described as an example of a base sequence for that purpose (paragraph 0162, FIG. 3). , 4 etc.).
I) RNA polymerase II system promoter-loxP-arbitrary sequence region in which expression is controlled-loxP-shRNA precursor coding region II) RNA polymerase II system promoter-loxP-shRNA precursor coding region-loxP-arbitrary in which expression is controlled Array area

  However, the sequence as described above in Patent Document 1 does not change the length of the expression cassette of the “shRNA precursor coding region” involved in RNAi, and the effect of RNAi depends on the length of the expression cassette. There is no description or suggestion that it is possible to regulate the strength of the.

JP 2007-104969 A

  An object of the present invention is to provide a means for regulating the knockdown effect of a target gene by RNAi.

  In order to find a gene expression construct exhibiting a high knockdown effect, the inventors conducted a comparative analysis of the knockdown effect between constructs by creating multiple types of artificial miRNA expression constructs and introducing them into cultured cells. . As a result, the knockdown effect is not affected by the position of the artificial miRNA present in the construct (eg, on the 3'-UTR or in the intron), but is enhanced when the miRNA expression cassette is short, It was found that it was suppressed more strongly. It is possible to control (improve or reduce) the knockdown effect of RNAi by changing (shortening or extending) the length of the expression cassette, for example, after introducing a vector into a cell, a reporter gene It has been found that the RNAi effect can be improved by deleting the above from the expression cassette as compared with the case where it is not deleted, and the present invention has been completed.

  That is, the present invention provides, in one aspect, an expression vector that can regulate the RNAi effect of the vector, and in another aspect, an expression vector and members related thereto for carrying out the method. Each invention specifically includes the following inventions.

[1] Region (I) encoding RNAi-related transcript, site-specific recombination enzyme recognition sequence (r) placed in two locations in the same direction, or transposase recognition sequence placed in two locations in the opposite direction (T), and the region (R) containing the arbitrary sequence (n) sandwiched between the two recognition sequences, and the transcription of the region containing both the regions (I) and (R) An expression vector for RNAi comprising an expression cassette comprising a pair of promoters (P) and a polyA addition signal (A).
[2] The expression vector for RNAi according to [1], wherein the arbitrary sequence (n) is a sequence encoding a reporter gene and / or a drug resistance gene.
[3] The promoter (P), the region (I), the region (R) and the first polyA addition signal (A) are arranged in this order from the 5 ′ end to the 3 ′ end, The RNAi according to [1] or [2], wherein the arbitrary sequence (n) contained in the region (R) contains a second polyA addition signal (A) that is the same as the first polyA addition signal Expression vector.

[4] A recognition sequence (rr) of a site-specific recombinase in which the expression cassette is different from the recognition sequence (r) or (t) of the region (R) and arranged in two places in the same direction. Alternatively, the RNAi expression vector according to any one of [1] to [3], which is sandwiched between transposase recognition sequences (tt) arranged at two positions in opposite directions.
[5] Region (I) encoding the RNAi-related transcription product provided in the RNAi expression vector according to any one of [1] to [4], site-specific sets arranged in two locations in the same direction A recognition enzyme sequence (r) or a transposase recognition sequence (t) placed at two positions in the opposite direction and a region (R) containing an arbitrary sequence (n) sandwiched between these two sequences, and restriction A double-stranded oligonucleotide comprising an enzyme site.
[6] The region (I) encoding the RNAi-related transcript, and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same. The expression vectors for RNAi in multiple states, or the vectors for producing them, differing in the length of the entire expression cassette due to the different lengths, and thus the knockdown efficiency of the target gene is different. A kit for RNAi, comprising:

[7] The RNAi expression vector according to any one of [1] to [4] or a vector for producing the RNAi expression vector according to any one of [1] to [4] as a vector for producing the plurality of RNAi expression vectors. RNAi kit.
[8] The RNAi according to [7], further comprising a site-specific recombinant enzyme expression vector or a transposase expression vector corresponding to the recognition sequence (r) or (t) included in the region (R). For kit.
[9] A region further comprising a recognition sequence (r) that is the same as or close to the region (R) and a region (R ′) that includes an arbitrary sequence (n ′) having a length different from that of the region (R) The kit for RNAi according to [7] or [8], comprising a vector for R recombination or a vector for producing the same.
[10] The method according to any one of [7] to [9], further comprising a site-specific recombinant enzyme expression vector or a transposase expression vector corresponding to the recognition sequence (rr) or (tt). RNAi kit.

[11] The region (I) encoding the RNAi-related transcription product and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same, but other nucleotide sequences The target gene is characterized by using an expression vector for RNAi in a plurality of states in which the length of the entire expression cassette is different due to the difference in length, and thereby the knockdown efficiency of the target gene is different. Knockdown method.
[12] Regions (I) in which the RNAi expression vector in the plurality of states encodes an RNAi-related transcription product, recognition sequences (r) of site-specific recombinant enzymes arranged in two places in the same direction, or reverse directions A region (R) comprising a transposase recognition sequence (t) arranged at two positions in FIG. 5 and an arbitrary sequence (n) sandwiched between the two positions, and both the regions (I) and (R) Produced by an expression vector for RNAi comprising an expression cassette comprising a set of promoter (P) and polyA addition signal (A) for controlling the transcription of a region containing the RNAi. After the introduction, a step (regulation) of expressing a site-specific recombinase or transposase corresponding to the recognition sequence contained in the region (R) and changing the base length of the expression cassette. The step), to change the knockdown of the target gene before and after this step, the method described in [11].

[13] The method according to [12], wherein the regulating step is a step of excising the region (R) by allowing the site-specific recombinase or transposase to act.
[14] The method according to [13], wherein the base length of the expression cassette is shortened by deleting the region (R), thereby improving the knockdown efficiency of the target gene as compared to before the step.
[15] The promoter (P), the region (I), the region (R) and the first polyA addition signal (A) are arranged in this order from the 5 ′ end to the 3 ′ end, The arbitrary sequence (n) contained in the region (R) contains a second polyA addition signal (A) that is the same as the first polyA addition signal, and together with the deletion of the region (R), the second sequence By removing the polyA addition signal (A), the base length of the expression cassette is extended by changing the end of the expression cassette to the first polyA addition signal (A), thereby knocking down the target gene. The method according to [13], wherein the efficiency is decreased as compared with that before the step.

[16] The region (R ′), wherein the regulatory step includes a recognition sequence (r) having the same or close mutant type as the region (R) and an arbitrary sequence (n ′) having a length different from that of the region (R) Recombination of the arbitrary sequence (n) and the arbitrary sequence (n ′) contained in the region (R) by allowing the site-specific recombinase to act in the presence of a vector comprising The method according to [12], which is a step of extending or shortening the base length of the cassette, thereby reducing or improving the knockdown efficiency of the target gene, respectively, as compared to before the step.
[17] As an RNAi expression vector, when the region (R) contains a recognition sequence (r) or (t), the expression cassette is of a different type and is located at two locations in the same direction A specific recombination enzyme recognition sequence (rr) or a transposase recognition sequence (tt) arranged at two positions in the opposite direction is used and corresponds to the recognition sequence (rr) or (tt). The method according to any one of [11] to [16], further comprising the step of integrating the expression cassette into a host cell chromosome by expressing a site-specific recombinase or transposase.

  In the present invention, “RNAi-related transcript” is a generic term for transcripts that can be finally induced by RNAi by being subjected to predetermined processing in the cell after being transcribed. In the present invention, RNAi mainly involving miRNA is assumed, and pri-miRNA, pre-miRNA, and artificial miRNA having a hairpin-like structure similar to these, which can finally produce mature miRNA or shRNA and the like are included in “RNAi (miRNA) -related transcripts”. However, the present invention does not exclude the possibility of siRNA involving RNAi. For example, transcripts such as long hairpin RNA produced by multiple siRNAs are included in “RNAi (siRNA) -related transcripts”. Also good.

  By using the expression vector for RNAi provided by the present invention and the technique for adjusting the length of the RNAi expression cassette included therein, the level of knockdown of the target gene by RNAi can be arbitrarily controlled. become. As a result, it is improved when the knockdown effect is weak, while it is reduced when the knockdown effect is strong, making it easier to achieve the intended purpose, and various functional analyzes of genes through the strength of the knockdown effect. It will become possible to expand the range of application of RNAi.

1 is an overview of Example 1. (A) Configuration of miRNA expression vectors (1) to (4). (B) FACS analysis result of eGFP expression intensity of host cells (eGFP-expressing ES cells). Overview of Example 2. (A) Configuration of miRNA expression vectors (1) to (7) and eGFP expression vector. (B) FACS analysis result of eGFP expression intensity in host cells after introduction of these vectors. (C) Photographed by a fluorescence microscope. Overview of Example 3. (A) Configuration of miGFP expression vector and eGFP expression construct inserted in host cell (eGFP expression ES cell). (1) before Dre action, (2) after Dre action. (B) FACS analysis result of eGFP expression intensity in host cells.

-RNAi expression vector-
The expression vector of the present invention comprises a region (I) consisting of a sequence encoding an RNAi-related transcription product, a predetermined recognition sequence, and a sandwiched therebetween in an initial state before site-specific recombinase is allowed to act on the region (R). The RNAi expression cassette includes a region (R) composed of an arbitrary sequence, a promoter (P), and a polyA addition signal (A).

  The position where the region I and the region R are arranged is not particularly limited as long as the base length of the expression cassette for RNAi that transcribes the sequence encoding the RNAi-related transcription product can be changed as long as the action and effect of the present invention are exhibited. It is not limited. For example, the order of P- [region I]-[region R] -A from the 5 'end to the 3' end is the order of P- [region R]-[region I] -A. May be. Region I may be present in the intron or may be present in the 3'UTR. There may be a spacer sequence of appropriate length between each region and element.

  Further, only one region R may exist in the RNAi expression vector, or a plurality of regions R may exist so that the length of the expression cassette via the region R can be changed in multiple stages. For example, when the expression cassette has the structure of P- [region I]-[region R1]-[region R2] -A, the site-specific corresponding to the recognition sequence (r1) contained in [region R1] at the first stage By acting the recombination enzyme, the length of the expression cassette is shortened, and in the second stage, expression is made by acting a site-specific recombination enzyme corresponding to the recognition sequence (r2) contained in [region R2]. It is possible to further reduce the length of the cassette. In such a case, it is appropriate that the site-specific recombination systems at each stage are different from each other.

  In addition to the RNAi expression cassette, the expression vector of the present invention may contain elements of a general expression vector (for example, a multiple cloning site, a replication origin for E. coli, and a selection marker) as necessary. . The appropriate length of the expression vector varies depending on the type of plasmid, virus, etc. used. Therefore, it is appropriate to adjust the length of the expression cassette for RNAi (particularly region I and region R) while taking this into consideration. .

  The expression vector may be a non-viral vector typified by a plasmid or a viral vector, and may be circular or non-circular (linear) such as an adenoviral genome (double-stranded DNA). ). An expression vector suitable for the expression vector may be prepared in consideration of the use of the expression vector.

  The details of the basic configuration and production method, the introduction method into the host cell, the expression method, etc. relating to the vector used in the present invention can be said to be a well-known and commonly used technical matter for those skilled in the art. Such technical matters can be applied mutatis mutandis after appropriately modifying the characteristic portions.

[Region I]
Region I, that is, a sequence encoding an RNAi-related transcript, can be designed by a known technique for miRNA and siRNA, depending on the target gene whose expression is to be suppressed (knocked down). Generally, in region I, i) a base sequence homologous to at least a part of the base sequence of the target gene (sense sequence), ii) the sense sequence and the antisense sequence described below are connected, and cleaved by Dicer. A hairpin-like structure comprising a base sequence forming a loop structure (loop sequence), and iii) a base sequence complementary to the sense sequence (antisense sequence) is included. Only one such structure may be included in the region I, or a plurality of such structures may be included as seen in pri-miRNA.

[Region R]
The region R includes a site-specific recombination enzyme recognition sequence (r) arranged at two positions in the same direction, or a transposase recognition sequence (t) arranged at two positions in the reverse direction, and the recognition sequence (r ) Or (t) and consists of an arbitrary sequence (n).

  When shortening the length of the expression cassette for RNAi according to the first embodiment (first regulation method) of the method for regulating RNAi effect in the present invention (in the absence of the region R replacement vector described below) Region R is excised from the expression cassette by the action of an appropriate site-specific recombinase or transposase.

  In the second embodiment (second regulation method) of the method for regulating RNAi effect in the present invention, when the length of the expression cassette for RNAi is extended or shortened, an appropriate site-specificity is present in the presence of the region R replacement vector. Region R 'provided in the vector for replacement of region R (longer than region R in the case of extension, shorter than region R in the case of shortening) Replaced.

  Note that the region R ′ included in the region R replacement vector used in the second regulatory method can be configured in the same manner as the region R of the expression vector for RNAi, and therefore the site-specific recombination enzyme recognition sequence and arbitrary sequence will be described later. The matters to be applied can be applied not only to the expression vector for RNAi but also to the region R replacement vector.

  In the present invention, the site-specific recombination enzyme recognition sequence and the transposase recognition sequence may be used not only for sandwiching an arbitrary sequence in the region R but also for sandwiching the entire RNAi expression cassette. Therefore, the matters described below for the site-specific recombination enzyme recognition sequence and the transposase recognition sequence as recognition sites (r) and (t) for sandwiching an arbitrary sequence in the region R are the recognition sites for sandwiching the entire expression cassette ( rr) and (tt) can be applied mutatis mutandis.

  However, the site-specific recombinant enzyme recognition sequence (r) or transposase recognition sequence (t) used to sandwich an arbitrary sequence in region R and the site-specific recombinant enzyme used to sandwich the entire RNAi expression cassette The recognition sequence (rr) or transposase recognition sequence (tt) corresponds to a different site-specific recombination enzyme or transposase in order to prevent unintentional recombination and the like and achieve a desired purpose. It is appropriate to use the same site-specific recombinase or transposase that do not act simultaneously.

(R) Site-specific recombinase recognition sequence In the first control method, when a site-specific recombinase was allowed to act, it was sandwiched between site-specific recombinase recognition sequences placed in two locations in the same direction. A fragment containing one of the arbitrary sequence (n) and the recognition sequence is excised (in this case, the excised fragment is ligated in a loop and separated from the vector), and the other remaining of the recognition sequence is excised. The fragment pieces are packed and reconnected to the vector. At this time, the length of the RNAi expression cassette is usually shortened, but in a special manner as will be described later, the length of the RNAi expression cassette is extended.

  When the site-specific recombination enzyme recognition sequence is placed in the reverse direction, when the site-specific recombination enzyme is allowed to act, an inversion of any sequence sandwiched between them occurs (after being cleaved once, However, the length of the expression cassette for RNAi is not shortened in the first regulation method.

  In the second regulation method, when an appropriate site-specific recombination enzyme is allowed to act in the presence of the region R replacement vector, a combination is established between the region R of the RNAi expression vector and the region R ′ of the region R replacement vector. Since replacement occurs, the length of the expression cassette for RNAi can be extended or shortened depending on the length of these regions.

  A system comprising a combination of a site-specific recombination enzyme and its recognition sequence may be any one that functions in a host cell, and is not particularly limited. For example, in mammalian cells, Dre-rox system, Cre-loxP system, FLP-FRT system, φC31 integrase system (by attP, attB), etc. can be used. Recognition sequences can be placed at both ends of region R.

  Here, the recognition sequences of the site-specific recombinase (R) usually have variations (normal type and several variants), but if the same site-specific recombinase can be recognized, one Multiple types of recognition sequences can be used in one site-specific recombination system.

For the Cre-loxP system, as a recognition sequence for site-specific recombinase (Cre), sequences such as mutant JT15 / JTZ17 and lox2272 can be used in addition to normal loxP sequences. JT15 and JTZ17 are “close variants” and can recombine between them. As site-specific recombinase, in addition to normal Cre, improved iCre can also be used.
Regarding the FLP-FRT system, as a recognition sequence for site-specific recombinase (FLP), in addition to a normal FRT sequence, sequences such as mutant F14 and F15 can be used.

(T) Transposase recognition sequence A transposase recognition sequence is an inverted repeat sequence located at both ends of a transposon found in various eukaryotic cells. The transposase recognizes this sequence and identifies the transposon as a specific sequence. Move to another part that you have. Therefore, the region R composed of the transposase recognition sequence and any sequence sandwiched between the transposase recognition sequence can be excised from the expression cassette (moved to another site) by the action of the transposase.

  The transposase recognition sequence is not particularly limited as long as it functions in the host cell. For example, in mammalian cells, transposon recognition sequences such as piggyBac, Sleeping Beauty (SB), and Tol2 can be used.

(N) Arbitrary sequence The “arbitrary sequence” as used herein refers to other elements contained in the expression cassette, that is, region I, site-specific recombinase, unless otherwise specified (for example, except in the specific embodiments described below). It is a sequence different from the recognition sequence (t), transposase recognition sequence (r), promoter (P), and polyA addition signal (a sequence not including them).

  Arbitrary sequences are not particularly limited, but fluctuations in the RNAi effect are affected by the length of the arbitrary sequences attached to and detached from the expression cassette. Therefore, select an arbitrary sequence having such a length that a desired change appears. Is appropriate.

  An example of the arbitrary sequence includes a base sequence encoding a “reporter gene” or “drug resistance gene”, which is an indicator that a vector has been successfully introduced and an RNAi-related transcript is expressed. The arbitrary sequence may include either one of the reporter gene and the drug resistance gene or both.

  These genes may be used as so-called marker genes for selecting cells that stably express RNAi-related transcripts, for example, but after completing the process of selecting such cells, Since the gene is unnecessary and it does not matter if the gene encoding it is deleted from the expression cassette, it can be used as an arbitrary sequence included in the region R.

  Examples of the reporter gene and drug resistance gene include: a) a gene encoding a fluorescent protein (GFP, eGFP, tdTomoato, etc.) that emits specific fluorescence when irradiated with excitation light, and b) a coloring substance from a specific substrate. Genes encoding the enzymes to be produced (firefly, renilla, luciferase such as Gaussia), β-galactosidase, alkaline phosphatase, chloramphenicol acetyltransferase (CAT), etc. c) specific drug (host cell is eukaryotic) In the case of neomycin, hygromycin, puromycin, blasticidin, etc.).

  Alternatively, a base sequence that does not have a functional meaning such as the above-described marker gene and does not adversely affect cell survival or RNAi even if transcribed may be an arbitrary sequence included in the region R. Such an arbitrary sequence is different from a sequence that cannot be deleted from the expression cassette until the predetermined function is completed, such as a marker gene, and can be excised from the expression cassette at an arbitrary timing or conversely inserted into the expression cassette. Can be. For example, analysis is performed using an expression cassette that intentionally exhibits a weak knockdown effect, and then the region R is excised to enhance the knockdown effect, thereby functioning in a two-stage (or multistage) knockdown effect. An analysis can be considered.

[P: Promoter]
A promoter (P) that controls (starts) transcription of a region including both of these is arranged upstream (on the 5 ′ end side) of the regions I and R. If necessary, an enhancer for enhancing the activity of the promoter may be further arranged.

  An appropriate promoter may be selected according to the cell type (human, mouse, rat, etc.) and tissue into which the vector is introduced while considering the purpose of RNAi and the strength of the expected effect. For example, it may be a promoter for constitutive expression in a wide range of cultured cells, or a promoter for specific expression of tissue and time when administered in vivo. In the present invention, since a relatively long sequence including an arbitrary sequence is transcribed together with an RNAi-related transcription product (for example, pri-miRNA), a promoter (CMV, SV40, etc.) for RNA Polymerase II having such activity is used. Preferably (RNA Polymerase III promoters (U6, human H1, mouse H1, etc.) have a relatively short RNA strand transcription activity, but are not suitable for transcription of relatively long sequences as described above. Sometimes).

[A: polyA addition signal]
A polyA addition signal (polyadenylation signal) that controls (terminates) transcription of a region including both of these is disposed downstream (on the 3 ′ end side) of the regions I and R. When a gene is expressed in eukaryotic cells, a polyA addition signal is required in most cases, except when a special viral vector is used, to normally finish transcription of mRNA and polyadenylate the 3 ′ end. Because.

The polyA addition signal is not particularly limited, and an appropriate signal may be selected from known ones. Examples thereof include a SV40-derived polyA addition signal.
A CAG promoter expression cassette comprising a combination of a chicken β-actin gene promoter and cytomegalovirus IE enhancer and a rabbit β-globin gene polyA addition signal is known as a strong expression cassette. These promoter, enhancer and polyA Additional signals are also suitable in the vectors of the present invention.

  Except for the case of the special embodiment described below, the RNAi expression vector of the present invention is basically a promoter upstream of both the regions (I) and (R) in the initial state. (P) (when there are a plurality of such promoters on the vector, the one closest to regions (I) and (R)) and downstream of both regions (I) and (R) Other promoter and polyA addition in the region between the polyA addition signal (A) (when there are a plurality of such polyA addition signals on the vector, those closest to the regions (I) and (R)) Does not contain signal.

(One embodiment for extending the expression cassette)
As one embodiment of the expression vector for RNAi of the present invention, the promoter (P), region (I), region (R) and first polyA addition signal (A) are directed from the 5 ′ end to the 3 ′ end. In which the arbitrary sequence (n) contained in the region (R) contains the second polyA addition signal (A) identical to the first polyA addition signal. It is done. The reason why the first polyA addition signal is the same as the first polyA addition signal is to allow the knockdown effect to be accurately evaluated by maintaining the conditions.

  When such an RNAi expression vector is used, in the regulation step described later, the second polyA addition signal (A) is removed together with the deletion of the region (R), thereby removing the end of the expression cassette. By changing to the polyA addition signal (A), the base length of the expression cassette can be extended, whereby the knockdown efficiency of the target gene can be reduced as compared with that before the step.

  In this case, the region (R) substantially consists of only the second polyA addition signal (A) and the recognition sequence (r) or (t) sandwiching it, that is, the arbitrary sequence (n) of the region (R) is Substantially only the second polyA addition signal (A) may be present.

  Further, in consideration of how much the length of the expression cassette should be extended between the region (R) and the first polyA addition signal (downstream side), a second arbitrary sequence having an appropriate length ( n2) may be arranged.

(Expression vector production method)
The method for producing the expression vector is not particularly limited as long as it is a method capable of producing a vector having all the elements required in the present invention.

  For example, various vectors having a basic configuration consisting of a promoter, a polyA addition signal, a restriction enzyme site (multicloning site) and the like are commercially available and can be easily obtained. Can be produced. That is, a double-stranded oligonucleotide containing region I, region R and a restriction enzyme site (for example, restriction enzyme site- [region I]-[region R] -restriction enzyme site) is synthesized separately and corresponds to the restriction enzyme site. Using the restriction enzyme, the double-stranded oligonucleotide may be ligated between the promoter of the vector and the polyA addition signal.

  Furthermore, in addition to a promoter, a polyA addition signal, and a restriction enzyme site, an expression vector containing a region encoding a library-made RNAi-related transcription product in advance is also commercially available. When the expression vector of the present invention is produced using these, a double-stranded oligonucleotide containing a region R and a restriction enzyme site (restriction enzyme site- [region R] -restriction enzyme site) is separately synthesized and its restriction is performed. Using a restriction enzyme corresponding to the enzyme site, the double-stranded oligonucleotide is ligated upstream (between the promoter) or downstream (between the polyA addition signal) of the region encoding the RNAi-related transcription product of the vector. do it.

(RNAi kit)
In the kit for RNAi of the present invention, the region (I) encoding the RNAi-related transcription product and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same. The expression cassette for RNAi in multiple states with different lengths of the base sequences other than the above and the length of the entire expression cassette is different. It contains the vector for doing. Such a kit is suitable for carrying out the target gene knockdown method according to the present invention.

  The first embodiment of the kit for RNAi of the present invention can be constructed using the expression vector for RNAi of the present invention as described above. This kit is a completed expression vector for RNAi of the present invention, that is, an expression vector for RNAi comprising an expression cassette comprising region (I), region (R), promoter (P) and polyA addition signal (A). , One or more may be included. Alternatively, an RNAi expression vector can be completed by ligating a double-stranded oligonucleotide containing a region (I) and / or region (R) consisting of an arbitrary sequence prepared separately. One or more vectors may be included. By using such an expression vector for RNAi or a vector for producing the same, a plurality of states as described above can be generated.

  In the second embodiment of the kit for RNAi of the present invention, the region (I) encoding an RNAi-related transcription product and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same. However, a plurality of expression vectors for RNAi, in which the length of the entire expression cassette is different due to the difference in the length of the base sequences other than them, and thereby the knockdown efficiency of the target gene is different, or It is comprised by the vector for producing them. Such a kit for RNAi produces a plurality of states as described above without using the region (R) provided in the expression vector for RNAi of the present invention.

  Further, the first embodiment of the kit of the present invention is a site-specific recombinase corresponding to the recognition sequence (r) contained in the region (R) of the RNAi expression vector or a transposase corresponding to the recognition sequence (t). It is preferable that the vector for expression is also included. These expression vectors are used in the regulation step in the target gene knockdown method according to the present invention.

  The first embodiment of the kit of the present invention further includes a recognition sequence (r) that is the same as or close to the region (R) and an arbitrary sequence (n ′) having a length different from that of the region (R). A region R recombination vector comprising (R ′) or a vector for its production may be included. The region R recombination vector is used in the second embodiment of the regulation step in the target gene knockdown method according to the present invention.

  If the RNAi expression cassette is further sandwiched between the recognition sequences (rr) or (tt) as necessary, the kit of the present invention can be used to express site-specific recombinant enzymes corresponding to those recognition sequences or A vector for expression of transposase may be included. These expression vectors are used in the chromosome recombination step in the target gene knockdown method according to the present invention.

  In addition, the kit of the present invention includes various members used in carrying out the present invention, such as introduction reagents used in the introduction process into host cells, luminescent substrates and drugs corresponding to selection markers, and expression vectors. Protocols for packaging and envelope vectors, control vectors for measuring the knockdown efficiency of target genes, and methods of using the kit of the present invention (target gene knockdown method of the present invention) A manual describing the above may be included as necessary.

-Target gene knockdown method-
In the target gene knockdown method of the present invention, the region (I) encoding an RNAi-related transcript, and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same. However, the length of the entire expression cassette is different due to the difference in the length of the base sequences other than those, and thus the expression vector for RNAi in multiple states is used, in which the knockdown efficiency of the target gene is different. It is characterized by that.

  In the first embodiment of the knockdown method of the present invention as described above, the RNAi expression vector in a plurality of states is preferably the RNAi expression vector of the present invention as described above, preferably the first embodiment of the RNAi kit. Produced using form. Therefore, in the first embodiment, a “regulation step” for adjusting the length of the expression cassette included in the expression vector for RNAi is performed, and in one host cell, different states can be created before and after the step. . In order to carry out this preparation step, an “introduction step” for introducing the RNAi expression vector of the present invention into a host cell is performed in advance. In addition, if necessary, the RNAi expression cassette can be integrated into the host cell chromosome before the regulation step (integrated with the introduction step or between the introduction step and the regulation step) or after the regulation step. A “chromosomal recombination step” may be performed.

  According to the second embodiment of the knockdown method of the present invention, the expression vector for RNAi in a plurality of states is preferably used without using the expression vector for RNAi of the present invention, preferably using the second embodiment of the RNAi kit. It is created. Individual expression vectors for RNAi can be prepared by a known technique while making the lengths of the expression cassettes different from each other. In such a second embodiment, the “regulation step” as in the first embodiment is not performed, and the “introduction step” for introducing a desired plurality of RNAi expression vectors into respective host cells is performed. . Further, if necessary, a “chromosome recombination step” for integrating the RNAi expression cassette into the chromosome of the host cell may be carried out either integrated with the introduction step or after the introduction step.

[Introduction process]
The introduction step commonly performed in the first embodiment and the second embodiment of the knockdown method of the present invention includes, for example, a step of performing an operation for introducing an expression vector for RNAi into a host cell, and RNAi by this step. For example, a step for selecting cells that are stably expressed after the expression vector is introduced.

(Host cell)
The host cell is not particularly limited as long as it is a eukaryotic cell that can exert the RNAi effect. For example, humans and mammals other than humans (experimental animals such as mice and rats) can assume a wide range of uses related to RNAi, and are suitable as the application target of the present invention. Non-mammalian cells such as nematodes and Drosophila, or plant cells are also applicable as the application target of the present invention.

  The cell type is not particularly limited. For example, in the case of mammals, fertilized eggs, ES cells (embryonic stem cells), various stem cells including iPS cells, or non-dividing cells (somatic cells other than hepatocytes). Is mentioned. The cell may be in vitro (ex vivo) or in vivo.

  Here, an aspect in which a transgenic animal is produced in order not to make the RNAi effect by the expression vector locally transient, but to exert the RNAi effect over a long period of time or over generations in the whole body of one individual The expression vector may be introduced into a host cell.

  As a method for producing a transgenic animal, a known method can be used. For example, microinjection (microinjection) in which a plasmid is injected into a fertilized egg (in the male pronucleus) with a microcapillary, viral infection in which a fertilized egg is infected with a lentivirus or retrovirus, or electroporation of an ES cell. Examples include a method of introducing a plasmid.

(Expression vector introduction method)
The method for introducing the expression vector for RNAi is not particularly limited as long as it is a method suitable for introduction into a host cell (ie, a eukaryotic cell expressing a target gene). In order to exert the effect of continuously knocking down the target gene, a method that can be integrated into the chromosome of the host cell is preferable. An appropriate method can be employed depending on the use of the expression vector for RNAi.

  For example, a method using a viral vector, a cell fusion method, a microinjection (microinjection) method, an electroporation method, a transfection using a liposome or polyethyleneimine, and the like can be mentioned. Details of each method and specific protocols are known to those skilled in the art.

  Among these, adenovirus vectors, herpes simplex virus vectors, lentivirus vectors, (gamma) retrovirus vectors and the like are exemplified as virus vectors, each having different characteristics.

  Adenovirus vectors are highly anticipated for gene therapy because they have a high gene transfer capacity for a wide range of cell types, including non-dividing cells, and are capable of recovering high-titer viruses. Although it is one of the vectors, the expression of the gene (RNAi effect) is transient because it does not have the property of incorporating the gene into the chromosome. Herpes simplex virus vectors are mainly used for gene transfer into nerve cells because the virus infects nerve cells. However, since adenovirus vectors and herpes simplex virus vectors do not have the property of incorporating genes into chromosomes, gene expression (RNAi effect) is transient. Therefore, as described later, it is preferable to maintain the RNAi effect by incorporating an RNAi expression cassette into a chromosome using a transposon.

  On the other hand, (gamma) retroviral vectors are unsuitable for introducing genes into non-dividing cells, but can continuously exert RNAi effects on dividing cells by integrating the genes into chromosomes. Lentiviral vectors are based on human immunodeficiency viruses and simian immunodeficiency viruses belonging to retroviralization. Unlike (gamma) retroviral vectors, genes are integrated into chromosomes even for non-dividing cells. be able to. However, since ordinary lentiviral vectors have a high risk of incorporating a gene in the vicinity of an endogenous gene whose transcription is activated, an integrase deficiency that eliminates this risk instead of losing the property of incorporating the gene into the chromosome Lentiviral vectors are also used. When using an integrase-deficient lentiviral vector, it is preferable to use a transposon in combination with the adenoviral vector.

[Adjustment process]
In the regulation step performed only in the first embodiment of the knockdown method of the present invention, an operation for shortening or extending the length of the region R of the expression cassette for RNAi is performed. Specifically, depending on whether a site-specific recombination enzyme recognition sequence or a transposase recognition sequence is used in the region R of the RNAi expression cassette, a site-specific recombination enzyme expression vector, respectively. Alternatively, a transposase expression vector is introduced into the host cell.

  When the second regulation method described above is carried out, the region R replacement vector is introduced into the host cell together with the site-specific recombinant enzyme expression vector in the regulation step. These vectors may be different from each other, or may be a single vector (including both a site-specific recombinase expression cassette and a region R substitution sequence (region R ′)). There may be.

  A method for producing and introducing a vector used in the regulation step can be performed according to a known procedure. In particular, for the introduction method, the introduction method of the expression vector as described above in the introduction step can be applied mutatis mutandis.

[Chromosome recombination process]
In common with the first embodiment and the second embodiment of the knockdown method of the present invention, in order to integrate an RNAi expression cassette into the chromosome of a host cell when necessary depending on the purpose of analysis, the method of introducing an expression vector, etc. The “chromosomal recombination step” may be performed. When performing a chromosome recombination step, an RNAi vector in which an RNAi expression cassette is sandwiched between a site-specific recombination enzyme recognition site (rr) or a transposase recognition site (tt) is used in the introduction step, The corresponding site-specific recombinase or transposase is used in this step.

  When the chromosome recombination step is integrated with the introduction step, a site-specific recombination enzyme or transposase expression vector may be introduced into the host cell together with the RNAi expression vector of the present invention in the introduction step. When the chromosome recombination step is performed after the introduction step and before the regulation step, or after the regulation step, a site-specific recombination enzyme or transposase expression vector is introduced into the host cell at such a stage. What is necessary is just to introduce.

  By the action of a site-specific recombination enzyme or transposase expressed from such an expression vector, a predetermined region containing the RNAi expression cassette is integrated into the host cell chromosome.

  A method for producing and introducing a vector used in the chromosome recombination step can also be performed according to a known procedure. In particular, for the introduction method, the introduction method of the expression vector as described above in the introduction step can be applied mutatis mutandis.

[Evaluation process]
If the RNAi effect is changed before and after (presence / absence) of the regulation step in the RNAi effect regulation method of the present invention through the above-described technique, it can be evaluated that the effect of the present invention has been achieved. For example, after introducing a predetermined expression vector into a host cell, a sample that has undergone a regulatory process of shortening the RNAi expression cassette using a site-specific recombinant enzyme expression vector, etc., and such a regulatory process is not performed. It is expected that the former RNAi effect will be higher when compared with the control. In other words, various conditions according to the present invention can be set as appropriate so that an evaluation result can be obtained that the effects of the present invention are achieved when the above-described method is used.

  In order to evaluate the target gene knockdown effect by RNAi, various known techniques such as cell-based assays, enzyme-based assays, array analysis, and the like can be used. The above effects may be evaluated at the mRNA level of the target gene or may be evaluated at the protein level.

  The use of improving or reducing the knockdown effect of a target gene using the present invention and evaluating it is not particularly limited. For example, when analyzing hypomorphs at the individual level (such as reducing the gene expression level to see the effect on the phenotype), create individuals with different gene expression levels at various levels to see the effect on the expression level. Can be used for

(Comparison method of knockdown efficiency)
Each comprising the same region (I) encoding an RNAi-related transcript, and a promoter (P) and a polyA addition signal (A) for controlling the transcription of the region (I); Comparing the knockdown efficiencies of target genes by using multiple expression vectors for RNAi that differ in the length of the entire expression cassette due to the different lengths of the expression cassettes, or on the assumption that these knockdown efficiencies are different Or set up an experimental system.

  As described above, expression vectors having different lengths of the entire expression cassette may be prepared by excising or recombining the region R using an expression vector that also includes the region R in advance. It is also possible to prepare the base portion by making the lengths of the base sequences different from each other. That is, an expression vector comprising an expression cassette comprising region I, region R, promoter (P) and polyA addition signal (A), and an expression cassette comprising region I, promoter (P) and polyA addition signal (A) are provided. In combination with one or more expression vectors.

  In such a comparison experiment, the RNAi expression cassette may be in a state of being integrated into the chromosome of the host cell, or may be in a transient state without being integrated.

[Example 1] Comparison of knockdown effect between artificial miRNA expression constructs using eGFP-expressing ES cells (see Fig. 1)
Expression vectors (1) to (4) containing an artificial miRNA for the eGFP gene were prepared and introduced into eGFP-expressing ES cells. Two days after the introduction, eGFP expression intensity was compared by FACS analysis. As a result, the highest knockdown effect was obtained with the construct (2) containing no reporter gene.

  As an artificial miRNA for the eGFP gene, a sequence derived from 'BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP' (K4936-00, Invitrogen) is used. This uses the miR155 miRNA backbone, and this kit is used to insert an oligo, and then clone it back into its own expression vector. The miRNA sequence for eGFP was designed using BLOCK-iT RNAi Designer on the website, and two types are connected in tandem (using a total of four types of oligos). Its sequence is as follows (eGFP_123_top and bottom have been published in Miura et al. Anal Biochem 2011):

eGFP_123_top:
5′-TGCTGATGAACTTCAGGGTCAGCTTGGTTTTGGCCACTGACTGACCAAGCTGACTGAAGTTCAT-3 ′
eGFP_123_bottom:
5′-CCTGATGAACTTCAGTCAGCTTGGTCAGTCAGTGGCCAAAACCAAGCTGACCCTGAAGTTCATC-3 ′
eGFP_419_top:
5′-TGCTGTGTAGTTGTACTCCAGCTTGTGTTTTGGCCACTGACTGACACAAGCTGGTACAACTACA-3 ′
eGFP_419_bottom:
5′-CCTGTGTAGTTGTACCAGCTTGTGTCAGTCAGTGGCCAAAACACAAGCTGGAGTACAACTACAC-3 ′.

[Example 2] Comparison of knockdown effect between artificial miRNA expression constructs using wild-type ES cells
Expression vectors (1) to (5) containing an artificial miRNA for the eGFP gene and an eGFP expression vector were co-introduced into wild-type ES cells. Two days after introduction, eGFP expression intensity was compared by FACS analysis and a fluorescence microscope. As a result, the highest knockdown effect was obtained with the constructs (2) and (5) containing no reporter gene as in Example 1. (See Figure 2)

[Example 3] Comparison of knockdown effect by reporter gene removal (see FIG. 3)
From the results obtained in Examples 1 and 2, it is possible that (A) a difference due to a difference in transfection efficiency or (B) a possibility due to the length of a transcription unit as a possibility that a difference in knockdown effect occurs. Two were considered. In order to clarify the influence of (B), the following experiment was conducted.

  First, create an expression cassette that contains an artificial miRNA, a tdTomato reporter gene sandwiched between two rox sequences, and a polyA addition signal (pA) directly under the CAG promoter, and construct it so that it is sandwiched between piggyBac transposon sequences (back arrow). Designed and produced. Next, this vector was introduced into an eGFP-expressing ES cell together with a piggyBac transposase expression vector and a neomycin resistance gene expression transposon vector. After G418 selection, cell clones in which this knockdown construct was inserted on the genome were selected from the stably expressing cell lines obtained using tdTomato gene expression (with or without red fluorescence) as an index (1). Regarding the obtained cell clone, a Dre expression plasmid was introduced to obtain a cell from which the tdTomato gene part was removed (2). The site-specific recombination enzyme Dre recognizes the rox sequence and functions to catalyze the recombination between the two rox sequences. Finally, comparative analysis of eGFP expression intensity in each of the cells (1) and (2) was performed by FACS. As a result, it was confirmed that the cell (2) from which the tdTomato reporter gene was removed had a higher eGFP knockdown effect than the cell (1) from which the tdTomato reporter gene was not removed.

Claims (17)

Region encoding RNAi-related transcripts (I),
A recognition sequence (r) for site-specific recombinase placed in two places in the same direction, or a recognition sequence (t) for transposase placed in two places in the opposite direction, and sandwiched between these two recognition sequences A region (R) containing any arbitrary sequence (n), and a set of promoter (P) and polyA addition signal (A) for controlling transcription of the region containing both the regions (I) and (R)
An expression vector for RNAi, comprising an expression cassette comprising   The expression vector for RNAi according to claim 1, wherein the arbitrary sequence (n) is a sequence encoding a reporter gene and / or a drug resistance gene.   The promoter (P), region (I), region (R) and first polyA addition signal (A) are arranged in this order from the 5 ′ end to the 3 ′ end, and the region (R 3. The expression vector for RNAi according to claim 1, wherein the arbitrary sequence (n) contained in (1) contains a second polyA addition signal (A) that is the same as the first polyA addition signal.   The recognition cassette (rr) or the reverse direction of the site-specific recombinase in which the expression cassette is of a type different from the recognition sequence (r) or (t) of the region (R) and arranged in two locations in the same direction. The expression vector for RNAi according to any one of claims 1 to 3, which is sandwiched between transposase recognition sequences (tt) arranged in two places.   A region (I) encoding an RNAi-related transcription product provided in the expression vector for RNAi according to any one of claims 1 to 4, and a recognition sequence for site-specific recombinase arranged in two locations in the same direction (R) or a region (R) containing a recognition sequence (t) of transposase arranged at two positions in the reverse direction and an arbitrary sequence (n) sandwiched between the two sequences, and a restriction enzyme site A double-stranded oligonucleotide characterized by   The region (I) encoding the RNAi-related transcript and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same, but the length of the other base sequences is It includes different expression vectors for RNAi, or vectors for producing them, which differ in the length of the entire expression cassette due to differences in the knockdown efficiency of the target gene. Characteristic kit for RNAi.   The RNAi kit according to claim 6, comprising the RNAi expression vector according to any one of claims 1 to 4 or a vector for producing the RNAi expression vector as a vector for producing the plurality of RNAi expression vectors.   The kit for RNAi according to claim 7, further comprising a site-specific recombinant enzyme expression vector or transposase expression vector corresponding to the recognition sequence (r) or (t) contained in the region (R).   Furthermore, a region R recombination comprising a recognition sequence (r) having a mutation type identical or close to that of the region (R) and a region (R ′) comprising an arbitrary sequence (n ′) having a length different from that of the region (R) The RNAi kit according to claim 7 or 8, comprising a vector for use or a vector for producing the vector.   The RNAi kit according to any one of claims 7 to 9, further comprising a site-specific recombinant enzyme expression vector or a transposase expression vector corresponding to the recognition sequence (rr) or (tt).   The region (I) encoding the RNAi-related transcript and the promoter (P) and polyA addition signal (A) for controlling the transcription of the region (I) are the same, but the length of the other base sequences is Target gene knockdown, characterized by using multiple expression vectors for RNAi, which differ in the length of the entire expression cassette due to differences in the target gene knockdown efficiency Method. The expression vectors for RNAi in the plurality of states are
Region encoding RNAi-related transcripts (I),
A recognition sequence (r) for site-specific recombinase placed in two places in the same direction, or a recognition sequence for transposase (t) placed in two places in the opposite direction, and sandwiched between these two sequences A region (R) containing an arbitrary sequence (n), and a set of promoter (P) and polyA addition signal (A) for controlling transcription of the region containing both the regions (I) and (R)
Produced by an expression vector for RNAi equipped with an expression cassette containing
A step (regulation step) of introducing a site-specific recombination enzyme or transposase corresponding to a recognition sequence contained in the region (R) after introducing the RNAi expression vector into a cell and changing the base length of the expression cassette ) To change the knockdown efficiency of the target gene before and after the step.   The method according to claim 12, wherein the regulating step is a step of excising the region (R) by allowing the site-specific recombinase or transposase to act.   14. The method according to claim 13, wherein the deletion of the region (R) shortens the base length of the expression cassette, thereby improving the knockdown efficiency of the target gene compared to before the step. The promoter (P), the region (I), the region (R) and the first polyA addition signal (A) are arranged in this order from the 5 ′ end to the 3 ′ end,
The arbitrary sequence (n) contained in the region (R) contains a second polyA addition signal (A) identical to the first polyA addition signal;
By removing the second polyA addition signal (A) together with the deletion of the region (R), the terminal of the expression cassette is changed to the first polyA addition signal (A) by changing the end of the expression cassette. 14. The method of claim 13, wherein the length is increased, thereby reducing the knockdown efficiency of the target gene compared to before the step.   A vector comprising the regulatory step comprising a recognition sequence (r) that is the same as or close to that of the region (R) and a region (R ′) containing an arbitrary sequence (n ′) having a length different from that of the region (R) In the presence of the gene, the site-specific recombinase is allowed to act to recombine the arbitrary sequence (n) and the arbitrary sequence (n ′) contained in the region (R), and the base of the expression cassette The method according to claim 12, wherein the method is a step of extending or shortening the length, thereby reducing or improving the knockdown efficiency of the target gene, respectively, compared to before the step. As an expression vector for RNAi, the expression cassette is a recognition sequence (rr) of a site-specific recombination enzyme arranged in two places in the same direction or a recognition sequence (tt) of a transposase arranged in two places in the reverse direction. Use what is sandwiched,
17. The method of any one of claims 11 to 16, further comprising the step of integrating the expression cassette into a host cell chromosome by expressing a site-specific recombinase or transposase corresponding to the recognition sequence (rr) or (tt). The method according to claim 1, wherein when the region (R) contains a recognition sequence (r) or (t), the recognition sequence (rr) or (tt) Corresponding to the replacement enzyme or transposase).