WO2007032428A1 - NOVEL LOOP SEQUENCE EFFECTIVE FOR EXPRESSION OF shRNA - Google Patents

NOVEL LOOP SEQUENCE EFFECTIVE FOR EXPRESSION OF shRNA Download PDF

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WO2007032428A1
WO2007032428A1 PCT/JP2006/318247 JP2006318247W WO2007032428A1 WO 2007032428 A1 WO2007032428 A1 WO 2007032428A1 JP 2006318247 W JP2006318247 W JP 2006318247W WO 2007032428 A1 WO2007032428 A1 WO 2007032428A1
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sequence
loop
shrna
rna
seq
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PCT/JP2006/318247
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French (fr)
Japanese (ja)
Inventor
Junichi Mineno
Takashi Ueno
Sachiko Okamoto
Hideto Chono
Tatsuya Ando
Hiroyuki Izu
Ikunoshin Kato
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Takara Bio Inc.
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Priority to JP2007535531A priority Critical patent/JP5139067B2/en
Publication of WO2007032428A1 publication Critical patent/WO2007032428A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • the present invention relates to a method that makes it possible to improve the effect of gene suppression by RNA, and a series of techniques related thereto, which are useful in fields such as medicine, cell engineering, genetic engineering, and developmental engineering.
  • miRNA which plays a major role in expression control in many stages such as development, moves to the cytoplasm with a short hairpin structure (pre-miRNA) and is cleaved by Dicer to become mature miRNA. miRNAs are generally said to suppress expression at the translational stage by binding complementarily to the 3 'untranslated region.
  • Non-patent literature l Fire A, 5 others, Nature, 1998, Vol. 391, p.
  • Non-patent literature 2 Zamore PO, 3 others, Cell, 2000, Vol, 101, p. 25- 33
  • Non-Patent Document 3 Hammond SM, 3 others, Nature, 2000, Vol. 404, p. 293-3 96
  • the first invention of the present invention is an shRNA useful for gene suppression of a target gene.
  • the present invention relates to a loop sequence screening method comprising the following steps.
  • a second invention of the present invention relates to a nucleic acid containing a shRNA loop sequence useful for gene suppression obtained by the method of the first invention of the present invention.
  • the loop sequence may be a sequence that is selected as follows:
  • a third invention of the present invention relates to a vector for expressing an shRNA containing the nucleic acid of the second invention of the present invention.
  • the fourth invention of the present invention relates to a kit comprising the nucleic acid of the second invention of the present invention or the vector of the third invention of the present invention. The invention's effect
  • the shRNA expression vector containing the sequence of the present invention can provide a higher gene suppression effect than a loop sequence that has been frequently used in the past.
  • FIG. 1 is a diagram showing the screening method of the present invention and the effect of nucleic acid gene suppression.
  • FIG. 2 is a view showing the screening method of the present invention and the effect of nucleic acid gene suppression.
  • FIG. 5 is a view showing the screening method of the present invention and the effect of nucleic acid gene suppression.
  • stem loop hairpin loop structure
  • stem loop refers to a double-stranded portion (system) generated by hydrogen bonding between inverted repeats present on single-stranded RNA or DNA. ) And the partial force structure of the loop between them. That is, in the stem region, the base sequences of the two regions are complementary to each other and exist in opposite directions.
  • the hydrogenated inverted repeats may be fully complementary or partially complementary.
  • the structure of the loop portion may be a bulge inserted in a stem region that may have a stem region in part. Stem loop structure can be predicted and confirmed by the secondary structure prediction algorithm of nucleic acid.
  • the genome information before and after the low molecular RNA base sequence of the living organism is obtained. It is considered that the sequence of the loop region of the stem-loop structure is included in this low molecular RNA base sequence and the sequences before and after that.
  • the length of the low molecular RNA base sequence for selecting the loop sequence and the length of the sequence before and after the RNA sequence are not particularly limited. For example, 20 to 500 bases, 40 to 300 bases, 60 to 260 bases, 80 to 220 Bases, 110-200 bases are preferred.
  • the free energy is calculated by the Zuker method, and the region where the free energy is low, preferably the region where the free energy is the lowest, is selected.
  • a region of a certain length for example, a region of 110 bases
  • Energy is calculated, for example, a region having the lowest free energy is selected.
  • the lowest free energy is ⁇ 25.
  • Okcal / mol or less preferably one 30.
  • OkcalZmol or less more preferably one 85.0 to one 30. OkcalZmol or less.
  • shRNA comprising a selected loop sequence and a stem region containing a target gene to be suppressed or a partial sequence thereof and a complementary sequence in the reverse direction.
  • This shRNA may be chemically synthesized or may be generated by an RNA transcription system by preparing DNA that is transcribed by RNA polymerase.
  • a shRNA comprising a loop sequence obtained by the screening method of the present invention and a sequence having a part of a nucleic acid encoding the fluorescent protein as a stem region in a cell expressing the fluorescent protein.
  • the effect of the loop region on gene repression can be evaluated by quantifying the fluorescent protein in the cells.
  • An shRNA comprising a loop sequence selected by the screening method of the present invention can effectively suppress gene expression by the mechanism of siRNA or miRNA, and is useful for gene function analysis, gene therapy, and the like. .
  • Nucleic acids, vectors, and kits containing shRNA loop sequences useful for gene suppression are obtained by the screening method described in 1. above.
  • the length of the loop sequence of the shRNA of the present invention is 2 to 200 bases, preferably 4 to 50 bases, particularly preferably 8 to 20 bases. Examples of such a base sequence include loop sequences described in SEQ ID NOs: 12 to 20 in the sequence listing.
  • Examples thereof include those represented by sequences having at least one of deletion, addition, insertion or substitution, and useful for gene suppression.
  • sequences having at least one of deletion, addition, insertion or substitution of one or more bases in the sequence described in any one of SEQ ID NOs: 12 to 20 in the sequence listing for example, SEQ ID NO: 12 to 20 Nucleic acid sequences having 50% or more homology in any nucleotide, preferably nucleic acid sequences having 70% or more homology in the nucleotide, particularly preferably 90% or more homology in the nucleotide The nucleic acid sequence which has is illustrated.
  • a person skilled in the art can easily create and order an shRNA containing a loop sequence based on the nucleic acid sequence of the present invention, and introduce the prepared shRNA into a cell or the like expressing a target gene for easy gene suppression. Since the effect can be confirmed, a nucleic acid in which at least one of deletion, addition, insertion or substitution of one or more base groups has been performed based on the nucleic acid of the present invention should be construed as being included in the present invention.
  • the nucleic acid of the present invention includes a nucleic acid that can be hybridized under stringent conditions to the nucleic acid and is useful for gene suppression.
  • the stringent conditions are as follows: 1989, Cold 'Spring' Nova 1 Laboratories published, edited by J. Sambrook et al., Molecular Cloning: The Laboratory Manual 2nd Edition (Molecular Cloning: 7 conditions described in A Laboratory Manual 2nd ed., Etc. Specifically, for example, 0.5% SDS, 5 X Denharz solution, 0.01 % Incubate with probe in 6 X SSC containing denatured salmon sperm DNA at 65 ° C for 12-20 hours.
  • the nucleic acid hybridized to the probe can be detected after removing non-specifically bound probe by washing at 37 ° C in 0.1 X SSC containing 0.5% SDS, for example.
  • RNA in which a base sequence of a target gene or a part of the base sequence and a complementary sequence thereof are arranged in opposite directions with the nucleic acid of the present invention interposed therebetween effectively suppresses the target gene. Therefore, it is useful for gene suppression using dsRNA.
  • the length of the stem region is not particularly limited as long as it generates dsRNA that can be used in gene suppression, but for example, 10 to 200 bases, preferably 14 to 30 bases, and particularly preferably 19 to 24 bases It is.
  • the present invention includes a vector useful for gene suppression in which the nucleic acid of the present invention is inserted into an appropriate expression vector.
  • the vector of the present invention can be used as long as the shRNA containing the loop sequence of the present invention is transcribed, that is, a transcriptional regulator such as a promoter that functions in a gene-suppressing organism, 3, UTR, or 5 ′ UTR. Any vector with a transcription termination sequence, such as sequence, terminator, poly A signal, etc.
  • pSINsi—hU6 and other pSINsi vector series (Takara Bio), pBAsi vector series (Takara Bio), piGENE vector (iGENE), and pSIREN vector (Clontech)
  • a vector into which a loop sequence has been inserted is useful because it immediately becomes a shRNA expression vector when the target gene or a partial sequence thereof is incorporated.
  • the vector may be constructed so that the target gene or a part of the sequence becomes an inverted repeat so as to form the stem region of the shRNA and the loop sequence is sandwiched.
  • kits containing at least one nucleic acid containing the shRNA loop sequence of the present invention or the vector of the present invention is also included in the present invention.
  • the kit of the present invention may further contain a vector and a nucleic acid construct for transcription and expression of the target gene in these hosts, which may contain organisms, cells, tissues, and organs as hosts.
  • a transformation reagent for introducing the nucleic acid into the host may be included.
  • Example [0031] The ability of the present invention to be described more specifically with reference to the following examples The present invention is not limited to the following examples.
  • E. coli TOPlO dnvitrogen E. coli TOPlO dnvitrogen
  • LB medium tryptone 1%, yeast extract 0.5%, NaCl 0.5%, pH 7.0
  • the cells were aerobically cultured at 37 ° C. using LB-chloramphee-coal plates containing 10% agar.
  • RNA was electrophoresed on 15% TBE-urea gel (Invtrogen). After electrophoresis, 21 to 27 bases were excised and low molecular RNA was separated from the gel.
  • This synthetic oligo-linked low molecular weight RNA was subjected to T4 polynucleotide kinase (Takara Bio Inc.) to cause phosphate at the 5 ′ end. After that, the 5'-terminal 6 bases are RNA and the others are DNA! /
  • the synthetic DNA / RNA chimera oligo shown in SEQ ID NO: 2 in the sequence listing is T4 RNA Ligase (manufactured by Tacarano). Connected. Furthermore, electrophoresis was performed with 15% TBE-urea gel (manufactured by Invtrogen). After electrophoresis, the target product was removed from the gel.
  • the purified PCR product was digested with SfaNI (manufactured by NEB) to purify the DNA.
  • SfaNI manufactured by NEB
  • the DNA was electrophoresed on a 15% acrylamide gel, the band of the desired DNA fragment was cut out, and the DNA fragment was extracted from the gel.
  • PCR was performed using the above tag library as a cage. PCR uses 5'-methylated dCTP, dATP, dGTP, and dTTP as substrates, and the primer is the oligonucleotide shown in SEQ ID NO: 6 in the sequence listing, the FAM-labeled oligonucleotide shown in SEQ ID NO: 7 and the sequence number in the sequence listing
  • the reaction was carried out with Ex Taq Hot Start Version (manufactured by Takara Bio Inc.) using a mixture of 9: 1 biotinic oligonucleotides.
  • T4 DNA ligase was allowed to act on the washed microbeads to form a covalent bond between the target DNA fragment and the anti-tag. Then, bind to 600 g Dynabeads M-280 Streptavidin (Magnetic Streptavidin Beads, manufactured by Dynal) for 30 minutes at 25 ° C, and allow to stand for 1 minute on MPC (manufactured by Dynal), then remove the supernatant. did. 10ml Tris—HCl (pH8), ImM EDTA, 0.01% Tween 20 Resuspend in 1ml, let stand on MPC (Dynal), and then repeat the washing procedure to remove the supernatant. Only microbeads carrying the strand-tagged target DNA fragment were isolated.
  • the 4737 sequences obtained by the above MPSS analysis were subjected to a homology search in the UCSC genome database (http: ⁇ genome.ucsc.edu), and 906 sequences that matched the genome sequences were extracted.
  • Sanger micro RNA data base http://microrna.sanger.ac.uk/
  • NCBI Refseq data base http://www.ncbi.nlm.nih.gov/ReSeq/
  • the European nbosomal RNA ⁇ Tahe ' ⁇ http: / www.psb.ugent.be/rRNA/
  • homologous search in the Genomic tRNA database http://lowelab.ucsc.edu/GtRNAdb/
  • Example 4 Preparation of shRNA expression vector having various loop sequences
  • a shRNA expression vector for target sequence A (GGAGTTGTCCCAATTCTTG) (SEQ ID NO: 27) and target sequence B (GACACGTGCTGAAGTCAAG) (SEQ ID NO: 28) of green fluorescent protein rsGFP was prepared by the following procedure.
  • Stem loop shRNA having a loop sequence of SEQ ID NOs: 12 to 20 i.e., a sequence of SEQ ID NO: 27 or 28 and a complementary sequence in the opposite direction are linked to both ends of the loop sequence.
  • a synthetic oligo DNA for expressing the RNA was inserted downstream of the hU6 promoter of the expression vector pSINsi-hU 6 (manufactured by Takara Bio Inc.) according to the procedure described in the product instructions to construct a plasmid vector.
  • a vector expressing a loop of the sequence shown in SEQ ID NO: 21 (Brummelkamp et al. Science. 2002 296: 550-553.), which has been used in the loop structure of the shRNA expression vector so far, was prepared.
  • As a negative control a vector into which nothing was inserted was prepared.
  • the various vectors obtained were transformed into E. coli JM109, and the plasmid DNA was purified using QIAGEN Plasmid Midi Kit (manufactured by Kagen) and used as DNA for transfection.
  • HT1080 cells (ATCC CCL-121) and K562 cells (ATCC CCL-243) stably expressing the target gene rsGFP were prepared by the following procedure.
  • the rsGFP expression vector pQBI25 (Qbiogene) was digested with restriction enzymes Nhel and Notl to obtain a 775 bp GFP gene fragment.
  • pQBI ⁇ (manufactured by Qbiogene) was cleaved with restriction enzymes Nhel and Notl to remove the rsGFP-NeoR fusion gene.
  • the 775 bp rsGFP gene fragment obtained earlier was inserted, and the rsGFP gene under the control of the ⁇ promoter.
  • the vector pQBI polll (neo ⁇ ) expressing is obtained.
  • the ends were smoothed using a DNA blunting kit (Takara Bio).
  • Vector fragment obtained by digesting the retroviral vector plasmid pDON—AI (Takara Bio) with restriction enzymes Xhol and Sphl 4.
  • Use the DNA blunting kit (Takara Bio) to smooth the ends of 58 kbp. After soaking, dephosphorylation was performed using alkaline phosphatase (manufactured by Takara Bio Inc.).
  • HT1080 cells were seeded on a 6-well tissue culture plate (Iwaki Glass Co., Ltd.) at 5 X 10 4 per well, and 5% CO was present in DMEM medium containing 10% urinary fetal serum (FBS). 37
  • the cells were cultured at ° C for 24 hours.
  • the unphoto-mouth pick DOG virus solution was serially diluted and infected in the presence of 8 ⁇ g Zml of polyprene (hexadimethrine bromide; Sigma). Culturing for 3 days after infection, analyzing GFP expression with a flow cytometer (FACS Vantage, manufactured by Becton Dickinson), collecting GFP-positive cells with a sample power of 20% or less of introduction efficiency by sorting, culturing, and GFP Stable expressing cells HT1080-GFP.
  • polyprene hexadimethrine bromide
  • the unphoto mouth pick DOG- ⁇ virus solution was serially diluted and infected by a standard method using RetroNectin (registered trademark, manufactured by Takara Bio Co., Ltd.). Incubate for 3 days after infection, analyze GFP expression with a flow cytometer, collect GFP positive cells with sorting efficiency of 20% or less by sorting, culture, and GFP stable expression cells ⁇ 562-GFP did.
  • RetroNectin registered trademark, manufactured by Takara Bio Co., Ltd.
  • HT1080-GFP and K562-GFP prepared as described above were infected with the retroviral vector prepared in Example 5.
  • the virus vector was diluted 10-fold and 100-fold into HT1080-GFP and infected in the presence of polyprene 8 g / ml.
  • K562-GFP was infected by a standard method using retronectin after diluting the virus vector 10 times with the stock solution.
  • 24 hours after infection HT1080-GFP was replaced with a growth medium containing 500 ⁇ g / ml of G418 (dienetin; manufactured by Invitrogen), and K562-GFP was replaced with a growth medium containing 1000 g / ml of G418. Selection culture was performed for 2 weeks.
  • FIG. 1 shows the results for HT1080-GFP at target sequence A
  • Figure 2 shows the results for K562-GFP at target sequence A
  • the result of HT1080-GFP at the target sequence B is shown in FIG. 3
  • the result of K562-GFP at the target sequence B is shown in FIG.
  • the vertical axis shows the relative intensity of GFP fluorescence intensity when the negative control is 100 (Relative GFP me an (%)).
  • the numbers on the horizontal axis indicate the sequence numbers. As shown in the figure, it is compared with the norpe IJ shown in SEQ ID NO: 21 that compared to the ILE IJ numbers 12, 13, 14, 15, 16, 18, 19, 20 It was shown that the gene sequence has a high gene suppression effect.
  • An shRNA expression vector for human integrin ⁇ 4 target sequence (GAGTGTTTGTGTACATCAA) (SEQ ID NO: 29) was prepared by the following procedure. Regarding the target sequence SEQ ID NOs: 13 and 19 used in Example 4, and the stem loop sequence of microRNA-30, which has recently been used with high siRNA effect (Boden et al.
  • a plasmid vector having the loop of the target sequence A of rsGFP shown in SEQ ID NO: 27 shown in SEQ ID NO: 27 and the sequence shown in SEQ ID NO: 21 was used.
  • Escherichia coli JM109 was transformed with the various vectors obtained, and the plasmid DNA was purified using QIA GEN Plasmid Midi Kit (manufactured by Qiagen) and used as DNA for transfection.
  • the plasmid vector prepared in Example 7 was transferred to retrovirus preparation cells G3T-hi (Takara Bio) using Retorovirus Packaging Kit Ampho (Takara Bio) according to the product protocol, and various amphoteric mouths were used.
  • the supernatant of the pick virus was obtained, filtered through a 0.45 ⁇ m filter (Milex HV, manufactured by Millipore), and stored in an 80 ° C ultra-low temperature freezer until use. Each virus was prepared in 2 cases.
  • the retroviral vector prepared in Example 8 was serially diluted, and HT1080 cells (ATCC CCL-121) were infected in the presence of polybrene 8 ⁇ gZml. After 24 hours from the infection, G418 (Dieticin; manufactured by Invitrogen) was used. The culture medium was replaced with a growth medium containing 500 ⁇ g Zml, and selective culture was performed for 2 weeks. The retroviral vector titer was calculated from the number of colonies formed.
  • HL60 (ATCC CCL-240) cells were infected with the retroviral vector prepared in Example 8 and titered in Example 9 using MOI2 by a standard method using retronectin. After 24 hours from the infection, G418 was replaced with a growth medium containing 1000 / z gZml, and selective culture was performed for 2 weeks.
  • the amount of total RNA was corrected using the GAPDH gene amplification primers of SEQ ID NOs: 25 and 26.
  • the gene suppression effect was evaluated by calculating the ratio of the expression relative value in each experimental group to the integrin ex 4 expression relative value in the control experimental group.
  • the results are shown in FIG.
  • the vertical axis indicates the integrin ⁇ 4 expression level and shows the relative value when the negative control is set to 100 (Relative integrin ⁇ 4 mean (%)).
  • the numbers on the horizontal axis indicate sequence numbers.
  • FIG. 5 compared to the loop sequence shown in SEQ ID NO: 21 and the microRNA-30 loop sequence shown in SEQ ID NO: 22, the gene suppression effect of the loop sequence shown in SEQ ID NO: 13, 19 was shown to be high.
  • the order of the gene suppression effect of the loop sequences compared this time coincided with the results of the gene suppression effect on GFP performed in Examples 4, 5, and 6.
  • MiQ ID N ⁇ l: Synthetic chimera oligonucleotide, nucleotide ⁇ to are are nbonucle otide— other nucleotides are deoxyribonucleotides
  • SEQ ID NO: 2 Synthetic chimera oligonucleotide, "nucleotide 17 to 22 are ribonuc leotide— other nucleotides are deoxyribonucleotides
  • SEQ ID N ⁇ : 3 Synthetic primer for revearse transcription.
  • SEQ ID NO: 4 Synthetic primer.
  • SEQ ID NO: 5 Synthetic primer.
  • SEQ ID NO: 6 Synthetic primer.
  • SEQ ID N ⁇ : 7 Synthetic primer.
  • SEQ ID NO: 9 Synthetic oligonucleotide for adaptor DNA.
  • SEQ ID NO: 10 Synthetic oligonucleotide for adaptor DNA.
  • SEQ ID NO: l l Synthetic oligonucleotide for adaptor DNA.
  • SEQ ID NO: 12 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 13 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 15 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 16 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 17 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 20 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 21 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 22 Nucleotide sequence for loop region of shRNA.
  • SEQ ID NO: 23 Synthetic primer for amplification of integrin alpha 4 gene
  • SEQ ID NO: 24 Synthetic primer for amplification of integrin alpha 4 gene
  • SEQ ID NO: 26 Synthetic primer for amplification of GAPDH gene
  • SEQ ID NO: 27 Green fluorescence protein rsGFP target sequence A
  • SEQ ID NO: 28 Green fluorescence protein rsGFP target sequence B
  • SEQ ID NO: 29 Human integrin alpha 4 target sequence

Abstract

Disclosed is a loop sequence having high gene-suppressive effect by utilizing shRNA. Short RNA expressed in a human cell is purified and screened by a comprehensive gene expression analysis technique (MPSS) using microbeads, and the sequence of the RNA is analysed. Based on the sequence information given by the analysis, a database analysis is made. Among the sequences showing 100% matching to the human genome, a sequence which does not hit known miRNA, rRNA or tRNA is extracted. Based on the prediction of secondary sequence, a loop sequence is obtained.

Description

明 細 書  Specification
shRNA発現のための有効な新規ループ配列  A novel novel loop sequence for shRNA expression
技術分野  Technical field
[0001] 本発明は、医学、細胞工学、遺伝子工学、発生工学などの分野において有用な、 RNAによる遺伝子抑制の効果を向上させることを可能にする方法、およびそれに関 連する一連の技術に関する。  [0001] The present invention relates to a method that makes it possible to improve the effect of gene suppression by RNA, and a series of techniques related thereto, which are useful in fields such as medicine, cell engineering, genetic engineering, and developmental engineering.
背景技術  Background art
[0002] 近年、小さな RNAによる遺伝子発現抑制が次々と発見されている。 siRNA (short interfering RNA)と呼ばれる 21mer〜23merの二本鎖 RNAは配列特異的に 遺伝子発現を抑制する。 1998年に Fireら (非特許文献 1)が線虫にて二本鎖 RNA が配列特異的に遺伝子のサイレングを引き起こすことを発見して以来、 21〜23mer にプロセッシングされた RNAが mRNAを切断する機構 (非特許文献 2)や RISC (R NA- induced silencing complex)の存在（非特許文献 3)、 Dicerのクローニン グ (非特許文献 4)を経て、 2001年に Elbashirら (非特許文献 5)により哺乳類細胞で も siRNAによる配列特異的な発現抑制が可能であることが証明され、 siRNAを用い た研究は一気に盛んになった。  [0002] In recent years, gene expression suppression by small RNAs has been discovered one after another. A 21-mer to 23-mer double-stranded RNA called siRNA (short interfering RNA) suppresses gene expression in a sequence-specific manner. Since 1998, Fire et al. (Non-Patent Document 1) discovered that double-stranded RNA causes sequence silencing in C. elegans, and RNA processed by 21-23mer cleaves mRNA. After the mechanism (Non-Patent Document 2), the existence of RISC (RNA-induced silencing complex) (Non-Patent Document 3), Dicer Cloning (Non-Patent Document 4), Elbashir et al. (2001) As a result, it was proved that siRNA can suppress sequence-specific expression in mammalian cells, and research using siRNA became very popular.
一方、発生など多くのステージでの発現制御に大きな役割を果たして 、る miRNA (microRNA)は、 short hairpin構造（pre— miRNA)で細胞質に移行し、 Dicer により切断され成熟 miRNAになる。 miRNAは、一般に 3'非翻訳領域に相補的に 結合することにより、発現を翻訳段階で抑制するといわれている。  On the other hand, miRNA (microRNA), which plays a major role in expression control in many stages such as development, moves to the cytoplasm with a short hairpin structure (pre-miRNA) and is cleaved by Dicer to become mature miRNA. miRNAs are generally said to suppress expression at the translational stage by binding complementarily to the 3 'untranslated region.
[0003] 哺乳類細胞にお!、て、 siRNAや miRNAを人為的に細胞内で発現させる場合、発 現ベクターから shRNA (short hairpin RNA)を発現させることが一般的である。 このとき、センス配列とアンチセンス配列の間のループ配列力 細胞質への移行や、 Dicerによる認識に重要であると言われており、最近の報告では shRNAのループ配 列に miRNA由来の配列がよく使用されている。しかしながら、 shRNAにおけるルー プ配列の重要性を示唆する報告があるにも拘らず、効果的なループ配列の検索は Miyagishiらの研究（非特許文献 6)を除けばほとんど行われて ヽな 、。 [0004] 非特許文献 l :Fire A、他 5名、 Nature、 1998年、 Vol. 391、 p. 806— 811 非特許文献 2 :Zamore PO、他 3名、 Cell, 2000年、 Vol, 101、 p. 25- 33 非特許文献 3 : Hammond SM、他 3名、 Nature, 2000年、 Vol. 404、 p. 293- 2 96 [0003] When siRNA or miRNA is artificially expressed in a mammalian cell, it is common to express shRNA (short hairpin RNA) from the expression vector. At this time, it is said that the loop arrangement force between the sense sequence and the antisense sequence is important for cytoplasmic transition and recognition by Dicer. In recent reports, miRNA-derived sequences are often used in the shRNA loop sequence. in use. However, despite the reports suggesting the importance of the loop sequence in shRNA, effective loop sequence search is almost done except for the research by Miyagishi et al. (Non-Patent Document 6). [0004] Non-patent literature l: Fire A, 5 others, Nature, 1998, Vol. 391, p. 806-811 Non-patent literature 2: Zamore PO, 3 others, Cell, 2000, Vol, 101, p. 25- 33 Non-Patent Document 3: Hammond SM, 3 others, Nature, 2000, Vol. 404, p. 293-3 96
非特許文献 4: Bernstein E、他 3名、 Nature, 2001年、 Vol. 409、 p. 363- 366 非特許文献 5 :Elbashir SM、他 5名、 Nature, 2001年、 Vol. 411、 p. 494-49 8  Non-patent literature 4: Bernstein E, 3 others, Nature, 2001, Vol. 409, p. 363-366 Non-patent literature 5: Elbashir SM, 5 others, Nature, 2001, Vol. 411, p. 494 -49 8
非特許文献 6 : Miyagishi M、他 4名、 Gene Med.、 2004年、 Vol. 6、 p. 715 - 723  Non-Patent Document 6: Miyagishi M and 4 others, Gene Med., 2004, Vol. 6, p. 715-723
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0005] 本発明の目的は、上記従来技術を鑑みて行われたものであり、 shRNAを利用した 遺伝子抑制における効果の高いループ (loop)配列を提供することにある。本発明の ループ配列を用いて、より強い抑制効果が得られれば、例えば遺伝子の機能解析実 験などに大きな影響を及ぼすと考えられる。また、 shRNAを利用した遺伝子抑制を 利用した治療においては、その治療効果を左右する可能性がある。 [0005] An object of the present invention has been made in view of the above-described conventional technology, and is to provide a loop sequence that is highly effective in gene suppression using shRNA. If a stronger inhibitory effect can be obtained using the loop sequence of the present invention, it is considered to have a great influence on, for example, gene functional analysis experiments. In addition, in the treatment using gene suppression using shRNA, the therapeutic effect may be affected.
課題を解決するための手段  Means for solving the problem
[0006] 本発明者らは鋭意研究の結果、次のような網羅的なループ配列検索を実施し、効 果的なループ配列を見出した。これらの新規ループ配列は、従来使用されている配 列と比べて高い遺伝子抑制効果を示すことを確認し、発明を完成した。すなわち、ヒ ト細胞で発現して 、る短 、RNAを精製し、マイクロビーズを用いた網羅的遺伝子発 現解析技術 (MPSS)を利用してスクリーニングし、その配列を解析した。また、配列 情報を元にデータベースでの解析を行い、ヒトゲノムに 100%マッチする配列の中か ら、既知の miRNA、 rRNA、 tRNAにヒットしない配列を抽出し、二次構造予測から 新規 microRNAの候補を得た。得られた新規 microRNA候補より無作為に 9種類 の配列を選択し、ステムループ配列よりループ配列を得た。こうして選択したループ 配列を含む shRNA発現ベクターを構築し、本発明を完成させた。  [0006] As a result of intensive studies, the present inventors conducted the following comprehensive loop sequence search and found an effective loop sequence. These novel loop sequences were confirmed to show a higher gene suppression effect than the conventionally used sequences, and the invention was completed. That is, RNA expressed in human cells was purified, RNA was purified, screened using a comprehensive gene expression analysis technique (MPSS) using microbeads, and the sequence was analyzed. In addition, database analysis is performed based on sequence information, and sequences that do not hit known miRNAs, rRNAs, or tRNAs are extracted from sequences that match 100% of the human genome, and new microRNA candidates are predicted from secondary structure prediction. Got. Nine types of sequences were randomly selected from the obtained novel microRNA candidates, and a loop sequence was obtained from the stem loop sequence. An shRNA expression vector containing the loop sequence thus selected was constructed, and the present invention was completed.
[0007] すなわち、本発明の第 1の発明は、標的遺伝子の遺伝子抑制に有用な shRNAの ループ配列のスクリーニング方法であって、以下の工程を包含する方法に関する。[0007] That is, the first invention of the present invention is an shRNA useful for gene suppression of a target gene. The present invention relates to a loop sequence screening method comprising the following steps.
(1)生体試料力も低分子の RNAを抽出する工程、 (1) A process for extracting RNA with low molecular weight,
(2) (1)で得られた低分子 RNAの塩基配列を決定する工程、  (2) determining the base sequence of the low molecular weight RNA obtained in (1),
(3)前記生体のゲノム情報を基に、 (2)で得られた塩基配列の前後のゲノム配列を 取得し、 (2)で得られた塩基配列およびその前後のゲノム配列力 なる一連の配列 から、以下の (A)かつ (B)に該当する配列を選択する工程、  (3) Based on the genome information of the living body, the genome sequence before and after the base sequence obtained in (2) is obtained, and the base sequence obtained in (2) and a series of sequences comprising the genome sequence power before and after the base sequence. A step of selecting a sequence corresponding to the following (A) and (B):
(A)—定の長さ当たりの自由エネルギーが最も低い領域の配列であって、ステムル ープ構造を形成する配列、  (A) —the arrangement of the region with the lowest free energy per fixed length, which forms a stem-loop structure,
(B) (2)で得られた塩基配列がステムループ構造のステム領域に存在する配列、 (B) a sequence in which the base sequence obtained in (2) is present in the stem region of the stem-loop structure,
(4) (3)で得られたゲノム配列から、ループ配列を決定し、当該ループ配列の両端に 標的遺伝子配列およびその逆方向の相補配列力 Sステム領域を形成するように連結さ れた shRNAを構築する工程、 (4) Determine the loop sequence from the genomic sequence obtained in (3), and connect the target gene sequence and the complementary sequence in the opposite direction at both ends of the loop sequence. Building process,
(5) (4)で得られた shRNAを用いて標的遺伝子の遺伝子抑制効果を確認する工程  (5) The process of confirming the gene suppression effect of the target gene using the shRNA obtained in (4)
[0008] 本発明の第 2の発明は、本発明の第 1の発明の方法により得られる、遺伝子抑制に 有用な shRNAのループ配列を含有する核酸に関する。 [0008] A second invention of the present invention relates to a nucleic acid containing a shRNA loop sequence useful for gene suppression obtained by the method of the first invention of the present invention.
[0009] 本発明の第 2の発明にお ヽて、ループ配列が以下力 選択される塩基配列力 選 択される配列であってもよ 、： [0009] In the second invention of the present invention, the loop sequence may be a sequence that is selected as follows:
(1)配列表の配列番号 12〜20のいずれ力 1つに記載の塩基配列；または  (1) the nucleotide sequence according to any one of SEQ ID NOS: 12 to 20 in the sequence listing; or
(2)配列表の配列番号 12〜20のいずれか 1つに記載の塩基配列において 1個以上 の塩基の欠失、付加、挿入もしくは置換の少なくとも 1つを有する塩基配列。  (2) A base sequence having at least one of deletion, addition, insertion or substitution of one or more bases in the base sequence described in any one of SEQ ID NOS: 12 to 20 in the sequence listing.
[0010] 本発明の第 3の発明は、本発明の第 2の発明の核酸を含有する shRNAを発現す るためのベクターに関する。  [0010] A third invention of the present invention relates to a vector for expressing an shRNA containing the nucleic acid of the second invention of the present invention.
[0011] 本発明の第 4の発明は、本発明の第 2の発明の核酸、もしくは本発明の第 3の発明 のベクターを含むキットに関する。 発明の効果  [0011] The fourth invention of the present invention relates to a kit comprising the nucleic acid of the second invention of the present invention or the vector of the third invention of the present invention. The invention's effect
[0012] 本発明の配列を含む shRNA発現ベクターは、従来良く使用されているループ配 列と比較して高い遺伝子抑制効果を提供することが可能となる。 図面の簡単な説明 [0012] The shRNA expression vector containing the sequence of the present invention can provide a higher gene suppression effect than a loop sequence that has been frequently used in the past. Brief Description of Drawings
[0013] [図 1]本発明のスクリーニング方法および核酸の遺伝子抑制の効果を示す図である。  FIG. 1 is a diagram showing the screening method of the present invention and the effect of nucleic acid gene suppression.
[図 2]本発明のスクリーニング方法および核酸の遺伝子抑制の効果を示す図である。  FIG. 2 is a view showing the screening method of the present invention and the effect of nucleic acid gene suppression.
[図 3]本発明のスクリーニング方法および核酸の遺伝子抑制の効果を示す図である。  FIG. 3 is a view showing the screening method of the present invention and the effect of nucleic acid gene suppression.
[図 4]本発明のスクリーニング方法および核酸の遺伝子抑制の効果を示す図である。  FIG. 4 is a view showing the screening method of the present invention and the effect of nucleic acid gene suppression.
[図 5]本発明のスクリーニング方法および核酸の遺伝子抑制の効果を示す図である。 発明を実施するための最良の形態  FIG. 5 is a view showing the screening method of the present invention and the effect of nucleic acid gene suppression. BEST MODE FOR CARRYING OUT THE INVENTION
[0014] 本明細書にぉ 、て「ステムループ（ヘアピンループ）構造」とは、一本鎖 RNA又は DNA上に存在する逆方向反復配列間で水素結合によって生じる二本鎖の部分 (ス テム）とそれに挟まれたループの部分力 なる構造を示す。つまりステム領域は、 2つ の領域の塩基配列が互いに相補的であり、かつ逆方向に存在している。水素結合す る逆方向反復配列は完全に相補的であっても良ぐ部分的に相補的であってもよい 。ループ部分の構造は一部にステム領域があっても良ぐステム領域にバルジが揷 入されていても良い。ステムループ構造は、核酸の二次構造予測アルゴリズムにより 予測、確認することができる。このアルゴリズムは、例えば、 Vienna RNA Packag e (Hof acker Iら、 Nucleic Acids Research, Vol. 31 (13) , p. 3429— 31 (20 03) )、 MFOLD (Zuker Mら、 Nucleic Acids Research, Vol. 31 (13) , p. 34 06 - 15 (2003) )力 S挙げられる。  As used herein, the term “stem loop (hairpin loop) structure” refers to a double-stranded portion (system) generated by hydrogen bonding between inverted repeats present on single-stranded RNA or DNA. ) And the partial force structure of the loop between them. That is, in the stem region, the base sequences of the two regions are complementary to each other and exist in opposite directions. The hydrogenated inverted repeats may be fully complementary or partially complementary. The structure of the loop portion may be a bulge inserted in a stem region that may have a stem region in part. Stem loop structure can be predicted and confirmed by the secondary structure prediction algorithm of nucleic acid. This algorithm is described in, for example, Vienna RNA Package (Hof acker I et al., Nucleic Acids Research, Vol. 31 (13), p. 3429-31 (20 03)), MFOLD (Zuker M et al., Nucleic Acids Research, Vol. 31 (13), p. 34 06-15 (2003)) Force S.
[0015] 本明細書にぉ 、て「shRNA」とは、ステムループ構造を有する RNAを示す。 shRN Aは一本鎖 RNAであり、一本鎖 RNA上に存在する逆方向反復配列がそれぞれァ ニーリングし、ステム領域と呼ばれる二本鎖 RNA (double stranded RNA: dsRN A)部分を形成する。前記逆方向反復配列に挟まれる部分をループ領域という。ルー プ領域の配列をループ配列と 、う。  As used herein, “shRNA” refers to RNA having a stem-loop structure. shRN A is a single-stranded RNA, and each inverted repeat sequence present on the single-stranded RNA is annealed to form a double-stranded RNA (dsRNA) portion called a stem region. The portion sandwiched between the inverted repeat sequences is called a loop region. The loop region array is called a loop array.
[0016] 本明細書において「遺伝子抑制に有用」とは、遺伝子の発現抑制を行う際に使用 することが可能であることを示し、特に遺伝子抑制を強く誘導すること、つまり遺伝子 の発現を強く抑制することをいう。遺伝子抑制に有用な shRNAのループ配列とは、 遺伝子抑制を行うときに利用可能である shRNAのループ配列であり、特に限定はさ れないが例えば、一般に shRNA発現ベクターのループ構造に利用されていた、配 列番号 21に示す配列のループ領域（Brummelkamp et al. Science. 2002 296 : 550— 553. )と比較して、遺伝子抑制の効果が高いループ配列が遺伝子抑 制に特に有用である。 [0016] In the present specification, "useful for gene suppression" indicates that it can be used for gene expression suppression, and particularly strongly induces gene suppression, that is, strongly increases gene expression. It means to suppress. The shRNA loop sequence useful for gene suppression is a shRNA loop sequence that can be used for gene suppression. For example, although not particularly limited, it was generally used for the loop structure of shRNA expression vectors. , Arrangement Compared with the loop region of the sequence shown in column number 21 (Brummelkamp et al. Science. 2002 296: 550-553.), A loop sequence having a higher gene suppression effect is particularly useful for gene suppression.
[0017] 本明細書において「自由エネルギー」とは、 Zuker法（Zuker Mら、 RNA bioche mistry and biotechnology, (Kluwer Academic Publishers; , p. l l— 43 ( 1999) )により与えられる値であり、 RNAの高次構造を予測するために使用される。  In this specification, “free energy” is a value given by the Zuker method (Zuker M et al., RNA biochemistry and biotechnology, (Kluwer Academic Publishers;, p. Ll—43 (1999)) Used to predict the higher order structure of
[0018] 1.遺伝子抑制に有用な shRNAのループ配列のスクリーニング方法  [0018] 1. Screening method of shRNA loop sequence useful for gene suppression
本発明の遺伝子抑制に有用な shRNAのループ配列のスクリーニング方法は以下 の工程を包含する。  The screening method of the loop sequence of shRNA useful for gene suppression of the present invention includes the following steps.
(1)生体試料力も低分子の RNAを抽出する工程、  (1) A process for extracting RNA with low molecular weight,
(2) (1)で得られた低分子 RNAの塩基配列を決定する工程、  (2) determining the base sequence of the low molecular weight RNA obtained in (1),
(3)前記生体のゲノム情報を基に、 (2)で得られた塩基配列の前後のゲノム配列を 取得し、 (2)で得られた塩基配列およびその前後のゲノム配列力 なる一連の配列 から、以下の (A)かつ (B)に該当する配列を選択する工程、  (3) Based on the genome information of the living body, the genome sequence before and after the base sequence obtained in (2) is obtained, and the base sequence obtained in (2) and a series of sequences comprising the genome sequence power before and after the base sequence. A step of selecting a sequence corresponding to the following (A) and (B):
(A)—定の長さ当たりの自由エネルギーが最も低い領域の配列であって、ステムル ープ構造を形成する配列、  (A) —the arrangement of the region with the lowest free energy per fixed length, which forms a stem-loop structure,
(B) (2)で得られた塩基配列がステムループ構造のステム領域に存在する配列、 (B) a sequence in which the base sequence obtained in (2) is present in the stem region of the stem-loop structure,
(4) (3)で得られたゲノム配列から、ループ配列を決定し、当該ループ配列の両端に 標的遺伝子配列およびその逆方向の相補配列力 Sステム領域を形成するように連結さ れた shRNAを構築する工程、 (4) Determine the loop sequence from the genomic sequence obtained in (3), and connect the target gene sequence and the complementary sequence in the opposite direction at both ends of the loop sequence. Building process,
(5) (4)で得られた shRNAを用いて標的遺伝子の遺伝子抑制効果を確認する工程  (5) The process of confirming the gene suppression effect of the target gene using the shRNA obtained in (4)
[0019] 本発明のスクリーニング方法において、生体試料は RNAを含有すると考えられる 試料であれば特に限定は無ぐ培養細胞、組織、器官、体液などいずれの由来であ つても良い。さらに、遺伝子抑制を行う生体由来の生体試料であることが好ましいが、 遺伝子抑制に有用であれば遺伝子抑制を行う生体以外の生物由来の生体試料であ つても良い。これらの生体試料より RNAを抽出する方法は、当業者に公知の方法を 使用すればよぐ生体試料に最適化されたキットが数多く市販されている。 [0020] 本発明のスクリーニング方法において、低分子の RNAは一般的な mRNA、 tRNA 、 rRNAよりも低分子量の RNAを意味し、 miRNA程度の長さの RNAが特に好適で ある。特に限定はされないが例えば、 10〜： LOO塩基、好ましくは 15〜50塩基、より好 ましくは 17〜30塩基、特に好ましくは 21〜27塩基の長さの RNAである。低分子の RNAを抽出する方法は、 RNAを分子量により分画できる方法であれば特に限定さ れないが、例えばゲル電気泳動法による切り出し、カラム分画法による分取、フィルタ 一によるろ過法などが挙げられる。 In the screening method of the present invention, the biological sample may be derived from any source such as cultured cells, tissues, organs, body fluids, and the like, as long as the sample is considered to contain RNA. Furthermore, a biological sample derived from a living body that performs gene suppression is preferable, but a biological sample derived from a living organism other than the living body that performs gene suppression may be used as long as it is useful for gene suppression. As a method for extracting RNA from these biological samples, there are many commercially available kits optimized for biological samples by using methods known to those skilled in the art. [0020] In the screening method of the present invention, low-molecular-weight RNA means RNA having a lower molecular weight than general mRNA, tRNA, and rRNA, and RNA having a length of about miRNA is particularly suitable. Although not particularly limited, for example, RNA having a length of 10 to: LOO base, preferably 15 to 50 bases, more preferably 17 to 30 bases, and particularly preferably 21 to 27 bases. The method for extracting low molecular weight RNA is not particularly limited as long as it can fractionate RNA according to molecular weight. For example, it is excised by gel electrophoresis, fractionated by column fractionation, or filtered by a single filter. Is mentioned.
[0021] 前記低分子 RNAの塩基配列を決定する方法は、一般の RNA配列決定方法を使 用することがきる。特に限定はされないが、例えば RNAより cDNAを合成し、チェ一 ンターミネーシヨン法、 MPSS法（日本特許公開第 2000— 515006号公報および Br enner S.他 23名 Nature Biotechnology 2000年 vol. 18、 p630— 634) 等により配列を決定すればよい。配列が決定された低分子 RNAの塩基配列は、由 来する生体のゲノムデータベースなどを用いてゲノム配列に一致する配列を選択す ることが好ましい。つまり、ゲノム塩基配列に一致しない低分子 RNA配列は、除外す ることが好ましい。さらに、 mRNA、 tRNA, rRNAである配列をデータベースと比較 して除外することが好ましい。前記検討を行うことによりノイズを除去することができる ため、後に行う選択の効率を向上させることができる。前記データベースは、特に限 定はされないが例えば、 UCSCゲノムデータベース、 NCBI Refseqデータベース、 the European ribosomal RNAデータベース、 the Genomic tRNAテータ ベースが利用できる。  [0021] As a method for determining the base sequence of the low molecular RNA, a general RNA sequencing method can be used. Although not particularly limited, for example, cDNA is synthesized from RNA, and the chain termination method, MPSS method (Japanese Patent Publication No. 2000-515006 and Brener S. et al., 23 others, Nature Biotechnology 2000 vol. 18, p630) — 634) The sequence may be determined according to the above. As the base sequence of the low molecular weight RNA whose sequence has been determined, it is preferable to select a sequence that matches the genomic sequence using the genome database of the living organism. That is, it is preferable to exclude low-molecular RNA sequences that do not match the genomic base sequence. Furthermore, it is preferable to exclude sequences that are mRNA, tRNA, and rRNA in comparison with the database. Since noise can be removed by performing the above-described examination, the efficiency of selection performed later can be improved. The database is not particularly limited, and for example, UCSC genome database, NCBI Refseq database, the European ribosomal RNA database, and the Genomic tRNA database can be used.
[0022] 次に、決定した低分子 RNA塩基配列を基に、由来する生体のゲノム情報力 低分 子 RNA塩基配列の前後のゲノム配列を取得する。この低分子 RNA塩基配列および その前後の配列にステムループ構造のループ領域の配列が含まれることが考えられ る。ループ配列を選択する低分子 RNA塩基配列およびその前後の配列の長さは、 特に限定 ίまされな ヽカ 列えば、、 20〜500塩基、 40〜300塩基、 60〜260塩基、 80 〜220塩基、 110〜200塩基が好適である。この低分子 RNA塩基配列を含む前後 のゲノム配列から、（A) Zuker法により自由エネルギーを計算し、自由エネルギーが 低くなる領域、好ましくは自由エネルギーが最も低くなる領域の配列を選択する。つ まり、前記低分子 RNA塩基配列を含む前後のゲノム配列の中で、一定の長さの領 域 (例えば 110塩基の領域)を、端より 1塩基ずつずらした領域につ!、てそれぞれ自 由エネルギーを算出し、例えば最も自由エネルギーが低い領域を選択する。最も低 い自由エネルギーは、 - 25. Okcal/mol以下、好ましくは一 30. OkcalZmol以下 、より好ましくは一 85. 0〜一 30. OkcalZmol以下であることが望ましい。さらに、最 も自由エネルギーが低い領域であって、ステムループ構造を形成すると予想される 配列を選択する。また、（B)低分子 RNAの塩基配列がステムループ構造のステム領 域に存在する配列を選択する。これらの選択は、例えば、 Vienna RNA Package の RNA fold, mf oldを利用することができる。すなわち、特定の配列と、その逆方 向の相補配列を含むゲノム配列はステムループ構造を形成すると予想され、前記 2 つの配列にはさまれた配列がループ配列である。 Next, based on the determined low molecular RNA base sequence, the genome information before and after the low molecular RNA base sequence of the living organism is obtained. It is considered that the sequence of the loop region of the stem-loop structure is included in this low molecular RNA base sequence and the sequences before and after that. The length of the low molecular RNA base sequence for selecting the loop sequence and the length of the sequence before and after the RNA sequence are not particularly limited. For example, 20 to 500 bases, 40 to 300 bases, 60 to 260 bases, 80 to 220 Bases, 110-200 bases are preferred. From the genomic sequences before and after this low-molecular RNA base sequence, (A) the free energy is calculated by the Zuker method, and the region where the free energy is low, preferably the region where the free energy is the lowest, is selected. One In other words, in the genomic sequences before and after the low-molecular RNA base sequence, a region of a certain length (for example, a region of 110 bases) is shifted by one base from the end! Energy is calculated, for example, a region having the lowest free energy is selected. The lowest free energy is −25. Okcal / mol or less, preferably one 30. OkcalZmol or less, more preferably one 85.0 to one 30. OkcalZmol or less. Furthermore, a sequence that is the region with the lowest free energy and is expected to form a stem-loop structure is selected. In addition, (B) a sequence in which the base sequence of low molecular RNA is present in the stem region of the stem loop structure is selected. For this selection, for example, RNA fold, mf old of Vienna RNA Package can be used. That is, a genomic sequence including a specific sequence and a complementary sequence in the opposite direction is expected to form a stem-loop structure, and a sequence sandwiched between the two sequences is a loop sequence.
[0023] 次に、ゲノム配列力 選択したループ配列と、遺伝子抑制を行う標的遺伝子または その一部の配列およびその逆方向の相補配列を含有するステム領域とからなる shR NAを構築する。この shRNAは化学的に合成してもよぐ RNAポリメラーゼにより転 写されるような DNAを作製し、 RNA転写システムにより生成してもよい。  [0023] Next, a shRNA comprising a selected loop sequence and a stem region containing a target gene to be suppressed or a partial sequence thereof and a complementary sequence in the reverse direction is constructed. This shRNA may be chemically synthesized or may be generated by an RNA transcription system by preparing DNA that is transcribed by RNA polymerase.
[0024] 前記で構築した shRNAの遺伝子抑制における効果を確認することにより、ループ 領域の遺伝子抑制に与える影響を評価することができる。強い遺伝子抑制を起こす s hRNAのループ配列は、遺伝子抑制に有用なループ配列である。遺伝子抑制の効 果を確認する方法は、特に限定はされないが例えば、 shRNAを直接細胞に導入す る方法、 shRNAが細胞内で転写される DNA構築物を細胞内に導入する方法があり 、細胞内の標的遺伝子の転写、翻訳を検出することにより確認することができる。遺 伝子抑制を確認するためのベクター、宿主、およびそれらのキットが数多く市販され ている。本発明のスクリーニング方法において、これらのキットを好適に使用すること ができる。特に限定はされないが、例えば、蛍光タンパク質を発現している細胞に、 本発明のスクリーニング方法により得られたループ配列と、蛍光タンパク質をコードす る核酸の一部をステム領域とする配列を含む shRNAを導入し、細胞の蛍光タンパク 質を定量することによりループ領域の遺伝子抑制に与える影響を評価することができ る。 [0025] 本発明のスクリーニング方法により選択されたループ配列を含む shRNAは、 siRN Aや miRNAの機構により、効果的に遺伝子発現を抑制することができ、遺伝子機能 解析、遺伝子治療などに有用である。 [0024] By confirming the effect of shRNA constructed as described above in gene repression, the effect of the loop region on gene repression can be evaluated. The loop sequence of sRNA that causes strong gene suppression is a useful loop sequence for gene suppression. The method for confirming the effect of gene suppression is not particularly limited. For example, there are a method of directly introducing shRNA into a cell, and a method of introducing a DNA construct into which shRNA is transcribed into the cell. This can be confirmed by detecting transcription and translation of the target gene. A number of vectors, hosts, and kits for confirming gene suppression are commercially available. These kits can be suitably used in the screening method of the present invention. Although not particularly limited, for example, a shRNA comprising a loop sequence obtained by the screening method of the present invention and a sequence having a part of a nucleic acid encoding the fluorescent protein as a stem region in a cell expressing the fluorescent protein. The effect of the loop region on gene repression can be evaluated by quantifying the fluorescent protein in the cells. [0025] An shRNA comprising a loop sequence selected by the screening method of the present invention can effectively suppress gene expression by the mechanism of siRNA or miRNA, and is useful for gene function analysis, gene therapy, and the like. .
[0026] 2.遺伝子抑制に有用な shRNAのループ配列を含有する核酸、ベクター、キット 本発明の遺伝子抑制に有用な shRNAのループ配列を含有する核酸は、前記 1. 記載のスクリーニング方法により得られたループ配列を含有する核酸である。本発明 の shRNAのループ配列の長さは、 2〜200塩基、好ましくは 4〜50塩基、特に好ま しくは 8〜20塩基である。このような塩基配列は例えば、配列表の配列番号 12〜20 に記載のループ配列が挙げられる。また、配列表の配列番号 12〜20のいずれかに 記載の配列において 1個以上、好ましくは 1個又は複数個、特に好ましくは 1個又は 数個、さらに好ましくは 1〜10個の塩基の欠失、付加、挿入もしくは置換の少なくとも 1つを有する配列で示され、かつ遺伝子抑制に有用なものが挙げられる。ここで、配 列表の配列番号 12〜20のいずれかに記載の配列において 1個以上の塩基の欠失 、付加、挿入もしくは置換の少なくとも 1つを有する配列としては、例えば配列番号 12 〜20のいずれかに記載のヌクレオチドに 50%以上のホモロジ一を有する核酸配列、 好ましくは前記のヌクレオチドに 70%以上のホモロジ一を有する核酸配列、特に好ま しくは前記のヌクレオチドに 90%以上のホモロジ一を有する核酸配列が例示される。 当業者は、本発明の核酸配列を基に、容易にループ配列を含有する shRNAを作製 、発注することができ、用意した shRNAを標的遺伝子を発現する細胞などに導入し て容易に遺伝子抑制の効果が確認できるため、本発明の核酸を基に 1個以上の塩 基の欠失、付加、挿入もしくは置換の少なくとも 1つを行った核酸も本発明に含まれる と解釈されるべきである。  [0026] 2. Nucleic acids, vectors, and kits containing shRNA loop sequences useful for gene suppression Nucleic acids containing shRNA loop sequences useful for gene suppression of the present invention are obtained by the screening method described in 1. above. A nucleic acid containing a loop sequence. The length of the loop sequence of the shRNA of the present invention is 2 to 200 bases, preferably 4 to 50 bases, particularly preferably 8 to 20 bases. Examples of such a base sequence include loop sequences described in SEQ ID NOs: 12 to 20 in the sequence listing. In addition, in the sequence described in any one of SEQ ID NOS: 12 to 20 in the sequence listing, the lack of one or more, preferably one or more, particularly preferably one or several, more preferably 1 to 10 bases. Examples thereof include those represented by sequences having at least one of deletion, addition, insertion or substitution, and useful for gene suppression. Here, as a sequence having at least one of deletion, addition, insertion or substitution of one or more bases in the sequence described in any one of SEQ ID NOs: 12 to 20 in the sequence listing, for example, SEQ ID NO: 12 to 20 Nucleic acid sequences having 50% or more homology in any nucleotide, preferably nucleic acid sequences having 70% or more homology in the nucleotide, particularly preferably 90% or more homology in the nucleotide The nucleic acid sequence which has is illustrated. A person skilled in the art can easily create and order an shRNA containing a loop sequence based on the nucleic acid sequence of the present invention, and introduce the prepared shRNA into a cell or the like expressing a target gene for easy gene suppression. Since the effect can be confirmed, a nucleic acid in which at least one of deletion, addition, insertion or substitution of one or more base groups has been performed based on the nucleic acid of the present invention should be construed as being included in the present invention.
[0027] さらに、本発明の核酸は、前記の核酸にストリンジェントな条件でハイブリダィズ可 能であり、遺伝子抑制に有用な核酸を包含する。前記のストリンジ ントな条件として は、 1989年、コールド 'スプリング'ノヽーバ一'ラボラトリー発行、 J.サムブルック (J. Sambrook)ら編集、モレキュラー ·クローユング：ァ ·ラボラトリー ·マニュアル第 2版（ Molecular Cloning ： A Laboratory Manual 2nd ed.リ等に記載され 7こ 条件が例示される。具体的には、例えば 0. 5% SDS、 5 Xデンハルツ溶液、 0. 01 % 変性サケ精子 DNAを含む 6 X SSC中、プローブとともに 65°Cにて 12〜20時間 インキュベートする条件が挙げられる。プローブにハイブリダィズした核酸は、例えば 0. 5% SDSを含む 0. 1 X SSC中、 37°Cで洗浄して非特異的に結合したプローブ を除去した後に検出することができる。 [0027] Furthermore, the nucleic acid of the present invention includes a nucleic acid that can be hybridized under stringent conditions to the nucleic acid and is useful for gene suppression. The stringent conditions are as follows: 1989, Cold 'Spring' Nova 1 Laboratories published, edited by J. Sambrook et al., Molecular Cloning: The Laboratory Manual 2nd Edition (Molecular Cloning: 7 conditions described in A Laboratory Manual 2nd ed., Etc. Specifically, for example, 0.5% SDS, 5 X Denharz solution, 0.01 % Incubate with probe in 6 X SSC containing denatured salmon sperm DNA at 65 ° C for 12-20 hours. The nucleic acid hybridized to the probe can be detected after removing non-specifically bound probe by washing at 37 ° C in 0.1 X SSC containing 0.5% SDS, for example.
[0028] 本発明の核酸を挟んで標的遺伝子の塩基配列またはその一部の塩基配列と、そ の相補的な配列が互いに逆方向に配置された RNAは、前記標的遺伝子を効果的 に抑制することができるため、 dsRNAを利用する遺伝子抑制に有用である。ステム 領域の長さは、遺伝子抑制において利用できる dsRNAを生成する長さであれば特 に限定はないが、例えば、 10〜200塩基、好ましくは 14〜30塩基、特に好ましくは 1 9〜24塩基である。 [0028] RNA in which a base sequence of a target gene or a part of the base sequence and a complementary sequence thereof are arranged in opposite directions with the nucleic acid of the present invention interposed therebetween effectively suppresses the target gene. Therefore, it is useful for gene suppression using dsRNA. The length of the stem region is not particularly limited as long as it generates dsRNA that can be used in gene suppression, but for example, 10 to 200 bases, preferably 14 to 30 bases, and particularly preferably 19 to 24 bases It is.
[0029] また、本発明の核酸を適切な発現ベクターに挿入した、遺伝子抑制に有用なベタ ターも本発明に含まれる。本発明のベクターは、本発明のループ配列を含有する sh RNAが転写されるものであれば、つまり、遺伝子抑制を行う生物で機能するプロモ 一ター、 3，UTR、 5 ' UTRなどの転写調節配列、ターミネータ一、ポリ Aシグナルなど 転写終結配列を有するベクターであれば何でもよ 、。例えば pSINsi— hU6などの p SINsiベクターシリーズ (タカラバイオ社製）、 pBAsiベクターシリーズ (タカラバイオ社 製）、 piGENEベクター（iGENE社製）、 pSIRENベクター（クローンテック）のプロモ 一ター下流に本発明のループ配列を挿入したベクターは、標的とする遺伝子または その一部の配列を組み込めば直ちに shRNA発現ベクターとなるため、有用である。 その場合、標的遺伝子またはその一部の配列は、 shRNAのステム領域を形成する ように逆方向反復配列となり、かつループ配列を挟むようにベクターを構築すればよ い。  [0029] Also, the present invention includes a vector useful for gene suppression in which the nucleic acid of the present invention is inserted into an appropriate expression vector. The vector of the present invention can be used as long as the shRNA containing the loop sequence of the present invention is transcribed, that is, a transcriptional regulator such as a promoter that functions in a gene-suppressing organism, 3, UTR, or 5 ′ UTR. Any vector with a transcription termination sequence, such as sequence, terminator, poly A signal, etc. For example, pSINsi—hU6 and other pSINsi vector series (Takara Bio), pBAsi vector series (Takara Bio), piGENE vector (iGENE), and pSIREN vector (Clontech) A vector into which a loop sequence has been inserted is useful because it immediately becomes a shRNA expression vector when the target gene or a partial sequence thereof is incorporated. In that case, the vector may be constructed so that the target gene or a part of the sequence becomes an inverted repeat so as to form the stem region of the shRNA and the loop sequence is sandwiched.
[0030] さらに、本発明の shRNAのループ配列を含有する核酸、または本発明のベクター を少なくとも 1つ含むキットも本発明に含まれる。本発明のキットは、さらに宿主となる 生物、細胞、組織、器官を含んでいてもよぐこれらの宿主で標的遺伝子が転写、発 現するためのベクター、核酸構築物を含んでいてもよい。また、宿主へ核酸を導入す るための形質転換用試薬を含んで 、てもよ 、。  [0030] Furthermore, a kit containing at least one nucleic acid containing the shRNA loop sequence of the present invention or the vector of the present invention is also included in the present invention. The kit of the present invention may further contain a vector and a nucleic acid construct for transcription and expression of the target gene in these hosts, which may contain organisms, cells, tissues, and organs as hosts. Also, a transformation reagent for introducing the nucleic acid into the host may be included.
実施例 [0031] 以下に実施例を挙げて本発明を更に具体的に説明する力 本発明は以下の実施 例のみに限定されるものではない。 Example [0031] The ability of the present invention to be described more specifically with reference to the following examples The present invention is not limited to the following examples.
また、本明細書に記載の操作のうち、基本的な操作については 2001年、コールド スプリング ハーバー ラボラトリー発行、 T.マニアテイス（T. Maniatis)ら編集、モ レキユラ一 クロー-ング：ァ ラボラトリー マ-ユアル第 3版（Molecular Cloning : A Laboratory Manual 3rd ed. )に記載の方法によった。  Among the operations described in this specification, the basic operations are published in 2001 by Cold Spring Harbor Laboratory, edited by T. Maniatis et al., Molecular Cloning: Laboratory Laboratory. According to the method described in the third edition (Molecular Cloning: A Laboratory Manual 3rd ed.).
[0032] さらに、以下に示す大腸菌を用いたプラスミドの構築には、特に記載のない限り大 腸菌 TOPlO dnvitrogen社製)を宿主として使用した。また、形質転換された大腸 菌は 30 μ gZmlのクロラムフエ-コールを含む LB培地（トリプトン 1%、酵母エキス 0. 5%, NaCl 0. 5%, pH7. 0)、あるいは上記培地に 1. 5%の寒天をカロえ固ィ匕 させた LB—クロラムフエ-コールプレートを用いて 37°Cで好気的に培養した。 [0032] Furthermore, in the following plasmid construction using E. coli, E. coli TOPlO dnvitrogen) was used as a host unless otherwise specified. In addition, transformed Escherichia coli can be added to LB medium (tryptone 1%, yeast extract 0.5%, NaCl 0.5%, pH 7.0) containing 30 μgZml of chloramfecol, or 1.5% in the above medium. The cells were aerobically cultured at 37 ° C. using LB-chloramphee-coal plates containing 10% agar.
[0033] 実施例 1 試料の調製 Example 1 Sample Preparation
(l)RNAの抽出  (l) RNA extraction
ヒト胎児腎臓由来 293TZ17細胞 (ATCC CRL— 11268)を、 10% ゥシ胎児血 清 (FBS ;ギブコネ土製)含有 DMEM培地中で 5%CO存在下、 37°Cで 7日間培養し  Human fetal kidney-derived 293TZ17 cells (ATCC CRL-11268) were cultured for 7 days at 37 ° C in the presence of 5% CO in DMEM medium containing 10% Ushi fetal serum (FBS; Gibconnet).
2  2
た。細胞を回収し、トリゾル試薬 (ギブコネ土製）により全 RNAを抽出した。  It was. Cells were collected, and total RNA was extracted with Trizol reagent (Gibcone).
[0034] (2)低分子 RNAの分離 [0034] (2) Isolation of small RNA
293TZ17細胞由来全 RNA lmgを Microcon 30 (ミリポア社製）にて低分子 R 293TZ17 cell-derived total RNA lmg with Microcon 30 (Millipore)
NAの濃縮を行った。濃縮した低分子 RNAを 15% TBE—ゥレアゲル（Invtrogen 社製）にて電気泳動を行った。電気泳動後、 21塩基から 27塩基付近を切り出し、低 分子 RNAをゲルより分離した。 Concentration of NA was performed. The concentrated low molecular weight RNA was electrophoresed on 15% TBE-urea gel (Invtrogen). After electrophoresis, 21 to 27 bases were excised and low molecular RNA was separated from the gel.
[0035] (3)低分子 RNA タグライブラリー作製 [0035] (3) Preparation of small RNA tag library
米国公開公報第 2004Z0002104号記載のタグベクター pMBSlを BamHIおよ び Bbsl (いずれも-ユー イングランド ノィォラブ (NEB)社製）により消化したあと、 ゥシ小腸アルカリホスファターゼ (CIAP、タカラバィォ社製）により脱リン酸化処理を 行った。  After digesting the tag vector pMBSl described in US Publication No. 2004Z0002104 with BamHI and Bbsl (both-Eu England Neolab (NEB)), dephosphorylation was carried out with urinary intestinal alkaline phosphatase (CIAP, Takarabio) Oxidation treatment was performed.
[0036] 293TZ17細胞由来、低分子 RNA 30ngに大腸菌 C75アルカリホスファタ一ゼ（ BAP C75、タカラバィォ社製)を作用させ、脱リン酸化を行った。続いて、この低分 子 RNAの 3'末端に 5'側の 6塩基分が RNA、その他が DNAとなっている配列表の 配列番号 1に示される合成 RNAZDNAキメラオリゴを T4 RNA Ligase (タカラバ ィォ社製)で連結した。その後 15% TBE -ゥレアゲル (Invtrogen社製)で電気泳 動を行った。電気泳動後、 目的産物の切り出しを行いゲルより分離した。 [0036] Dephosphorylation was performed by allowing E. coli C75 alkaline phosphatase (BAP C75, manufactured by Takara Bio Co., Ltd.) to act on 30 ng of low molecular weight RNA derived from 293TZ17 cells. Then this low minute Synthetic RNAZDNA chimera oligo shown in SEQ ID NO: 1 in the sequence listing in which 6 bases on the 5 'side are RNA and DNA in the others is linked to the 3' end of the child RNA with T4 RNA Ligase (Takara Bio) . Thereafter, electrophoretic exercise was performed with 15% TBE-Ureagel (Invtrogen). After electrophoresis, the target product was excised and separated from the gel.
この合成オリゴ連結低分子 RNAを T4 polynucleotide kinase (タカラバイオ 社製)を作用させることにより、 5'末端のリン酸ィ匕を行った。その後 5'末端に 3'側の 6 塩基分が RNA、その他が DNAとなって!/、る配列表の配列番号 2に示される合成 D NA/RNA キメラオリゴを T4 RNA Ligase (タカラノィォ社製)で連結した。さら に、 15% TBE—ゥレアゲル （Invtrogen社製）で電気泳動を行った。電気泳動後 、 目的産物のゲル抜きを行いゲルより分離した。  This synthetic oligo-linked low molecular weight RNA was subjected to T4 polynucleotide kinase (Takara Bio Inc.) to cause phosphate at the 5 ′ end. After that, the 5'-terminal 6 bases are RNA and the others are DNA! / The synthetic DNA / RNA chimera oligo shown in SEQ ID NO: 2 in the sequence listing is T4 RNA Ligase (manufactured by Tacarano). Connected. Furthermore, electrophoresis was performed with 15% TBE-urea gel (manufactured by Invtrogen). After electrophoresis, the target product was removed from the gel.
[0037] この両末端へ合成オリゴを連結させた低分子 RNAを铸型とし、配列表の配列番号 3で示されるプライマーを用いて dCTP、 dATP、 dGTP、 dTTPを基質として M— M LV RTase (タカラバイオ社製)で逆転写反応を行った。この DNAを铸型とし、配列 表の配列番号 4で示される FAM標識オリゴヌクレオチドと、配列表の配列番号 5で示 される FAM標識オリゴヌクレオチドをプライマーに用いて、 PCRを行った。 PCR反応 は、 Pyrobest DNA polymerase (タカラバイオ社製）を使用し、 5， 一メチル化一 d CTP、 dATP、 dGTP、 dTTPを基質として行った。この PCR産物をフエノール処理、 クロ口ホルム処理、エタノール沈殿によって DN Aを精製した。  [0037] This low molecular weight RNA having synthetic oligos linked to both ends is used as a saddle type, and M- M LV RTase (with the primers shown in SEQ ID NO: 3 in the sequence listing, using dCTP, dATP, dGTP, and dTTP as substrates. The reverse transcription reaction was performed with Takara Bio. PCR was performed using this DNA as a saddle and using the FAM-labeled oligonucleotide represented by SEQ ID NO: 4 in the Sequence Listing and the FAM-labeled oligonucleotide represented by SEQ ID NO: 5 in the Sequence Listing as primers. Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) was used for PCR reaction, and 5, monomethylated dCTP, dATP, dGTP, and dTTP were used as substrates. This PCR product was purified by DNA treatment, phenol form treatment, and ethanol precipitation.
[0038] 精製した PCR産物を SfaNI (NEB社製）にて消化を行 、、 DNAを精製した。この D NAについて 15% アクリルアミドゲルによる電気泳動を行い、 目的の DNA断片の バンドを切り出し、ゲルより DNA断片の抽出を行った。  [0038] The purified PCR product was digested with SfaNI (manufactured by NEB) to purify the DNA. The DNA was electrophoresed on a 15% acrylamide gel, the band of the desired DNA fragment was cut out, and the DNA fragment was extracted from the gel.
前記の方法により得られた cDNA断片と前述の直鎖化した pMBSlとを、 T4 DN A Ligase (タカラバイオ社製）にて連結し、得られた組換えプラスミドを用いたエレク トロポレーシヨンにより大腸菌 TOP10を形質転換した。形質転換体の一部を LB—ク 口ラムフエ-コールプレートに接種し、生じたコロニー数力 独立したクローン数を算 出するとともに、残りの形質転換体を LB—クロラムフエ-コール含有 LB培地に接種 し、クローン数 64万相当の培養物から QIAGEN Plasmid Midi Kit (キアゲン社 製)を用いてプラスミド DNAを精製し、タグライブラリーを得た。 [0039] 実施例 2 マイクロビーズの調製 The cDNA fragment obtained by the above method and the linearized pMBSl described above were ligated with T4 DN A Ligase (manufactured by Takara Bio Inc.), and E. coli TOP10 was obtained by electroporation using the obtained recombinant plasmid. Transformed. A part of the transformant is inoculated into LB-Culamphae-Cole plate, and the resulting colony strength is calculated. The number of independent clones is calculated, and the remaining transformant is inoculated into LB medium containing LB-chloramphee-coal. Then, plasmid DNA was purified from a culture corresponding to 640,000 clones using QIAGEN Plasmid Midi Kit (Qiagen) to obtain a tag library. [0039] Example 2 Preparation of microbeads
上記のタグライブラリーを铸型にして PCRを行った。 PCRは 5'—メチル化 dCTP 、 dATP、 dGTP、 dTTPを基質とし、プライマーには配列表の配列番号 6で示される オリゴヌクレオチドと、配列番号 7で示される FAM標識オリゴヌクレオチドと配列表の 配列番号 7で示されるピオチンィ匕オリゴヌクレオチドを 9： 1の比率で混合したものを用 い、 Ex Taq Hot Start Version (タカラバイオ社製）にて反応した。 PCR産物 を精製した後、制限酵素 Pad (NEB社製）による消化を行い、さら〖こ、 dGTP存在下 で T4 DNAポリメラーゼ (NEB社製)を作用させ、タグ部分の 1本鎖化を行った後、 DNAを精製した。  PCR was performed using the above tag library as a cage. PCR uses 5'-methylated dCTP, dATP, dGTP, and dTTP as substrates, and the primer is the oligonucleotide shown in SEQ ID NO: 6 in the sequence listing, the FAM-labeled oligonucleotide shown in SEQ ID NO: 7 and the sequence number in the sequence listing The reaction was carried out with Ex Taq Hot Start Version (manufactured by Takara Bio Inc.) using a mixture of 9: 1 biotinic oligonucleotides. After the PCR product was purified, it was digested with the restriction enzyme Pad (NEB), and then Sareko and T4 DNA polymerase (NEB) were allowed to act in the presence of dGTP to form a single-stranded tag. Later, the DNA was purified.
[0040] 1本鎖タグつき標的 DNA断片 30 μ gと、アンチタグが結合したマイクロビーズ (Sol exa社製） 7. 2 X 107個を混合し、 100 μ l( 500mM NaCl、 11. 6 mM リン酸ナ トリウム、 0. 01% Tween20、 3. 5% デキストラン硫酸中で 69°C、 3日間ハイブリ ダイズさせた。反応は 2本分行った。マイクロビーズを 10mM Tris— HCl (pH8)、 1 mM EDTA、 0. 01% Tween 20で洗浄し、 2本分のマイクロビーズを 1本にまと めた。 [0040] 30 μg of single-stranded tagged target DNA fragment and anti-tag-bound microbeads (Sol exa) 7.2 2 × 10 7 were mixed, and 100 μl (500 mM NaCl, 11.6 mM) Hybridization was carried out in sodium phosphate, 0.01% Tween20, 3.5% dextran sulfate for 3 days at 69 ° C. The reaction was carried out twice, and the microbeads were added with 10 mM Tris—HCl (pH 8), 1 Washed with mM EDTA, 0.01% Tween 20, and combined two microbeads into one.
[0041] 洗浄後のマイクロビーズに T4 DNAリガーゼを作用させることによって標的 DNA 断片とアンチタグの間に共有結合を形成させた。その後、 600 gダイナビーズ M— 280ストレプトアビジン（磁性ストレプトアビジンビーズ、ダイナル社製）に 25°Cで 30分 間結合させ、 MPC (ダイナル社製）に 1分間静置した後、上清を除去した。 10mM Tris— HCl (pH8)、 ImM EDTA、 0. 01% Tween 20 1mlにて再懸濁し、 M PC (ダイナル社製）に静置した後、上清を除去するという洗浄操作を繰り返し、 1本鎖 タグつき標的 DNA断片の載ったマイクロビーズのみを分離した。  [0041] T4 DNA ligase was allowed to act on the washed microbeads to form a covalent bond between the target DNA fragment and the anti-tag. Then, bind to 600 g Dynabeads M-280 Streptavidin (Magnetic Streptavidin Beads, manufactured by Dynal) for 30 minutes at 25 ° C, and allow to stand for 1 minute on MPC (manufactured by Dynal), then remove the supernatant. did. 10ml Tris—HCl (pH8), ImM EDTA, 0.01% Tween 20 Resuspend in 1ml, let stand on MPC (Dynal), and then repeat the washing procedure to remove the supernatant. Only microbeads carrying the strand-tagged target DNA fragment were isolated.
[0042] 分離したマイクロビーズを Mbol (タカラバイオ社製）にて消化し、ダイナビーズ M - 280ストレプトアビジンより切り出した。さらに dGTP存在下 Klenow Fragmentをマ イク口ビーズに作用させた後、 2等分し、 2種類のアダプター DNAを T4 DNAリガ一 ゼを用いて連結した。なお、アダプター DNAは、配列表の配列番号 8で示されるオリ ゴヌクレオチドと配列表の配列番号 9で示されるオリゴヌクレオチドをァニールさせた もの、および配列表の配列番号 10で示されるオリゴヌクレオチドと配列表の配列番号 11で示されるオリゴヌクレオチドをァニールさせたものである。 [0042] The separated microbeads were digested with Mbol (manufactured by Takara Bio Inc.) and cut out from Dynabeads M-280 streptavidin. Furthermore, Klenow Fragment was allowed to act on the mic bead in the presence of dGTP, and then divided into two equal parts, and the two types of adapter DNAs were ligated using T4 DNA ligase. The adapter DNA is prepared by annealing an oligonucleotide represented by SEQ ID NO: 8 in the sequence listing and an oligonucleotide represented by SEQ ID NO: 9 in the sequence listing, and an oligonucleotide represented by SEQ ID NO: 10 in the sequence listing. Sequence number of column table An oligonucleotide represented by 11 is annealed.
[0043] 実施例 3. MPSS解析による microRNA候補配列の抽出 [0043] Example 3. Extraction of microRNA candidate sequences by MPSS analysis
(l) MPSS解析  (l) MPSS analysis
上記のマイクロビーズを、 日本特許公開第 2000 - 515006号公報および Brenne r S.他 23名 Nature Biotechnology 2000年 vol. 18、 p630— 634に開示 された技術を用いて、マイクロビーズ上に固定化された標的 DNAの配列 22塩基分 を読み取り、同じ配列をまとめて個数を算出した。次に、算出した個数を合計し、個 々の配列の個数を合計個数で割り算して 100万を掛け、 100万個あたりの個々の配 列の個数を算出した。  The above microbeads were immobilized on the microbeads using the technology disclosed in Japanese Patent Publication No. 2000-515006 and Brenner S. et al. 23 Nature Biotechnology 2000 vol. 18, p630-634. The target DNA sequence of 22 bases was read and the number of the same sequences was calculated. Next, the calculated numbers were summed, and the number of individual arrays was divided by the total number and multiplied by 1 million to calculate the number of individual arrays per million.
[0044] (2) MPSSデータよりの新規 microRNA候補配列の抽出  [0044] (2) Extraction of novel microRNA candidate sequences from MPSS data
上記の MPSS解析により得られた 4737の配列を、 UCSCゲノムデータベース（htt p：〃 genome.ucsc.edu)にホモロジ一検索を行 、、ゲノム配列にマッチした 906の配列 を抽出した。その後、 Sangerマイクロ RNAテータベース (http://microrna.sanger.ac. uk/)、 NCBI Refseqテータベース (http://www.ncbi.nlm.nih. gov/ReSeq/)、 the European nbosomal RNAァ ~~タへ' ~~ (http:/ www.psb.ugent.be/rRNA/)、th e Genomic tRNAデータベース（http://lowelab.ucsc.edu/GtRNAdb/)にホモロジ 一検索を行い、どのデータベースにもマッチしない 243の配列を抽出した。その後、 抽出した配列がマッチしたゲノム配列情報よりその配列の両末端を 88塩基分延ばし 、 198塩基の配列を取得し、この配列の端から 110塩基の配列を 1塩基ずつずらして 、 88の互いに 1塩基異なる 110塩基の配列を作成した。この 110塩基の配列の 2次 構造予 S!Jを Vienna RNA Package (http://www.tbi.univie.ac.at/~ivo/RNA/)の R NA foldを用いて行い、自由エネルギーが最小で、かつ、ステムループを形成し、 かつ、ステム部分にある MPSS解析により得られた 22塩基の配列中 16塩基以上が 相補鎖側と相補的であるものを抽出し、 42の新規 microRNAの候補を得た。得られ た新規 microRNA候補より無作為に 9種類の配列を選択し、ステムループ配列より ループ配列を得た（配列表の配列番号 12〜20に示す)。選択した 9種類の自由エネ ルギ一は、—84. 1〜一 32. 7kcal/molであった。  The 4737 sequences obtained by the above MPSS analysis were subjected to a homology search in the UCSC genome database (http: 〃genome.ucsc.edu), and 906 sequences that matched the genome sequences were extracted. After that, Sanger micro RNA data base (http://microrna.sanger.ac.uk/), NCBI Refseq data base (http://www.ncbi.nlm.nih.gov/ReSeq/), the European nbosomal RNA ~~ Tahe '~~ (http: / www.psb.ugent.be/rRNA/), homologous search in the Genomic tRNA database (http://lowelab.ucsc.edu/GtRNAdb/) We extracted 243 sequences that did not match the database. After that, both ends of the sequence are extended by 88 bases from the genome sequence information with which the extracted sequences match, and a 198 base sequence is obtained. 110 base sequences differing by one base were prepared. This secondary structure prediction S! J of the 110 base sequence was performed using the RNA fold of the Vienna RNA Package (http://www.tbi.univie.ac.at/~ivo/RNA/). Extract the smallest of the stem loops, and the 22 base sequences obtained by MPSS analysis in the stem part, with 16 bases or more complementary to the complementary strand side. Got a candidate. Nine types of sequences were randomly selected from the obtained novel microRNA candidates, and a loop sequence was obtained from the stem loop sequence (shown in SEQ ID NOs: 12 to 20 in the sequence listing). The nine selected free energies ranged from -84.1 to 12.7 kcal / mol.
[0045] 実施例 4 各種ループ配列を有する shRNA発現ベクターの作製 緑色蛍光タンパク質 rsGFPの標的配列 A (GGAGTTGTCCCAATTCTTG) (配 列番号 27)および標的配列 B (GACACGTGCTGAAGTCAAG) (配列番号 28) に対する shRNA発現ベクターを以下の手順で作製した。標的配列にっ 、て配列番 号 12から 20のループ配列を有するステムループ構造の shRNA (すなわち、いずれ かのループ配列の両端に配列番号 27または 28の配列およびその逆方向の相補配 列が連結した RNA)を発現するための合成オリゴ DNAを発現ベクター pSINsi - hU 6 (タカラバイオ社製)の hU6プロモーターの下流に、製品説明書の手順に従って挿 入しプラスミドベクターを構築した。対照として、これまで shRNA発現ベクターのルー プ構造に利用されていた、配列番号 21に示す配列のループ（Brummelkamp et al. Science. 2002 296 : 550— 553. )を発現するベクターを作製した。ネガテ イブコントロールとして、何も挿入しないベクターを作製した。得られた各種ベクターは 大腸菌 JM109に形質転換し、プラスミド DNAを QIAGEN Plasmid Midi Kit (キ ァゲン社製)を用いて精製し、トランスフエクシヨン用 DNAとして供した。 Example 4 Preparation of shRNA expression vector having various loop sequences A shRNA expression vector for target sequence A (GGAGTTGTCCCAATTCTTG) (SEQ ID NO: 27) and target sequence B (GACACGTGCTGAAGTCAAG) (SEQ ID NO: 28) of green fluorescent protein rsGFP was prepared by the following procedure. Stem loop shRNA having a loop sequence of SEQ ID NOs: 12 to 20 (i.e., a sequence of SEQ ID NO: 27 or 28 and a complementary sequence in the opposite direction are linked to both ends of the loop sequence. A synthetic oligo DNA for expressing the RNA) was inserted downstream of the hU6 promoter of the expression vector pSINsi-hU 6 (manufactured by Takara Bio Inc.) according to the procedure described in the product instructions to construct a plasmid vector. As a control, a vector expressing a loop of the sequence shown in SEQ ID NO: 21 (Brummelkamp et al. Science. 2002 296: 550-553.), Which has been used in the loop structure of the shRNA expression vector so far, was prepared. As a negative control, a vector into which nothing was inserted was prepared. The various vectors obtained were transformed into E. coli JM109, and the plasmid DNA was purified using QIAGEN Plasmid Midi Kit (manufactured by Kagen) and used as DNA for transfection.
[0046] 実施例 5 各種ループ配列を有する shRNA発現レトロウイルスベクターの作製 Example 5 Preparation of shRNA-expressing retroviral vector having various loop sequences
実施例 4で調製したプラスミドベクターを 293TZ17細胞に、 Retorovirus Packa ging Kit Ampho (タカラバイオ社製）を用いて製品プロトコールに従 、トランスフエ クシヨンし、各種アンフォト口ピックウィルス上清液を獲得し、 0. 45 mフィルター（Mi lex HV、ミリポア社製）にてろ過し、使用するまで— 80°C超低温フリーザーで保存 した。  Transfect the plasmid vector prepared in Example 4 into 293TZ17 cells using the Retorovirus Packing Kit Ampho (manufactured by Takara Bio Inc.) according to the product protocol, and obtain supernatants of various amphoteric mouth pick virus supernatants. Filtered through a 0.45 m filter (Mi lex HV, manufactured by Millipore) and stored in an 80 ° C ultra-low temperature freezer until use.
[0047] 実施例 6 レトロウイルスベクターによる遺伝子抑制効果の確認  [0047] Example 6 Confirmation of gene repression effect by retroviral vector
標的遺伝子 rsGFPを安定に発現している HT1080細胞（ATCC CCL— 121)お よび K562細胞（ATCC CCL- 243)は以下の手順で作製した。  HT1080 cells (ATCC CCL-121) and K562 cells (ATCC CCL-243) stably expressing the target gene rsGFP were prepared by the following procedure.
rsGFP発現ベクター pQBI25 (Qbiogene社製）を制限酵素 Nhel及び Notlで切断 し、 775bpの GFP遺伝子断片を得た。次に pQBI ροΙΠ (Qbiogene社製）を制限酵 素 Nhel及び Notlで切断して rsGFP— NeoR融合遺伝子を除去し、先に得た 775b pの rsGFP遺伝子断片を挿入し、 ροΙΠプロモーター制御下で rsGFP遺伝子が発現 するベクター pQBI polll (neo - )を得た。 pQBI polll (neo - )を制限酵素 Xholで 消化し、 polllプロモーター制御下、 GFP発現ユニットを含む DNA断片を得、その末 端を DNA blunting kit (タカラバイオ社製）を用いて平滑ィ匕した。レトロウイルスべ クタ一プラスミド pDON— AI (タカラバイオ社製)を制限酵素 Xholと Sphlで消化して 得られたベクター断片 4. 58kbpの末端を DNA blunting kit (タカラバイオ社製） を用いて平滑ィ匕したのち、アルカリフォスファターゼ (タカラバイオ社製)を用いて脱リ ン酸化した。この平滑化したベクターに先の平滑化した ροΙΠプロモーター制御下 rs GFP発現ユニットを含む DNA断片を DNA Ligation Kit (タカラバイオ社製）を用 いて挿入し、 rsGFP発現組換えレトロウイルスベクター pDOG— polllを得た。また、 平滑ィ匕したベクターに平滑ィ匕した GFP遺伝子を挿入して rsGFP発現組換えレトロゥ ィルスベクター pDOGを得た。 The rsGFP expression vector pQBI25 (Qbiogene) was digested with restriction enzymes Nhel and Notl to obtain a 775 bp GFP gene fragment. Next, pQBI ροΙΠ (manufactured by Qbiogene) was cleaved with restriction enzymes Nhel and Notl to remove the rsGFP-NeoR fusion gene. The 775 bp rsGFP gene fragment obtained earlier was inserted, and the rsGFP gene under the control of the ροΙΠ promoter. The vector pQBI polll (neo −) expressing is obtained. Digest pQBI polll (neo-) with the restriction enzyme Xhol to obtain a DNA fragment containing the GFP expression unit under the control of the polll promoter. The ends were smoothed using a DNA blunting kit (Takara Bio). Vector fragment obtained by digesting the retroviral vector plasmid pDON—AI (Takara Bio) with restriction enzymes Xhol and Sphl 4. Use the DNA blunting kit (Takara Bio) to smooth the ends of 58 kbp. After soaking, dephosphorylation was performed using alkaline phosphatase (manufactured by Takara Bio Inc.). Insert the DNA fragment containing the previously smoothed rs GFP expression unit under the control of the ροΙΠ promoter into this smoothed vector using the DNA Ligation Kit (Takara Bio Inc.), and insert the rsGFP-expressing recombinant retroviral vector pDOG-poll. Obtained. In addition, a smoothed GFP gene was inserted into a smoothed vector to obtain an rsGFP-expressing recombinant retrovirus vector pDOG.
[0048] これらプラスミドベクターをレトロウイルス調製用細胞 G3T— hi (タカラバイオ社製） に、 Retorovirus Packaging Kit Amphoを用いて製品プロトコ一ノレに従いトラン スフエクシヨンし、各種アンフォト口ピックウィルス上清液を獲得し、 0. 45 μ mフィルタ 一 (Milex HV、ミリポア社製）にてろ過し、使用するまで— 80°C超低温フリーザー で保存した。 [0048] Transform these plasmid vectors into G3T-hi (manufactured by Takara Bio Inc.) for retrovirus preparation according to the product protocol using the Retorovirus Packaging Kit Ampho to obtain supernatants of various unphotopic mouth pick virus. It was then filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore) and stored in an 80 ° C ultra-low temperature freezer until use.
HT1080細胞を 6穴組織培養用プレート (岩城硝子社製）に、 1ゥエルあたり 5 X 10 4個播種し、 10% ゥシ胎児血清 (FBS)含有 DMEM培地中で 5%CO存在下、 37  HT1080 cells were seeded on a 6-well tissue culture plate (Iwaki Glass Co., Ltd.) at 5 X 10 4 per well, and 5% CO was present in DMEM medium containing 10% urinary fetal serum (FBS). 37
2  2
°Cで 24時間培養した。  The cells were cultured at ° C for 24 hours.
[0049] アンフォト口ピック DOGウィルス液を段階希釈し、ポリプレン（臭化へキサジメトリン； シグマ社製) 8 μ gZml存在下で感染を行った。感染後 3日間培養し、 GFP発現をフ ローサイトメーター（FACS Vantage, Becton Dickinson社製）で分析し、導入 効率 20%以下のサンプル力も GFP陽性細胞をソーティングにより回収し、培養を行 い、 GFP安定発現細胞 HT1080— GFPとした。  [0049] The unphoto-mouth pick DOG virus solution was serially diluted and infected in the presence of 8 μg Zml of polyprene (hexadimethrine bromide; Sigma). Culturing for 3 days after infection, analyzing GFP expression with a flow cytometer (FACS Vantage, manufactured by Becton Dickinson), collecting GFP-positive cells with a sample power of 20% or less of introduction efficiency by sorting, culturing, and GFP Stable expressing cells HT1080-GFP.
K562細胞は 10% ゥシ胎児血清（FBS)含有 RPMI1640培地中で 5%CO存在  K562 cells are present in 5% CO in RPMI1640 medium containing 10% ushi fetal serum (FBS)
2 下、 37°Cで培養した。  2 The cells were cultured at 37 ° C.
[0050] アンフォト口ピック DOG— ροΙΠウィルス液を段階希釈し、レトロネクチン（登録商標、 タカラバィォ社製)を用いた標準的な方法で感染を行った。感染後 3日間培養し、 G FP発現をフローサイトメーターで分析し、導入効率 20%以下のサンプル力も GFP陽 性細胞をソーティングにより回収し、培養を行い、 GFP安定発現細胞 Κ562— GFPと した。 [0050] The unphoto mouth pick DOG-ροΙΠ virus solution was serially diluted and infected by a standard method using RetroNectin (registered trademark, manufactured by Takara Bio Co., Ltd.). Incubate for 3 days after infection, analyze GFP expression with a flow cytometer, collect GFP positive cells with sorting efficiency of 20% or less by sorting, culture, and GFP stable expression cells Κ562-GFP did.
[0051] このようにして調製した HT1080— GFPおよび K562— GFPに実施例 5で調製し たレトロウイルスベクターを感染した。 HT1080— GFPにはウィルスベクターを 10倍 および 100倍希釈し、ポリプレン 8 g/ml存在下で感染を行った。 K562— GFPに はウィルスベクターを原液および 10倍希釈し、レトロネクチンを用いた標準的な方法 で感染を行った。感染から 24時間経過した後、 HT1080— GFPは G418 (ジエネテ イシン;インビトロジェン社製）を 500 μ g/ml含有する増殖培地に、 K562— GFPは G418を 1000 g/ml含有する増殖培地に交換し、 2週間選択培養を行った。  [0051] HT1080-GFP and K562-GFP prepared as described above were infected with the retroviral vector prepared in Example 5. The virus vector was diluted 10-fold and 100-fold into HT1080-GFP and infected in the presence of polyprene 8 g / ml. K562-GFP was infected by a standard method using retronectin after diluting the virus vector 10 times with the stock solution. 24 hours after infection, HT1080-GFP was replaced with a growth medium containing 500 μg / ml of G418 (dienetin; manufactured by Invitrogen), and K562-GFP was replaced with a growth medium containing 1000 g / ml of G418. Selection culture was performed for 2 weeks.
2週間選択培養し、 siRNAを安定に発現している細胞を獲得後、フローサイトメ一 ターで分析し、 GFPの蛍光強度を算出した。対照実験群の蛍光強度に対する各実 験群での蛍光強度の割合を算出することによって遺伝子抑制効果を評価した。標的 配列 Aでの HT1080— GFPの結果を図 1に、標的配列 Aでの K562— GFPの結果 を図 2に示す。標的配列 Bでの HT1080— GFPの結果を図 3に、標的配列 Bでの K 562— GFPの結果を図 4に示す。図中、縦軸は GFPの蛍光強度についてネガティ ブコントロール（Negative control)を 100としたときの相対値で示す（Relative GFP me an (%))。横軸の数字は配列番号を示す。図に示されるように、配列番号 21に示され るノレープ酉己歹 IJと it較して、酉己歹 IJ番号 12、 13、 14、 15、 16、 18、 19、 20に示されるノレ ープ配列の遺伝子抑制効果が高いことが示された。  After selective culture for 2 weeks, cells stably expressing siRNA were obtained and analyzed by a flow cytometer to calculate the fluorescence intensity of GFP. The gene suppression effect was evaluated by calculating the ratio of the fluorescence intensity in each experimental group to the fluorescence intensity in the control experimental group. Figure 1 shows the results for HT1080-GFP at target sequence A, and Figure 2 shows the results for K562-GFP at target sequence A. The result of HT1080-GFP at the target sequence B is shown in FIG. 3, and the result of K562-GFP at the target sequence B is shown in FIG. In the figure, the vertical axis shows the relative intensity of GFP fluorescence intensity when the negative control is 100 (Relative GFP me an (%)). The numbers on the horizontal axis indicate the sequence numbers. As shown in the figure, it is compared with the norpe IJ shown in SEQ ID NO: 21 that compared to the ILE IJ numbers 12, 13, 14, 15, 16, 18, 19, 20 It was shown that the gene sequence has a high gene suppression effect.
[0052] 実施例 7 各種ループ配列を有する shRNA発現ベクターの作製  [0052] Example 7 Construction of shRNA expression vector having various loop sequences
ヒトインテグリン α 4の標的配列（GAGTGTTTGTGTACATCAA) (配列番号 29 )に対する shRNA発現ベクターを以下の手順で作製した。標的配列について実施 例 4にて使用した配列番号 13、 19、及び近年 siRNA効果が高いとされよく使用され ている microRNA— 30のステムループ配列（Boden et al. Nucleic Acids R esearch 2004 32 (3) : 1154— 1158)である配列番号 22のループ配列を有する shRNA (すなわち、いずれかのループ配列の両端に配列番号 29およびその逆方向 の相補配列が連結した RNA)を発現するための合成オリゴ DNAを発現ベクター pSI Nsi-hU6 (タカラバイオ社製)の hU6プロモーターの下流に、製品説明書の手順に 従って挿入しプラスミドベクターを構築した。対照として、これまで shRNA発現べクタ 一のループ構造に利用されて 、た、配列番号 21に示す配列のループ（Brummelk amp et al. Science. 2002 296 : 550— 553. )を発現するベクターを作製し た。ネガティブコントロールとして、実施例 4で作製した配列番号 27に示す rsGFPの 標的配列 Aと配列番号 21に示す配列のループを有するプラスミドベクターを使用し た。得られた各種ベクターにて大腸菌 JM109を形質転換し、プラスミド DNAを QIA GEN Plasmid Midi Kit (キアゲン社製）を用いて精製し、トランスフエクシヨン用 DNAとして供した。 An shRNA expression vector for human integrin α4 target sequence (GAGTGTTTGTGTACATCAA) (SEQ ID NO: 29) was prepared by the following procedure. Regarding the target sequence SEQ ID NOs: 13 and 19 used in Example 4, and the stem loop sequence of microRNA-30, which has recently been used with high siRNA effect (Boden et al. Nucleic Acids Research 2004 32 (3) : 1154—1158) Synthetic oligo DNA for expressing the shRNA having the loop sequence of SEQ ID NO: 22 (that is, RNA having SEQ ID NO: 29 and its complementary sequence ligated to both ends of either loop sequence) Was inserted into the downstream of the hU6 promoter of the expression vector pSI Nsi-hU6 (manufactured by Takara Bio Inc.) in accordance with the procedure described in the product instructions to construct a plasmid vector. As a control, so far shRNA expression vector A vector expressing the loop of the sequence shown in SEQ ID NO: 21 (Brummelk amp et al. Science. 2002 296: 550-553.) Was prepared using one loop structure. As a negative control, a plasmid vector having the loop of the target sequence A of rsGFP shown in SEQ ID NO: 27 shown in SEQ ID NO: 27 and the sequence shown in SEQ ID NO: 21 was used. Escherichia coli JM109 was transformed with the various vectors obtained, and the plasmid DNA was purified using QIA GEN Plasmid Midi Kit (manufactured by Qiagen) and used as DNA for transfection.
[0053] 実施例 8 各種ループ配列を有する shRNA発現レトロウイルスベクターの作製  Example 8 Preparation of shRNA-expressing retroviral vector having various loop sequences
実施例 7で調製したプラスミドベクターをレトロウイルス調製用細胞 G3T— hi (タカラ バイオ社製）に、 Retorovirus Packaging Kit Ampho (タカラバイオ社製）を用 いて製品プロトコールに従いトランスフエクシヨンし、各種アンフォト口ピックウィルス上 清液を獲得し、 0. 45 μ mフィルター（Milex HV、ミリポア社製）にてろ過し、使用す るまで 80°C超低温フリーザーで保存した。各ウィルス作製は 2例ずつ行った。  The plasmid vector prepared in Example 7 was transferred to retrovirus preparation cells G3T-hi (Takara Bio) using Retorovirus Packaging Kit Ampho (Takara Bio) according to the product protocol, and various amphoteric mouths were used. The supernatant of the pick virus was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and stored in an 80 ° C ultra-low temperature freezer until use. Each virus was prepared in 2 cases.
[0054] 実施例 9 レトロウイルスベクターのタイター測定  Example 9 Titer measurement of retroviral vector
実施例 8で調製したレトロウイルスベクターを段階希釈し、 HT1080細胞 (ATCC CCL- 121)へポリブレン 8 μ gZml存在下で感染させ、感染から 24時間経過した 後、 G418 (ジエネティシン;インビトロジェン社製）を 500 μ gZml含有する増殖培地 に交換し、 2週間選択培養を行った。形成されたコロニー数よりレトロウイルスベクター のタイターを算出した。  The retroviral vector prepared in Example 8 was serially diluted, and HT1080 cells (ATCC CCL-121) were infected in the presence of polybrene 8 μgZml. After 24 hours from the infection, G418 (Dieticin; manufactured by Invitrogen) was used. The culture medium was replaced with a growth medium containing 500 μg Zml, and selective culture was performed for 2 weeks. The retroviral vector titer was calculated from the number of colonies formed.
[0055] 実施例 10 レトロウイルスベクターによる遺伝子抑制効果の確認  [0055] Example 10 Confirmation of gene repression effect by retroviral vector
HL60 (ATCC CCL— 240)細胞に実施例 8で調製し、実施例 9にてタイター測 定を行ったレトロウイルスベクターを MOI2にてレトロネクチンを用いた標準的な方法 で感染を行った。感染から 24時間経過した後、 G418を 1000 /z gZml含有する増 殖培地に交換し、 2週間選択培養を行った。  HL60 (ATCC CCL-240) cells were infected with the retroviral vector prepared in Example 8 and titered in Example 9 using MOI2 by a standard method using retronectin. After 24 hours from the infection, G418 was replaced with a growth medium containing 1000 / z gZml, and selective culture was performed for 2 weeks.
[0056] 2週間選択培養し、 siRNAを安定に発現している細胞を回収し、 QIAGEN RNe asy Mini Kit (キアゲン社製)にて全 RN Aの抽出及び DNAsel処理を行った。抽 出した全 RNAをランダムプライマー（6mer)を用いて、 Reverse Transcriptase M— MLV (タカラバイオ社製）にて逆転写反応を行い、 SYBR Premix Ex Taq ( タカラバィォ社製)及び配列番号 23、 24のインテグリンひ 4増幅用プライマーを用い てリアルタイム PCRを行 ヽ、インテグリン a 4の遺伝子発現量の相対値を算出した。 全 RNA量の補正は配列番号 25、 26の GAPDH遺伝子増幅用プライマーを用いて 行った。 [0056] After selective culture for 2 weeks, cells stably expressing siRNA were recovered, and total RNA was extracted and treated with DNAsel using QIAGEN RNeasy Mini Kit (manufactured by Qiagen). The extracted total RNA was subjected to reverse transcription with Reverse Transcriptase M—MLV (Takara Bio) using a random primer (6mer), and SYBR Premix Ex Taq ( Real-time PCR was performed using the integrin 4 amplification primers of SEQ ID NOS: 23 and 24, and the relative value of the integrin a4 gene expression level was calculated. The amount of total RNA was corrected using the GAPDH gene amplification primers of SEQ ID NOs: 25 and 26.
[0057] 対照実験群のインテグリン ex 4発現相対値に対する各実験群での発現相対値の割 合を算出することによって遺伝子抑制効果を評価した。結果を図 5に示す。図中、縦 軸はインテグリン α 4発現量につ!、てネガティブコントロール（Negative control)を 10 0としたときの相対値で示す（Relative integrin α 4 mean (%))。横軸の数字は配列番 号を示す。図 5に示されるように、配列番号 21に示されるループ配列、配列番号 22 に示される microRNA— 30のループ配列と比較して、配列番号 13、 19、に示され るループ配列の遺伝子抑制効果が高 、ことが示された。さらに今回比較したループ 配列の遺伝子抑制効果の高さの順番は、実施例 4、 5、 6にて行った GFPに対する遺 伝子抑制効果の結果と一致した。  [0057] The gene suppression effect was evaluated by calculating the ratio of the expression relative value in each experimental group to the integrin ex 4 expression relative value in the control experimental group. The results are shown in FIG. In the figure, the vertical axis indicates the integrin α4 expression level and shows the relative value when the negative control is set to 100 (Relative integrin α4 mean (%)). The numbers on the horizontal axis indicate sequence numbers. As shown in FIG. 5, compared to the loop sequence shown in SEQ ID NO: 21 and the microRNA-30 loop sequence shown in SEQ ID NO: 22, the gene suppression effect of the loop sequence shown in SEQ ID NO: 13, 19 Was shown to be high. Furthermore, the order of the gene suppression effect of the loop sequences compared this time coincided with the results of the gene suppression effect on GFP performed in Examples 4, 5, and 6.
産業上の利用可能性  Industrial applicability
[0058] 本発明により、遺伝子抑制に有用な shRNAのループ領域をコードする核酸を簡便 にスクリーニングすることが可能となる。さらに、遺伝子抑制に有用で、従来使用され て!、る shRNAのループ配列よりも遺伝子抑制作用の大き!/、shRNAのループ領域 をコードする核酸、該核酸を含有するベクター、前記核酸またはベクターを含有する キットを提供することが可能となる。 [0058] According to the present invention, a nucleic acid encoding a loop region of shRNA useful for gene suppression can be easily screened. Furthermore, it is useful for gene suppression and has been used in the past. The gene suppressive action is larger than the loop sequence of shRNA !, a nucleic acid encoding the loop region of shRNA, a vector containing the nucleic acid, the nucleic acid or the vector It becomes possible to provide the containing kit.
配列表フリーテキスト  Sequence listing free text
[0059] MiQ ID N〇：l : Synthetic chimera oligonucleotide, nucleotide丄 toり are nbonucle otide— other nucleotides are deoxyribonucleotides [0059] MiQ ID N〇 ： l: Synthetic chimera oligonucleotide, nucleotide 丄 to are are nbonucle otide— other nucleotides are deoxyribonucleotides
SEQ ID NO:2: Synthetic chimera oligonucleotide, "nucleotide 17 to 22 are ribonuc leotide— other nucleotides are deoxyribonucleotides  SEQ ID NO: 2: Synthetic chimera oligonucleotide, "nucleotide 17 to 22 are ribonuc leotide— other nucleotides are deoxyribonucleotides
SEQ ID N〇：3: Synthetic primer for revearse transcription.  SEQ ID N〇: 3: Synthetic primer for revearse transcription.
SEQ ID NO:4: Synthetic primer.  SEQ ID NO: 4: Synthetic primer.
SEQ ID NO:5: Synthetic primer.  SEQ ID NO: 5: Synthetic primer.
SEQ ID NO:6: Synthetic primer. SEQ ID N〇:7: Synthetic primer. SEQ ID NO: 6: Synthetic primer. SEQ ID N〇: 7: Synthetic primer.
SEQ ID N0:8: Synthetic oligonucleotide for adaptor DNA.  SEQ ID N0: 8: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO:9: Synthetic oligonucleotide for adaptor DNA.  SEQ ID NO: 9: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO: 10: Synthetic oligonucleotide for adaptor DNA.  SEQ ID NO: 10: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO:l l: Synthetic oligonucleotide for adaptor DNA.  SEQ ID NO: l l: Synthetic oligonucleotide for adaptor DNA.
SEQ ID NO: 12: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 12: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 13: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 13: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 14: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 14: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 15: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 15: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 16: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 16: Nucleotide sequence for loop region of shRNA.
SEQ ID N〇：17: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 17: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 18: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 18: Nucleotide sequence for loop region of shRNA.
SEQ ID NO: 19: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 19: Nucleotide sequence for loop region of shRNA.
SEQ ID NO:20: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 20: Nucleotide sequence for loop region of shRNA.
SEQ ID NO:21: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 21: Nucleotide sequence for loop region of shRNA.
SEQ ID NO:22: Nucleotide sequence for loop region of shRNA.  SEQ ID NO: 22: Nucleotide sequence for loop region of shRNA.
SEQ ID NO:23: Synthetic primer for amplification of integrin alpha 4 gene SEQ ID NO: 23: Synthetic primer for amplification of integrin alpha 4 gene
SEQ ID NO:24: Synthetic primer for amplification of integrin alpha 4 geneSEQ ID NO: 24: Synthetic primer for amplification of integrin alpha 4 gene
SEQ ID NO:25: Synthetic primer for amplification of GAPDH geneSEQ ID NO: 25: Synthetic primer for amplification of GAPDH gene
SEQ ID NO:26: Synthetic primer for amplification of GAPDH geneSEQ ID NO: 26: Synthetic primer for amplification of GAPDH gene
SEQ ID NO:27: Green fluorescence protein rsGFP target sequence ASEQ ID NO: 27: Green fluorescence protein rsGFP target sequence A
SEQ ID NO:28: Green fluorescence protein rsGFP target sequence BSEQ ID NO: 28: Green fluorescence protein rsGFP target sequence B
SEQ ID NO:29: Human integrin alpha 4 target sequence SEQ ID NO: 29: Human integrin alpha 4 target sequence

Claims

請求の範囲 [1] 標的遺伝子の遺伝子抑制に有用な shRNAのループ配列のスクリーニング方法で あって、以下の工程を包含する方法： Claims [1] A method for screening shRNA loop sequences useful for gene suppression of a target gene, which comprises the following steps:
(1)生体試料力も低分子の RNAを抽出する工程、  (1) A process for extracting RNA with low molecular weight,
(2) (1)で得られた低分子 RNAの塩基配列を決定する工程、  (2) determining the base sequence of the low molecular weight RNA obtained in (1),
(3)前記生体のゲノム情報を基に、 (2)で得られた塩基配列の前後のゲノム配列を 取得し、 (2)で得られた塩基配列およびその前後のゲノム配列力 なる一連の配列 から、以下の (A)かつ (B)に該当する配列を選択する工程、  (3) Based on the genome information of the living body, the genome sequence before and after the base sequence obtained in (2) is obtained, and the base sequence obtained in (2) and a series of sequences comprising the genome sequence power before and after the base sequence. A step of selecting a sequence corresponding to the following (A) and (B):
(A)—定の長さ当たりの自由エネルギーが最も低い領域の配列であって、ステムル ープ構造を形成する配列、  (A) —the arrangement of the region with the lowest free energy per fixed length, which forms a stem-loop structure,
(B) (2)で得られた塩基配列がステムループ構造のステム領域に存在する配列、 (B) a sequence in which the base sequence obtained in (2) is present in the stem region of the stem-loop structure,
(4) (3)で得られたゲノム配列から、ループ配列を決定し、当該ループ配列の両端に 標的遺伝子配列およびその逆方向の相補配列力 Sステム領域を形成するように連結さ れた shRNAを構築する工程、 (4) Determine the loop sequence from the genomic sequence obtained in (3), and connect the target gene sequence and the complementary sequence in the opposite direction at both ends of the loop sequence. Building process,
(5) (4)で得られた shRNAを用いて標的遺伝子の遺伝子抑制効果を確認する工程  (5) The process of confirming the gene suppression effect of the target gene using the shRNA obtained in (4)
[2] 請求項 1記載の方法により得られる、遺伝子抑制に有用な shRNAのループ配列を 含有する核酸。 [2] A nucleic acid containing the shRNA loop sequence useful for gene suppression obtained by the method according to claim 1.
[3] ループ配列が以下力 選択される塩基配列からなる請求項 2記載の核酸：  [3] The nucleic acid according to claim 2, wherein the loop sequence comprises a nucleotide sequence selected as follows:
(1)配列表の配列番号 12〜20のいずれ力 1つに記載の塩基配列；または  (1) the nucleotide sequence according to any one of SEQ ID NOS: 12 to 20 in the sequence listing; or
(2)配列表の配列番号 12〜20のいずれ力 1つに記載の塩基配列において 1個また は数個の塩基の欠失、付加、挿入もしくは置換の少なくとも 1つを有する塩基配列。  (2) A base sequence having at least one of deletion, addition, insertion or substitution of one or several bases in the base sequence described in any one of SEQ ID NOS: 12 to 20 in the sequence listing.
[4] 請求項 2または請求項 3記載の核酸を含有する shRNAを発現するためのベクター  [4] A vector for expressing the shRNA containing the nucleic acid according to claim 2 or claim 3.
[5] 請求項 2または請求項 3記載の核酸、もしくは請求項 4記載のベクターを含むキット [5] A kit comprising the nucleic acid according to claim 2 or claim 3, or the vector according to claim 4.
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