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WO2007032428A1 - NOUVELLE SÉQUENCE EN BOUCLE EFFICACE POUR L’EXPRESSION DE shARN - Google Patents

NOUVELLE SÉQUENCE EN BOUCLE EFFICACE POUR L’EXPRESSION DE shARN Download PDF

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Publication number
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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Prior art keywords
sequence
loop
shrna
rna
seq
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PCT/JP2006/318247
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English (en)
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/ja
Publication of WO2007032428A1 publication Critical patent/WO2007032428A1/fr

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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 nucleic acids [NA]
    • 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

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Abstract

L’invention concerne une séquence en boucle ayant un fort effet répressif sur les gènes en utilisant du shARN. Le petit ARN exprimé dans une cellule humaine est purifié et dépisté par une technique complète d'analyse d'expression de gènes (MPSS) utilisant des microbilles, et la séquence d'ARN est analysée. Sur la base des informations de séquence que procure l'analyse, on effectue une analyse de base de données. Parmi les séquences qui sont à 100% identiques au génome humain, on extrait une séquence qui ne contient pas de miARN, rARN ou tARN connu. Sur la base de la prédiction d’une séquence secondaire, on obtient une séquence en boucle.
PCT/JP2006/318247 2005-09-14 2006-09-14 NOUVELLE SÉQUENCE EN BOUCLE EFFICACE POUR L’EXPRESSION DE shARN WO2007032428A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010007797A1 (fr) * 2008-07-18 2010-01-21 国立大学法人 東京大学 Arnsh hautement actif et son procédé de fabrication
JP5687188B2 (ja) * 2009-03-31 2015-03-18 Ｄｅｌｔａ−Ｆｌｙ Ｐｈａｒｍａ株式会社 チミジル酸合成酵素に対するＲＮＡｉ分子およびその用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMBROS V. ET AL.: "MicroRNAs and other tiny endogenous RNAs in C. elegans", CURREN BIOLOGY, vol. 13, 2003, pages 807 - 818, XP009058745 *
BEREZIKOV E. ET AL.: "Phylogenetic shadowing and computational identification of human microRNA genes", CELL, vol. 120, January 2005 (2005-01-01), pages 21 - 24, XP002370262 *
BODEN D. ET AL.: "Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins", NUCLEIC ACIDS RES., vol. 32, no. 3, 2004, pages 1154 - 1158, XP003005126 *
MIYAGISHI M. ET AL.: "Optimization of an siRNA-expression systems with an improved hairpin and its significant suppressive effects in mammalian cells", J. GENE MED., vol. 6, 2004, pages 715 - 723, XP003010166 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010007797A1 (fr) * 2008-07-18 2010-01-21 国立大学法人 東京大学 Arnsh hautement actif et son procédé de fabrication
JP5687188B2 (ja) * 2009-03-31 2015-03-18 Ｄｅｌｔａ−Ｆｌｙ Ｐｈａｒｍａ株式会社 チミジル酸合成酵素に対するＲＮＡｉ分子およびその用途

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