WO2005040385A1 - METHODE D’INDUCTION D’UNE ACTIVITE ARNi SPECIFIQUE DANS DES CELLULES ET ACIDES NUCLEIQUES POUR SA MISE EN OEUVRE - Google Patents
METHODE D’INDUCTION D’UNE ACTIVITE ARNi SPECIFIQUE DANS DES CELLULES ET ACIDES NUCLEIQUES POUR SA MISE EN OEUVRE Download PDFInfo
- Publication number
- WO2005040385A1 WO2005040385A1 PCT/FR2004/002673 FR2004002673W WO2005040385A1 WO 2005040385 A1 WO2005040385 A1 WO 2005040385A1 FR 2004002673 W FR2004002673 W FR 2004002673W WO 2005040385 A1 WO2005040385 A1 WO 2005040385A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- tat protein
- rnai
- cells
- nucleic acid
- Prior art date
Links
Classifications
-
- 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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
-
- 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
Definitions
- the invention relates to the field of biology and more particularly the preparation of double stranded oligonucleotides intended for use in an RNA interference process (RNAi or RNAi) in order to induce the degradation of a target RNA .
- RNA interference also designated “RNAi” or co-suppression, has been demonstrated in plants, where it has been observed that the introduction of a long double-stranded RNA, corresponding to a gene, induces specific repression and efficient expression of the targeted gene.
- the mechanism of this interference involves the degradation of double-stranded RNA into short duplexes of oligonucleotides of approximately 20 to 22 nucleotides called siRNAs.
- RNA interference has now been applied to mammals to specifically inhibit gene expression for applications in functional genetics.
- siRNAs make it possible to identify the function of the genes revealed by the sequencing of the human genome, either in cell culture models, or in animal models in particular in mice.
- RNA interference is also useful in the therapeutic field for the treatment or prevention of cancers, infectious diseases and more generally of diseases involving a mutated heterologous or homologous gene (Elbashir, SM, Harborth, J., Lendeckel, W .,
- SiRNAs are short double-stranded RNA sequences, which can be introduced into cells in the form of synthetic oligonucleotides or in the form of vectors allowing the expression of these siRNAs.
- siRNA is synthesized in the form of a precursor which is a shRNA (short hairpin RNA), formed of a "loop rod" in which the rod represents the siRNA and the loop any sequence which will be degraded by cellular RNAases, which will give rise to mature siRNA.
- shRNA short hairpin RNA
- the implementation of siRNA expression vectors has many advantages, in particular for functional genetics applications. It makes it possible to express double stranded RNA in a stable manner in cells, and therefore to more easily inhibit the expression of proteins with a long half-life. Indeed, synthetic siRNAs have a half-life of 3 days in mammalian cells. In addition, siRNAs are diluted during cell divisions. It also makes it possible to analyze long-term effects.
- siRNA activity is meant its ability to recognize and induce the degradation of its target RNA. It is described in the prior art a vector allowing the stable and inducible expression of siRNA. This system is based on the inhibition of transcription of the siRNA precursor.
- the vector derived from the vector pSUPER (Brummelkamp T. R et al., Www.sciencexpress.org/21 March 2002 / page 1 / 10.1126 / science.1068999), contains a known sequence, "the tetracycline operator"("TEToperator”), positioned between the promoter directing the transcription of siRNA and the sequence encoding said siRNA.
- the tetracycline operator ("TEToperator”)
- TOTAT repressor binds to the "TET operator” and thus blocks the transcription of siRNA.
- doxycycline it ⁇
- the invention aims precisely to overcome these drawbacks by providing a system allowing to induce the activity of a siRNA at will.
- This object is achieved by the present invention through the use of the TAT protein-TAR RNA sequence couple to block the activity of an siRNA in mammalian cells.
- the principle of the invention is based on the TAT / TAR interaction.
- siRNA must be recognized in the cell by a protein complex, called RISC complex (RNA-Induced Silencing Complex), which will make it undergo a maturation step and use it as a guide to recognize and degrade the target mRNA.
- RISC complex RNA-Induced Silencing Complex
- the TAT protein can be used to block this last step.
- the TAT protein of HIV plays a crucial role in viral replication.
- TAT regulates the speed of replication of the virus.
- TAT is a secreted protein capable of diffusing into uninfected cells and of preparing an environment conducive to viral infection.
- the TAT protein exists in two forms (71 and 101 amino acids) encoded by two exons separated by the env gene.
- TAT exerts its activator function of the HIV promoter by binding to the TAR RNA sequence present in 5 'of all HIV RNAs.
- the binding of TAT and its cellular transcriptional co-activators to TAR RNA will stimulate the elongation of transcription by increasing the processivity of RNA polymerase II.
- the presence of double stranded RNAs in a cell represents a signal: it involves an RNaselll called Dicer. This ribonuclease cuts the long double stranded RNA into small fragments of double stranded RNA, the siRNAs, of precise size and structure, usually 21 to 23 nucleotides.
- RISC RNA-Induced Silencing Complex
- RISC is therefore a ribonucleoprotein complex containing a siRNA molecule associated with proteins belonging to the Argonaute family.
- RISC guides, thanks to the 5'Phosphate end of a single-stranded siRNA, the small siRNAs for specific recognition of the target messenger RNA sequence and catalyzes its cleavage; this reaction takes place in the cytoplasm. This results in inhibition of translation and thus inhibition of expression of the target gene.
- the loop of the siRNA precursor (shRNA), normally consisting of any sequence, is replaced by a nucleotide sequence coding for at least one nucleotide sequence contained in the TAR sequence, minimum and sufficient to be recognized by the protein TAT.
- Said sequence referenced in the sequence list submitted in the appendix under the number SEQ ID No. 1, is composed of 11 nucleotides and has the following sequence: ATCTGAGCTCT (or AUCUGAGCUCU in its RNA form). It is also possible to use, according to the invention, a sequence encoding the entire wild-type or mutated TAR sequence or any fragment thereof containing said minimal non-mutated sequence in nucleotides described above.
- This sequence (DNA or RNA) is also known in the text as a separator sequence.
- the separator sequence used according to the invention can be natural or synthetic.
- the TAT protein interacts with the TAR loop on the shRNA, and the interaction of siRNA with the protein maturation complex (RISC complex) cannot take place and the siRNA does not undergo the stages of maturation induced by said complex and therefore remains inactive, that is to say in the shRNA form.
- the TAR loop of the shRNA is degraded and the resulting siRNA is accessible to the RISC maturation complex and the process of degradation of the target RNA by the RNAi can thus be carried out.
- the subject of the invention is therefore a method of inducing the activity of an RNAi in cells in which: - the protein TAT and a nucleic acid encoding the sense and antisense sequences of a protein are introduced into eukaryotic cells RNAi of interest, said sense and antisense sequences being separated by a nucleotide sequence comprising at least the sequence referenced under the number SEQ ID No.
- the separator sequence may consist of any nucleotide sequence provided that it comprises at least the sequence SEQ ID No 1.
- the separator sequence consists of a sequence coding for the complete TAR sequence, wild-type or mutated, or of a fragment of the latter comprising a sequence coding for the sequence SEQ ID No. 1.
- said nucleic acid coding for the sequences comprising the sense and antisense sequences of an RNAi of interest separated by the separator sequence described above can be introduced into the cell in the form of an expression vector, particularly in the form of a plasmid or a viral vector.
- the RNAi of interest will be transcribed from the vector coding for the shRNA which will give rise to the siRNA after cleavage of the single-stranded separator sequence.
- the vector comprises at least one nucleotide sequence coding for the sense and siRNA antisense separated by a nucleotide sequence comprising at least the sequence referenced under the number SEQ ID No. 1, under the dependence of a transcription promoter.
- Said nucleic acid described above allows the implementation of the method of inducing the activity of an RNAi in eukaryotic cells.
- the vector may further include an antibiotic resistance gene.
- the antibiotic resistance gene is a neomycin resistance gene. The presence of an antibiotic resistance gene, such as neomycin, makes it possible to select the transfected cells.
- the TAT protein when the cells are cells in culture, the TAT protein can be introduced into said cells by simple culture of the cells in a medium comprising the TAT protein.
- an advantage of the present invention resides in the fact that the TAT protein spontaneously penetrates into the cells when the latter are cultured in a culture medium containing the TAT protein. It is therefore easy to block the action of siRNA, comprising the TAR separator sequence as described above, by culturing the cells containing siRNA in a culture medium containing TAT protein, and to stimulate the activity of RNAi by cultivating said cells in a culture medium without TAT protein, which will lead to the degradation of the target RNA by the RNAi.
- the culture medium can contain the TAT protein in a concentration of between 0.1 ⁇ g / ml and 5 ⁇ g / ml, preferably 0.5 ⁇ g / ml and 1.5 ⁇ g / ml and very preferably 1 ⁇ g / ml.
- the TAT protein can also be introduced into the cell in the form of an inducible expression vector comprising a nucleotide sequence coding for the TAT protein. In this case, the expression of the protein may be induced, which will have the consequence of inhibiting the activity of the RNAi and therefore blocking the degradation of the target RNA by the RNAi.
- the cells transfected with the nucleic acid according to the invention are mammalian cells.
- the method is applicable both to the transfection of cells in culture and directly in animals.
- the invention makes it possible to reliably analyze genes, particularly human genes from a functional point of view, in cultured cells or in animals, in particular in mice.
- the invention also relates to a cell or a cell line into which a nucleic acid according to the invention, as described previously, has been introduced.
- the invention also relates to animals comprising cells into which the nucleic acid according to the invention as described above has been introduced.
- compositions in particular pharmaceutical compositions, comprising as active substance at least one nucleic acid according to the invention as described above or cells containing said nucleic acid, as described above, optionally combined in the composition with a compatible excipient.
- FIG. 1 represents the inhibition of the GFP marker by RNAi.
- Example 1 Construction of the plasmid pTATOF-siRNAGFP coding for an RNAi whose target is the messenger RNA coding for the green fluorescent protein (Green Fluorescent Protein: GFP): This plasmid is constructed from the plasmid pSUPER, allowing constitutive expression from siRNA and described by Brummelkamp et al.
- the DNA sequence (oligonucleotide synthetic DNA synthesized in order), containing in sequence the sequences SEQ ID N ° 2 (coding for the siRNA sense) -SEQ ID N ° 1 (coding for the separating sequence) -SEQ ID N ° 3 (coding for antisense siRNA) is introduced immediately downstream of the H1 promoter of plasmid pSuper at Bgl II sites in 5 ', and Hind III in 3'.
- the expression vector pTATOF-siRNAGFP is thus obtained.
- SEQ ID NO. 2 coding for siRNA sense 5 'GCAAGCTGACCCTGAAGTTC 3' 3 'CGTTCGACTGGGACTTCAAG 5' sequence coding for the separating sequence (SEQ ID NO.
- sequences coding for the sense and antisense siRNAs are separated by a minimum TAR loop, coded by the separator sequence called SEQ ID NO.1, coding for the minimum sequence recognized by the TAT protein (SEQ ID NO. 4) .
- Example 2 inhibition of the expression of GFP by the siRNAGFP expressed by the vector of Example 1: Mammalian cells COS-7 are transfected with polyfect (Qiagen) with 4 ⁇ g of vectors siRNA expression system (pTATOFsiRNAGFP, part A, or pSuper siRNAGFP without TAR loop, part B), as well as a Green Fluorescent Protein or GFP expression vector (500 ng). Sixty hours after the transfection, a western blot was produced from the total extracts using an antibody directed against GFP (Santa cruz) or cellular tubulin (Sigma) in order to evaluate the quantity of proteins used for this test. The cells are cultured in DMEM medium (Gibco) containing
- Figure 1 shows the results of this experiment: Part A: In the absence of TAT protein and vector pTATOF-siRNAGFP, GFP is present in the cells (column 1). In the absence of TAT protein and in the presence of the vector pTATOF-siRNAGFP, the GFP is degraded (column 2). In the presence of wild-type TAT protein and of the vector pTATOF- siRNAGFP, GFP is present in the cells (column 3) in an amount comparable to that present in cells which have not received a TAT protein or of vector pTATOF-siRNAGFP (column 1) ), and is therefore not degraded.
- GFP is degraded in the cells (columns 4 and 5).
- Part B In the absence of TAT protein and of vector pSupersiRNAGFP (without TAR sequence), GFP is present in the cells (column 1). In the absence of TAT protein and in the presence of the vector pSupersiRNAGFP, the GFP is degraded (column 2). In the presence of wild or mutated TAT protein and of the vector pSupersiRNAGFP,) the GFP is degraded in the cells (column 1, 4 and 5).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002543002A CA2543002A1 (fr) | 2003-10-20 | 2004-10-19 | Methode d'induction d'une activite arni specifique dans des cellules et acides nucleiques pour sa mise en oeuvre |
EP04805237A EP1678306A1 (fr) | 2003-10-20 | 2004-10-19 | METHODE D'INDUCTION D'UNE ACTIVITE ARNi SPECIFIQUE DANS DES CELLULES ET ACIDES NUCLEIQUES POUR SA MISE EN OEUVRE |
US10/576,390 US20070082864A1 (en) | 2003-10-20 | 2004-10-19 | Method for inducing a specific rnai activity in cells and nucleic acids for carrying out said method |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0312250A FR2861086B1 (fr) | 2003-10-20 | 2003-10-20 | Methode d'induction d'une activite arni specifique dans des cellules et acides nucleiques pour sa mise en oeuvre |
FR0312250 | 2003-10-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005040385A1 true WO2005040385A1 (fr) | 2005-05-06 |
Family
ID=34385311
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2004/002673 WO2005040385A1 (fr) | 2003-10-20 | 2004-10-19 | METHODE D’INDUCTION D’UNE ACTIVITE ARNi SPECIFIQUE DANS DES CELLULES ET ACIDES NUCLEIQUES POUR SA MISE EN OEUVRE |
Country Status (5)
Country | Link |
---|---|
US (1) | US20070082864A1 (fr) |
EP (1) | EP1678306A1 (fr) |
CA (1) | CA2543002A1 (fr) |
FR (1) | FR2861086B1 (fr) |
WO (1) | WO2005040385A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014196931A1 (fr) * | 2013-06-06 | 2014-12-11 | Agency For Science, Technology And Research | Transposon pour la manipulation du génome |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8192938B2 (en) | 2005-02-24 | 2012-06-05 | The Ohio State University | Methods for quantifying microRNA precursors |
IT1397569B1 (it) | 2009-12-10 | 2013-01-16 | Icgeb | Peptidi e loro derivati che inibiscono il rilascio extracellulare della proteina tat di hiv-1 e la replicazione di hiv-1. |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002059300A2 (fr) * | 2000-12-28 | 2002-08-01 | J & J Research Pty Ltd | Suppression de gene mediee par arn bicatenaire |
-
2003
- 2003-10-20 FR FR0312250A patent/FR2861086B1/fr not_active Expired - Fee Related
-
2004
- 2004-10-19 CA CA002543002A patent/CA2543002A1/fr not_active Abandoned
- 2004-10-19 EP EP04805237A patent/EP1678306A1/fr not_active Withdrawn
- 2004-10-19 US US10/576,390 patent/US20070082864A1/en not_active Abandoned
- 2004-10-19 WO PCT/FR2004/002673 patent/WO2005040385A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002059300A2 (fr) * | 2000-12-28 | 2002-08-01 | J & J Research Pty Ltd | Suppression de gene mediee par arn bicatenaire |
Non-Patent Citations (3)
Title |
---|
ALLIKIAN MICHAEL J ET AL: "Doxycycline-induced expression of sense and inverted-repeat constructs modulates phosphogluconate mutase (Pgm) gene expression in adult Drosophila melanogaster.", GENOME BIOLOGY, vol. 3, no. 5, 15 April 2002 (2002-04-15), pages RESEARCH0021 1 - 0021 10, XP002282710, ISSN: 1465-6914 * |
FRITSCH LAURIANE ET AL: "Conditional gene knock-down by CRE-dependent short interfering RNAs.", EMBO REPORTS, vol. 5, no. 2, February 2004 (2004-02-01), pages 178 - 182, XP002275072, ISSN: 1469-221X * |
MATSUKURA S ET AL: "Establishment of conditional vectors for hairpin siRNA knockdowns", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 31, no. 15, 1 August 2003 (2003-08-01), pages e77, XP002270940, ISSN: 0305-1048 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014196931A1 (fr) * | 2013-06-06 | 2014-12-11 | Agency For Science, Technology And Research | Transposon pour la manipulation du génome |
Also Published As
Publication number | Publication date |
---|---|
US20070082864A1 (en) | 2007-04-12 |
FR2861086A1 (fr) | 2005-04-22 |
FR2861086B1 (fr) | 2005-12-30 |
CA2543002A1 (fr) | 2005-05-06 |
EP1678306A1 (fr) | 2006-07-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | Design of extended short hairpin RNAs for HIV-1 inhibition | |
Rohr et al. | Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas | |
Prucca et al. | Antigenic variation in Giardia lamblia is regulated by RNA interference | |
Bramsen et al. | Improved silencing properties using small internally segmented interfering RNAs | |
Bauer et al. | Prevention of interferon-stimulated gene expression using microRNA-designed hairpins | |
Capodici et al. | Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference | |
KR101605144B1 (ko) | Cns 질환의 치료 | |
Maeda et al. | In vitro and in vivo suppression of GJB2 expression by RNA interference | |
EP1453962B1 (fr) | Oligonucleotides inhibiteurs et leur utilisation pour reprimer specifiquement un gene codant pour un recepteur aux androgenes | |
JP2013532973A (ja) | Rna分子およびその使用 | |
JP2004261002A (ja) | siRNAの製造方法 | |
Bidari et al. | Targeting the microRNA pathway core genes, Dicer 1 and Argonaute 1, negatively affects the survival and fecundity of Bemisia tabaci | |
Tsang et al. | Ectopic expression of systemic RNA interference defective protein in embryonic stem cells | |
EP1678306A1 (fr) | METHODE D'INDUCTION D'UNE ACTIVITE ARNi SPECIFIQUE DANS DES CELLULES ET ACIDES NUCLEIQUES POUR SA MISE EN OEUVRE | |
EP2007882B1 (fr) | Procede de preparation de cellules aviaires differenciees et genes impliques dans le maintien de la pluripotence | |
Kim et al. | Targeted gene silencing by RNA interference in Chlamydomonas | |
Holen | Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC | |
WO2006130976A1 (fr) | Arn interferents, procedes d'elaboration et utilisation | |
US7947823B2 (en) | siRNA expression system | |
FR2847588A1 (fr) | METHODES D'EXPRESSION INDUCTIBLE D'ARNi DANS LES CELLULES, LES MOLECULES D'ACIDE NUCLEIQUE POUR SA MISE EN OEUVRE ET LES CELLULES TRANSFORMEES PAR CES MOLECULES | |
US7741469B2 (en) | Compositions for treating hearing loss and methods of use thereof | |
JP2006288321A (ja) | 変異対立遺伝子に対する特異的なRNAiの評価方法 | |
WO2020097630A1 (fr) | Inactivation de la réplication d'un génome viral dans la partie médiane de l'intestin de vecteurs d'insectes par activation de voies de défense anti-virale endogènes | |
Kunath | Transgenic RNA interference to investigate gene function in the mouse | |
의학과 et al. | Knockdown of nfa1 Gene Cloned from Naegleria fowleri by Antisense RNA |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2543002 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004805237 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2007082864 Country of ref document: US Ref document number: 10576390 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 2004805237 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 10576390 Country of ref document: US |