WO2001079853A2 - Systeme de test pour caracteriser des modulateurs du processus d'epissage des arn messagers dans des cellules vivantes (in vivo), sa realisation et son utilisation - Google Patents
Systeme de test pour caracteriser des modulateurs du processus d'epissage des arn messagers dans des cellules vivantes (in vivo), sa realisation et son utilisation Download PDFInfo
- Publication number
- WO2001079853A2 WO2001079853A2 PCT/EP2001/002235 EP0102235W WO0179853A2 WO 2001079853 A2 WO2001079853 A2 WO 2001079853A2 EP 0102235 W EP0102235 W EP 0102235W WO 0179853 A2 WO0179853 A2 WO 0179853A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- test system
- substance
- mrna
- nucleic acid
- vivo
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Definitions
- Test system for the characterization of modulators of the splicing process of mRNA in living cells (in vivo), its production and use
- the present invention relates to an in vivo test system comprising:
- Genome interrupted by one or more sequences (introns) not coding for the protein.
- these non-coding areas are transferred to the primary transcript.
- this precursor mRNA in order to generate a correct form of the mRNA, this precursor mRNA (pre-mRNA) must be processed.
- the pre-mRNA is processed by removing the introns and fusing the coding regions (exons). Only then can a continuously read nucleotide strand be made available for translation in the cytoplasm.
- the formation of mRNA in eukaryotes therefore requires a so-called
- the splicing takes place in the core before the mRNA is transported out of the core. It is generally carried out in a two-stage mechanism in which one transesterification step is involved (Moore, JM et al., (1993) Splicing of precursors to messenger RNAs by the Spliceosome. In The RNA world, Edited by Gesteland RF, Gesteland, JF, Cold Spring Harbor Laboratory Press, 303-358) , The first step generates a free 5 ' exon and a so-called lariat structure of the intron, which is still connected to the 3 exon.
- the lariat structure contains a branched RNA, which is caused by esterification of the 5 ' end of the intron with a 2 ' hydroxyl group of a ribose in an adenosine, which is approximately 20-40 nucleotides upstream of the 3 ' end of the intron is arises.
- the second catalytic step leads to ligation of the exons and release of the intron. Although no nucleotides are incorporated during these reactions, an energy source, for example ATP, is necessary for this catalysis (Guthrie, C. (1991) Science, 253, 157).
- snRNPs small nuclear ribonucleoprotein particles
- the second class consists of so far little characterized proteins, which are not firmly bound to the snRNPs and are therefore called non-snRNP splice factors (Lamm, GM & Lamond, AJ (1993) Biochim. Biophys. Acoph. 1173, 247; Beggs , JD (1995), Yeast splicing factors and genetic strategies for their analysis, In: Lamond, Al (ed) PremRNA Processing Austin, RG Company, Texas, pp. 79-95. Krämer, A. (1995), The biochemistry of pre-mRNA splicing. In:
- composition of the snRNPs is best examined in HeLa cells (Will, C.L. et al., (1995) Nuclear pre-mRNA splicing. In: Eckstein, F. and Lilley, D.M.J.
- the snRNPs lie in a 12S U1 snRNP, a 17S U2 snRNP and a 25S [U4 / U6.U5] t - snRNP Complex before.
- the tri-snRNP complex dissociates into a 20S U5 and a 12S U4 / U6 particle.
- the U4 and U6 RNAs are base-paired in the U4 / U6 snRNP via two intermolecular helices (Bringmann, P. et al. (1984) EMBO J., 3, 1357; Hashimoto, C. & Steitz, JA (1984) Nucleic Acids Res., 12, 3283; Rinke, J. et al., (1985) J. Mol. Biol., 185, 721; Brow,
- the snRNPs consist of two groups of proteins.
- the group of general proteins (B / B ' , D1, D2, D3, E, F and G) is contained in all snRNPs.
- each snRNP contains specific proteins that are only contained in this.
- the U1 snRNP contains three additional proteins (70K, A and C) and the U2 snRNP contains eleven additional proteins.
- the 20S U5 snRNP carries nine additional proteins with a molecular weight of 15, 40, 52, 100, 102, 110, 116, 200 and 220 kDa
- the 12S U4 / U6 snRNP carries two additional proteins with a molecular weight of approx. Contains 60 and 90 kDa
- the 25S tri-snRNP [U4 / U6.U5] contains five additional proteins with a molecular weight of approx. 15.5, 20, 27, 61 and 63 kDa.
- the individual components pre-mRNA, snRNPs and non-snRNP proteins
- pre-mRNA, snRNPs and non-snRNP proteins are brought together in a step-by-step process. This is achieved not only through interactions of the pre-mRNA with the protein-containing components, but also through numerous interactions between the protein-containing components themselves (Moore, JM (1993) supra; Madhani, HD & Guthrie, C. (1994) Annu. Rev. Genetics, 28, 1; Nilsen, TW (1994) Cell, 65, 115).
- the sequence of the pre-mRNA carries specific recognition sequences for the different splice components.
- the U1 snRNP binds to the 5 ' splicing region of the intron of the pre-mRNA via these recognition sequences.
- an as yet undetermined number of various other factors eg SF2 / ASF, U2AF, SC35, SF1
- the U2 snRNP particle interacts with the so-called branch site in the intron area (Krämer, A. & Utans, U. (1991) EMBO J., 10, 1503; Fu, XD & Maniatis, T. (1992) Proc. Natl. Acad. Sci USA, 89, 1725;
- the [U4 / U6.U5] tri-snRNP and a number of previously unidentified proteins interact with the pre-spliceosome to form the mature spliceosome (Moore, JM et al., (1993 ) supra).
- alternative splicing By alternative splicing, different mature mRNAs can be formed from one and the same primary transcript, which code for different proteins. This alternative splicing is regulated in many cases. This mechanism can e.g. can be used to switch from a non-functional to a functional protein (e.g. transposase in Drosophila). It is also known that alternative splicing is carried out in a tissue-specific manner. For example, the tyrosine kinase, which is encoded by the src proto-oncogene, is synthesized in nerve cells by alternative splicing in a special form.
- CD44 splice variants of the membrane-bound molecule CD44 seem to play a decisive role.
- the CD44 gene contains several exons, 10 of which are adjacent exons in a different arrangement in the mRNA
- Globin can lead to ß + thalemia.
- the point mutation creates an incorrect splice location, which leads to an altered reading frame and premature termination of the peptide chain (Weatherall, D. & Clegg, JB (1982) Cell, 29, 7; Fukumaki, Y. et al. (1982) Cell , 28, 585).
- Arabidopsis thaliana mutants z. B. a point mutation at the 5 ' splice of the phytochrome B gene to an incorrect expression of the gene. This change does not remove an intron that contains a stop codon in its sequence. The development of the plants is disturbed because the gene is involved in phytomorphogenesis (Bradley, JM et al. (1995) Plant Mol. Biol, 27, 1133).
- the NS1 protein which is encoded by the genome of the influenza virus, can also interfere with the splicing by binding to the U6 snRNA.
- the protein binds to nucleotides 27-46 and 83-101 of human U6 snRNA and thus prevents U6 from interacting with partners U2 and U4 during the splicing process (Fortes, P. et al. (1994) EMBO J. , 13, 704; Qiu, Y. & Krug, RM (1995) J. Virol., 68, 2425.
- the NS1 protein also appears to prevent export from the nucleus by binding to the poly-A tail of the mRNA formed (Fortes, P. et al. (1994), supra; Qiu, Y. & Krug, RM (1994), supra.) Similar effects are derived from a herpes simplex virus type 1 gene product
- RNA polymerase II a model RNA (ß-globin pre-mRNA) in in vitro experiments.
- peptides generated from the C-terminal domain of the large subunit of RNA polymerase II also appear to be able to intervene in the splicing processes (Yurvey, A. et al. (1996) Proc. Natl. Acad. Sei USA, 93, 6975; WO97 / 20031).
- Oligonucleotides on splicing The ratio of two different splice products of the rat c-erb oncogene mRNA (c-erbA-alpha 1 and 2) appears to be regulated by another mRNA, rev-ErbA-alpha.
- Rev-ErbA-alpha is a naturally occurring anti-sense RNA that pairs with the c-erbA-alpha 2 mRNA but not with the c-erbA-alpha 1 mRNA.
- RNase H can cause (Hodges, D. & Crooke ST (1995) Mol. Pharmacol., 48, 905).
- a more detailed analysis of the pre-mRNA sequences required for the splicing showed that 19 nucleotides upstream from the branch point adenosine and 25 nucleotides around the 3 ' and 5 ' splice site are suitable sequences for generating antisense RNAs (Dominski, Z. & Kole, R.
- an mRNA is generally first produced by in vitro transcription. Genetic constructs from viruses, for example adenoviruses, or cellular structural genes are used for this purpose. Such mRNAs contain all the important structural elements which are necessary for the recognition of the mRNA by the spliceosome and The splicing process is necessary In general, the mRNA is radiolabelled so that, after separation on a denaturing urea-polyacrylamide gel, it can be judged on the basis of the characteristic band pattern whether a splicing reaction has occurred or in which reaction step a fault has occurred.
- test systems are very useful Time-consuming and labor-intensive and therefore not suitable for the systematic detection of substances that can modulate or even inhibit splicing
- Buckler et al (WO92 / 13071) identify coding sequences by inserting unidentified genomic sucker DNA into a plasmid intron with subsequent transfection into sucker cells, which were detected after the in vivo splicing with the aid of antisense oligonucleotides
- WO98 / 08953 describes a screen of inhibitors and stimulators of the ORF P gene from herpes simplex virus, which interacts with splicing factors, recombinant ORF P inhibiting splicing of the viral DNA
- Kang et al (Kang, S-H, Cho, M-J & Kole R, Biochemsistry 37, 6235-6239, 1998) describe transferred Heia tet-off cells, the plasmid being a luciferase
- Carries genes which has an intron inserted, which in turn carries a mutation, which prevents correct splicing or translation of the luciferase becomes.
- antisense oligonucleotide treatment of the cells the luciferase activity can be restored.
- the splicing activity in cultivated maize cells could be determined by Carle-Uroste et al. (Carle-Urioste et al., Plant Molecular Biology 26, 1785-1795 (1994)) with two vectors, whereby the luciferase is only translated if a) splicing is omitted and b) if the splicing process was carried out correctly.
- a start codon was cloned into the intron sequence located in front of the luciferase gene in frame with the luciferase sequence; in b) the start codon was in front of the intron sequence, which in this case contained a stop codon.
- German patent application 199 09 56.0 discloses a technical teaching which makes it possible to effectively identify a large number of compounds from chemical or natural substance libraries based on their effects when splicing nucleic acids in a high throughput system "in vitro".
- the present invention expressly makes reference to this disclosure content.
- Substances of interest are particularly preferably used in the "in vivo" test system which have already been successfully identified as a modulator in an "in vitro" assay.
- the present invention therefore relates to a test system
- the splice and translation product according to c) is at least one protein which can be expressed or secreted in the cell and in cell compartments as well as on the cell surface and which induces a signal.
- This induced signal can be expanded in a simple manner, preferably optically, biochemically, chemically or physically
- Senses can be detected or analyzed.
- the detection includes, for example, an optically detectable changed wavelength (color change), changed light intensity and excitability by means of distinct wavelengths (UV, fluorescence). Morphological changes (cell size, shape, surface structure, growth behavior) are also optically detectable.
- optically detectable changed wavelength color change
- changed light intensity changed light intensity
- excitability by means of distinct wavelengths
- Morphological changes cell size, shape, surface structure, growth behavior
- detection options based on the induced signal by means of primary or secondary reactions. Adequate criterion is the decisive causality between the signal induction and the
- the detection can be carried out using a specific probe. If the signal to be detected is, for example, in a cell surface structure, the detection can be carried out by the reaction of this structure with another biological or chemical molecule.
- the detection can be carried out by the reaction of this structure with another biological or chemical molecule.
- the detectable signal can therefore also be an element of intracellular signal transduction.
- the expression of oncogenic Ras preferably in its constitutively active mutant Ras (G12V) produces a morphological change in the cell in the form of neurite outgrowth after binding of the growth factor NGF (nerve growth factor).
- the signal can be considered morphological
- Rho G12V
- Rac G12V
- Cdc42 G12V
- splicable nucleic acid means a nucleic acid which is recognized in a cellular "in vivo" system and is subject to the splicing process.
- the splicable nucleic acids therefore contain at least one intron, which is recognized and spliced as such depending on the cellular in vivo system used. Therefore, intron means a non-coding sequence which is spliced due to the recognizing consensus sequences in the cellular "in vivo" system.
- Such consensus sequences are in yeast: / GUAUGU in the 5 'splice, YAG / G in the 3' splice and UACUAAC in the branch point; in humans: AG / GURAGU in the 5'Spllicing parts, YAG / in the 3'-splice point and YNYURAC in the branching point (branchpoint).
- the underlined nucleotides are strictly preserved.
- An intron which is ligated terminally via consensus sequences with two exons is very particularly preferred.
- “in vivo” is understood to mean a conversion, ie a reaction, which takes place within a living cell.
- Cellular "in vivo" systems in the sense of the invention are splicable eukaryotes and preferably single cells. Cell lines from mammals - including human cell lines (such as HeLa cell line) or yeasts are very particularly preferably used.
- reporter molecules such as the fluorescent proteins GFP (green fluorescent protein from Aequorea victoria) and its variants YFP (yellow fluorescent protein), BFP (blue fluorescent protein), CFP (cyan fluorescent) are particularly preferred protein) and others encoded by reporter genes.
- GFP green fluorescent protein from Aequorea victoria
- YFP yellow fluorescent protein
- BFP blue fluorescent protein
- CFP cyan fluorescent
- Splicable nucleic acids according to features (a) - (c), which represent feature (a) and code for an optically detectable signal - (b) and (c) - are representative of such reporter molecules:
- SEQ ID No. 1 represents a nucleic acid coding for GFP actin, containing an exon sequence of nucleotide 1-171, an intron sequence of 172-483 and an exon sequence of 483-1201, and consensus sequences of 172-177 and 478-481 and 357-363.
- SEQ ID No. 2 represents a nucleic acid coding for GFP-MINX, containing an exon sequence from nucleotide 1-171, an intron sequence from 172-290 and an exon sequence from 291-837, and consensus sequences from 171-177, and 259- 263 and 288 - 290.
- the claimed splicable nucleic acids contain at least one artificial intron, as is the case with SEQ ID No. 1 (containing actin) in yeast and in
- Case SEQ ID No. 2 can be recognized by mammalian cells and human cells (containing MINX).
- MINX mammalian cells
- other usable introns including consensus sequences those that are already included in the genomic DNA are explicitly included of an organism are contained, preferably those of the cellular in vivo system used.
- nucleic acids can exist as both RNA and DNA.
- an inducible expression system is preferably used, which is commercially available, for example: the tet-off & tet -on® expression system from Clontech (the “Tet System “US. 5,464,758) or the Ecdysone inducible Expression System®. (Invitrogen BV, Groningen, NL) for the inducible
- the substance of interest or to be investigated can be selected from a large number of chemical or natural compounds
- Substance libraries for example natural substances in the broadest sense, herbicides, insecticides, pesticides, antibiotics, pharmaceuticals, combinatorial substance libraries.
- the substance to be examined can be selected from a naturally occurring, naturally occurring and chemically modified and / or synthetic substance. Such substances of interest are introduced into the cellular in vivo system using conventional methods.
- the invention therefore relates to a method for finding an active substance, characterized in that the substance according to feature (d) modulates the splicing process and inhibits and / or activates it.
- the invention relates to a method for producing the test system, wherein one or more, identical or different splicable nucleic acids according to feature (a) in the presence of at least one substance of interest in a cellular "in vivo" system by means of an inducible
- the invention therefore also relates to a method for diagnosing a disease, wherein at least one splicable nucleic acid according to (a) having the features (b) and (c) are incubated in the presence of at least one substance of interest and, if appropriate, further auxiliaries in a cellular in vivo system and the splice product is detected.
- the mRNA to be spliced consists of at least two exons that are separated by an intron.
- the exons code for a protein that is expressed after the splicing process has been carried out successfully and forms a signal that allows detection of the splicing reaction by specific methods.
- RNA constructs with signals that can be detected optically without aids
- GFP-Exon 1 - MINX-Intron - GFP-Exon2 (corresponding to SEQ ID No. 2 and Figure 2) 2 constructs for detection by means of a probe
- cell cultures from mammalian cells were used.
- 5x10 4 frozen Heia cells were transferred to 10 ml medium, sedimented, then taken up in 7.5 ml medium and in a 25 cm2 culture bottle at 37 ° C with gassing 5% CO 2 incubated Dulbecco's Modified Eagle Medium (Gibco) was used as the medium, to which 10U penicil n / streptavidin per ml, 580 mg L-glutamine per ml, 10% fetal calf serum and non-essential amino acids were added. The value was set to 7.3. The medium of the incubated cell cultures was renewed daily. The cells grew to a 90% confluent density over a period of 48 hours and began to detach from the background
- the cells were transferred to new incubation bottles of 75 cm 2.
- the cells were first washed in phosphate-buffered cream and then with a solution of 0.5 mg / ml trypsin and 0.2 mg / ml EDTA in phosphate-buffered cream from Dissolved underground, then stopping the reaction with 10 ml of medium.
- the cells were sedimented at 500 ⁇ g, washed with medium and taken up in 10 ml of medium and transferred to 75 cm 2 culture flasks at a dilution of 150 in a volume of 30 ml of medium 5.
- the recombinant vectors of the inducible expression system were introduced into the cells by cotrection with the aid of the Superfect transfection system from Qiagen. One day before the transfection process, 2.5 x 10 5
- Serum still containing proteins and antibiotics, dissolved and 10 ⁇ l Superfect Transfection reagent added. This mixture was incubated for 10 min at room temperature to form a complex. During this time, the growth medium was removed from the adherent cells by suction and the cells were washed once with 2 ml of PBS. Then 600 ul medium 10U penicillin / streptavidin per and
- the main culture was carried out in a culture volume of 100 ml and was inoculated with 0.01%, 0.25%, 0.05% and 0.1% with the preculture and again incubated at 28 ° C. overnight.
- the cells were pelleted in the logarhythmic growth phase (corresponding to a cell density of 0.5 at OD 60 o) and washed once with 10 ml 0.1 M LiAc in TE pH 8.0, then in 2 ml 0.1 M LiAc in TE pH 8.0 added.
- 100 ⁇ l cells were mixed with 5 ⁇ g recombinant vector DNA, 5 ⁇ l He ngssperm DNA (10 mg / ml) and 600 ⁇ l PEG solution (40% PEG 4000; 0.1M LiAc in TE pH 8.0) and 30 min at 42 ° C.
- the expression of the recombinant genes in the Heia cells was induced on the day after the transfection by replacing the culture medium with fresh medium which contained 5 ⁇ M Ponasterone A and then incubating at 37 ° C. for 20 hours.
- Rhodamine-phalloidin Rhodamine-phalloidin.
- the observation of the neurite outgrowth of the NGF-treated Ras (G12V) expressing cells was carried out with the Axiovert S100 (ZEISS) in phase contrast.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2001242439A AU2001242439A1 (en) | 2000-04-14 | 2001-02-28 | Test system for characterizing modulators of the splicing process of mrna in living cells (in vivo), production thereof and use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10018464A DE10018464A1 (de) | 2000-04-14 | 2000-04-14 | Testsystem zur Charakterisierung von Modulatoren des Spleißprozesses von mRNA in lebenden Zellen (in vivo), dessen Herstellung und Verwendung |
DE10018464.2 | 2000-04-14 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001079853A2 true WO2001079853A2 (fr) | 2001-10-25 |
WO2001079853A3 WO2001079853A3 (fr) | 2002-12-19 |
Family
ID=7638697
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/002235 WO2001079853A2 (fr) | 2000-04-14 | 2001-02-28 | Systeme de test pour caracteriser des modulateurs du processus d'epissage des arn messagers dans des cellules vivantes (in vivo), sa realisation et son utilisation |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU2001242439A1 (fr) |
DE (1) | DE10018464A1 (fr) |
WO (1) | WO2001079853A2 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1302543A1 (fr) * | 2001-10-15 | 2003-04-16 | Bayer CropScience AG | Procédé d'épissage comme cible pour l'identification de préparations pharmaceutiques |
GB2382577A (en) * | 2001-11-28 | 2003-06-04 | Talat Nasim | A reporter-based assay for assessing relative RNA processing activity in mammaliian cells |
WO2004028464A2 (fr) * | 2002-09-27 | 2004-04-08 | University Of North Carolina At Chapel Hill | Procedes et compositions servant a modifier l'epissage d'arn pre-messager |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102014207507B4 (de) | 2014-04-17 | 2021-12-16 | Kennametal Inc. | Zerspanungswerkzeug sowie Verfahren zum Herstellen eines Zerspanungswerkzeugs |
-
2000
- 2000-04-14 DE DE10018464A patent/DE10018464A1/de not_active Withdrawn
-
2001
- 2001-02-28 WO PCT/EP2001/002235 patent/WO2001079853A2/fr active Application Filing
- 2001-02-28 AU AU2001242439A patent/AU2001242439A1/en not_active Abandoned
Non-Patent Citations (5)
Title |
---|
BLENCOWE B J B J: "Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases" TIBS TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER PUBLICATION, CAMBRIDGE, EN, Bd. 25, Nr. 3, M{rz 2000 (2000-03), Seiten 106-110, XP004202539 ISSN: 0968-0004 * |
CARLE-URIOSTE JOSE C ET AL: "In vivo analysis of intron processing using splicing-dependent reporter gene assays." PLANT MOLECULAR BIOLOGY, Bd. 26, Nr. 6, 1994, Seiten 1785-1795, XP001073994 ISSN: 0167-4412 in der Anmeldung erw{hnt * |
COOPER THOMAS A ET AL: "The regulation of splice-site selection, and its role in human disease." AMERICAN JOURNAL OF HUMAN GENETICS, Bd. 61, Nr. 2, 1997, Seiten 259-266, XP002204084 ISSN: 0002-9297 * |
KANG SHIN-HONG ET AL: "Up-regulation of luciferase gene expression with antisense oligonucleotides: Implications and applications in functional assay development." BIOCHEMISTRY, Bd. 37, Nr. 18, 5. Mai 1998 (1998-05-05), Seiten 6235-6239, XP002204083 ISSN: 0006-2960 in der Anmeldung erw{hnt * |
NIEDZ R P ET AL: "GREEN FLUORESCENT PROTEIN: AN IN VIVO REPORTER OF PLANT GENE EXPRESSION" PLANT CELL REPORTS, SPRINGER VERLAG, DE, Bd. 14, Nr. 7, 1995, Seiten 403-406, XP000571886 ISSN: 0721-7714 & DATABASE EMBL / GENBANK [Online] U28417, 10. Juli 1995 (1995-07-10) NIEDZ ET AL.: "Cloning vector p35S-GFP" * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1302543A1 (fr) * | 2001-10-15 | 2003-04-16 | Bayer CropScience AG | Procédé d'épissage comme cible pour l'identification de préparations pharmaceutiques |
WO2003033711A1 (fr) * | 2001-10-15 | 2003-04-24 | Bayer Cropscience Ag | Epissage en tant que cible pour l'identification de nouveaux principes actifs |
GB2382577A (en) * | 2001-11-28 | 2003-06-04 | Talat Nasim | A reporter-based assay for assessing relative RNA processing activity in mammaliian cells |
WO2004028464A2 (fr) * | 2002-09-27 | 2004-04-08 | University Of North Carolina At Chapel Hill | Procedes et compositions servant a modifier l'epissage d'arn pre-messager |
WO2004028464A3 (fr) * | 2002-09-27 | 2004-07-08 | Univ North Carolina | Procedes et compositions servant a modifier l'epissage d'arn pre-messager |
Also Published As
Publication number | Publication date |
---|---|
DE10018464A1 (de) | 2001-10-18 |
AU2001242439A1 (en) | 2001-10-30 |
WO2001079853A3 (fr) | 2002-12-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Tabara et al. | A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans | |
DE69535428T2 (de) | Verfahren zum Auffinden von differentiel exprimierte Gene | |
Pankratz et al. | Krüppel requirement for knirps enhancement reflects overlapping gap gene activities in the Drosophila embryo | |
CN101437933B (zh) | 作为药物靶标的天然反义和非编码的rna转录物 | |
Kuhl et al. | Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER. | |
Dworkin et al. | Functions of maternal mRNA in early development | |
Himi et al. | Differential localization of SCG10 and p19/stathmin messenger RNAs in adult rat brain indicates distinct roles for these growth-associated proteins | |
Sbisa et al. | The complete and symmetric transcription of the main non coding region of rat mitochondrial genome: in vivo mapping of heavy and light transcripts | |
CN103173447B (zh) | 新的β-肌动蛋白和RPS21 启动子及其应用 | |
DE60126115T2 (de) | Oberflächentransfektion und expressionsverfahren | |
Kou et al. | Transcriptional activation of the insulin-like growth factor-II gene during myoblast differentiation. | |
WO2001079853A2 (fr) | Systeme de test pour caracteriser des modulateurs du processus d'epissage des arn messagers dans des cellules vivantes (in vivo), sa realisation et son utilisation | |
US20050164282A1 (en) | Synthetic cell-or tissue-specific transcriptional regulatory regions | |
DE60009530T2 (de) | Genetische toxizitätsmarker, herstellung und verwendung | |
Fukunaga et al. | Novel kruppel‐like factor is induced by neuronal activity and by sensory input in the central nervous system of the terrestrial slug Limax valentianus | |
Komine et al. | A novel type of non-coding RNA expressed in the rat brain | |
Wieczorek | Regulation of alternatively spliced alpha-tropomyosin gene expression by nerve extract. | |
DE69734143T2 (de) | Neue methoden zur charakterisierung von verbindungen, welche die stf-1 expression in inselzellen der bauchspeicheldrüse stimuliert | |
DE112004002318T5 (de) | Transkriptionsregulatoren und Verfahren dazu | |
DE19740578A1 (de) | Verfahren zur Identifizierung von Zielgenen von Transkriptionsfaktoren | |
DE19710159A1 (de) | In vitro Transkriptionsverfahren zum Screening von Naturstoffen und anderen chemischen Substanzen | |
WO2000052201A2 (fr) | Systeme de test pour mettre en evidence une reaction d'epissure et utilisation dudit systeme | |
DE19838837A1 (de) | Zellspezifische Promoter des Entkopplungsproteins 3 | |
DE69918260T2 (de) | Methoden zur Identifizierung und Validierung von funktionellen Zielen mittels Intrameren oder in vivo Selektion | |
DE102004063265B4 (de) | Verfahren zur Bestimmung der Funktion von Nukleinsäuresequenzen und den dadurch kodierten Expressionsprodukten |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE 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 NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE 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 NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |