WO1999050401A1 - Procede pour detecter les changements de l'expression genique par le traitement avec un compose de test - Google Patents

Procede pour detecter les changements de l'expression genique par le traitement avec un compose de test Download PDF

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Publication number
WO1999050401A1
WO1999050401A1 PCT/JP1999/001574 JP9901574W WO9950401A1 WO 1999050401 A1 WO1999050401 A1 WO 1999050401A1 JP 9901574 W JP9901574 W JP 9901574W WO 9950401 A1 WO9950401 A1 WO 9950401A1
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WIPO (PCT)
Prior art keywords
cdna
labeled
gene
compound
specific
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PCT/JP1999/001574
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English (en)
Japanese (ja)
Inventor
Masaaki Muramatsu
Hiroshi Wakao
Rika Wakao
Kazuhiro Yano
Teruhisa Noguchi
Akira Suyama
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Helix Research Institute
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Publication of WO1999050401A1 publication Critical patent/WO1999050401A1/fr

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    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention relates to a method for detecting a change in gene expression due to a compound treatment, a method for screening a gene whose expression level changes by a compound treatment, and a method for screening a compound that changes the expression level.
  • Methods for detecting differences in gene expression between cells (or tissues) in two different states include a differential hybridization method (Gene 145: 313-314 (1994) Cloning and sequence analysis of The human S NAP25 cDNA. N. Zhao, H. Hashida, N. Takahashi & Y. Sakaki) and the differential display (DD) method (FEBS Lett 351: 231-236 (1994) Fluore scent differential display: arbitrarily primed. RT-PCR fingerprinting on an automated DNA sequencer. T. I to, K. Kito, N. Adati, Y. Mitsui, H. Hagiwara & Y. Sakaki) have been developed.
  • the present invention provides a method for simply and efficiently detecting a change in gene expression due to compound treatment, a method for simply and efficiently screening for a gene whose expression level changes due to compound treatment, and a method for simple and efficient Another object of the present invention is to provide a method for screening a compound that changes the expression level of a gene.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, extracted intracellular mRNA from cells treated with a specific compound and cells not treated (control). By comparing the configurations, they found that it was possible to detect changes in the state of gene expression in cells due to treatment with a specific compound.
  • the present inventors have proposed a method for detecting a difference in the expression level of a specific gene in different cells using fluorescence (Nature Biotechnology 14: 1675-1680 (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. David J. Lockhart, Helin Dong, Michael C. Bynel, Maximillian T. Follettiel, Michael V. Gallol, Mark S.
  • the present inventors have shown that using this detection method, it is possible to screen for a gene whose expression is changed by treatment with a specific compound or to screen a compound which changes the expression of a specific gene. That is, the present invention provides a method for detecting a change in gene expression due to a compound treatment by a change in fluorescent color, a method for screening a gene whose expression level changes by a specific compound treatment using the same, and a method for screening the gene. Regarding a method for screening a compound that changes the expression level of a gene, more specifically,
  • the present invention provides: (a) a step of isolating “mRNA in a cell treated with a specific compound” and “mRNA in a cell not treated with a specific compound”; Reverse transcription of each mRNA to obtain cDNA group, (c) applying different fluorescent labeling to each cDNA group, (d) identifying each labeled cDNA group (E) hybridizing the difference in the amount of cDNA that hybridizes to a specific probe DNA in each of the labeled cDNA groups to the specific probe DNA.
  • a method of detecting a change in the state of gene expression of a cell by the specific compound which comprises a step of detecting the fluorescent compound by a fluorescent color emitted from the cDNA.
  • mRNA in a cell treated with a specific compound and “mRNA in a cell not treated with a specific compound” are first isolated.
  • the compound used in the detection method of the present invention is not particularly limited, and it is possible to use a desired compound for which a change in gene expression due to its action is desired to be detected.
  • the compound may be of natural origin or artificially synthesized.
  • the cells subjected to the compound treatment there is no particular limitation on the cells subjected to the compound treatment, and for example, various animal and plant cells, microbial cells, and the like can be used.
  • animal cells include 3T3L1 cells, COS cells, NT-2 cells, Ba / F3 cells and the like.
  • the present invention The cells to be treated with the compound include not only cultured cells but also cells in animals and plants.
  • the treatment of the compound with respect to the cells can be performed by, for example, adding cultured cells or microorganisms of animals and plants to the medium in which they are cultured.
  • it can be administered by an administration method known to those skilled in the art, such as intravascular administration, intraperitoneal administration, intraventricular administration, and oral administration.
  • MRNA expressed in the cells treated with the compound can be obtained by a method known to those skilled in the art, for example, a method using a guanidinium method or an oligo dT column (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons).
  • reverse transcription is performed on mRNA isolated from cells treated with the test compound and control cells to obtain a cDNA group.
  • mRNA isolated from cells treated with the test compound and control cells generally, the whole mRNA in the isolated cell is used, but mRNA fractionated by various methods can be used according to the purpose.
  • Reverse transcription of mRNA is performed, for example, by using oligo dT as a primer. (1987) Publish. John Wiley & Sons).
  • the fluorescent label is not particularly limited.
  • rhodamine, FITC® Texas Red, Cy2, Cy3, Cy5, Cy7 and the like can be used.
  • Fluorescent labels include, for example, nick transfusion (for example, kits manufactured by GIBC0-BRL) and random primer-DNA labeling (for example, kits manufactured by GIBC0-BRL) are available.
  • nick transfusion for example, kits manufactured by GIBC0-BRL
  • random primer-DNA labeling for example, kits manufactured by GIBC0-BRL
  • the labeled cDNAs are then hybridized to a particular probe DNA.
  • the specific probe DNA to be used it is possible to use a probe DNA for a desired gene for which a change in expression due to compound treatment is to be detected. There are no restrictions. Hybridization between a labeled cDNA group and a specific probe DNA can be performed by a method known to those skilled in the art (for example, “Current protocols in Molecular Biology edition. Ausubel et al. (1987) Publish. John Wiley & Sonsj or“ (1995) Science 270, 467-470 ").
  • the difference in the amount of hybridized cDNA relative to the specific probe DNA in each of the labeled cDNA groups is detected by the fluorescent color of the cDNA hybridized to the specific probe DNA.
  • rhodamine is included in the DNA that hybridizes to the specific probe DNA.
  • Fluorescent color can be detected using, for example, a scanning laser confocal microscope (Olympus 1X70 type), FMB I0 (Hitachi Soft).
  • a large number of probe DNAs can be used.
  • a chip in which a large number of probe DNAs are bonded on a substrate hereinafter, referred to as a “genome chip” (see US Pat. No. 5,405,783)
  • a gene chip in which a large number of probe DNAs are bonded on a substrate
  • the change in expression of can be easily and efficiently detected.
  • detection method using fluorescent color for example, hybridization is performed on a large number of probe DNAs fixed on a base glass of a genome chip, and the fluorescence intensity of cDNA hybridized to each probe is determined by a scanning laser. By detecting with a microscope, it is possible to detect many different amounts of cDNA at the same time.
  • the detection method using a genome chip has the advantages that iterative operations can be performed easily and inexpensively. If a fluorescent color indicating a difference in the amount of cDNA is detected by the detection method of the present invention, a probe DNA corresponding to the cDNA is selected, and a gene corresponding to the probe DNA is selected. Genes whose expression level changes in cells can be screened.
  • the present invention also provides: (a) a step of separately isolating “mRNA in a cell treated with a specific compound” and “mA in a cell not treated with a specific compound”; (B) a step of performing reverse transcription on each of the isolated mAs to obtain cDNA groups; (c) a step of applying different fluorescent labels to the respective cDNA groups; and (d) a step of applying different fluorescent labels.
  • Genes whose expression level in cells changes depending on the compound Regarding offspring screening method In the screening method of the present invention, a desired gene (for example, an increase in the expression level of a certain value or more was observed) among the genes whose expression was changed by the method of the present invention was detected. Gene or a gene whose expression level has been reduced by more than a certain value). If the hybridized probe DNA is known, the selected gene can be identified as having the same nucleotide sequence as the probe DNA or having a similar nucleotide sequence.
  • the gene is inserted into an appropriate vector, and this vector is introduced into a host cell to obtain the obtained form.
  • COS cells and NT-2 cells use “pME18S vector one” (Mol Ce 11 Biol 8: 466-72 (1988) SR alpha promoter: an efficient and versati le mammal ian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cel l leukemia virus type 1 long terminal repeat.Y. Takebe, M. Seiki, J. Fuj isawa, P. Hoy, K.
  • PAT Vector-1 Yukara
  • BacPac Vector-1 BacPac Vector-1 (Clontech) are preferably used for insect cells.
  • Vectors can be introduced into host cells in a conventional manner, for example, by the electroporation method for Escherichia coli, the ribofectamine (GIBC0-BRL) method for COS cells, and the baculovirus infection for insect cells. It can be performed by the method used.
  • Purification of the recombinant protein from the transformant that has expressed the recombinant protein can be carried out by appropriately combining various conventional methods such as chromatography, electrophoresis, and gel filtration depending on the properties of the protein. It is also possible to purify columns by the His-tag method or HA-tag method.
  • the test compound obtained by treating the cells from which the cDNA has been isolated can be selected to be intracellular.
  • Compounds that alter the expression of a particular gene can be screened.
  • the present invention also provides (a) a step of separately isolating mRNA j in a cell treated with a test compound and mA in a cell not treated with the test compound, (B) performing reverse transcription on each of the isolated mRNAs to obtain a group of cDNAs; (c) applying different fluorescent labels to the respective groups of cDNAs; A step of hybridizing the labeled cDNA group to the probe DNA; (e) the difference in the amount of cDNA that hybridizes to the probe DNA in each of the labeled cDNA groups is determined by the amount of the cDNA hybridized to the probe DNA.
  • the present invention relates to a method for screening a compound to be converted.
  • a compound library is used as a compound for treating cells.
  • the compound library can be prepared by methods known to those skilled in the art (for example, Applicatio n of combinatorial l ibrary methods in cancer Research and Drug Disco very. Lann, KS (1997) Anticancer Drug Desl2: 145-167). It is possible.
  • the isolated compound will Can be a candidate for a therapeutic drug.
  • FIG. 1 is a diagram showing the results of detecting a gene whose expression is induced in 3T3L1 cells treated with retinoic acid and FCS.
  • On the nylon membrane 48 clones, each of which was considered to be specific for retinoic acid obtained by differential screening and 3T3L1 cells treated with FCS, were bound two by two.
  • Oligo DNAs (Table 1) of sequences 1 to 6 (each described in SEQ ID NO: 1 to 6) were bound on a glass substrate by the following photolithographic method (Hermanson, G.T., et al.). Immobilized Affinity Ligand Techniques ", Academic Press, Inc., San Diego, CA (1992); Robertson, SA, et al., J. Am. Chem. Soc., 113, 2722-2729 (1991)). table 1
  • the surface of a slide glass (ATSUNAMI, S0313) was treated with 0.05M HC1 for 1 hour, washed several times with ultrapure water, and then immersed in ethanol.
  • the slide glass immersed in the solution C was put together with the container in a desiccator overnight and replaced with nitrogen gas, and the aminosilanization reaction was performed overnight.
  • the slide was washed several times with ethanol, and further washed with ultrapure water for 2 hours. After that, it was dried under vacuum in a dessicator (see Hermanson, GT et al., Academic Press, Inc., San Diego, California (1992) j). Dimethoxy-2-nitrobenzyl chloride mouth formate) 10 mg / ml (dissolved in tetrahydrofuran) was placed on the silanized plate and reacted for 10 minutes, then washed with THF and ultrapure water in that order, and then nitrogen gas was added. (See literature "Robertson, SA et al., J. Am. Chem. So 113, 2722-2729 (1991)").
  • ImM Potassium acetate (pH 4.5) was removed. ⁇ Mounted on the slide, irradiated with light from a mercury lamp under a microscope (330-380 fill light) for 30 minutes, and decapped. After the reaction, the resultant was washed several times with ultrapure water. The slide was coated with 10 mM DSS (Disuccinimidyl suberate), reacted for 10 minutes, washed, and dried by blowing nitrogen gas. Further, 0.2 ⁇ 1 of oligo DNA (U ⁇ 00 ⁇ M) was placed on the surface and reacted for 3 minutes.
  • DSS Disuccinimidyl suberate
  • Cultured cells Ba / F3 were supplemented with 1 0M of 12-0-tetradecanoylformol-13-acetate (TPA) and DMS0 as a control (these were compared with those in groups (a) and (b)). Do). Thirty minutes later, the cells were collected, and mRNA was prepared using “Quick prep micro mRNA purification kit j (manufactured by Pharmacia). The 0D value of mRNA obtained from the groups (a) and (b) was measured. After unifying the concentrations, reverse transcriptase reaction was performed using "cDNA first strand kit" (Pharmacia) to prepare cDNA.
  • Fluorescently labeled cDNA was detected using a glass plate reader.
  • This reader is a combination of an Olympus epi-illumination fluorescence microscope, a holder and an STI high-sensitivity camera (Hamamatsu Photonics) for detection.
  • a green blue wavelength generated by FluorX was observed.
  • yellow interference wavelengths generated from FluorX and Cy-S were observed at the site where the sequences 4, 5, and 6, which are probes for detecting actin, were bound. From this, it was found that the amount of /?-Actin! NRNA was the same in the samples of the groups (a) and (b), but the amount of C-fos mRNA was significantly higher in the group A.
  • the present invention provides a method for detecting a change in gene expression due to a compound treatment, a method for screening a gene whose expression level is changed by a compound treatment, and a method for screening a compound whose expression level is changed.
  • a different fluorescent label is used for each cDNA group used for detecting a change in expression, it is possible to easily detect a change in gene expression using the fluorescent color as an index.
  • a genome chip to which a large number of probe DNAs are linked is used, it is possible to efficiently detect a change in gene expression.

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  • Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne un procédé pour détecter les changements de l'état d'expression génique provoqués par le traitement avec un composé spécifique, ledit procédé consistant à extraire les ARNm intracellulaires depuis les cellules qui ont été traitées avec le composé en question et les cellules non traitées (cellules de contrôle), respectivement, et à comparer la constitution des ces ARNm. Selon ce procédé, on peut mesurer les différences au niveau des contenus d'ADNc hybridés avec un capteur spécifique par le marquage au moyen de substances fluorescentes des ADNc obtenus à partir d'ARNm isolés, par le mélangeage de ces ADNc dans des proportions déterminées et par l'hybridation de ces ADNc au moyen du capteur. Ce procédé permet de sélectionner un gène dont l'expression est modifiée par le traitement avec un composé spécifique ou de sélectionner un composé capable de modifier l'expression d'un gène spécifique.
PCT/JP1999/001574 1998-03-27 1999-03-26 Procede pour detecter les changements de l'expression genique par le traitement avec un compose de test WO1999050401A1 (fr)

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JP10/100096 1998-03-27
JP10009698 1998-03-27

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033228A2 (fr) * 1999-11-03 2001-05-10 Oncotech, Inc. Methodes de prognostic et de diagnostic du cancer
WO2001053534A3 (fr) * 2000-01-21 2002-04-18 Curagen Corp Procede d'identification de la fonction d'un agent d'essai
US6998234B2 (en) 2000-11-03 2006-02-14 Oncotech, Inc. Methods for cancer prognosis and diagnosis relating to tumor vascular endothelial cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05192199A (ja) * 1991-09-23 1993-08-03 Pfizer Inc 細胞中の特異的ｍＲＮＡおよびＤＮＡの検出法
JPH10304880A (ja) * 1997-03-07 1998-11-17 Shiseido Co Ltd 核酸の測定方法及びそのための試薬
JPH1146772A (ja) * 1997-07-31 1999-02-23 Masayuki Yamamoto 新規スクリーニング方法及び新規非ヒト動物の作出方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05192199A (ja) * 1991-09-23 1993-08-03 Pfizer Inc 細胞中の特異的ｍＲＮＡおよびＤＮＡの検出法
JPH10304880A (ja) * 1997-03-07 1998-11-17 Shiseido Co Ltd 核酸の測定方法及びそのための試薬
JPH1146772A (ja) * 1997-07-31 1999-02-23 Masayuki Yamamoto 新規スクリーニング方法及び新規非ヒト動物の作出方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"TAMPAKU KOUGAKU SERIES TAMPAKU KOUGAKU NI OKERU IDENSHI HATSUGEN NOCHOUSETSU", TAMPAKU KOUGAKU SERIES, XX, XX, 20 May 1991 (1991-05-20), XX, pages 129 - 131, XP002920084 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033228A2 (fr) * 1999-11-03 2001-05-10 Oncotech, Inc. Methodes de prognostic et de diagnostic du cancer
WO2001033228A3 (fr) * 1999-11-03 2002-11-14 Oncotech Inc Methodes de prognostic et de diagnostic du cancer
US6511806B1 (en) 1999-11-03 2003-01-28 Oncotech, Inc. Methods for cancer prognosis and diagnosis
US7323300B2 (en) 1999-11-03 2008-01-29 Oncotech, Inc. Methods for cancer prognosis and diagnosis
WO2001053534A3 (fr) * 2000-01-21 2002-04-18 Curagen Corp Procede d'identification de la fonction d'un agent d'essai
US6998234B2 (en) 2000-11-03 2006-02-14 Oncotech, Inc. Methods for cancer prognosis and diagnosis relating to tumor vascular endothelial cells

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