WO1996012011A1 - Lignee cellulaire a reporteurs - Google Patents

Lignee cellulaire a reporteurs Download PDF

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Publication number
WO1996012011A1
WO1996012011A1 PCT/EP1995/004032 EP9504032W WO9612011A1 WO 1996012011 A1 WO1996012011 A1 WO 1996012011A1 EP 9504032 W EP9504032 W EP 9504032W WO 9612011 A1 WO9612011 A1 WO 9612011A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression
gene
reporter gene
die
cells
Prior art date
Application number
PCT/EP1995/004032
Other languages
English (en)
Inventor
Stefan Nilsson
Original Assignee
Karo Bio Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Karo Bio Ab filed Critical Karo Bio Ab
Priority to AU38420/95A priority Critical patent/AU3842095A/en
Priority to JP8512938A priority patent/JPH10509584A/ja
Publication of WO1996012011A1 publication Critical patent/WO1996012011A1/fr

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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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention relates to a reporter cell line and is particularly, though not exclusively,
  • Glucocorticoids exert profound effects on the inflammatory and immune responses. They
  • Agonistic glucocorticoids inhibit transcription of these genes by interfering with the API transcription factor, the
  • GR hormone activated glucocorticoid receptor
  • Glucocorticoid induces a combination of high levels of parathyroid hormone, PTH, and
  • a cell line including a
  • first reporter gene arranged to express an assayable first gene product, expression of the
  • first reporter gene being mediated by a transcription factor
  • second reporter gene
  • the invention provides a cell line including a first reporter gene arranged to express an assayable first gene product, expression of d e first reporter gene being AP-1 mediated, and a second reporter gene arranged to express a second assayable gene
  • the present invention provides a convenient one cell line-based assay system which
  • the cell line allows the testing of two activities in a single cell whereas previously two cell lines would be required.
  • the cell line is derived from human, more preferably HeLa cells.
  • One of the reporter genes may be arranged to express an alkaline phosphatase such as
  • human placental alkaline phosphatase and the other reporter gene may be arranged to
  • a method of testing a compound for glucocorticoid-like activity comprising providing cells in accordance with
  • API activity may be stimulated by TPA or EGF.
  • Fig. 1A shows the nucleotide sequence of d e five API responsive elements inserted in tandem upstream of the mouse mammary tumour virus long terminal repeat (MMTN
  • ALP phosphatase
  • Fig IB shows the ALP reporter vector 5AP ⁇ T-ALP containing the nucleotide sequence
  • Fig 2 shows the glucocorticoid receptor controlled reporter vector MMTV-hGH2
  • hGH human growth hormone polypeptide
  • Fig.3 illustrates the effect of dexamethasone on API -dependent ALP expression and GR dependent hGH reporter gene transactivation, respectively and the effects of Epidermal
  • EGF Growth Factor
  • Fig. 4 illustrates the effect of different corticosteroids on glucocorticoid receptor dependent transactivation of hGH expression and repression of AP-1 -dependent ALP
  • HeLa tk " cells were initially stably transformed using conventional techniques with a
  • reporter vector 5APNT-ALP comprising five API response elements arranged in tandem
  • Figs. 1A and B show that secretion of the ALP protein into the medium is indicative of AP-1 mediated transcription.
  • Fig. 1A shows that secretion of the ALP protein into the medium is indicative of AP-1 mediated transcription.
  • MMTV terminal repeat
  • alkaline phosphatase ALP
  • 5APNT-ALP alkaline phosphatase
  • ALP is underlined.
  • the resulting cells were termed GRAP cells.
  • the level of ALP reporter protein expressed and secreted into d e medium can be determined indirectly by a chemiluminescence assay.
  • the facility to use a chemiluminescence assay The facility to use a chemiluminescence assay.
  • me ALP based reporter assay particularly convenient to use compared to intracellular reporters such as chloramphenicol acetyltransferase (CAT) and luciferase
  • CAT chloramphenicol acetyltransferase
  • the reporter vector MMTV-hGH2 comprises a GR-regulated promoter (MMTV) fused to a reporter
  • hGH human growth hormone
  • the level of glucocorticoid- induced hGH expression is determined immunologically by a Delfia assay (Wallac OY,
  • the GRAPF cells contain two exogenous transcription units whose reporter genes
  • the ALP transcription unit is controlled by, and responds to,
  • d e hGH transcription unit responds to ligand activated GR resulting in an
  • TPA or EGF induced API activity indirectly determined by increased ALP
  • glucocorticoids as well as dieir potency as inhibitors of API -dependent transcription, indirectly reflecting their potency as anti-inflammatory drugs.
  • the expression of the ALP reporter protein in these cells is controlled by the API transcription factor, induced by phorbol esters such as TPA or die growth factor EGF.
  • inflammatories was demonstrated using die synthetic glucocorticoid dexamethasone.
  • GRAPF cells were seeded in 96-well microtiter plates in
  • the relative levels of ALP expressed were determined by a chemiluminescent assay as follows: a lO ⁇ l aliquot of die cell culture medium was mixed wid 200 il of assay buffer (lOmM die ianolamine pH 10.; ImM MgCl 2 and 0.5mM AMPPD) in accordance with
  • Luminoskan Labsy stems, Finland The setting of the Luminoskan luminometer was
  • the alkaline phosphatase activity is expressed in light units (LU).
  • hGH expression was monitored immunologically with the Delfia assay mentioned above.
  • the cells were treated wid lOOng/ml EGF to induce the /as and jun genes (and
  • reporter expression is induced in a hormone-dependent manner irrespective of the
  • the reporter cell line and process of die invention may be used to test a wide variety of

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On décrit une lignée cellulaire comprenant un premier gène reporteur conçu pour exprimer un premier produit génique dosable, l'expression de ce premier gène reporteur étant induite par AP-1, ainsi qu'un second gène reporteur conçu pour exprimer un second produit génique dosable, l'expression de ce second gène reporteur étant induite par le récepteur glucocorticoïde (GR). On décrit également un procédé de recherche d'un composé pour l'activité de type glucocorticoïde de celui-ci, consistant à fournir des cellules selon le premier aspect de l'invention, à stimuler l'activité AP-1 dans ces cellules, à mettre celles-ci en contact avec le composé puis à contrôler l'expression des produits des premier et second gènes.
PCT/EP1995/004032 1994-10-14 1995-10-12 Lignee cellulaire a reporteurs WO1996012011A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU38420/95A AU3842095A (en) 1994-10-14 1995-10-12 Reporter cell line
JP8512938A JPH10509584A (ja) 1994-10-14 1995-10-12 リポーター細胞株

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9420735.4 1994-10-14
GB9420735A GB9420735D0 (en) 1994-10-14 1994-10-14 Reporter cell line

Publications (1)

Publication Number Publication Date
WO1996012011A1 true WO1996012011A1 (fr) 1996-04-25

Family

ID=10762844

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1995/004032 WO1996012011A1 (fr) 1994-10-14 1995-10-12 Lignee cellulaire a reporteurs

Country Status (4)

Country Link
JP (1) JPH10509584A (fr)
AU (1) AU3842095A (fr)
GB (1) GB9420735D0 (fr)
WO (1) WO1996012011A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE29916160U1 (de) 1999-09-14 2000-03-09 Cardiogene Gentherapeutische Systeme AG, 40699 Erkrath Modulation der Transkription von Genen in vaskulären Zellen
WO2000023581A1 (fr) * 1998-10-22 2000-04-27 Signal Pharmaceuticals, Inc. Systeme a double rapporteur et techniques d'utilisation
KR20030046896A (ko) * 2001-12-07 2003-06-18 학교법인 포항공과대학교 당질코르티코이드에 의해 발현이 조절되는 재조합 리포터유전자를 가지는 형질전환 세포주 및 이를 이용한당질코르티코이드 유사물질 및 저해물질의 생물학적검색방법
US6599741B1 (en) 1999-09-14 2003-07-29 Avontec Gmbh Modulating transcription of genes in vascular cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005447A1 (fr) * 1990-09-21 1992-04-02 The Salk Institute For Biological Studies ANTAGONISME FONCTIONNEL ENTRE LA PROTO-ONCOPROTEINE c-JUN ET DES RECEPTEURS D'HORMONES
WO1994001584A1 (fr) * 1992-07-06 1994-01-20 President And Fellows Of Harvard College Procedes et necessaires de diagnostic pour determiner la toxicite utilisant des promoteurs de stress bacteriens fusionnes a des genes rapporteurs
WO1994017208A1 (fr) * 1993-01-21 1994-08-04 President And Fellows Of Harvard College Methodes et trousses de diagnostic faisant appel aux promoteurs de stress des mammiferes pour determiner la toxicite d'un compose
WO1994023041A2 (fr) * 1993-04-02 1994-10-13 Ribogene, Inc. Procede d'inactivation selective de replication virale

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005447A1 (fr) * 1990-09-21 1992-04-02 The Salk Institute For Biological Studies ANTAGONISME FONCTIONNEL ENTRE LA PROTO-ONCOPROTEINE c-JUN ET DES RECEPTEURS D'HORMONES
WO1994001584A1 (fr) * 1992-07-06 1994-01-20 President And Fellows Of Harvard College Procedes et necessaires de diagnostic pour determiner la toxicite utilisant des promoteurs de stress bacteriens fusionnes a des genes rapporteurs
WO1994017208A1 (fr) * 1993-01-21 1994-08-04 President And Fellows Of Harvard College Methodes et trousses de diagnostic faisant appel aux promoteurs de stress des mammiferes pour determiner la toxicite d'un compose
WO1994023041A2 (fr) * 1993-04-02 1994-10-13 Ribogene, Inc. Procede d'inactivation selective de replication virale

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALKSNIS ET AL.: "High level expression of functional full length and truncated glucocorticoid receptor in chinese hamster ovary cells", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 16, 1991, MD US, pages 10078 - 10085, XP002000023 *
JONAT ET AL.: "Antitumor promotion and atiinflamation: downmodulation of AP-1 (FOS/JUN) activity by glucocorticoid hormone", CELL, vol. 62, 1990, NA US, pages 1189 - 1204, XP002000021 *
SCHÜLE ET AL.: "Functional antagonism between oncoprotein c-Jun and the GCR", CELL, vol. 62, 1990, NA US, pages 1217 - 1226, XP002000022 *
YANG-YEN ET AL.: "Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction", CELL, vol. 62, 1990, NA US, pages 1205 - 1215, XP002000020 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023581A1 (fr) * 1998-10-22 2000-04-27 Signal Pharmaceuticals, Inc. Systeme a double rapporteur et techniques d'utilisation
DE29916160U1 (de) 1999-09-14 2000-03-09 Cardiogene Gentherapeutische Systeme AG, 40699 Erkrath Modulation der Transkription von Genen in vaskulären Zellen
US6599741B1 (en) 1999-09-14 2003-07-29 Avontec Gmbh Modulating transcription of genes in vascular cells
US7186556B2 (en) 1999-09-14 2007-03-06 Avontec Gmbh Modulating transcription of genes in vascular cells
KR20030046896A (ko) * 2001-12-07 2003-06-18 학교법인 포항공과대학교 당질코르티코이드에 의해 발현이 조절되는 재조합 리포터유전자를 가지는 형질전환 세포주 및 이를 이용한당질코르티코이드 유사물질 및 저해물질의 생물학적검색방법

Also Published As

Publication number Publication date
AU3842095A (en) 1996-05-06
JPH10509584A (ja) 1998-09-22
GB9420735D0 (en) 1994-11-30

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