WO2002012474A2 - Procede pour la selection concernant des cellules hotes a sequences d'adn eliminees - Google Patents

Procede pour la selection concernant des cellules hotes a sequences d'adn eliminees Download PDF

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Publication number
WO2002012474A2
WO2002012474A2 PCT/DE2001/002511 DE0102511W WO0212474A2 WO 2002012474 A2 WO2002012474 A2 WO 2002012474A2 DE 0102511 W DE0102511 W DE 0102511W WO 0212474 A2 WO0212474 A2 WO 0212474A2
Authority
WO
WIPO (PCT)
Prior art keywords
recombinase
dna sequence
host cells
inducible promoter
peptide
Prior art date
Application number
PCT/DE2001/002511
Other languages
German (de)
English (en)
Other versions
WO2002012474A3 (fr
Inventor
Lorenz Bülow
Klaus Düring
Original Assignee
Mpb Cologne Gmbh
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 Mpb Cologne Gmbh filed Critical Mpb Cologne Gmbh
Priority to AU2001279561A priority Critical patent/AU2001279561A1/en
Publication of WO2002012474A2 publication Critical patent/WO2002012474A2/fr
Publication of WO2002012474A3 publication Critical patent/WO2002012474A3/fr

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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems

Definitions

  • he has a DNA which is under the control of an inducible promoter and which codes for a toxic protein or peptide next to the desired DNA sequence within of the 5 'and 3' recombination DNA sequences, whereby the toxic protein or peptide can be expressed after induction of the promoter, provided that no or incomplete elimination via the 5 'and 3' recombination DNA Sequences have taken place, killing those host cells in which d The controlled elimination of the desired DNA sequence has not taken place.
  • an inducible promoter codes for a toxic protein or peptide next to the desired DNA sequence within of the 5 'and 3' recombination DNA sequences, whereby the toxic protein or peptide can be expressed after induction of the promoter, provided that no or incomplete elimination via the 5 'and 3' recombination DNA Sequences have taken place, killing those host cells in which d
  • the controlled elimination of the desired DNA sequence has not taken place.
  • the present invention thus relates to a method for selection on host cells in which there is a controlled elimination of a desired DNA sequence and for the destruction of host cells in which this does not take place via the expression of a toxic protein or peptide, the method being characterized in that is that in step (I)
  • step (b) the host cells are transformed with a recombinase-encoding recombinase-encoding DNA sequence under the control of an inducible promoter under conditions under which the inducible promoter is repressed, the inducible promoters of (a) and ( b) are not the same or have the same inductors.
  • step (II) the desired DNA sequence is eliminated via the expression of the recombinase by activating the inducible promoter of (b), and in step (III) the host cells in which step (II) is not or not completely carried out is done via the expression of the toxic protein or peptide by activating the inducible promoter of (a).
  • the plants which can be used in the process according to the invention can in principle be plants of any plant species, i.e. both monocot and dicotyledonous plants. It is preferably gramineae, chenopodia, legumes, brassicaceae, solanaceae, algae, moss and mushrooms, in particular wheat, barley, rice, maize, sugar beet, sugar cane, rapeseed, mustard, turnip, flax, pea, bean, lupine, Tobacco and potato.
  • the parts of plants desired for the elimination of the corresponding DNA sequence principally relate to any part of the plant, e.g. Plant cells, at least propagation material and harvest products of these plants, e.g. Fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc.
  • the recombinase is expressed by activating the promoter of (b), as a result of which a site-specific recombination takes place between the 5 'and 3' recombination DNA sequences and thus an excision of the DNA sequence to be eliminated.
  • the DNA sequence to be eliminated is preferably between a promoter and a gene and prevents the transcription and / or translation of this gene. It is only through the excision of the DNA sequence to be eliminated that the desired gene is located directly downstream of the promoter, which initiates the transcription and translation and thus the foreign protein biosynthesis.
  • inducible promoters suitable for the process according to the invention and the inducers (or inhibitors) (gaseous, liquid or solid, volatile compounds) which are suitable for this purpose.
  • inducers gaseous, liquid or solid, volatile compounds
  • gases gaseous, liquid or solid, volatile compounds
  • suitable gaseous inductors and conditions for the most efficient exchange of the gas phase are also known to the person skilled in the art. It reference is made to the Anaerocult system (Merck, Darmstadt, Germany), which creates an anaerobic environment in which oxygen is bound and C0 2 is released. In this system, the GapC4 promoter from maize is induced anaerobically by the CO 2 atmosphere (Bülow, L. et al., Molecular Plant-Microbe Interactions (1999), 182-188).
  • the inducible promoter suitable for the method according to the invention is preferably an anaerobic condition, a chemical or a physically inducible promoter, such as the GapC4 promoter or the Adhl promoter, and the induction takes place via a change in the gas phase in this way that it is an oxygen deprivation; see also the explanations above.
  • the anaerobically inducible GapC4 promoter (DE 195 47 272), e.g. in connection with harvested transgenic plant tissue, e.g. transgenic potato tubers.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé pour la sélection concernant des cellules hôtes dans lesquelles une élimination commandée d'une séquence d'ADN désirée a lieu et pour la destruction de cellules hôtes dans lesquelles cette élimination commandée n'a pas lieu, au moyen de l'expression d'une protéine ou d'un peptide toxique. Le procédé selon l'invention est caractérisé en ce que, à l'étape (I), les cellules hôtes sont transformées à l'aide (a) de la séquence d'ADN à éliminer ultérieurement, flanquée en 5' et 3' de séquences d'ADN de recombinaison un ADN codant pour la protéine ou le peptide toxique et sous le contrôle d'un promoteur inductible étant également présent entre les séquences d'ADN de recombinaison , dans des conditions dans lesquelles le promoteur inductible est réprimé, et les cellules hôtes sont transformées à l'aide (b) d'une séquence d'ADN reconnaissant ces séquences d'ADN de recombinaison, codant pour une recombinase et sous le contrôle d'un promoteur inductible, dans des conditions dans lesquelles le promoteur inductible est réprimé, les promoteurs inductibles de (a) et (b) étant différents ou n'ayant pas les mêmes inducteurs. A l'étape (II) a lieu l'élimination de la séquence d'ADN désirée au moyen de l'expression de la recombinase par activation du promoteur inductible de (b). A l'étape (III) se produit la destruction des cellules hôtes dans lesquelles l'étape (II) n'a pas été exécutée ou a été exécutée de manière incomplète, au moyen de l'expression de la protéine ou du peptide toxique par activation du promoteur inductible de (a). Selon un mode de réalisation préféré de l'invention, la recombinase est une protéine hybride recombinase-domaine de liaison du ligand (Rec-LBD). On préfère également que la séquence d'ADN désirée code pour un gène marqueur ou agisse comme séquence d'arrêt de transcription ou de translation. L'invention concerne également les vecteurs et les cellules hôtes contenant les séquences précédentes (a) et (b) ; il s'agit de préférence de cellules végétales transgéniques ou de plantes transgéniques.
PCT/DE2001/002511 2000-08-03 2001-07-04 Procede pour la selection concernant des cellules hotes a sequences d'adn eliminees WO2002012474A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001279561A AU2001279561A1 (en) 2000-08-03 2001-07-04 Method of selection in host cells with eliminated dna sequences

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2000138573 DE10038573A1 (de) 2000-08-03 2000-08-03 Verfahren zur Selektion auf Wirtszellen mit eliminierten DNA-Sequenzen
DE10038573.7 2000-08-03

Publications (2)

Publication Number Publication Date
WO2002012474A2 true WO2002012474A2 (fr) 2002-02-14
WO2002012474A3 WO2002012474A3 (fr) 2002-04-25

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2001/002511 WO2002012474A2 (fr) 2000-08-03 2001-07-04 Procede pour la selection concernant des cellules hotes a sequences d'adn eliminees

Country Status (3)

Country Link
AU (1) AU2001279561A1 (fr)
DE (1) DE10038573A1 (fr)
WO (1) WO2002012474A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7183097B1 (en) 1998-05-07 2007-02-27 Universite Libre De Bruxelles Cytotoxin-based biological containment
WO2009150441A1 (fr) * 2008-06-13 2009-12-17 University Of Stavanger Transformation mitochondriale
US8318497B2 (en) 2002-09-03 2012-11-27 Universite Libre De Bruxelles Reversible, parallel and multitask cloning method and kit
US8470580B2 (en) 2001-02-23 2013-06-25 Universite Libre De Bruxelles Method for the selection of recombination clones comprising a sequence encoding an antidote protein to a toxic molecule
US9309518B2 (en) 2002-09-03 2016-04-12 Universite Libre De Bruxelles Reversible, parallel and multitask cloning method and kit
US9333227B2 (en) 2013-08-19 2016-05-10 Syngulon Sa. Controlled growth of microorganisms
US11932672B2 (en) 2017-12-19 2024-03-19 Syngulon S.A. Fermentation process

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050260585A1 (en) * 2002-03-19 2005-11-24 Cedric Szpirer Poison/antidote genetic systems for the selection of genetically modified eucaryote cells or organisms
DE10256042B4 (de) * 2002-11-30 2006-06-08 Universität Potsdam Verfahren zur Selektion von Zellen, die spezifisch bindende Moleküle produzieren
US8293503B2 (en) 2003-10-03 2012-10-23 Promega Corporation Vectors for directional cloning

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5527695A (en) * 1993-01-29 1996-06-18 Purdue Research Foundation Controlled modification of eukaryotic genomes
EP0632054A1 (fr) * 1993-06-28 1995-01-04 European Molecular Biology Laboratory Régulation de la recombinaison à site spécifique par des protéines de fusion de recombinase à site spécifique/récepteur nucléaire
EG23907A (en) * 1994-08-01 2007-12-30 Delta & Pine Land Co Control of plant gene expression
WO1997013401A1 (fr) * 1995-10-13 1997-04-17 Purdue Research Foundation Procede de production de plantes hybrides
DE19547272C1 (de) * 1995-12-19 1997-03-27 Ruediger Prof Dr Cerff Ein Expressionssystem für die anaerobe Genexpression in höheren Pflanzen
AR006928A1 (es) * 1996-05-01 1999-09-29 Pioneer Hi Bred Int Una molecula de adn aislada que codifica una proteina fluorescente verde como marcador rastreable para la transformacion de plantas, un metodo para laproduccion de plantas transgenicas, un vector de expresion, una planta transgenica y celulas de dichas plantas.
DE19834430C2 (de) * 1998-07-30 2000-05-31 Harald Von Melchner Selbstdeletierende Vektoren für die Krebstherapie
EP1218488B1 (fr) * 1999-09-21 2007-11-21 Rutgers, The State University Of New Jersey Systeme de recombinaison specifique au site permettant de manipuler le genome du plaste de plantes superieures
DE10024740A1 (de) * 2000-05-19 2001-11-29 Mpb Cologne Gmbh Molecular Pla Verfahren zur gesteuerten Eliminierung gewünschter DNA-Sequenzen in Wirtsorganismen

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7183097B1 (en) 1998-05-07 2007-02-27 Universite Libre De Bruxelles Cytotoxin-based biological containment
US7595186B2 (en) 1998-05-07 2009-09-29 Université Libre de Bruxelles Cytotoxin-based biological containment
US7595185B2 (en) 1998-05-07 2009-09-29 Université Libre de Bruxelles Cytotoxin-based biological containment
US8470580B2 (en) 2001-02-23 2013-06-25 Universite Libre De Bruxelles Method for the selection of recombination clones comprising a sequence encoding an antidote protein to a toxic molecule
US8877504B2 (en) 2001-02-23 2014-11-04 Universite Libre De Bruxelles Method for the selection of recombinant clones comprising a sequence encoding an antidote protein to toxic molecule
US8318497B2 (en) 2002-09-03 2012-11-27 Universite Libre De Bruxelles Reversible, parallel and multitask cloning method and kit
US9309518B2 (en) 2002-09-03 2016-04-12 Universite Libre De Bruxelles Reversible, parallel and multitask cloning method and kit
WO2009150441A1 (fr) * 2008-06-13 2009-12-17 University Of Stavanger Transformation mitochondriale
US9333227B2 (en) 2013-08-19 2016-05-10 Syngulon Sa. Controlled growth of microorganisms
US10188114B2 (en) 2013-08-19 2019-01-29 Syngulon Sa Controlled growth of microorganisms
US11427800B2 (en) 2013-08-19 2022-08-30 Syngulon Sa Controlled growth of microorganisms
US11932672B2 (en) 2017-12-19 2024-03-19 Syngulon S.A. Fermentation process

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Publication number Publication date
WO2002012474A3 (fr) 2002-04-25
AU2001279561A1 (en) 2002-02-18
DE10038573A1 (de) 2002-02-21

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