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WO2003068971A1 - Procede d'expression d'une sequence de codage par des cellules xenopus - Google Patents

Procede d'expression d'une sequence de codage par des cellules xenopus Download PDF

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Publication number
WO2003068971A1
WO2003068971A1 PCT/NL2003/000123 NL0300123W WO03068971A1 WO 2003068971 A1 WO2003068971 A1 WO 2003068971A1 NL 0300123 W NL0300123 W NL 0300123W WO 03068971 A1 WO03068971 A1 WO 03068971A1
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WIPO (PCT)
Prior art keywords
cell
nucleotide
promoter
sequence
construct
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/NL2003/000123
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English (en)
Inventor
Gerardus Julianus Maria Martens
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Multigen Holding SA
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Multigen Holding SA
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Filing date
Publication date
Application filed by Multigen Holding SA filed Critical Multigen Holding SA
Priority to AU2003211329A priority Critical patent/AU2003211329A1/en
Publication of WO2003068971A1 publication Critical patent/WO2003068971A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/50—Amphibians, e.g. Xenopus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to a method of expressing a nucleotide sequence wherein a cell is made transgenic with i) a nucleotide construct comprising a promoter that is functionally linked to the nucleotide sequence to be expressed; or ii) the complementary sequence of the nucleotide construct.
  • the method according to the invention is characterised in that the cell is a Xenopus cell and is made transgenic using a nucleotide construct comprising a POMC promoter, which is functionally linked to the nucleotide se- quence to be expressed; or the complementary sequence of the nucleotide construct.
  • the present invention makes it possible to bring about expression in intermediate pituitary melantrope cells.
  • An in- teresting aspect of the invention is that the activity of these intermediate pituitary melantrope cells can be altered (increased or decreased) , without administering a non-self compound. All that is required is for the animal to be placed on a black or white surface. In contrast to induction by par- enterally administrating (i.e. injecting) an inducing compound, this can be done without any stress to the animals, especially if the animals are used to being handled. Alternatively, the surroundings of the animal can be adapted, for example using one or several electrochrome walls (including the floor) .
  • the colour of the wall can be changed, that is to say without any physical contact with the animal.
  • the moment in time that cell activation takes place is further very well defined.
  • Intermediate pituitary melantrope cells can be isolated and biochemically examined using a large variety of techniques well known in the art.
  • the present invention makes it possible to study the function of a gene in an embryonic stage (when the promoter induces expression) and in later stages of development, in which, depending on the modification of the POMC promoter (see below) , expression is made possible to a lesser or greater extent.
  • a POMC promoter is understood to be a promoter of the prohormone "proopiomelanocor- tin” or a modified nucleotide sequence thereof being expressed in the intermediate pituitary melantrope cells only and exhibiting an activity that depends on the colour of the surface.
  • the nucleotide sequence of this promoter is known and described in Deen, P.M.T. et al. (Mol. Biol. Evol . 9 , pp. 483-494 (1992)).
  • the modified POMC promoter one or more nucleotides of the POMC promoter nucleotide sequence may be deleted, replaced or inserted without loss of the typical POMC promoter activity as expressed in the tissue specificity.
  • the modified POMC promoter according to the invention has a length of at least 100 nucleotides.
  • the modified POMC promoter according to the invention has one or several domains exhibiting homology with the POMC promoter, which ho- mology is at least 40%, preferably at least 60%, more preferably at least 80% and most preferably at least 95%. When a construct is made of these homologous domains, it should result in a construct exhibiting substantially the same tissue specificity as the POMC promoter.
  • the homology may be discon- tinuous, i.e. one or several non-homologous segments may be present in the POMC promoter or in the modified POMC promoter.
  • the POMC promoter facilitates very strong expression of the nucleotide sequence. This opens the possibility of very simply, for example by deletion, limiting the POMC pro- moter activity to a level at which expression is not so strong as to inevitably disrupt the physiological processes in the cell, irrespective of which sequence is being ex- pressed.
  • heterologous gene is a gene that in nature is not controlled by a POMC promoter.
  • the heterologous gene will originate from an organism other than the host cell that is being made transgenic, for example a human gene.
  • the term "to bring to expression” is the subjection to transcription resulting in an RNA molecule.
  • This may a) undergo translation resulting in a polypeptide or a functional or non-functional protein; b) hybridise with another RNA or DNA molecule, thereby disrupting the transcription or translation of the other RNA or DNA molecule; or c) form an intramolecular RNA hybrid processed by the cell and resulting in RNA interference, thereby dis- rupting the transcription or translation of the other RNA or DNA molecule.
  • nucleotide construct is not necessarily a desoxynucleotide sequence, that the same may be double-stranded or single- stranded, and that in the case of a single-strand sequence, the same may be the complementary, i.e. the antisense sequence. Since the present application was written to be understood by the ordinary person skilled in the art, the interested layman may refer to standard works such as Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York, USA (1989) ) and a university at home or abroad teaching molecular biology.
  • the nucleotide construct further comprises a second promoter, which is functionally linked to an indicator gene; or comple- mentary sequence of the nucleotide construct.
  • the second promoter may be any promoter with a broad tissue specificity. This could conceivably be an elongation factor (EF) l promoter for expressing the indicator gene (the marker) in all cells of an embryo, a neural tubulin pro- moter for expression of the marker in brain cells of the embryo, or a muscle promoter for expressing the marker in tail muscle cells of the embryo.
  • EF elongation factor
  • the indicator organ encodes for Green Fluorescent Protein (GFP) .
  • GFP Green Fluorescent Protein
  • the POMC promoter makes it possible to specifically express the gene in the intermediate pituitary melantrope of Xenopus .
  • a cell from the South African clawed toad frog Xenopus laevis or Xenopus tropicalis
  • This provides a simple model system for obtaining numerous progenies, all of which have the inserted nucleotide construct in the same site.
  • the cell is preferably an egg cell.
  • the nucleus of a transgenic cell is removed and transferred to a cell from which the nucleus has been removed.
  • the nuclei may be isolated from cells of an early transgenic embryo. These nu- clei can then be transferred to empty eggs. In this way a large number of essentially identical animals can be obtained. Since the original embryo was transgenic it is also certain that all the animals obtained will be transgenic as well .
  • the invention also relates to a vector comprising a
  • POMC promoter and a nucleotide sequence that may or may not be encoding and that is under the control of the POMC promoter; or the complementary sequence of the POMC promoter and the nucleotide sequence that may or may not be encoding.
  • Such a vector may conveniently be used for making a cell transgenic, for example, by using the technique described by Sparrow, D.B. et al. Nucleic Acids Res. 2_8: E12 (2000) .
  • the invention relates to E. Coli strain DH10-B with plasmid pPOMC-GFP2, as registered with the Cen- traal Bureau voor Schimmelcultures under number CBS110047.
  • This strain comprises a vector affording an excel- lent starting point for the ordinary person skilled in the art for the application of the method according to the invention, wherein the expression of the nucleotide sequence is adjusted to a level that does not inevitably disrupt physiological processes in the host cell.
  • the present invention will now be elucidated with reference to the exemplary embodiments below and the drawing in which the only figure represents a Western blot.
  • a DNA fragment of 529 base pairs containing the nucleotides -487 to +41 of the Xenopus POMC gene A promoter (wherein +1 is the transcription initiation site) was ampli- fied with the aid of PCR using the Xenopus genomic clone AXPA5 (Deen, P.M.T. et al . , Mol . Biol. Evol. 9, pp 483-494 (1992)) as a template.
  • the primers used were pPOMC5 ' : 5 ' - ACGCGTCGACGGTACCCCGTGTAAATGTCCCTCTCC-3 ' , and pPOMC3 ' : 5 ' - TAAGAAGCTTCACTAGTCCCAAGCTGTGC-3' .
  • This fragment was incorpo- rated in a pCSGFP2 construct (a gift from Dr. E. Amaya, Wellcome, Cambridge, Great Britain) by replacing its CMV promoter with the Xenopus POMC promoter (Sall/Hindlll fragment) obtained by PCR.
  • the pCSGFP2 construct contains SV40 pA behind the GFP cDNA for coupling a SV40 poly-A tail to the GFP mRNA.
  • the nucleotide sequence of the pPOMCGFP construct obtained were checked with the aid of a Big Dye Ready Reaction system (Perkin Elmer, Norwalk, CT, USA) and the pPOMC5 ' primer.
  • a Big Dye Ready Reaction system Perkin Elmer, Norwalk, CT, USA
  • an E. coli strain DH10-B was registered under number CBS110047, which strain contains a plasmid of 4319 base pairs with a nucleotide construct wherein the gene coding for GFP is under the control of the POMC promoter.
  • the gene for GFP may be replaced by cutting with the restriction enzymes BamHI and Xbal.
  • Sperm nuclei were purified using a Percoll gradient as described by Sparrow, D.B. et al . (supra), using digitonin for permeabilisation of sperm cells and the REMI reaction was omitted.
  • a Sall/Notl fragment of 1557 base pairs containing the pPOMCGFP construct and the SV40 pA signal were purified using the Qiaex II Gel Extraction Kit (Qiagen, Valencia, CA, USA) . 100 ng/2 ⁇ l of this fragment were mixed with sperm nu- clei (2.5xl0 5 in 2.5 ⁇ l "Sperm storage buffer" (Amaya E. et al. Mol. Biol. 97 pp 393-414 (1999)). Using the method described by Sparrow et al. (supra) the mixture obtained was diluted to 500 ⁇ l with "sperm dilution buffer" (Amaya E. et al., supra), and per egg ⁇ 10 nl were injected at a tempera- ture of 18 * C.
  • cleaving embryos were selected at the 4- cell stage and cultured in 0.1xMMR/ ⁇ % Ficoll-400 with 50 ⁇ g/ml Gentamycin at 18°C until gastrulation (stage 12) was reached. From that moment on culturing of the embryos was continued in 0. lxMMR with 50 ⁇ g/ml Gentamycin and the temperature was raised to 22°C. From stage 45 onward the tadpoles were raised in tap water at 22°C.
  • transgenic frogs were generated that express a fusion protein.
  • the fusion protein was a combination of so-called Xp24 ⁇ 2 protein and Green Fluorescent Protein (GFP) .
  • the fusion protein was expressed in the neurointermediate lobe (NIL) .
  • NIL neurointermediate lobe
  • WT and WT transgenic animals were placed on a white (WA) or a black (BA) surface for three weeks and then sacrificed, after which the NIL and the AL were removed. A Western blot was carried out.
  • the fusion protein was only found in the NIL, and not in the AL (anterior lobe) , again emphasising the tissue specificity.
  • the Western blot shows that in the NIL the expression of the fusion protein was 15 times higher in the black animals (placed on a black surface) than in the white animals (placed on a white surface) .

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Abstract

L'invention concerne un procédé d'expression d'une séquence nucléotidique. Ce procédé consiste à rendre une cellule xénopus transgénique par I) une construction nucléotidique comprenant un promoteur qui est fonctionnellement lié à la séquence nucléotidique à exprimer ou par ii) la séquence complémentaire de la construction nucléotidique. Selon l'invention, la cellule est rendue transgénique à l'aide d'une construction nucléotidique comprenant un promoteur POMC qui est fonctionnellement lié à la séquence nucléotidique à exprimer, ou de la séquence complémentaire de la construction nucléotidique.
PCT/NL2003/000123 2002-02-18 2003-02-18 Procede d'expression d'une sequence de codage par des cellules xenopus Ceased WO2003068971A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003211329A AU2003211329A1 (en) 2002-02-18 2003-02-18 Method of expressing an encoding sequence by xenoplus cells

Applications Claiming Priority (2)

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NL1019997 2002-02-18
NL1019997A NL1019997C2 (nl) 2002-02-18 2002-02-18 Werkwijze voor het tot expressie brengen van een coderende volgorde.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107980710A (zh) * 2016-10-26 2018-05-04 张伟 一种蟾蜍脱皮收集六步法
CN109258570A (zh) * 2018-10-10 2019-01-25 安徽省农业科学院水产研究所 一种蟾蜍亲本营养强化培育方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103782957B (zh) * 2014-02-14 2017-12-19 山东康源堂中药饮片股份有限公司 一种林下养殖蟾蜍的管理方法

Citations (1)

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Non-Patent Citations (9)

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Title
CHALFIE MARTIN ET AL: "Green fluorescent protein as a marker for gene expression.", SCIENCE (WASHINGTON D C), vol. 263, no. 5148, 1994, pages 802 - 805, XP002219685, ISSN: 0036-8075 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1996, CHAN AGNES PUI-YEE ET AL: "Nuclear transplantation from stably transfected cultured cells of Xenopus.", XP002219689, Database accession no. PREV199699131476 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; December 2001 (2001-12-01), CONE R D ET AL: "The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis.", XP002219688, Database accession no. PREV200200196467 *
DEEN P M ET AL: "Comparative structural analysis of the transcriptionally active proopiomelanocortin genes A and B of Xenopus laevis.", MOLECULAR BIOLOGY AND EVOLUTION. UNITED STATES MAY 1992, vol. 9, no. 3, May 1992 (1992-05-01), pages 483 - 494, XP002219687, ISSN: 0737-4038 *
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JANSEN ERIC J R ET AL: "Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis.", FEBS LETTERS. NETHERLANDS 10 APR 2002, vol. 516, no. 1-3, 10 April 2002 (2002-04-10), pages 201 - 207, XP002242501, ISSN: 0014-5793 *
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TREMBLAY Y ET AL: "PITUITARY-SPECIFIC EXPRESSION AND GLUCOCORTICOID REGULATION OF A PROOPIOMELANOCORTIN FUSION GENE IN TRANSGENIC MICE", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 85, no. 23, 1988, 1988, pages 8890 - 8894, XP002219684, ISSN: 0027-8424 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107980710A (zh) * 2016-10-26 2018-05-04 张伟 一种蟾蜍脱皮收集六步法
CN109258570A (zh) * 2018-10-10 2019-01-25 安徽省农业科学院水产研究所 一种蟾蜍亲本营养强化培育方法

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AU2003211329A1 (en) 2003-09-04
NL1019997C2 (nl) 2003-08-19

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