WO2009015623A1 - Monitorage d'effets biologiques au moyen d'hydres transgéniques - Google Patents

Monitorage d'effets biologiques au moyen d'hydres transgéniques Download PDF

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Publication number
WO2009015623A1
WO2009015623A1 PCT/DE2008/001086 DE2008001086W WO2009015623A1 WO 2009015623 A1 WO2009015623 A1 WO 2009015623A1 DE 2008001086 W DE2008001086 W DE 2008001086W WO 2009015623 A1 WO2009015623 A1 WO 2009015623A1
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Prior art keywords
hydra
transgenic
reporter gene
stress factor
seq
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PCT/DE2008/001086
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German (de)
English (en)
Inventor
Thomas C. G. Bosch
Konstantin Khalturin
Original Assignee
Christian-Albrechts-Universität Zu Kiel
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Publication of WO2009015623A1 publication Critical patent/WO2009015623A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2520/00—Use of whole organisms as detectors of pollution

Definitions

  • the invention relates to biomonitors, in particular for the control of drinking water and water quality, usable transgenic hydra, integrable in common vectors expression cassettes for their production and a method for the detection of pollutants by means of transgenic hydra.
  • DE 698 25 873 T2 describes a method for automatic biological monitoring of the water quality by evaluating the respiratory behavior and the body movement of the fish used in this case.
  • the freshwater polyp Hydra is a long established toxicological test system. So z. B. the high regenerative capabilities of this basal multicellular in the production of "artificial embryos" from cell aggregates used, which can be used in particular for the detection of teratogenic (teratogenic) toxicological effects of chemicals, as described, for example, in US 4,346,070 is also the use of Hydra as a biomonitor in drinking water, wastewater and water monitoring z Arkhipchuk et al. Environ Pollut, 2006 JuI; 142 (2): 200-11), Pardos et al.
  • transgenic mouse model described in WO 99/11772 is based on a similar principle.
  • the transgenic mice or the cell cultures derived therefrom express a reporter gene that can be detected by laboratory tests (a human hormone).
  • the freshwater polyp Hydra is a classic freshwater organism and is very sensitive to even the slightest traces of impurities and is therefore often superior to the widespread Microtox test in terms of sensitivity (Pardos et al., Acute toxicity assessment of polish (waste) water with a microplate-based Hydra attenuata assay: a comparison with the Microtox test "Be Total Environ., 1999 Dec 15; 243-244: 141-8), but so far it is not state of the art using either Hydra or any of the others It is therefore possible to differentiate the individual stress factors (pollutants, germs and other harmful environmental factors) acting on the respective biomonitor from one another, and there is therefore a need for biomonitoring systems which are suitable for this purpose.
  • the object of the present invention is therefore to provide a practicable, sensitive and cost-effective biomonitoring system for drinking water and water monitoring, which provides immediate information about the type of a detected stress factor.
  • the object is achieved by the provision of transgenic hydra having the features listed in claim 1.
  • the subclaims indicate advantageous embodiments of the invention.
  • Hydra is a common organism found in all aquatic habitats. The polyps live in all fresh waters and react very rapidly to a deterioration of the water quality (heavy metal pollution, etc.) or the water temperature with a stress response (Bosch et al., Thermotolerance and synthesis of heat shock proteins: Hydra attenuata but absent in Hydra oligactis. "Proc Natl Acad Sci USA, 1988 Nov; 85 (21): 7927-31).
  • the inventors have discovered genes in Hydra that are specifically regulated by different environmental signals (stress factors) such as temperature, presence of toxic substances and microbial germs. These are the Hydra gene hsp70.1 (Acces- sion No. M84019, SEQ ID NO: 1) and periculin (SEQ ID NO: 2).
  • hsp70.1 encodes the Hydra Heat Shock Protein (HSP) 70.
  • HSP Hydra Heat Shock Protein
  • This gene is very sensitive to temperature variations in the water surrounding the hydrate (Gellner et al., "Cloning and expression of an inducible hsp70 gene in two species oihydra which Differ in their stress response "Eur J Biochem 1992, 210, 683-691.)
  • traces of toxic substances eg, sodium azide, heavy metals
  • promoter 5'-regulatory region of this gene is outstandingly suitable for the sensitive and specific regulation of a reporter gene for the detection of toxic substances.
  • the transcription of the hydra gene periculin is specifically induced by microbial water contaminants such as bacteria, fungi and viruses or by substances characteristic for them, for example lipopolysaccharides (LPS) from gram-negative bacteria or viral double-stranded RNA.
  • LPS lipopolysaccharides
  • the corresponding periculin promoter can therefore be used as a very sensitive and specific regulator for the transcription of a microbial contamination indicating reporter gene.
  • the respective promoters of these genes could be identified and integrated into different DNA constructs (expression cassettes). According to the invention, these are flanked by Nelt restriction sites and can be easily integrated into a vector backbone suitable for cloning and propagation in the bacterium Escherichia coli.
  • Each expression cassette contains unique restriction endonucleases for the restriction endonucleases ApaLI, XbaI, BamHI, Sbfl, Pacl, AsiSI, AscI and Fsel. These interfaces allow for easy replacement and combining of different promoters and reporter genes.
  • the expression cassettes each contain at least one of the above-described stress promoters and the respective reporter gene regulated by it (eg GFP, eGFP, YFP, eYFP, CFP, dsRED, BFP, mTFPl, Emerald, Citrine, mOrange, mApple, mCherry, mGrape ).
  • the reporter gene is followed by an expression termination sequence, preferably the hydra actin terminator.
  • the expression cassette may contain another fluorescent light detectable reporter gene, which is under the control of a strong constitutive promoter.
  • a strong constitutive promoter This can be z.
  • the promoter of the Hydra actin gene as shown in Fig. 2.
  • This additional constitutively expressible in Hydra reporter gene may optionally after microinjection of the expression vectors in early hydra embryos, as in Wittling et al. ("Transgenic Hydra Allow In Vivo Tracking of Individual Stem Cells During Morphogenesis.” Proc Natl Acad US Pat. No.
  • transgenic Hydra lines can be generated, each of which is suitable for indicating the presence of a specific stress factor via the stress-inducible expression of a suitable reporter gene and its, for example, fluorescence-detectable gene product 4 and 6), only one stress-inducible reporter gene per expression cassette is shown by way of example in each of Figures 1 and 2.
  • it is just as possible to integrate two different stress-inducible reporter genes into the expression cassette In this case, reporter genes selected whose gene products under fluorescent light color can be clearly distinguished from each other kö For example, GFP and dsRED.
  • the transgenic hydra prepared by means of these multiply-inducible reporter gene constructs are incubated under fluorescent light upon contact with heavy metals, eg. B. green and in the presence of bacteria z. B. glow red. This allows a distinction to be made between different environmental stress factors with one and the same transgenic hydra line.
  • multiply-inducible reporter gene constructs it may be advantageous to dispense with the additional constitutively expressed reporter genes which serve to control the transfection success in order to avoid negative interactions with the inducible reporter genes.
  • z. B. one of the inducible reporter genes by short-term incubation with the respective stress factor for controlling the transfection success and for the selection of the transfected animals are used.
  • the nucleic acid sequences of the different expression cassettes according to the invention are shown under SEQ ID: N0 3-5.
  • transgenic hydra-lines thus prepared as biomonitors, it is possible, for example, to provide automatic systems in which the transgenic hydra are continuously bathed in the water to be monitored.
  • the corresponding hydra cultures are irradiated either permanently, but preferably in order to avoid premature bleaching of the fluorescence, at short intervals with fluorescent light of a suitable excitation wavelength. This can be z. B. with the aid of a modified fluorescence microscope or preferably a fluorescence Auflichtbinokulars done.
  • the culture is analyzed for the expression of the respective reporter genes at each irradiation interval.
  • the transgenic hydra according to the invention are also suitable for the targeted analysis of water samples taken from fresh water.
  • the hydra cultures are incubated for a certain time with the respective water samples and checked at regular intervals, as described above, for the expression of the respective reporter gene products.
  • substances which may also be dissolved in water and are to be tested for toxicity can be tested for their biological effect with the transgenic hydra according to the invention.
  • transgenic Hydra which express a particular reporter gene in the surrounding medium when a certain stress factor is present, thus allows the practical kable, sensitive and cost-effective monitoring of the quality of drinking water and bodies of water. Furthermore, the use of transgenic Hydra also allows statements on functional and structural changes at the level of cells and tissues. These include the detection of oxidative stress, pathological tissue changes, enzyme inhibition, dysfunctions of cell differentiation, genotoxic effects and much more.
  • FIG. 2 is a more detailed illustration of an exemplary expression cassette.
  • FIG. 6 shows a transgenic hydra which expresses the reporter gene dsRED under regulation of the pericinol promoter.
  • Fig. 1 is a schematic general representation of the expression vector constructs is shown.
  • the group of these expression vectors for the preparation of transgenic hydra suitable as biomonitors was designated as pHyMON by the inventors.
  • the vector carries, as a bacterial selection marker, an ampicillin resistance gene (Amp R ) as well as SP6 and T7 transcription initiation sequences.
  • An expression cassette with two reporter genes was inserted.
  • FIG. 1 A more detailed representation of the exemplary expression cassette incorporated in the vector shown in FIG. 1 is shown in FIG.
  • further cut-off cells for the restriction endonucleases ApaLI, XbaI, BamHI, HindIII, PstI, Sbfl, Päd, AsiSI, EcoRI, Spei, AscI and Fsel are indicated.
  • Individual restriction sites used to exchange promoters and reporter genes are shown in gray.
  • the position and orientation of the stress promoter and the stress-inducible reporter gene (in this case GFP) controlled by it, as well as the position and orientation of the constitutively active actin promoter and the reporter gene constitutively expressed via it (here dsRED) are indicated.
  • 3 'of the respective reporter gene is a termination sequence corresponding to the termination region of the hydraacetin gene.
  • the GFP reporter gene is only expressed if a certain stress factor is present, the dsRED reporter gene product here serves to control the transfection efficiency and the selection of transgenic animals.
  • FIG. 3 shows an early hydra embryo which is sucked in via the fixation pipette (left) of the micromanipulator and thus fixed.
  • the injection cannula with the vector solution can be seen at the bottom right.
  • FIG. 4 shows a transgenic hydra which expresses the reporter gene GFP under the regulation of the heavy metal-sensitive hsp70.1 promoter.
  • To the medium surrounding the hydra was added 25 ⁇ mol / l cadmium chloride and the hydra incubated therein for 2 hours.
  • the GFP expressed by the Hydra in all body cells glows bright green under fluorescent light.
  • FIG. 5 shows the concentration-dependent induction of the wild-type gene periculin in Hydra vulgaris by lipopolysaccharide (LPS - a characteristic cell wall constituent of Gram-negative bacteria) and after transfection with double-stranded RNA, as frequently occurs in viruses.
  • FIG. 6 shows a transgenic hydra expressing the reporter gene dsRED under regulation of the periculin promoter. The water surrounding the hydra was added to 20 ng / ml LPS and the hydra incubated therein for 2 hours. The Hydra shines red in many places. The clonal selection has not progressed so far that the reporter gene is expressed in all body cells.
  • transgenic hydra were, as described in Wittsch et al. ("Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis.") Proc Natl Acad US Pat. No. 2006 Apr 18; 103 (16): 6208-l 1) is described in detail therefore briefly summarized:
  • Reporter gene constructs are used to transfect the Hyc / ra polyps.
  • the transfection is carried out by microinjection of H. vulgaris (AEP) embryos in the 2-8 cell stage. Before the microinjection, the embryos are detached from the maternal tissue.
  • the microinjection is performed under an inverted microscope (Zeiss Axiovert 100) and two micro-manipulators (Leitz, Eppendorf). Embryos are maintained with a micropipette using a CellTram Air pump (Eppendorf).
  • the respective construct (0.1 ⁇ l, 0.6 ⁇ g vector / ⁇ l) is injected with a CellTram vario pump (Eppendorf).
  • the glass needles for microinjection are made with a Vertical Pipette Puller 700 C (Kopf Instruments, Tujunga, CA). Between 20 and 30% of the embryos express the construct from day 2 onwards. After hatching, the polyps express the reporter gene as "mosaics" in small groups of cells, and clonal propagation produces polyps from such founder polyps that carry the expression construct in all cells of the body or cell line.

Abstract

L'invention concerne des hydres transgéniques qui, en présence d'au moins un facteur de stress dans le milieu qui les entoure, expriment au moins un gène rapporteur inductible par le facteur de stress, l'utilisation de ces hydres transgéniques comme système de biomonitorage, ainsi qu'un procédé de détermination de facteurs de stress dans des échantillons au moyen d'hydres transgéniques. Elle concerne en outre des cassettes d'expression qui, après intégration dans des vecteurs appropriés, peuvent être utilisées pour générer des hydres transgéniques utilisables en tant que biomoniteurs.
PCT/DE2008/001086 2007-07-27 2008-06-25 Monitorage d'effets biologiques au moyen d'hydres transgéniques WO2009015623A1 (fr)

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DE102007035238.9 2007-07-27
DE200710035238 DE102007035238A1 (de) 2007-07-27 2007-07-27 Biologisches Effektmonitoring mittels transgener Hydra

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4346070A (en) * 1980-04-11 1982-08-24 Thomas Jefferson University Test for teratogenic potential employing hydra
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
US5877398A (en) * 1993-01-29 1999-03-02 University Of British Columbia Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology
WO2003079967A2 (fr) * 2002-03-26 2003-10-02 Nanocyte Inc. Cellules urticantes exprimant un polynucleotide exogene qui code pour un agent therapeutique, diagnostic ou cosmetique, et procedes, compositions et dispositifs faisant intervenir l'utilisation de ces cellules urticantes ou de capsules derivees de celles-ci pour administrer l'agent therapeutique, diagnostic ou cosmetique a

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US6017722A (en) 1991-04-04 2000-01-25 Board Of Regents, The University Of Texas System Luminous bacteria and methods for the isolation, identification and quantitation of toxicants
DE4440320A1 (de) 1994-11-11 1995-03-30 Wilfried Dr Rer Nat Pauli Verfahren und Vorrichtung zur Ermittlung der Umweltbelastung durch Chemikalien in wäßrigen Proben mit Bioindikatoren
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US4346070A (en) * 1980-04-11 1982-08-24 Thomas Jefferson University Test for teratogenic potential employing hydra
US5877398A (en) * 1993-01-29 1999-03-02 University Of British Columbia Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO2003079967A2 (fr) * 2002-03-26 2003-10-02 Nanocyte Inc. Cellules urticantes exprimant un polynucleotide exogene qui code pour un agent therapeutique, diagnostic ou cosmetique, et procedes, compositions et dispositifs faisant intervenir l'utilisation de ces cellules urticantes ou de capsules derivees de celles-ci pour administrer l'agent therapeutique, diagnostic ou cosmetique a

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ARKHIPCHUK ET AL: "Use of hydra for chronic toxicity assessment of waters intended for human consumption", ENVIRONMENTAL POLLUTION, BARKING, GB, vol. 142, no. 2, 1 July 2006 (2006-07-01), pages 200 - 211, XP005394798, ISSN: 0269-7491 *
BOSCH TCG ET AL: "Uncovering the evolutionary history of innate immunity: The simple metazoan Hydra uses epithelial cells for host defence", DEVELOPMENTAL & COMPARATIVE IMMUNOLOGY; DOI:10.1016/J.DCI.2008.10.004, 12 November 2008 (2008-11-12), XP002506362, Retrieved from the Internet <URL:http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T5X-4TX1545-3&_user=987766&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000049880&_version=1&_urlVersion=0&_userid=987766&md5=9d405fcdeee0a668b06ab2ab9ffcd4c2> [retrieved on 20081202] *
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BRENNECKE THOMAS ET AL: "The lack of a stress response in Hydra oligactis is due to reduced hsp70 mRNA stability", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 255, no. 3, 1 August 1998 (1998-08-01), pages 703 - 709, XP002506297, ISSN: 0014-2956 *
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PARDOS MICHEL ET AL: "Acute toxicity assessment of Polish (waste)water with a microplate-based Hydra attenuata assay: A comparison with the Microtox(R) test", SCIENCE OF THE TOTAL ENVIRONMENT, no. 243-244, 15 December 1999 (1999-12-15), pages 141 - 148, XP009109433, ISSN: 0048-9697 *
REN S ET AL: "THE USE OF A GENETICALLY ENGINEERED PSEUDOMONAS SPECIES (SHK1) AS A BIOLUMINESCENT REPORTER FOR HEAVY METAL TOXICITY SCREENING IN WASTEWATER TREATMENT PLANT INFLUENT", RESEARCH JOURNAL OF THE WATER POLLUTION CONTROL FEDERATION, WATER POLLUTION CONTROL FEDERATION, ALEXANDRIA, VA, US, vol. 75, no. 1, 1 January 2003 (2003-01-01), pages 21 - 29, XP001144950, ISSN: 1047-7624 *
WITTLIEB JORG ET AL: "Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 103, no. 16, April 2006 (2006-04-01), pages 6208 - 6211, XP002506299, ISSN: 0027-8424 *

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